Insulin-Deficient Mouse β-Cells Do Not Fully Maturebut Can Be Remedied Through Insulin Replacementby Islet Transplantation

Insulin-Deficient Mouse β-Cells Do Not Fully Maturebut Can Be Remedied Through Insulin... Abstract Insulin receptor (IR) insufficiency in β-cells leads to impaired insulin secretion and reduced β-cell hyperplasia in response to hyperglycemia. Selective IR deficiency in β-cells in later embryological development may lead to compensatory β-cell hyperplasia. Although these findings suggest insulin signaling on the β-cell is important for β-cell function, they are confounded by loss of signaling by the insulinlike growth factors through the IR. To determine whether insulin itself is necessary for β-cell development and maturation, we performed a characterization of pancreatic islets in mice with deletions of both nonallelic insulin genes (Ins1−/−Ins2−/−). We immunostained neonatal Ins1−/−Ins2−/− and Ins1+/+Ins2+/+ pancreata and performed quantitative polymerase chain reaction on isolated neonatal islets. Insulin-deficient islets had reduced expression of factors normally expressed in maturing β-cells, including muscoloaponeurotic fibrosarcoma oncogene homolog A, homeodomain transcription factor 6.1, and glucose transporter 2. Ins1−/−Ins2−/−β-cells expressed progenitor factors associated with stem cells or dedifferentiated β-cells, including v-myc avian myolocytomatosis viral oncogene lung carcinoma derived and homeobox protein NANOG. We replaced insulin by injection or islet transplantation to keep mice alive into adulthood to determine whether insulin replacement was sufficient for the completed maturation of insulin-deficient β-cells. Short-term insulin glargine (Lantus®) injections partially rescued the β-cell phenotype, whereas long-term replacement of insulin by isogenic islet transplantation supported the formation of more mature β-cells. Our findings suggest that tightly regulated glycemia, insulin species, or other islet factors are necessary for β-cell maturation. Diabetes affects >400 million people worldwide (1) and imposes high financial, disability, and mortality costs for health care systems and patients around the world (2). Regardless the type of diabetes (type 1, type 2, gestational, or rare forms), all patients with diabetes develop an eventual insufficiency of the hormone insulin (3). In humans, β-cells containing insulin first begin to appear in the pancreas at week 7 of development, and insulin acts as a powerful anabolic signaling molecule starting in the 26th week of gestation (4). Uptake of glucose into cells is a fundamental biological process that is almost always dependent on the actions of insulin (5). Without insulin, subjects face elevated glucose and glucagon levels, increased stress hormones and ketogenesis, and eventual death from severe ketoacidosis (6). However, insulin may also play other roles independent of directly lowering blood glucose, including serving as an important signaling molecule on the β-cell itself. Mice with targeted disruption of insulin receptors (IRs) on β-cells have impaired glucose-stimulated insulin secretion and glucose intolerance (7). IRs on β-cells are necessary for β-cell hyperplasia in response to insulin resistance (8). These studies (7, 8) used a transgenic mouse model with the rat insulin promoter expressing Cre, with a floxed insulin receptor gene (Insr). Cre is expressed after E9.5 when insulin first appears in the mouse pancreas (9) and recombines to delete the IR from ~80% (10) of β-cells and regions of the brain (11). Delayed and incomplete recombination allows some insulin signaling in β-cells beyond E9.5, and impaired insulin action in the brain can promote obesity and insulin resistance (12). Therefore, these findings do not conclusively demonstrate that a loss of insulin signaling in β-cells is responsible for the observed phenotype. Recently, Trinder et al. (13) addressed the caveat of central recombination by using mice with an inducible Cre transgene driven by a mouse insulin I promoter to generate IR deficiency selectively in β-cells after E13. MIP-CreERT mice had reduced β-cell mass at birth, but as in previous studies using the RIP-Cre line, these mice retained alternative insulin signaling pathways and attenuated insulinlike growth factor (IGF) signaling in β-cells through the IR, making it challenging to conclude that a loss of insulin action in β-cells was entirely responsible for the phenotype. Alongside incomplete β-cell recombination, delayed loss of IRs until later in development, and the presence of a human growth hormone minigene in the MIP-CreERT transgene (14, 15), there are other important factors to consider when attempting to study a loss of insulin signaling by using a model with deletion of the IR. First, insulin may signal with low affinity through IGF receptors I and II (16, 17), and it is possible that there are other insulin signaling pathways not yet appreciated. Hyperinsulinemia in β-cell IR-deficient mice may increase the likelihood of insulin signaling via the IGF receptors (7). Second, IGF-I and IGF-II bind the IR and play essential roles in embryological mammalian development (18). In fact, binding of IGF-II to the IR appears to be the most important signal through the IR during development (19). Thus, deleting the IR may not completely block all actions of insulin and impairs the IR-mediated actions of the IGF hormones. We posit that the only model capable of defining the role of insulin in β-cell development and maturation is a model with a loss of the insulin gene itself. In 1997 Duvillié et al. (20) generated an absolute insulin-deficient mouse lacking both copies of the two nonallelic insulin genes, Ins1 and Ins2 (Ins1−/−Ins2−/−), with β-galactosidase (βGAL; gene lacZ) knocked in under control of the Ins2 promoter. Ins1−/−Ins2−/− mice are viable at birth, have a slightly (20%) lower body weight, and though euglycemic at the time of parturition quickly develop severe hyperglycemia after suckling and die on average within 48 hours (20, 21). This result aligns with findings in humans with insulin mutations who first present with growth retardation at 27 weeks’ gestation, a similar developmental stage to mice at birth (22). Pancreatic islets of neonatal Ins1−/−Ins2−/− mice are enlarged but have low levels of endocrine hormone messenger RNA (mRNA) [glucagon (Gcg), somatostatin (Sst), and pancreatic polypeptide (Ppy)] (23). The pancreas of Ins1−/−Ins2−/− mice is full of aggregates of βGAL-expressing cells, but apart from positive immunoreactivity for pancreatic and duodenal homeobox 1 (PDX1) in βGAL-expressing cells (23), there are no reports of the functional maturity of these cells to determine the role of insulin signaling during β-cell development. In the current study, we determined the role of insulin during β-cell development and maturation by characterizing pancreatic islets of neonatal Ins1−/−Ins2−/− mice and mice maintained into adulthood by insulin replacement therapy. We found that alongside a complete loss of insulin production, β-cells of Ins1−/−Ins2−/− mice lacked many markers of mature β-cells and were present in islets of abnormal size and endocrine hormone composition. Additionally, insulin-deficient β-cells expressed markers of dedifferentiation and dysfunction, suggesting that insulin is a necessary signal for completely maturing β-cells or maintaining their maturity. Short-term replacement of insulin in the insulin knockout mice via exogenous injection facilitated a partial normalization of the β-cell phenotype but was associated with islet fibrosis. Long-term replacement of insulin by isogenic islet transplantation was sufficient for the maturation of insulin-deficient β-cells. This model provides direct evidence that insulin itself is needed for normal β-cell development and the maintenance of normal β-cell function. Materials and Methods Animal breeding and insulin therapy All experiments were approved by the University of British Columbia (UBC) Animal Care Committee and carried out in accordance with the Canadian Council on Animal Care Guidelines. C57Bl/6j Ins1+/+Ins2+/+ mice (Jackson Laboratories, Bar Harbor, ME) were used as controls. Ins1−/−Ins2+/− mice were generated in the laboratory of Dr. J. Jami (Institut Cochin, Paris, France) and supplied indirectly from the laboratory of Dr. J. Johnson (UBC, Vancouver, Canada). Animals were housed with a 12-hour light/dark cycle with ad libitum access to a standard chow diet (29018; Harlan Laboratories, Madison, WI). Ins1−/−Ins2+/− mice were bred to acquire Ins1−/−Ins2−/− mice. Upon first discovery of neonatal mice (P0 to P1), subsets of mice were euthanized by decapitation, and blood sugar was examined with a handheld glucometer (LifeScan, Burnaby, BC, Canada). Insulin therapy was immediately initiated in other cohorts of Ins1−/−Ins2−/− mice, initially identified by reduced body weight and confirmed by genotyping at an older age. We genotyped Ins2 by quantitative polymerase chain reaction (qPCR) assay (wild-type forward primer 5′-GGT CCT TGG TAG TAA CTT G, reverse primer 5′-GCC TCT AAA GCC TAC TCA TCT TC, and probe 5′-GCA GTG CTC TAT GAG GGC CCT AAA; lacZ knockin forward primer 5′-CTG TAT GAA CGG TCT GGT CTT T, reverse primer 5′-CGC TAT GAC GGA ACA GGT ATT, and probe 5′-TTG CCC GGA TAA ACG GAA CTG GAA) and Ins1 by qPCR assay (wild-type forward primer 5′-CCA TTG TTA GGT TGG ATG ATT, reverse primer 5′-CGG TTG CCT ACC TTC TT, and probe 5′-AGT ATC TGG AAT TCT GCT TCC TGC CC; knockout forward primer 5′-AAA CCA CAC TGC TCG AC, reverse primer 5′-CAG GAA GCA GAA TTC CAG ATA, and probe 5′-GGG CTG CAG GAA TTC GAT ATC AAG C). Briefly, animals received twice-daily subcutaneous injections of ~0.1 U insulin glargine diluted to 5 U/mL in F-10 media (Lantus®; Sigma-Aldrich, St. Louis, MO). Because regular blood glucose sampling is not possible with young pups, health of the animals was tracked by body weight and glycosuria, and when animals failed to gain weight or lost weight, insulin dosing was halved or skipped. When animals presented with severe glycosuria, we increased the insulin dose. As pups grew, we began regular blood sampling to better refine insulin doses. Therapy continued for up to 291 days. Mortality by this method was initially extremely high (>90%) but decreased with experience to <50% for animals analyzed in this study. Some animals received islet transplants into the anterior chamber of the eye at 14 days of age. In all experiments, we present findings from a mix of male and female animals. Islet isolation and transplantation Newborn Ins1−/−Ins2−/− or Ins1+/+Ins2+/+ mice were euthanized by decapitation upon first discovery (P0 to P1), and pancreatic islets were isolated by collagenase digestion (24). After euthanasia, we performed a rapid dissection and exposed the pancreatic duct. Approximately 1 mL of collagenase solution (1000 U/mL type XI collagenase; Sigma-Aldrich; diluted in Hanks buffer without CaCl2) was injected into the pancreatic duct. The pancreas was then excised and digested for 6 to 8 minutes at 37°C in 3 mL of collagenase. Islets were then handpicked three times in medium (Hams F10, 7.5% fetal bovine serum, and penicillin/streptomycin; Sigma-Aldrich) to increase purity to >90%. Islets were washed in phosphate-buffered saline (PBS) and immediately lysed by forceful suction into a 20-gauge needle 20 to 30 times in 50 µL of QIAzol Lysis Reagent (Qiagen, Toronto, ON, Canada) before storage at −80°C. Islet isolation from donor adult C57Bl/6 mice (used for transplantation) was completed in the same fashion, with slight changes (incubation in collagenase for 12 to 15 minutes at 37°C). After handpicking, islets were cultured overnight, visually inspected for purity, and washed in sterile PBS before transplantation. After 2 weeks of insulin therapy with insulin glargine (Lantus®), Ins1−/−Ins2−/− animals received an islet transplant into the anterior chamber of the eye (~100 to 150 islets) as previously described (25). This procedure is technically feasible at 2 weeks of age and provides reversal of diabetes within 24 hours. Animals were anesthetized by inhaled isoflurane (5%) and maintained at 1% to 3%. The cornea was punctured with a 27-gauge needle to gain access to the anterior chamber of the eye. Islets were loaded into a micropipette (MXL3-BP-IND-200; Origio MidAtlantic Devices, Mt. Laurel, NJ), the micropipette was passed through the opening in the cornea, and islets were deposited with a micromanipulator. After removal of the micropipette, animals received Isoptears (Alcon Canada, Mississauga, ON, Canada) with 0.3% wt/vol gentamycin to prevent infection. Immunohistochemistry and immunohistofluorescence Pancreata were dissected out of mice, washed in PBS, and fixed in 4% paraformaldehyde overnight before being transferred to 70% ethanol for long-term storage before paraffin embedding and sectioning (5 µm thickness; Wax-It Histology Services, Vancouver, Canada). Hematoxylin and eosin and Masson’s trichome staining was performed by standard protocol, and slides were scanned with the ScanScope CS system (Aperio, Vista, CA). Immunofluorescent staining was performed as previously described (26). Briefly, sections were deparaffinized in xylene (15 minutes) and rehydrated in graded ethanol (100% 2 × 5 minutes, 95% 5 minutes, 70% 5 minutes, and PBS 10 minutes) before heat-induced epitope retrieval in an EZ Retriever microwave oven (BioGenes, Fremont, CA) for 15 minutes at 95°C in 10 mM citrate buffer (0.5% Tween 20, pH 6.0; Thermo Fisher Scientific, Waltham, MA). Samples were blocked in DAKO Protein Block, Serum Free (Dako, Burlington, Canada) and incubated overnight in primary antibody diluted in Dako Antibody Diluent (Dako). The next day, slides were washed and incubated in secondary antibody for 1 hour at room temperature before mounting and counterstaining with nuclear stain 4′,6-diamidino-2-phenylindole (DAPI) and VECTASHIELD® Hard Set Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA). All images were captured and analyzed with an ImageXpress® Micro XLS System, controlled by MetaXpress® High-Content Image Acquisition & Analysis Software (Molecular Devices Corporation, Sunnyvale, CA) with a scientific CMOS camera, a Nikon 20× Plan Apo objective (NA = 0.75, 1-6300-0196; Nikon, Tokyo, Japan), and DAPI (DAPI-5060B), FITC (FITC-3540B), Cy3 (Cy3-4040B), Texas Red (TXRED-4040B), and Cy5 (Cy5-4040A) filter cubes. We performed quantification of histological images in the same software (MetaXpress®). We used islet amyloid polypeptide (IAPP), insulin, or βGAL to label β-cells, glucagon (GCG) to label α-cells, somatostatin (SST) to label δ-cells, and pancreatic polypeptide (PP) to label pancreatic polypeptide cells. Of note, although δ-cells are known to express some IAPP (27), given the smaller number of δ-cells, lower expression level, and limited colocalization of SST and IAPP (data not shown), we use bright IAPP immunoreactivity as a marker of β-cells. We immunostained for synaptophysin (SYN) to identify islet cells and quantify islet size and number. We calculated endocrine cell area of each cell type by quantifying immunoreactive surface area relative to total pancreas surface area and calculated the average of three immunostained sections 50 to 100 µM apart for each animal. We calculated the proportion of β-cells with positive immunoreactivity for proteins of interest or total immunoreactive area of proteins of interest by thresholding images and using a multiwavelength cell scoring journal in MetaXpress®, and we used image analysis to find the mean fluorescent intensity of positive signal in relevant cell compartments. RNA isolation and quantitative reverse transcription polymerase chain reaction After islet isolation, lysis, and storage in Qiazol Lysis Reagent (Qiagen), we performed acid guanidinium thiocyanate–phenol–chloroform extraction by standard methods (28). To ensure sufficient quantity of mRNA for downstream applications, we pooled lysed islets from three to five mice in each group of Ins1−/−Ins2−/−, Ins1−/−Ins2+/−, or Ins1+/+Ins2+/+ animals, for a total of 25 to 35 islets per sample. We generated complementary DNA from isolated islet RNA by using a RevertAid H Minus First Strand cDNA synthesis kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. We quantified mRNA levels of target genes via the 2−ΔΔCT method (glyceraldehyde 3-phosphate dehydrogenase used as housekeeping gene) by using the SsoFast EvaGreen Supermix (Bio-Rad, Mississauga, Canada) kit according to the manufacturer’s protocol (for primer details see Supplemental Table 1). Statistics Data were subject to the Shapiro–Wilk normality test, and when all groups passed the test for normality we analyzed them by Student t test, one-way analysis of variance with Tukey test for multiple comparisons, or two-way analysis of variance with Bonferroni post hoc testing. When one or more groups failed the test for normality, we used the nonparametric Mann–Whitney U test or Kruskal–Wallis test with the Dunn multiple comparisons test. Statistical analysis was performed in GraphPad Prism 7.01 (La Jolla, CA) with significance set at P < 0.05. Results Ins1−/−Ins2−/− mice are born with β-cells Upon discovery (P0 to P1), Ins−/−Ins2−/− mice had lower body weight compared with Ins1+/+Ins2+/+ controls (Fig. 1A). Ins−/−Ins2−/− mice were euglycemic at the time of delivery but quickly developed hyperglycemia after suckling. Therefore, blood glucose levels were highly variable at the time of blood sampling, unlike in the Ins1+/+Ins2+/+ controls. We immunostained pancreas from mice with reduced insulin gene copy number (Ins1−/−Ins2+/+, Ins1+/+Ins2−/−, or Ins1−/−Ins2+/−) for the prototypical mature β-cell marker muscoloaponeurotic fibrosarcoma oncogene homolog A (MAFA, a basic leucine zipper transcription factor; Supplemental Fig. 1). Given previous reports of abnormal β-cell size and islet structure (29) paired with reduced nuclear immunoreactivity for MAFA in mice with reduced insulin gene copy number, we compared the Ins1−/−Ins2−/− group with wild-type Ins1+/+Ins2+/+ controls in all subsequent experiments. Histological examination of neonatal pancreata revealed normal aggregates of IAPP+βGAL+INS− clusters resembling neonatal islets of Langerhans in Ins1−/−Ins2−/− mice and IAPP+βGAL− insulin-producing (INS+) β-cells in Ins1+/+Ins2+/+ mice (Fig. 1B). Figure 1. View largeDownload slide Ins1−/−Ins2−/− mice have IAPP-expressing β-cells. (A) Upon discovery (P0 to P1), wild-type Ins1+/+Ins2+/+ and insulin knockout Ins1−/−Ins2−/− mice were weighed and their blood glucose was measured (n = 10–31). Individual animal weight or blood glucose is shown in box-and-whisker plots. Statistical analysis to compare body weight was performed with a Student t test to assess significance. ***P < 0.001. (B) Immunostaining of P1 pancreata for INS, IAPP, βGAL, and DAPI in wild-type Ins1+/+Ins2+/+ controls (upper panels) and Ins1−/−Ins2−/− mice (lower panels), revealing isletlike clusters of INS−IAPP+βGAL+ cells in Ins1−/−Ins2−/− pancreata (representative micrographs from n = 3). Scale bar, 100 µm. Figure 1. View largeDownload slide Ins1−/−Ins2−/− mice have IAPP-expressing β-cells. (A) Upon discovery (P0 to P1), wild-type Ins1+/+Ins2+/+ and insulin knockout Ins1−/−Ins2−/− mice were weighed and their blood glucose was measured (n = 10–31). Individual animal weight or blood glucose is shown in box-and-whisker plots. Statistical analysis to compare body weight was performed with a Student t test to assess significance. ***P < 0.001. (B) Immunostaining of P1 pancreata for INS, IAPP, βGAL, and DAPI in wild-type Ins1+/+Ins2+/+ controls (upper panels) and Ins1−/−Ins2−/− mice (lower panels), revealing isletlike clusters of INS−IAPP+βGAL+ cells in Ins1−/−Ins2−/− pancreata (representative micrographs from n = 3). Scale bar, 100 µm. Insulin-deficient islets are enlarged and have an abnormal endocrine cell distribution We characterized the endocrine cell distribution of neonatal islets to determine whether a lack of insulin causes a shift in endocrine cell proportions. Ins1+/+Ins2+/+ and Ins1−/−Ins2−/− islets had a similar distribution of GCG+ α-cells and SST+ δ-cells (including cells in the core of the islet) and no obvious pathological immune infiltration or fibrosis by hematoxylin and eosin and Masson’s trichrome staining (Fig. 2A). Among all hormone-expressing cells, the relative proportion of GCG- and PP-expressing cells decreased and the proportion of SST-expressing cells increased in islets of Ins1−/−Ins2−/− mice compared with Ins1+/+Ins2+/+ controls (Fig. 2B). The proportion of total pancreas section area expressing IAPP and SST was larger and PP was smaller in Ins1−/−Ins2−/− pancreata compared with Ins1+/+Ins2+/+ controls (Fig. 2C) [measurement used as a surrogate of total endocrine cell mass (30)]. We immunostained for ghrelin and found extremely rare cells, as expected, in both controls and insulin-deficient islets (data not shown). Insulin-deficient islets were fewer in number (Fig. 2D) but more than three times as large as islets in pancreata of Ins1+/+Ins2+/+ control mice (Fig. 2E). We immunostained for the proliferating cell nuclear antigen (PCNA) as a marker of replicating cells and observed a pronounced increase in proportion of IAPP+PCNA+/IAPP+ cells in Ins1−/−Ins2−/− mice compared with controls (Fig. 2F). Figure 2. View largeDownload slide Ins1−/−Ins2−/− mice have enlarged islets with abnormal islet cell number and distribution. (A) P1 pancreata of Ins1+/+Ins2+/+ (top panels) and Ins1−/−Ins2−/− (bottom panels) mice were immunostained for IAPP and GCG or GCG, SST, and PP. Hematoxylin and eosin and Masson’s trichrome staining of P1 pancreas (representative micrographs from n = 3–5). Scale bars, 100 µm. (B) Percentage of total endocrine immunoreactive area that is immunoreactive for the islet hormones IAPP, GCG, SST, and PP (n = 4 or 5 animals, 3 sections quantified per animal). (C) β-cell, α-cell, δ-cell, and pancreatic polypeptide cell area relative to total pancreas area (n = 4 or 5 animals, 3 sections quantified per animal). (D) Number of islets identified by SYN immunoreactivity relative to pancreas section area (n = 4 or 5, 3 sections quantified per animal). (E) Average islet size by SYN immunoreactive area (n = 4 or 5, 3 sections quantified per animal). (F) Quantification of percentage of IAPP+ cells that were PCNA+; representative image shown (n = 5). Scale bar, 100 µm. Individual animals are shown on box-and-whisker plots. Statistical analysis was performed via Student t test to assess significance. *P < 0.05, **P < 0.01, ***P < 0.001 vs Ins1+/+Ins2+/+ controls. Figure 2. View largeDownload slide Ins1−/−Ins2−/− mice have enlarged islets with abnormal islet cell number and distribution. (A) P1 pancreata of Ins1+/+Ins2+/+ (top panels) and Ins1−/−Ins2−/− (bottom panels) mice were immunostained for IAPP and GCG or GCG, SST, and PP. Hematoxylin and eosin and Masson’s trichrome staining of P1 pancreas (representative micrographs from n = 3–5). Scale bars, 100 µm. (B) Percentage of total endocrine immunoreactive area that is immunoreactive for the islet hormones IAPP, GCG, SST, and PP (n = 4 or 5 animals, 3 sections quantified per animal). (C) β-cell, α-cell, δ-cell, and pancreatic polypeptide cell area relative to total pancreas area (n = 4 or 5 animals, 3 sections quantified per animal). (D) Number of islets identified by SYN immunoreactivity relative to pancreas section area (n = 4 or 5, 3 sections quantified per animal). (E) Average islet size by SYN immunoreactive area (n = 4 or 5, 3 sections quantified per animal). (F) Quantification of percentage of IAPP+ cells that were PCNA+; representative image shown (n = 5). Scale bar, 100 µm. Individual animals are shown on box-and-whisker plots. Statistical analysis was performed via Student t test to assess significance. *P < 0.05, **P < 0.01, ***P < 0.001 vs Ins1+/+Ins2+/+ controls. Insulin-deficient β-cells lack many markers of mature β-cells We examined the maturity of the insulin-deficient β-cells by immunostaining for select markers of mature β-cells. Interestingly, for some proteins the immunoreactivity appeared to coincide with the feeding and glycemia status of the animals. IAPP+ cells of hypoglycemic insulin-deficient mice had normal levels of PDX1 (Fig. 3A) but completely lacked immunoreactivity for homeodomain transcription factor 6.1 (NKX6.1), in contrast to Ins1+/+Ins2+/+ controls (Fig. 3B). We also detected less frequent immunoreactivity of homeodomain transcription factor 2.2 (NKX2.2) in β-cells (IAPP+) of Ins1−/−Ins2−/− mice compared with Ins1+/+Ins2+/+ controls (Fig. 3C). Ins1−/−Ins2−/− mice had a lower proportion of IAPP+ cells that expressed paired box 6 (PAX6), and IAPP+PAX6+ cells had lower intensity of PAX6 immunoreactivity than Ins1+/+Ins2+/+ controls (Fig. 3D), as did the remainder of the islet cells. MAFA immunoreactivity was observed in a lower proportion of βGAL+ β-cells in insulin knockouts compared with INS+ cells in control mice, and the intensity of immunoreactivity trended to be reduced (Fig. 3E). Glucose transporter 2 (GLUT2) immunoreactivity was detected in fewer IAPP+ cells in Ins1−/−Ins2−/− animals, and IAPP+GLUT2+ cells had lower intensity of GLUT2 immunoreactivity in Ins1−/−Ins2−/− animals compared with Ins1+/+Ins2+/+ controls (Fig. 4A). In hypoglycemic insulin-deficient neonates, immunoreactivity of sulfonylurea receptor 1 (SUR1) (Fig. 4B), prohormone convertase 1/3 (PC1/3) (Fig. 4C), and prohormone convertase 2 (PC2) (Fig. 4D) was comparable to that of Ins1+/+Ins2+/+ controls. In hyperglycemic Ins1−/−Ins2−/− neonates, NKX6.1, NKX2.2, PAX6, MAFA, and GLUT2 appeared to be completely lacking (Supplemental Fig. 2A), and there was a lower percentage of islet area with positive immunoreactivity for NKX6.1, NKX2.2, PAX6, MAFA, GLUT2, SUR1, and PC2 relative to control Ins1+/+Ins2+/+ mice (Supplemental Fig. 2B). Figure 3. View largeDownload slide β-cells in hypoglycemic Ins1−/−Ins2−/− mice lack expression of some β-cell transcription factors. P0 to P0.5 pancreata were immunostained for (A) PDX1, (B) NKX6.1, (C) NKX2.2, (D) PAX6, and (E) MAFA. Representative micrographs shown (n = 3). Quantification of percentage of (A–D) IAPP+ cells or (E) INS/βGAL+ cells immunoreactive for the selected transcription factor and fluorescent intensities relative to Ins1+/+Ins2+/+ controls in (A) PDX1+, (B) NKX6.1+, (C) NKX2.2+, (D) PAX6+, or (E) MAFA+ cells shown to the right of representative images. Individual animal values are shown on box-and-whisker plots. A Student t test [(A–E) target+ β-cells and (B, D, E) fluorescent intensity] or Mann–Whitney U test [(A, C) fluorescent intensity] was used to assess significance. *P < 0.05, **P < 0.01 vs Ins1+/+Ins2+/+ controls. Scale bar, 100 µm and, insets are magnified ×8. Figure 3. View largeDownload slide β-cells in hypoglycemic Ins1−/−Ins2−/− mice lack expression of some β-cell transcription factors. P0 to P0.5 pancreata were immunostained for (A) PDX1, (B) NKX6.1, (C) NKX2.2, (D) PAX6, and (E) MAFA. Representative micrographs shown (n = 3). Quantification of percentage of (A–D) IAPP+ cells or (E) INS/βGAL+ cells immunoreactive for the selected transcription factor and fluorescent intensities relative to Ins1+/+Ins2+/+ controls in (A) PDX1+, (B) NKX6.1+, (C) NKX2.2+, (D) PAX6+, or (E) MAFA+ cells shown to the right of representative images. Individual animal values are shown on box-and-whisker plots. A Student t test [(A–E) target+ β-cells and (B, D, E) fluorescent intensity] or Mann–Whitney U test [(A, C) fluorescent intensity] was used to assess significance. *P < 0.05, **P < 0.01 vs Ins1+/+Ins2+/+ controls. Scale bar, 100 µm and, insets are magnified ×8. Figure 4. View largeDownload slide Hypoglycemic Ins1−/−Ins2−/−β-cells lack expression of key β-cell factors. P0 to P0.5 pancreata were immunostained for (A) GLUT2, (B) SUR1, (C) PC1/3, and (D) PC2, Representative micrographs shown (n = 3). Quantification of percentage of (A, B) INS/βGAL+ cells or (C) IAPP+ cells immunoreactive for the selected protein and fluorescent intensities relative to Ins1+/+Ins2+/+ controls in (A) GLUT2+, (B) SUR1+, or (C) PC1/3+ cells shown to the right of representative images. (D) Percentage of GCG− islet cells expressing PC2 and relative fluorescent intensity of PC2 immunoreactivity in GCG−PC2+ cells. Scale bar, 100 µm, and insets are magnified ×8. (E) Relative mRNA quantification from isolated P0 to P05 islets from Ins1+/+Ins2+/+ and Ins1−/−Ins2−/− pups. Individual animals are shown on box-and-whisker plots. A Student t test [(B, D) target+ β-cells, (A–D) fluorescent intensity, and (E) Gcg, Nkx2.2, Slc2a2, Gck, Pcsk1, Amy] or Mann–Whitney U [(A, C) target+ β-cells and (E) Sst, Ppy, Pdx1, Nkx6.1, Pax6, Abcc8, Pcsk2] was used to assess significance. *P < 0.05, **P < 0.01, ***P < 0.001 vs controls. Figure 4. View largeDownload slide Hypoglycemic Ins1−/−Ins2−/−β-cells lack expression of key β-cell factors. P0 to P0.5 pancreata were immunostained for (A) GLUT2, (B) SUR1, (C) PC1/3, and (D) PC2, Representative micrographs shown (n = 3). Quantification of percentage of (A, B) INS/βGAL+ cells or (C) IAPP+ cells immunoreactive for the selected protein and fluorescent intensities relative to Ins1+/+Ins2+/+ controls in (A) GLUT2+, (B) SUR1+, or (C) PC1/3+ cells shown to the right of representative images. (D) Percentage of GCG− islet cells expressing PC2 and relative fluorescent intensity of PC2 immunoreactivity in GCG−PC2+ cells. Scale bar, 100 µm, and insets are magnified ×8. (E) Relative mRNA quantification from isolated P0 to P05 islets from Ins1+/+Ins2+/+ and Ins1−/−Ins2−/− pups. Individual animals are shown on box-and-whisker plots. A Student t test [(B, D) target+ β-cells, (A–D) fluorescent intensity, and (E) Gcg, Nkx2.2, Slc2a2, Gck, Pcsk1, Amy] or Mann–Whitney U [(A, C) target+ β-cells and (E) Sst, Ppy, Pdx1, Nkx6.1, Pax6, Abcc8, Pcsk2] was used to assess significance. *P < 0.05, **P < 0.01, ***P < 0.001 vs controls. We examined transcript levels by qPCR and found reduced expression of Ins, Gcg, Sst, and Ppy, as well as Pdx1, Nkx6.1, Nkx2.2, Pax6, Sur1, and Pcsk2 in isolated islets from hypoglycemic Ins1−/−Ins2−/− pups compared with Ins1+/+Ins2+/+ controls, and mRNA expression levels of Slc2a2,Gck, and Pcsk1 were similar to levels in islets from control mice (Fig. 4E). We found comparable expression of Amy in samples, indicating that the differences observed in expression levels of other target genes were unlikely to be a result of variable pancreatic exocrine contamination of samples. Insulin-deficient β-cells express progenitor markers We next determined whether insulin-deficient β-cells of Ins1−/−Ins2−/− mice resembled embryonic β-cell precursors or dedifferentiated β-cells. We immunostained pancreata from three neonatal Ins1+/+Ins2+/+ and three neonatal Ins1−/−Ins2−/− animals for factors associated with a progenitor state that have been detected in dedifferentiated β-cells (31). Many βGAL+ β-cells in Ins1−/−Ins2−/− mice were immunoreactive for v-myc avian myolocytomatosis viral oncogene lung carcinoma derived (L-MYC), homeobox protein NANOG (NANOG), and aldehyde dehydrogenase 1 family member A3 (ALDH1A3; Fig. 5A–5C). This finding contrasts with that of neurogenin-3 (NGN3), which was present in the perinuclear area of SYN+ islets cells in Ins1+/+Ins2+/+ controls but absent from Ins1−/−Ins2−/− islet cells (Fig. 5D). We repeated NGN3 immunostaining by using two commercially available antibodies (R&D Systems, catalog #AF3444; and Thermo Fisher Scientific, catalog #PA5-11893) and found similar patterns of immunoreactivity (Supplemental Fig. 3). The proportion of β-cells that were L-MYC+ or ALDH1A3+ was significantly higher in Ins1−/−Ins2−/− mice relative to controls. Ins1−/−Ins2−/−β-cells had higher intensity for immunoreactive NANOG and L-MYC compared with control samples. We attempted to quantify the corresponding gene levels by qPCR but could detect sufficient transcript only of Aldh1a3, which was about eight times higher in islets of in Ins1−/−Ins2−/− mice than in controls (Fig. 5C). Figure 5. View largeDownload slide Ins1−/−Ins2−/−β-cells are not fully differentiated. P0 to P0.5 pancreata from Ins1+/+Ins2+/+ and Ins1−/−Ins2−/− mice were immunostained for (A) L-MYC, (B) NANOG, (C) ALDH1A3, and (D) NGN3. Representative micrographs shown (n = 3). Quantification of percentage of (A–D) INS/βGAL+ cells or (E) SYN+ cells immunoreactive for the selected protein and fluorescent intensity relative to Ins1+/+Ins2+/+ controls in (A) L-MYC+, (B) NANOG+, (C) ALDH1A3+, or (D) NGN3+ cells shown to the right of representative images. Relative mRNA expression of (D) Aldh1a3 by 2−ΔΔCt method in isolated P0 to P0.5 islets from Ins1+/+Ins2+/+ and Ins1−/−Ins2−/− pups. Individual animals are shown on box-and-whisker plots. A Student t test [(A, C, D, and E) target+ β-cells, and (A–E) fluorescent intensity, and qPCR relative expression] or Mann–Whitney U [(B) target+ β-cells] was used to assess significance. *P < 0.05, ***P < 0.001 vs controls. Scale bar, 50 µm, and insets are magnified ×4. Figure 5. View largeDownload slide Ins1−/−Ins2−/−β-cells are not fully differentiated. P0 to P0.5 pancreata from Ins1+/+Ins2+/+ and Ins1−/−Ins2−/− mice were immunostained for (A) L-MYC, (B) NANOG, (C) ALDH1A3, and (D) NGN3. Representative micrographs shown (n = 3). Quantification of percentage of (A–D) INS/βGAL+ cells or (E) SYN+ cells immunoreactive for the selected protein and fluorescent intensity relative to Ins1+/+Ins2+/+ controls in (A) L-MYC+, (B) NANOG+, (C) ALDH1A3+, or (D) NGN3+ cells shown to the right of representative images. Relative mRNA expression of (D) Aldh1a3 by 2−ΔΔCt method in isolated P0 to P0.5 islets from Ins1+/+Ins2+/+ and Ins1−/−Ins2−/− pups. Individual animals are shown on box-and-whisker plots. A Student t test [(A, C, D, and E) target+ β-cells, and (A–E) fluorescent intensity, and qPCR relative expression] or Mann–Whitney U [(B) target+ β-cells] was used to assess significance. *P < 0.05, ***P < 0.001 vs controls. Scale bar, 50 µm, and insets are magnified ×4. Insulin replacement by injection leads to islet fibrosis and expanded islet cell area To determine whether restoration of insulin signaling by exogenous insulin therapy was sufficient for completed β-cell maturation, we treated insulin-deficient animals with insulin injections for 2 months. Insulin-injected Ins−/−Ins2−/− mice gained weight but at a slower rate than control animals (Supplemental Fig. 4A). After insulin therapy by injection or isogenic islet transplantation from Ins1+/+Ins2+/+ donors, animals retained clusters of IAPP+ cells, but the majority of IAPP+ cells were βGAL− (Supplemental Fig. 4B–4D). Seeking to clarify this observation, we examined INS and βGAL immunoreactivity in Ins1−/−Ins2+/− and Ins1+/+Ins2−/− islets. Evidently there can be unique expression of Ins1 compared with Ins2 within β-cells, because many INS+ cells were βGAL− in Ins1+/+Ins2−/− mice. Additionally, it appears there can be unique expression of the two Ins2 alleles within β-cells, because Ins1−/−Ins2+/− islets also had abundant INS+ cells that were βGAL− (Supplemental Fig. 4E). After 2 months of insulin therapy by injections, we found islet cell hyperplasia and obvious islet fibrosis by trichrome staining in insulin-deficient islets (Fig. 6A). Although a smaller proportion of endocrine cells were IAPP+ β-cells (Fig. 6B), we observed an expansion of all endocrine cell types relative to total pancreatic area in Ins1−/−Ins2−/− mice compared with Ins1+/+Ins2+/+ controls (Fig. 6C). Furthermore, Ins1−/−Ins2−/− islets trended to being larger (Fig. 66E), and there was a trend toward a higher proportion of IAPP+PCNA+/IAPP+ islet cells compared with controls (Fig. 6F). Figure 6. View largeDownload slide Adult Ins1−/−Ins2−/− mice treated by insulin injections have fibrosis and expanded islet cell area. (A) Pancreata of 2-month-old Ins1+/+Ins2+/+ (top panels) and insulin injection–treated Ins1−/−Ins2−/− (bottom panels) mice were immunostained for IAPP and GCG or GCG, SST, and PP. Hematoxylin and eosin and Masson’s trichrome staining of pancreata (representative micrographs from n = 3–5). Scale bars, 100 µm. (B) Percentage of total endocrine (SYN+) immunoreactive area that expressed the islet hormones IAPP, GCG, SST, or PP. (C) β-cell, α-cell, δ-cell, and pancreatic polypeptide cell area relative to total pancreas area (n = 4 animals, 3 sections quantified per animal). (D) Number of islets identified by SYN immunoreactivity relative to pancreas section area. (E) Average islet size by SYN immunoreactive area. (F) Quantification of percentage of IAPP+ cells that were PCNA+; representative image shown (n = 4). Scale bar, 100 µm. (B–F) Individual animal endocrine area is shown on box-and-whisker plots. Statistical analysis was performed with a Student t test [(B) IAPP, SST, PP, (C) IAPP, GCG, PP, (F)] or Mann–Whitney U test [(B) GCG, (C) SST, (D, E)] to assess significance. *P < 0.05, **P < 0.01, ***P < 0.001 vs Ins1+/+Ins2+/+ controls. Figure 6. View largeDownload slide Adult Ins1−/−Ins2−/− mice treated by insulin injections have fibrosis and expanded islet cell area. (A) Pancreata of 2-month-old Ins1+/+Ins2+/+ (top panels) and insulin injection–treated Ins1−/−Ins2−/− (bottom panels) mice were immunostained for IAPP and GCG or GCG, SST, and PP. Hematoxylin and eosin and Masson’s trichrome staining of pancreata (representative micrographs from n = 3–5). Scale bars, 100 µm. (B) Percentage of total endocrine (SYN+) immunoreactive area that expressed the islet hormones IAPP, GCG, SST, or PP. (C) β-cell, α-cell, δ-cell, and pancreatic polypeptide cell area relative to total pancreas area (n = 4 animals, 3 sections quantified per animal). (D) Number of islets identified by SYN immunoreactivity relative to pancreas section area. (E) Average islet size by SYN immunoreactive area. (F) Quantification of percentage of IAPP+ cells that were PCNA+; representative image shown (n = 4). Scale bar, 100 µm. (B–F) Individual animal endocrine area is shown on box-and-whisker plots. Statistical analysis was performed with a Student t test [(B) IAPP, SST, PP, (C) IAPP, GCG, PP, (F)] or Mann–Whitney U test [(B) GCG, (C) SST, (D, E)] to assess significance. *P < 0.05, **P < 0.01, ***P < 0.001 vs Ins1+/+Ins2+/+ controls. Insulin replacement by injection facilitates partial maturation of Ins1−/−Ins2−/−β-cells To determine whether insulin replacement by injection was sufficient for Ins1−/−Ins2−/−β-cells to complete maturation, gain expression of NKX6.1, NKX2.2, MAFA, PAX6, and GLUT2, and lose expression of L-MYC and NANOG, we immunostained for these factors in Ins1−/−Ins2−/− mice treated with insulin injections. After 2 months of twice-daily insulin injections, Ins1−/−Ins2−/−β-cells gained immunoreactive NKX2.2 (Fig. 7A) but still had lower intensity of NKX6.1 immunoreactivity compared with INS+ Ins1+/+Ins2+/+β-cells (Fig. 7B). Furthermore, Ins1−/−Ins2−/− islet cells were deficient in PAX6, MAFA, and GLUT2 compared with control islets (Fig. 7C–7E). There was cytoplasmic NANOG immunoreactivity in Ins1−/−Ins2−/− IAPP+ cells but no L-MYC+ cells (Fig. 77G). Rare ALDH1A3+ cells were observed (Fig. 7H), and pericytoplasmic immunoreactivity for NGN3 was frequent in Ins1−/−Ins2−/− IAPP+ β-cells and neighboring cells within the islet (Fig. 7I), similar to Ins1+/+Ins2+/+ neonatal β-cells (Fig. 5D). Figure 7. View largeDownload slide Insulin therapy by injection is not sufficient for the completed maturation of Ins1−/−Ins2−/−β-cells. Pancreata of 2-month-old Ins1+/+Ins2+/+ (left panels) and insulin injection–treated Ins1−/−Ins2−/− (right panels) mice were immunostained for factors associated with (A–E) mature β-cells and (F–J)a progenitor state. Ins1−/−Ins2−/−βGAL+ cells express (A) NKX2.2 but had less intense to absent (B) NKX6.1, (C) PAX6, (D) MAFA, and (E) GLUT2 immunoreactivity compared with INS+ Ins1+/+Ins2+/+β-cells. Ins1−/−Ins2−/−βGAL+ β-cells express (G) NANOG and (I) NGN3 but not (F) L-MYC and only (H) extremely rare ALDH1A3+IAPP+ cells. Scale bars, 100 µm, and insets are magnified (A–E) ×8 or (F–J) ×4. Figure 7. View largeDownload slide Insulin therapy by injection is not sufficient for the completed maturation of Ins1−/−Ins2−/−β-cells. Pancreata of 2-month-old Ins1+/+Ins2+/+ (left panels) and insulin injection–treated Ins1−/−Ins2−/− (right panels) mice were immunostained for factors associated with (A–E) mature β-cells and (F–J)a progenitor state. Ins1−/−Ins2−/−βGAL+ cells express (A) NKX2.2 but had less intense to absent (B) NKX6.1, (C) PAX6, (D) MAFA, and (E) GLUT2 immunoreactivity compared with INS+ Ins1+/+Ins2+/+β-cells. Ins1−/−Ins2−/−βGAL+ β-cells express (G) NANOG and (I) NGN3 but not (F) L-MYC and only (H) extremely rare ALDH1A3+IAPP+ cells. Scale bars, 100 µm, and insets are magnified (A–E) ×8 or (F–J) ×4. Insulin replacement by islet transplantation facilitates expansion and completed maturation of Ins1−/−Ins2−/−β-cells We next determined whether restoration of insulin signaling by isogenic islet transplantation into the anterior chamber of the eye was sufficient for completed β-cell maturation. We attempted to have a side-by-side comparison of the endocrine pancreas of animals treated for ~1 year by insulin injections alone or islet transplantation into the anterior chamber of the eye at 2 weeks of age (Fig. 8). Because of the major technical challenges in keeping Ins1−/−Ins2−/− animals alive with insulin injections for such a prolonged time, we were able to collect samples from only one mouse at 291 days of age. The mouse treated by injections had extreme islet fibrosis (Fig. 8A), with a nearly complete loss of IAPP+ cells, and islets were >50% GCG+ (Supplemental Fig. 5A). Compared with Ins1+/+Ins2+/+ controls, mice treated with islet transplantation (n = 4) had grossly normal-appearing islets without signs of fibrosis (Fig. 8A) and did not have significantly altered islet hormone proportions (Fig. 8B), but there was a slightly expanded α-cell area (Fig. 8C) and enlarged islets (Fig. 8E). Figure 8. View largeDownload slide Insulin replacement by islet transplantation leads to islet hyperplasia and preservation of β-cell area. (A) Pancreata of Ins1+/+Ins2+/+ (top panels; n = 3, 12 months of age), insulin injection–treated Ins1−/−Ins2−/− (middle panels; n = 1, 9 months of age), and islet transplantation–treated Ins1−/−Ins2−/− (bottom panels; n = 4, 12 to 14 months of age) mice were immunostained for IAPP and GCG or for GCG, SST, and PP. Arrows point to examples of polyhormonal cells that are GCG+PP+ or SST+PP+. Hematoxylin and eosin and Masson’s trichrome staining of pancreas. Scale bars, 100 µm. (B) Percentage of total endocrine immunoreactive area that is immunoreactive for the islet hormones IAPP, GCG, SST, or PP. (C) β-cell, α-cell, δ-cell, and pancreatic polypeptide cell area relative to total pancreas area (n = 1 to 4 animals, 3 sections quantified per animal). (D) Number of islets identified by SYN immunoreactivity relative to pancreas section area. (E) Average islet size by SYN immunoreactive area. (F) Percentage of IAPP+ cells that were PCNA+. Individual animal endocrine area is shown on box-and-whisker plots. Ins1+/+Ins2+/+ and islet-treated Ins1−/−Ins2−/− groups were compared by Student t test [(B) IAPP, GCG, SST, (C) IAPP, GCG, SST, (D, E)] or Mann–Whitney U test [(B) PP, (C) PP, (F)] to assess significance. *P < 0.05 vs Ins1+/+Ins2+/+ controls. Figure 8. View largeDownload slide Insulin replacement by islet transplantation leads to islet hyperplasia and preservation of β-cell area. (A) Pancreata of Ins1+/+Ins2+/+ (top panels; n = 3, 12 months of age), insulin injection–treated Ins1−/−Ins2−/− (middle panels; n = 1, 9 months of age), and islet transplantation–treated Ins1−/−Ins2−/− (bottom panels; n = 4, 12 to 14 months of age) mice were immunostained for IAPP and GCG or for GCG, SST, and PP. Arrows point to examples of polyhormonal cells that are GCG+PP+ or SST+PP+. Hematoxylin and eosin and Masson’s trichrome staining of pancreas. Scale bars, 100 µm. (B) Percentage of total endocrine immunoreactive area that is immunoreactive for the islet hormones IAPP, GCG, SST, or PP. (C) β-cell, α-cell, δ-cell, and pancreatic polypeptide cell area relative to total pancreas area (n = 1 to 4 animals, 3 sections quantified per animal). (D) Number of islets identified by SYN immunoreactivity relative to pancreas section area. (E) Average islet size by SYN immunoreactive area. (F) Percentage of IAPP+ cells that were PCNA+. Individual animal endocrine area is shown on box-and-whisker plots. Ins1+/+Ins2+/+ and islet-treated Ins1−/−Ins2−/− groups were compared by Student t test [(B) IAPP, GCG, SST, (C) IAPP, GCG, SST, (D, E)] or Mann–Whitney U test [(B) PP, (C) PP, (F)] to assess significance. *P < 0.05 vs Ins1+/+Ins2+/+ controls. Unlike those of mice treated with insulin injections for 2 months (Fig. 7), pancreatic β-cells of Ins1−/−Ins2−/− mice with healthy islets transplanted had normal immunoreactivity for PAX6, MAFA, and GLUT2 (Supplemental Fig. 5B), but we detected the abnormal presence of NANOG+ cells (Supplemental Fig. 6). NGN3 immunoreactivity was not detected in pancreata of Ins1−/−Ins2−/− mice with transplanted islets, similar to adult Ins1+/+Ins2+/+ controls (Supplemental Fig. 6). Discussion Mice lacking IRs on β-cells have impaired glucose-stimulated insulin secretion (7), and β-cell mass expands during development (13). IR-deficient β-cells are unable to sufficiently expand in response to hepatic insulin resistance, resulting in adult-onset diabetes (8). In the current study, we characterized pancreata in neonatal Ins1−/−Ins2−/− mice and Ins1−/−Ins2−/− mice kept alive into adulthood by using insulin therapy. By using the insulin knockout mouse model as an alternative to IR-deficient mice, we provide evidence that insulin itself is necessary for β-cell maturation and insulin deficiency alters islet development. However, we cannot rule out the possibility that IGF signaling though the IR is altered by changes to expression of the IR or insulinlike growth factor 1 receptor (IGF1R) and downstream signaling components. Additionally, it is possible that other defects secondary to the genetic deletion of insulin contribute to the observed β-cell phenotype. Independent of these caveats, we posit that the insulin-deficient mouse model provides the best evidence available that signaling by insulin itself is necessary for β-cell maturation. Certainly, the Ins1−/−Ins2−/− mouse model does not have any insulin signaling via an IR-independent pathway, and unlike in the IR-deficient model, there is no direct and complete loss of IGF signaling through the IR. At birth, insulin-deficient islets were enlarged with an expanded IAPP+ area. These findings are consistent with observations of expanded β-cell mass and increased β-cell replication in the MIP-CreER/IRflox/flox (tamoxifen at E13) model (13) and reduce the likelihood that those findings were attributable to the presence of the growth hormone minigene within the transgene (32, 33). We also observed a lack of mature β-cell factors PDX1, NKX6.1, MAFA, and membranous GLUT2 in insulin-deficient β-cells. These findings contrast with normal expression of PDX1, NKX6.1, MAFA, and GLUT2 in pancreata from MIP-CreER/IRflox/flox (tamoxifen at E13) mice. This discrepancy can be explained in three ways: insulin before E13 may be essential for the normal formation of maturing β-cells; although insulin signals through the IGF1R with low affinity (34), increased expression of the IGF1R in the islets of CreER/IRflox/flox (tamoxifen at E13) mice (13) may contribute to meaningful insulin signaling through the IGF1R; and despite normal blood glucose, Ins1−/−Ins2−/− pups have hypertriglyceridemia, which could contribute to loss of membranous GLUT2 (35) and β-cell dysfunction and loss of mature β-cell factors (36). Dedifferentiated β-cells in patients with type 2 diabetes (37, 38) express progenitor markers L-MYC and NANOG (31). Insulin-deficient β-cells in Ins1−/−Ins2−/− mice at birth appeared similar, with expression of both L-MYC and NANOG. Notably, we are unaware of any evidence of L-MYC or NANOG being present in the normal developing pancreas, suggesting that insulin-deficient β-cells may not be arrested at a developmental stage. Instead, insulin-deficient β-cells appear to dedifferentiate after failed maturation because of a lack of the β-cell defining protein, insulin. Furthermore, like β-cells of patients with type 2 diabetes, an elevated proportion of the insulin-deficient β-cells expressed ALDH1A3, a marker of dysfunctional β-cells (38). NGN3 was expressed in neonatal Ins1+/+Ins2+/+ islet cells but not in insulin-deficient islet cells. Although neurogenin3 was not detectable by in situ hybridization at birth (39), NGN3 is expressed in normal postnatal β-cells (40) and in non–fully differentiated β-cells that are still dividing (41) and β-cells replicate after birth (42). A lack of NGN3 in Ins1−/−Ins2−/−β-cells is consistent with reduced expression of the post-NGN3 factor NKX2.2 (43, 44). Because NGN3 is necessary for pancreatic endocrine cell formation (39), there must be NGN3 early in development to initiate the formation of pancreatic islets, but a lack of insulin results in a secondary loss of NGN3 expression later in development. Our results suggest that insulin signaling contributes to the maturation of β-cells, and in the absence of insulin, β-cells fail normal maturation and dedifferentiate to an early replicating embryonic state lacking NGN3 and downstream factors including NKX2.2 and MAFA and expressing pluripotency markers NANOG and L-MYC. Replacing insulin contributes to the further maturation of insulin-deficient β-cells. After insulin injection therapy, insulin-deficient β-cells did not express L-MYC or ALDH1A3, but we observed islet fibrosis. Additionally, β-cells of Ins1−/−Ins2−/− adults treated with insulin injection resembled neonatal wild-type β-cells, with perinuclear immunoreactivity for NGN3. This cytoplasmic localization of NGN3 has been previously reported as marker of newly forming β-cells: cytoplasmic NGN3 has been observed during β-cell regeneration after immunological destruction of β-cells (45) and has been observed in early phases of endocrine cell neogenesis in vitro by stimulation with a growth factor cocktail (46). Additionally, unlike the nuclear localization of NANOG observed in neonatal Ins1−/−Ins2−/− islet cells that is conventionally a marker of self-renewal in stem cells (47), insulin-treated mice (by insulin injection or islet transplantation) had cytoplasmic NANOG, which has been used as a marker of an epithelial-to-mesenchymal transition in nasopharyngeal carcinoma (48), cervical cancer (49), and pancreatic cancer (50). This finding contrasts with that of endogenous β-cells in Ins1−/−Ins2−/− animals treated with insulin replacement by islet transplantation, which had normal expression of all mature β-cell factors examined, including MAFA. Insulin-deficient β-cells of islet-treated adult mice also had a normal absence of L-MYC, ALDH1A3, and NGN3. Continued presence of cells with cytoplasmic NANOG immunoreactivity provides weak evidence for ongoing epithelial-to-mesenchymal transition of nonendocrine cells as a source of new islet cells contributing to islet hyperplasia. Because the primary deficit of Ins1−/−Ins2−/− mice is a loss of insulin, it is surprising that replacing insulin by injections alone or islet transplantation resulted in dramatically divergent outcomes for the endogenous β-cells. We proposed three variables that could contribute to the differences in β-cell phenotype between Ins1−/−Ins2−/− mice treated with islet transplantation and those treated with insulin injection: glycemic control was superior in mice treated with islet transplantation relative to those treated with insulin injections, native mouse insulin produced by transplanted islets may signal in β-cells with higher bioactivity than recombinant insulin (51, 52), or insulin-deficient islets may fail to produce other essential factors that are replaced by transplanted islets. Although we are unable to conclusively discern which, if any, of these variables contribute to the differences in β-cell phenotype, the striking resemblance in fibrotic islet phenotype between injection-treated Ins1−/−Ins2−/− mice and mice with diabetes as a result of inexcitable β-cells [adenosine triphosphate (ATP)–sensitive potassium channel gain of function] (53) suggests that differences in glycemia are at least partially responsible. Hyperglycemia leading to glucotoxicity has also been shown to cause reduced expression of Pdx1, Mafa, and Slc2a2 (36), and reprogramming of exocrine cells to insulin-producing cells was more complete and abundant from a viral therapy (expressing NGN3/PDX1/MAFA) when mice were treated with islet transplantation for good control of their toxin-induced diabetes compared with crude treatment with insulin pellets (54). Similarly, in models of neonatal diabetes (expression of an activating ATP-sensitive potassium channel), animals treated with islet transplantation did not develop islet fibrosis and retained glucose-stimulated insulin secretion, unlike the untreated hyperglycemic group (55). These findings also suggest that the crude glycemic regulation of insulin injections (or pellets) compared with the ideally regulated glycemia of mice treated with islet transplantation is probably an important contributor to the differences in β-cell phenotype. Transplanted islets also secrete additional peptides beyond insulin, including C-peptide, a byproduct of proinsulin processing that may contribute to preservation of islet health (56). Finally, in this study the mice treated by islet transplantation were kept alive for ~1 year, whereas injection-treated mice were kept alive for 2 months, with the exception of a single mouse that was kept alive by injections for nearly 1 year. The extreme challenge of keeping mice alive by multiple daily injections for a long time limited our ability to have age-matched groups, and the different duration of therapy may have contributed to the differences in phenotype. Ins1−/−Ins2−/− mice develop aggregates of endocrine cells in the pancreas resembling islets, but a loss of insulin alters the cellular composition. Ins1−/−Ins2−/− islets have a lower proportion of GCG+ and PP+ endocrine cells and greater IAPP+ and SST+ cell mass. Given the known contribution of paired box 4 to the β- and δ-cell lineages and ARX to the α-cell and pancreatic polypeptide cell lineages (57, 58), reduced GCG+ and PP+ and expanded IAPP+ and SST+ cell populations align with an overall reduction in ARX and increase in paired box 4 signaling. Additionally, there was a progressive expansion of GCG+ α-cells in mice treated with insulin injections that was not observed in mice treated with islet transplantation. A loss of insulin signaling in α-cells contributes to α-cell hyperplasia (59), and poor glycemic control in insulin-injected mice also may contribute to progressive α-cell expansion (60). Additionally, we made the surprising observation that the pan-endocrine factors NKX2.2 and PAX6 are absent from not only β-cells but also the remainder of the islet cells. A reduced number of PP+ cells in the pancreas aligns with the phenotype of the NKX2.2-deficient endocrine pancreas (44), but unlike in the PAX6 knockout mouse (61), we did not observe expanded ghrelin+ cells. There is probably an undefined paracrine effect inhibiting the normal maturation of non-β islet cells in the pancreas of insulin-deficient mice. MAFA immunoreactivity was diminished in β-cells of mice with reduced insulin gene copy number (Ins1−/−, Ins2−/−, and Ins1−/−Ins2+/−). Although we did not follow up on these findings, they raise the intriguing possibility that despite not having obvious severe abnormalities in glucose homeostasis (62), reduced insulin dosed β-cells may not be functionally mature because of MAFA insufficiency (63) and perhaps other unidentified factors. Additionally, we made an unexpected observation that there can be unique regulation of the Ins2 and Ins1 loci and the two Ins2 alleles in mouse β-cells. With lacZ knocked into the Ins2 locus, the majority of β-cells in adult Ins1−/−Ins2+/− or Ins1+/+Ins2−/− animals are INS+βGAL−. In the Ins1+/+Ins2−/− animals, cells that were INS+βGAL− had an active Ins1 gene, but the Ins2 knockin of βGAL was inactive. In the Ins1−/−Ins2+/− animals, cells that were INS+βGAL− had an active wild-type Ins2 allele, but the βGAL knockin allele was inactive. Similarly, loss of βGAL in most IAPP-expressing cells happens after birth in Ins1−/−Ins2−/− animals. Although chronic hyperglycemia has been shown to suppress insulin transcription (64), loss of βGAL cannot be attributed to a diabetic state because postnatal loss of βGAL expression also occurs in euglycemic Ins1−/−Ins2−/− mice treated by islet transplantation and Ins1+/+Ins2−/− and Ins1−/−Ins2+/− animals. Because expression of insulin is arguably the most important aspect of the pancreatic β-cell identity, loss of βGAL does not align with a mature β-cell identity. However, it is important to consider the model organism because age-dependent loss of βGAL in insulin-treated Ins1−/−Ins2−/− mice is consistent with reduced lacZ expression when knocked into the globin genes in mice (65). The distinct expression pattern of βGAL compared with insulin in the insulin knockout/lacZ knockin model may be a caveat of the mouse model because there can be selective CpG methylation to silence a foreign open reading frame such as the lacZ gene (66). Nevertheless, it may be worthwhile to further explore the possibility of differential regulation of the mouse insulin alleles. There have been many reported human cases of diabetes caused by mutations in the insulin gene. Most of these patients have a heterozygous dominant negative disease caused by misfolding of the mutated insulin, but there has been a report of patients with homozygous deletion of INS (68). Although we found no published histology of the pancreas from such patients, a patient with an intronic mutation causing altered splicing of INS had undetectable C-peptide but readily detectable IAPP, suggesting that the patient had insulin-deficient β-cells in the pancreas (69). This hypothesis is supported by the abundant INS−IAPP+ β-cells in Ins1−/−Ins2−/− mouse pancreas. Relatedly, a child with severe sulfonylurea-unresponsive permanent neonatal diabetes mellitus from an activating mutation in the ATP-sensitive potassium channel had severely reduced β-cell mass on postmortem histological examination (70). Patients with mutations in the ATP-sensitive potassium channel are less likely to become insulin independent with sulfonylurea therapy the longer the patient has been insulin dependent (71). Potentially there is a major change in β-cell maturity after transfer of therapy, but prolonged insulin replacement by injection causes progressive loss of β-cell mass, thus hindering the ability of sulfonylurea therapy to induce diabetic remission. This hypothesis aligns with our findings that although a lack of insulin during development initially results in an expanded β-cell mass, insulin replacement by injection is not sufficient to maintain β-cell mass. Like mouse β-cells, adult human β-cells also contain much of the necessary machinery for insulin signaling, including IRs and insulin receptor substrate (IRS)-1 and IRS-2 (72). Functionally, inhibition of IR by short hairpin RNA impaired glucose-stimulated insulin secretion (72), and human islets with an IRS-1 polymorphism have impaired glucose-stimulated insulin secretion, elevated proinsulin secretion, and reduced insulin content (73). Furthermore, there is evidence of reduced insulin signaling in β-cells of patients with type 2 diabetes: IRS-1, IRS-2, Tyr612 IRS-1, and Tyr612 IRS-2 expression are reduced in islets from patients with type 2 diabetes compared with islets from nondiabetic donors (74). Taken together, these findings lead us to propose that though usually attributed to reduced glucolipotoxicity (75), diabetic remission after intensive insulin therapy in patients with type 2 diabetes (76) may be attributed partially to augmented β-cell insulin signaling from increased circulating insulin. Increased insulin signaling in β-cells could lead to redifferentiation of dedifferentiated β-cells, a possibility that may warrant future investigation. Collectively, we provide evidence that insulin is a necessary signaling molecule for the maturation of β-cells. Replacement of insulin contributes to β-cell maturation, but the ability of insulin therapy to complete β-cell maturation may depend on euglycemia, replacement of the native species of insulin, or other factors secreted from islets. Without insulin during development in mice, β-cells appear not fully mature. Extremely rare mutations of the INS gene may also cause altered maturation of β-cells in patients. Understanding how insulin regulates β-cell maturation is relevant to research into the generation of β-cells in vitro and therapies attempting to induce diabetic remission in patients with dedifferentiated β-cells in early type 2 diabetes. Although there has been some debate about the potential that insulin signals in an autocrine fashion on β-cells (77), our findings provide evidence that insulin itself is an essential signaling molecule on β-cells. Appendix. Table of Antibodies Used for Immunofluorescent Staining Peptide/Protein Target  Antigen Sequence  Name of Antibody  Manufacturer or Name of Individual Providing the Antibody  Catalog No.  Species Raised in  Monoclonal or Polyclonal  Dilution Used  RRID  Rabbit IgG  Whole IgG  Goat anti-rabbit AF488  Life Technologies  A11034  Goat  Polyclonal  1:1000  AB_2576217  Rabbit IgG  Whole IgG  Goat anti-rabbit AF555  Life Technologies  A21429  Goat  Polyclonal  1:1000  AB_151761  Rabbit IgG  Whole IgG  Goat anti-rabbit AF594  Life Technologies  A11037  Goat  Polyclonal  1:1000  AB_2534095  Rabbit IgG  Whole IgG  Goat anti-rabbit AF647  Life Technologies  A21245  Goat  Polyclonal  1:1000  AB_2535813  Rabbit IgG  Whole IgG  Donkey anti-rabbit AF488  Life Technologies  A21206  Donkey  Polyclonal  1:1000  AB_141708  Rabbit IgG  Whole IgG  Donkey anti-rabbit AF555  Life Technologies  A31572  Donkey  Polyclonal  1:1000  AB_162543  Rabbit IgG  Whole IgG  Donkey anti-rabbit AF594  Life Technologies  A21207  Donkey  Polyclonal  1:1000  AB_141637  Rabbit IgG  Whole IgG  Donkey anti-rabbit AF647  Life Technologies  A31573  Donkey  Polyclonal  1:1000  AB_2536183  Mouse IgG  Whole IgG  Goat anti-mouse AF488  Life Technologies  A11029  Goat  Polyclonal  1:1000  AB_2534088  Mouse IgG  Whole IgG  Goat anti-mouse AF555  Life Technologies  A21424  Goat  Polyclonal  1:1000  AB_141780  Mouse IgG  Whole IgG  Goat anti-mouse AF594  Life Technologies  A11032  Goat  Polyclonal  1:1000  AB_141672  Mouse IgG  Whole IgG  Goat anti-mouse AF647  Life Technologies  A21236  Goat  Polyclonal  1:1000  AB_2535805  Mouse IgG  Whole IgG  Donkey anti-mouse AF488  Life Technologies  A21202  Donkey  Polyclonal  1:1000  AB_2535788  Mouse IgG  Whole IgG  Donkey anti-mouse AF555  Life Technologies  A31570  Donkey  Polyclonal  1:1000  AB_2536180  Mouse IgG  Whole IgG  Donkey anti-mouse AF594  Life Technologies  A21203  Donkey  Polyclonal  1:1000  AB_141633  Mouse IgG  Whole IgG  Donkey anti-mouse AF647  Life Technologies  A31571  Donkey  Polyclonal  1:1000  AB_162542  Goat IgG  Whole IgG  Donkey anti-goat AF594  Life Technologies  A11058  Donkey  Polyclonal  1:1000  AB_142540  Sheep IgG  Whole IgG  Donkey anti-sheep AF488  Life Technologies  A11015  Donkey  Polyclonal  1:1000  AB_141362  Guinea pig IgG  Whole IgG  Goat anti–guinea pig AF488  Life Technologies  A11073  Goat  Polyclonal  1:1000  AB_2534117  Guinea pig IgG  Whole IgG  Goat anti–guinea pig AF647  Life Technologies  A21450  Goat  Polyclonal  1:1000  AB_151882  Aldehyde dehydrogenase family member 1A3  Unknown  Rabbit anti-ALDH1A3  Novus Biologicals  NBP2-15339  Rabbit  Polyclonal  1:100  AB_2665496  Islet amyloid polypeptide  Unknown  Rabbit anti-IAPP  AbCam  ab15125  Rabbit  Polyclonal  1:50  AB_2295631  Beta-galactosidase  Whole β-galactosidase  Rabbit anti-βGAL  Thermo Scientific  A11132  Rabbit  Polyclonal  1:100  AB_221539  Beta-galactosidase  Unknown  Mouse anti-βGAL  DSHB  40-1a-c  Mouse  Monoclonal  1:50  AB_528100  Glucokinase  Recombinant glucokinase  Rabbit anti-GCK  Sigma  HPA007034  Rabbit  Polyclonal  1:50  AB_888431  Glucagon  Unknown  Mouse anti-GCG  Sigma  G 2654  Mouse  Monoclonal  1:1000  AB_259852  Glucose transporter 2  First extracellular loop of Glut2  Rabbit anti-GLUT2  Millipore  07-1402  Rabbit  Polyclonal  1:500  AB_1587076  Insulin  Full-length human insulin  Guinea pig anti-INS  Thermo Fisher Scientific  PA1-26938  Guinea pig  Polyclonal  1:100  AB_794668  Insulin  Unknown  Rabbit anti-INS  Cell Signaling  C27C9  Rabbit  Monoclonal  1:200  AB_2126503  Insulin  Residues surrounding Val36 of human insulin  Mouse anti-INS  Cell Signaling  L6B10  Mouse  Monoclonal  1:250  AB_10949314  v-myc Avian myolocytomatosis viral oncogene lung carcinoma derived  AA 105-154 of human l-myc  Rabbit anti-L-MYC  AbCam  Ab28739  Rabbit  Polyclonal  1:100  AB_2148730  V-maf muscoloapo-neurotic fibrosarcoma oncogene homolog A  Unknown  Rabbit anti-MAFA  Betalogics (Johnson & Johnson)  LP9872  Rabbit  Polyclonal  1:1000  AB_2665528  V-maf muscoloaponeurotic fibrosarcoma oncogene homolog B  AA 18-140 of MAFB  Rabbit anti-MAFB  Sigma Life Sciences  HPA005653  Rabbit  Polyclonal  1:100  AB_1079293  Homeobox protein NANOG  Mouse nanog  Rabbit anti-NANOG  AbCam  Ab80892  Rabbit  Polyclonal  1:100  AB_2150114  Homeodomain transcription factor 6.1  Human Nkx6.1  Goat anti-NKX6.1  R and D Systems  AF5857  Goat  Polyclonal  1:20  AB_1857045  Homeodomain transcription factor 2.2  Nkx2.2–GST fusion protein from Escherichia. coli  Mouse anti-NKX2.2  DSHB  74.5A5  Mouse  Monoclonal  1:100  AB_531794  Neurogenin-3  Met1-Leu214 of human Ngn3  Sheep anti-NGN3  R and D Systems  AF3444  Sheep  Polyclonal  1:20  AB_2149527  Neurogenin-3  AA40-69 of human Ngn3  Sheep anti-NGN3  Thermo Fisher Scientific  PA5-11893  Rabbit  Polyclonal  1:100  AB_2149526  Pancreatic polypeptide  Ala30-Leu95 of human PP  Goat anti-PP  R and D Systems  AF6297  Goat  Polyclonal  1:200  AB_10717571  Paired box 6  C-terminus of mouse PAX6  Rabbit anti-PAX6  Covance  PRB-278P  Rabbit  Polyclonal  1:250  AB_2313780  Prohormone convertase 1/3  Unknown  Rabbit anti-PC1/3  Lakshmi Devi  Gift  Rabbit  Polyclonal  1:500  AB_2665530  Prohormone convertase 1/3  Unknown  Mouse anti-PC1/3  Gunilla Westermark  Gift  Mouse  Monoclonal  Direct  AB_2665529  Prohormone convertase 2  E622-N638 of mouse PC2  Rabbit anti-PC2  Thermo Fisher Scientific  PA1-058  Rabbit  Polyclonal  1:500  AB_2158593  Proliferating cell nuclear antigen  Rat PCNA  Mouse anti-PCNA  Abcam  ab29  Mouse  Monoclonal  1:100  AB_303394  Pancreatic and duodenal homeobox 1  N-terminus of mouse PDX1  Guinea pig anti-PDX1  Abcam  ab47308  Guinea pig  Polyclonal  1:1000  AB_777178  Somatostatin  Human somatostatin  Mouse anti-SST  Β Cell Biology Consortium  AB1985  Mouse  Polyclonal  1:500  AB_10014609  Sulfonylurea receptor 1  Aa1560-1582  Rabbit anti-SUR1  Abcam  ab32844  Rabbit  Polyclonal  1:50  AB_2273320  Synaptophysin  C-terminus of human Syn  Rabbit anti-synaptophysin  Novus Biologicals  NB120-16659  Rabbit  Monoclonal  1:50  AB_792140  Peptide/Protein Target  Antigen Sequence  Name of Antibody  Manufacturer or Name of Individual Providing the Antibody  Catalog No.  Species Raised in  Monoclonal or Polyclonal  Dilution Used  RRID  Rabbit IgG  Whole IgG  Goat anti-rabbit AF488  Life Technologies  A11034  Goat  Polyclonal  1:1000  AB_2576217  Rabbit IgG  Whole IgG  Goat anti-rabbit AF555  Life Technologies  A21429  Goat  Polyclonal  1:1000  AB_151761  Rabbit IgG  Whole IgG  Goat anti-rabbit AF594  Life Technologies  A11037  Goat  Polyclonal  1:1000  AB_2534095  Rabbit IgG  Whole IgG  Goat anti-rabbit AF647  Life Technologies  A21245  Goat  Polyclonal  1:1000  AB_2535813  Rabbit IgG  Whole IgG  Donkey anti-rabbit AF488  Life Technologies  A21206  Donkey  Polyclonal  1:1000  AB_141708  Rabbit IgG  Whole IgG  Donkey anti-rabbit AF555  Life Technologies  A31572  Donkey  Polyclonal  1:1000  AB_162543  Rabbit IgG  Whole IgG  Donkey anti-rabbit AF594  Life Technologies  A21207  Donkey  Polyclonal  1:1000  AB_141637  Rabbit IgG  Whole IgG  Donkey anti-rabbit AF647  Life Technologies  A31573  Donkey  Polyclonal  1:1000  AB_2536183  Mouse IgG  Whole IgG  Goat anti-mouse AF488  Life Technologies  A11029  Goat  Polyclonal  1:1000  AB_2534088  Mouse IgG  Whole IgG  Goat anti-mouse AF555  Life Technologies  A21424  Goat  Polyclonal  1:1000  AB_141780  Mouse IgG  Whole IgG  Goat anti-mouse AF594  Life Technologies  A11032  Goat  Polyclonal  1:1000  AB_141672  Mouse IgG  Whole IgG  Goat anti-mouse AF647  Life Technologies  A21236  Goat  Polyclonal  1:1000  AB_2535805  Mouse IgG  Whole IgG  Donkey anti-mouse AF488  Life Technologies  A21202  Donkey  Polyclonal  1:1000  AB_2535788  Mouse IgG  Whole IgG  Donkey anti-mouse AF555  Life Technologies  A31570  Donkey  Polyclonal  1:1000  AB_2536180  Mouse IgG  Whole IgG  Donkey anti-mouse AF594  Life Technologies  A21203  Donkey  Polyclonal  1:1000  AB_141633  Mouse IgG  Whole IgG  Donkey anti-mouse AF647  Life Technologies  A31571  Donkey  Polyclonal  1:1000  AB_162542  Goat IgG  Whole IgG  Donkey anti-goat AF594  Life Technologies  A11058  Donkey  Polyclonal  1:1000  AB_142540  Sheep IgG  Whole IgG  Donkey anti-sheep AF488  Life Technologies  A11015  Donkey  Polyclonal  1:1000  AB_141362  Guinea pig IgG  Whole IgG  Goat anti–guinea pig AF488  Life Technologies  A11073  Goat  Polyclonal  1:1000  AB_2534117  Guinea pig IgG  Whole IgG  Goat anti–guinea pig AF647  Life Technologies  A21450  Goat  Polyclonal  1:1000  AB_151882  Aldehyde dehydrogenase family member 1A3  Unknown  Rabbit anti-ALDH1A3  Novus Biologicals  NBP2-15339  Rabbit  Polyclonal  1:100  AB_2665496  Islet amyloid polypeptide  Unknown  Rabbit anti-IAPP  AbCam  ab15125  Rabbit  Polyclonal  1:50  AB_2295631  Beta-galactosidase  Whole β-galactosidase  Rabbit anti-βGAL  Thermo Scientific  A11132  Rabbit  Polyclonal  1:100  AB_221539  Beta-galactosidase  Unknown  Mouse anti-βGAL  DSHB  40-1a-c  Mouse  Monoclonal  1:50  AB_528100  Glucokinase  Recombinant glucokinase  Rabbit anti-GCK  Sigma  HPA007034  Rabbit  Polyclonal  1:50  AB_888431  Glucagon  Unknown  Mouse anti-GCG  Sigma  G 2654  Mouse  Monoclonal  1:1000  AB_259852  Glucose transporter 2  First extracellular loop of Glut2  Rabbit anti-GLUT2  Millipore  07-1402  Rabbit  Polyclonal  1:500  AB_1587076  Insulin  Full-length human insulin  Guinea pig anti-INS  Thermo Fisher Scientific  PA1-26938  Guinea pig  Polyclonal  1:100  AB_794668  Insulin  Unknown  Rabbit anti-INS  Cell Signaling  C27C9  Rabbit  Monoclonal  1:200  AB_2126503  Insulin  Residues surrounding Val36 of human insulin  Mouse anti-INS  Cell Signaling  L6B10  Mouse  Monoclonal  1:250  AB_10949314  v-myc Avian myolocytomatosis viral oncogene lung carcinoma derived  AA 105-154 of human l-myc  Rabbit anti-L-MYC  AbCam  Ab28739  Rabbit  Polyclonal  1:100  AB_2148730  V-maf muscoloapo-neurotic fibrosarcoma oncogene homolog A  Unknown  Rabbit anti-MAFA  Betalogics (Johnson & Johnson)  LP9872  Rabbit  Polyclonal  1:1000  AB_2665528  V-maf muscoloaponeurotic fibrosarcoma oncogene homolog B  AA 18-140 of MAFB  Rabbit anti-MAFB  Sigma Life Sciences  HPA005653  Rabbit  Polyclonal  1:100  AB_1079293  Homeobox protein NANOG  Mouse nanog  Rabbit anti-NANOG  AbCam  Ab80892  Rabbit  Polyclonal  1:100  AB_2150114  Homeodomain transcription factor 6.1  Human Nkx6.1  Goat anti-NKX6.1  R and D Systems  AF5857  Goat  Polyclonal  1:20  AB_1857045  Homeodomain transcription factor 2.2  Nkx2.2–GST fusion protein from Escherichia. coli  Mouse anti-NKX2.2  DSHB  74.5A5  Mouse  Monoclonal  1:100  AB_531794  Neurogenin-3  Met1-Leu214 of human Ngn3  Sheep anti-NGN3  R and D Systems  AF3444  Sheep  Polyclonal  1:20  AB_2149527  Neurogenin-3  AA40-69 of human Ngn3  Sheep anti-NGN3  Thermo Fisher Scientific  PA5-11893  Rabbit  Polyclonal  1:100  AB_2149526  Pancreatic polypeptide  Ala30-Leu95 of human PP  Goat anti-PP  R and D Systems  AF6297  Goat  Polyclonal  1:200  AB_10717571  Paired box 6  C-terminus of mouse PAX6  Rabbit anti-PAX6  Covance  PRB-278P  Rabbit  Polyclonal  1:250  AB_2313780  Prohormone convertase 1/3  Unknown  Rabbit anti-PC1/3  Lakshmi Devi  Gift  Rabbit  Polyclonal  1:500  AB_2665530  Prohormone convertase 1/3  Unknown  Mouse anti-PC1/3  Gunilla Westermark  Gift  Mouse  Monoclonal  Direct  AB_2665529  Prohormone convertase 2  E622-N638 of mouse PC2  Rabbit anti-PC2  Thermo Fisher Scientific  PA1-058  Rabbit  Polyclonal  1:500  AB_2158593  Proliferating cell nuclear antigen  Rat PCNA  Mouse anti-PCNA  Abcam  ab29  Mouse  Monoclonal  1:100  AB_303394  Pancreatic and duodenal homeobox 1  N-terminus of mouse PDX1  Guinea pig anti-PDX1  Abcam  ab47308  Guinea pig  Polyclonal  1:1000  AB_777178  Somatostatin  Human somatostatin  Mouse anti-SST  Β Cell Biology Consortium  AB1985  Mouse  Polyclonal  1:500  AB_10014609  Sulfonylurea receptor 1  Aa1560-1582  Rabbit anti-SUR1  Abcam  ab32844  Rabbit  Polyclonal  1:50  AB_2273320  Synaptophysin  C-terminus of human Syn  Rabbit anti-synaptophysin  Novus Biologicals  NB120-16659  Rabbit  Monoclonal  1:50  AB_792140  Abbreviations: IgG, immunoglobulin G. View Large Abbreviations: ALDH1A3 aldehyde dehydrogenase 1 family member A3 ATP adenosine triphosphate βGAL β-galactosidase DAPI 4′,6-diamidino-2-phenylindole GCG glucagon GLUT2 glucose transporter 2 IAPP islet amyloid polypeptide IGF insulinlike growth factor IGF1R insulinlike growth factor 1 receptor INS insulin IR insulin receptor IRS insulin receptor substrate L-MYC v-myc avian myolocytomatosis viral oncogene lung carcinoma derived MAFA muscoloaponeurotic fibrosarcoma oncogene homolog A mRNA messenger RNA NGN3 neurogenin-3 NKX2.2 homeodomain transcription factor 2.2 NKX6.1 homeodomain transcription factor 6.1 PAX6 paired box 6 PBS phosphate-buffered saline PC1/3 prohormone convertase 1/3 PC2 prohormone convertase 2 PCNA proliferating cell nuclear antigen PDX1 pancreatic and duodenal homeobox 1 PP pancreatic polypeptide qPCR quantitative polymerase chain reaction SST somatostatin SUR1 sulfonylurea receptor 1 SYN synaptophysin UBC University of British Columbia. Acknowledgments We express our sincere gratitude to Ali Asadi for consultation on histological procedures, Shannon O’Dwyer for expert animal handling assistance, and Nazde Edeer for technical assistance. Financial Support: This work was supported by grants from the Canadian Diabetes Association and the Canadian Institutes of Health Research (CIHR Foundation Scheme). A.R. gratefully acknowledges studentship support from the Canadian Institutes of Health Research (Vanier Canada Graduate Scholarship) and Vancouver Coastal Health (CIHR-UBC MD/PhD Studentship). Author Contributions: A.R. and T.J.K. designed the experiments. A.R. performed the experiments. M.M. performed islet transplantation, optimized insulin therapy, and generated and maintained Ins1−/−Ins2−/− animals. A.R. analyzed data, and A.R. and T.J.K wrote the manuscript. All authors were involved in the discussion and revision of the manuscript. 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Insulin-Deficient Mouse β-Cells Do Not Fully Maturebut Can Be Remedied Through Insulin Replacementby Islet Transplantation

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Endocrine Society
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Copyright © 2018 Endocrine Society
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0013-7227
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1945-7170
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10.1210/en.2017-00263
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Abstract

Abstract Insulin receptor (IR) insufficiency in β-cells leads to impaired insulin secretion and reduced β-cell hyperplasia in response to hyperglycemia. Selective IR deficiency in β-cells in later embryological development may lead to compensatory β-cell hyperplasia. Although these findings suggest insulin signaling on the β-cell is important for β-cell function, they are confounded by loss of signaling by the insulinlike growth factors through the IR. To determine whether insulin itself is necessary for β-cell development and maturation, we performed a characterization of pancreatic islets in mice with deletions of both nonallelic insulin genes (Ins1−/−Ins2−/−). We immunostained neonatal Ins1−/−Ins2−/− and Ins1+/+Ins2+/+ pancreata and performed quantitative polymerase chain reaction on isolated neonatal islets. Insulin-deficient islets had reduced expression of factors normally expressed in maturing β-cells, including muscoloaponeurotic fibrosarcoma oncogene homolog A, homeodomain transcription factor 6.1, and glucose transporter 2. Ins1−/−Ins2−/−β-cells expressed progenitor factors associated with stem cells or dedifferentiated β-cells, including v-myc avian myolocytomatosis viral oncogene lung carcinoma derived and homeobox protein NANOG. We replaced insulin by injection or islet transplantation to keep mice alive into adulthood to determine whether insulin replacement was sufficient for the completed maturation of insulin-deficient β-cells. Short-term insulin glargine (Lantus®) injections partially rescued the β-cell phenotype, whereas long-term replacement of insulin by isogenic islet transplantation supported the formation of more mature β-cells. Our findings suggest that tightly regulated glycemia, insulin species, or other islet factors are necessary for β-cell maturation. Diabetes affects >400 million people worldwide (1) and imposes high financial, disability, and mortality costs for health care systems and patients around the world (2). Regardless the type of diabetes (type 1, type 2, gestational, or rare forms), all patients with diabetes develop an eventual insufficiency of the hormone insulin (3). In humans, β-cells containing insulin first begin to appear in the pancreas at week 7 of development, and insulin acts as a powerful anabolic signaling molecule starting in the 26th week of gestation (4). Uptake of glucose into cells is a fundamental biological process that is almost always dependent on the actions of insulin (5). Without insulin, subjects face elevated glucose and glucagon levels, increased stress hormones and ketogenesis, and eventual death from severe ketoacidosis (6). However, insulin may also play other roles independent of directly lowering blood glucose, including serving as an important signaling molecule on the β-cell itself. Mice with targeted disruption of insulin receptors (IRs) on β-cells have impaired glucose-stimulated insulin secretion and glucose intolerance (7). IRs on β-cells are necessary for β-cell hyperplasia in response to insulin resistance (8). These studies (7, 8) used a transgenic mouse model with the rat insulin promoter expressing Cre, with a floxed insulin receptor gene (Insr). Cre is expressed after E9.5 when insulin first appears in the mouse pancreas (9) and recombines to delete the IR from ~80% (10) of β-cells and regions of the brain (11). Delayed and incomplete recombination allows some insulin signaling in β-cells beyond E9.5, and impaired insulin action in the brain can promote obesity and insulin resistance (12). Therefore, these findings do not conclusively demonstrate that a loss of insulin signaling in β-cells is responsible for the observed phenotype. Recently, Trinder et al. (13) addressed the caveat of central recombination by using mice with an inducible Cre transgene driven by a mouse insulin I promoter to generate IR deficiency selectively in β-cells after E13. MIP-CreERT mice had reduced β-cell mass at birth, but as in previous studies using the RIP-Cre line, these mice retained alternative insulin signaling pathways and attenuated insulinlike growth factor (IGF) signaling in β-cells through the IR, making it challenging to conclude that a loss of insulin action in β-cells was entirely responsible for the phenotype. Alongside incomplete β-cell recombination, delayed loss of IRs until later in development, and the presence of a human growth hormone minigene in the MIP-CreERT transgene (14, 15), there are other important factors to consider when attempting to study a loss of insulin signaling by using a model with deletion of the IR. First, insulin may signal with low affinity through IGF receptors I and II (16, 17), and it is possible that there are other insulin signaling pathways not yet appreciated. Hyperinsulinemia in β-cell IR-deficient mice may increase the likelihood of insulin signaling via the IGF receptors (7). Second, IGF-I and IGF-II bind the IR and play essential roles in embryological mammalian development (18). In fact, binding of IGF-II to the IR appears to be the most important signal through the IR during development (19). Thus, deleting the IR may not completely block all actions of insulin and impairs the IR-mediated actions of the IGF hormones. We posit that the only model capable of defining the role of insulin in β-cell development and maturation is a model with a loss of the insulin gene itself. In 1997 Duvillié et al. (20) generated an absolute insulin-deficient mouse lacking both copies of the two nonallelic insulin genes, Ins1 and Ins2 (Ins1−/−Ins2−/−), with β-galactosidase (βGAL; gene lacZ) knocked in under control of the Ins2 promoter. Ins1−/−Ins2−/− mice are viable at birth, have a slightly (20%) lower body weight, and though euglycemic at the time of parturition quickly develop severe hyperglycemia after suckling and die on average within 48 hours (20, 21). This result aligns with findings in humans with insulin mutations who first present with growth retardation at 27 weeks’ gestation, a similar developmental stage to mice at birth (22). Pancreatic islets of neonatal Ins1−/−Ins2−/− mice are enlarged but have low levels of endocrine hormone messenger RNA (mRNA) [glucagon (Gcg), somatostatin (Sst), and pancreatic polypeptide (Ppy)] (23). The pancreas of Ins1−/−Ins2−/− mice is full of aggregates of βGAL-expressing cells, but apart from positive immunoreactivity for pancreatic and duodenal homeobox 1 (PDX1) in βGAL-expressing cells (23), there are no reports of the functional maturity of these cells to determine the role of insulin signaling during β-cell development. In the current study, we determined the role of insulin during β-cell development and maturation by characterizing pancreatic islets of neonatal Ins1−/−Ins2−/− mice and mice maintained into adulthood by insulin replacement therapy. We found that alongside a complete loss of insulin production, β-cells of Ins1−/−Ins2−/− mice lacked many markers of mature β-cells and were present in islets of abnormal size and endocrine hormone composition. Additionally, insulin-deficient β-cells expressed markers of dedifferentiation and dysfunction, suggesting that insulin is a necessary signal for completely maturing β-cells or maintaining their maturity. Short-term replacement of insulin in the insulin knockout mice via exogenous injection facilitated a partial normalization of the β-cell phenotype but was associated with islet fibrosis. Long-term replacement of insulin by isogenic islet transplantation was sufficient for the maturation of insulin-deficient β-cells. This model provides direct evidence that insulin itself is needed for normal β-cell development and the maintenance of normal β-cell function. Materials and Methods Animal breeding and insulin therapy All experiments were approved by the University of British Columbia (UBC) Animal Care Committee and carried out in accordance with the Canadian Council on Animal Care Guidelines. C57Bl/6j Ins1+/+Ins2+/+ mice (Jackson Laboratories, Bar Harbor, ME) were used as controls. Ins1−/−Ins2+/− mice were generated in the laboratory of Dr. J. Jami (Institut Cochin, Paris, France) and supplied indirectly from the laboratory of Dr. J. Johnson (UBC, Vancouver, Canada). Animals were housed with a 12-hour light/dark cycle with ad libitum access to a standard chow diet (29018; Harlan Laboratories, Madison, WI). Ins1−/−Ins2+/− mice were bred to acquire Ins1−/−Ins2−/− mice. Upon first discovery of neonatal mice (P0 to P1), subsets of mice were euthanized by decapitation, and blood sugar was examined with a handheld glucometer (LifeScan, Burnaby, BC, Canada). Insulin therapy was immediately initiated in other cohorts of Ins1−/−Ins2−/− mice, initially identified by reduced body weight and confirmed by genotyping at an older age. We genotyped Ins2 by quantitative polymerase chain reaction (qPCR) assay (wild-type forward primer 5′-GGT CCT TGG TAG TAA CTT G, reverse primer 5′-GCC TCT AAA GCC TAC TCA TCT TC, and probe 5′-GCA GTG CTC TAT GAG GGC CCT AAA; lacZ knockin forward primer 5′-CTG TAT GAA CGG TCT GGT CTT T, reverse primer 5′-CGC TAT GAC GGA ACA GGT ATT, and probe 5′-TTG CCC GGA TAA ACG GAA CTG GAA) and Ins1 by qPCR assay (wild-type forward primer 5′-CCA TTG TTA GGT TGG ATG ATT, reverse primer 5′-CGG TTG CCT ACC TTC TT, and probe 5′-AGT ATC TGG AAT TCT GCT TCC TGC CC; knockout forward primer 5′-AAA CCA CAC TGC TCG AC, reverse primer 5′-CAG GAA GCA GAA TTC CAG ATA, and probe 5′-GGG CTG CAG GAA TTC GAT ATC AAG C). Briefly, animals received twice-daily subcutaneous injections of ~0.1 U insulin glargine diluted to 5 U/mL in F-10 media (Lantus®; Sigma-Aldrich, St. Louis, MO). Because regular blood glucose sampling is not possible with young pups, health of the animals was tracked by body weight and glycosuria, and when animals failed to gain weight or lost weight, insulin dosing was halved or skipped. When animals presented with severe glycosuria, we increased the insulin dose. As pups grew, we began regular blood sampling to better refine insulin doses. Therapy continued for up to 291 days. Mortality by this method was initially extremely high (>90%) but decreased with experience to <50% for animals analyzed in this study. Some animals received islet transplants into the anterior chamber of the eye at 14 days of age. In all experiments, we present findings from a mix of male and female animals. Islet isolation and transplantation Newborn Ins1−/−Ins2−/− or Ins1+/+Ins2+/+ mice were euthanized by decapitation upon first discovery (P0 to P1), and pancreatic islets were isolated by collagenase digestion (24). After euthanasia, we performed a rapid dissection and exposed the pancreatic duct. Approximately 1 mL of collagenase solution (1000 U/mL type XI collagenase; Sigma-Aldrich; diluted in Hanks buffer without CaCl2) was injected into the pancreatic duct. The pancreas was then excised and digested for 6 to 8 minutes at 37°C in 3 mL of collagenase. Islets were then handpicked three times in medium (Hams F10, 7.5% fetal bovine serum, and penicillin/streptomycin; Sigma-Aldrich) to increase purity to >90%. Islets were washed in phosphate-buffered saline (PBS) and immediately lysed by forceful suction into a 20-gauge needle 20 to 30 times in 50 µL of QIAzol Lysis Reagent (Qiagen, Toronto, ON, Canada) before storage at −80°C. Islet isolation from donor adult C57Bl/6 mice (used for transplantation) was completed in the same fashion, with slight changes (incubation in collagenase for 12 to 15 minutes at 37°C). After handpicking, islets were cultured overnight, visually inspected for purity, and washed in sterile PBS before transplantation. After 2 weeks of insulin therapy with insulin glargine (Lantus®), Ins1−/−Ins2−/− animals received an islet transplant into the anterior chamber of the eye (~100 to 150 islets) as previously described (25). This procedure is technically feasible at 2 weeks of age and provides reversal of diabetes within 24 hours. Animals were anesthetized by inhaled isoflurane (5%) and maintained at 1% to 3%. The cornea was punctured with a 27-gauge needle to gain access to the anterior chamber of the eye. Islets were loaded into a micropipette (MXL3-BP-IND-200; Origio MidAtlantic Devices, Mt. Laurel, NJ), the micropipette was passed through the opening in the cornea, and islets were deposited with a micromanipulator. After removal of the micropipette, animals received Isoptears (Alcon Canada, Mississauga, ON, Canada) with 0.3% wt/vol gentamycin to prevent infection. Immunohistochemistry and immunohistofluorescence Pancreata were dissected out of mice, washed in PBS, and fixed in 4% paraformaldehyde overnight before being transferred to 70% ethanol for long-term storage before paraffin embedding and sectioning (5 µm thickness; Wax-It Histology Services, Vancouver, Canada). Hematoxylin and eosin and Masson’s trichome staining was performed by standard protocol, and slides were scanned with the ScanScope CS system (Aperio, Vista, CA). Immunofluorescent staining was performed as previously described (26). Briefly, sections were deparaffinized in xylene (15 minutes) and rehydrated in graded ethanol (100% 2 × 5 minutes, 95% 5 minutes, 70% 5 minutes, and PBS 10 minutes) before heat-induced epitope retrieval in an EZ Retriever microwave oven (BioGenes, Fremont, CA) for 15 minutes at 95°C in 10 mM citrate buffer (0.5% Tween 20, pH 6.0; Thermo Fisher Scientific, Waltham, MA). Samples were blocked in DAKO Protein Block, Serum Free (Dako, Burlington, Canada) and incubated overnight in primary antibody diluted in Dako Antibody Diluent (Dako). The next day, slides were washed and incubated in secondary antibody for 1 hour at room temperature before mounting and counterstaining with nuclear stain 4′,6-diamidino-2-phenylindole (DAPI) and VECTASHIELD® Hard Set Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA). All images were captured and analyzed with an ImageXpress® Micro XLS System, controlled by MetaXpress® High-Content Image Acquisition & Analysis Software (Molecular Devices Corporation, Sunnyvale, CA) with a scientific CMOS camera, a Nikon 20× Plan Apo objective (NA = 0.75, 1-6300-0196; Nikon, Tokyo, Japan), and DAPI (DAPI-5060B), FITC (FITC-3540B), Cy3 (Cy3-4040B), Texas Red (TXRED-4040B), and Cy5 (Cy5-4040A) filter cubes. We performed quantification of histological images in the same software (MetaXpress®). We used islet amyloid polypeptide (IAPP), insulin, or βGAL to label β-cells, glucagon (GCG) to label α-cells, somatostatin (SST) to label δ-cells, and pancreatic polypeptide (PP) to label pancreatic polypeptide cells. Of note, although δ-cells are known to express some IAPP (27), given the smaller number of δ-cells, lower expression level, and limited colocalization of SST and IAPP (data not shown), we use bright IAPP immunoreactivity as a marker of β-cells. We immunostained for synaptophysin (SYN) to identify islet cells and quantify islet size and number. We calculated endocrine cell area of each cell type by quantifying immunoreactive surface area relative to total pancreas surface area and calculated the average of three immunostained sections 50 to 100 µM apart for each animal. We calculated the proportion of β-cells with positive immunoreactivity for proteins of interest or total immunoreactive area of proteins of interest by thresholding images and using a multiwavelength cell scoring journal in MetaXpress®, and we used image analysis to find the mean fluorescent intensity of positive signal in relevant cell compartments. RNA isolation and quantitative reverse transcription polymerase chain reaction After islet isolation, lysis, and storage in Qiazol Lysis Reagent (Qiagen), we performed acid guanidinium thiocyanate–phenol–chloroform extraction by standard methods (28). To ensure sufficient quantity of mRNA for downstream applications, we pooled lysed islets from three to five mice in each group of Ins1−/−Ins2−/−, Ins1−/−Ins2+/−, or Ins1+/+Ins2+/+ animals, for a total of 25 to 35 islets per sample. We generated complementary DNA from isolated islet RNA by using a RevertAid H Minus First Strand cDNA synthesis kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. We quantified mRNA levels of target genes via the 2−ΔΔCT method (glyceraldehyde 3-phosphate dehydrogenase used as housekeeping gene) by using the SsoFast EvaGreen Supermix (Bio-Rad, Mississauga, Canada) kit according to the manufacturer’s protocol (for primer details see Supplemental Table 1). Statistics Data were subject to the Shapiro–Wilk normality test, and when all groups passed the test for normality we analyzed them by Student t test, one-way analysis of variance with Tukey test for multiple comparisons, or two-way analysis of variance with Bonferroni post hoc testing. When one or more groups failed the test for normality, we used the nonparametric Mann–Whitney U test or Kruskal–Wallis test with the Dunn multiple comparisons test. Statistical analysis was performed in GraphPad Prism 7.01 (La Jolla, CA) with significance set at P < 0.05. Results Ins1−/−Ins2−/− mice are born with β-cells Upon discovery (P0 to P1), Ins−/−Ins2−/− mice had lower body weight compared with Ins1+/+Ins2+/+ controls (Fig. 1A). Ins−/−Ins2−/− mice were euglycemic at the time of delivery but quickly developed hyperglycemia after suckling. Therefore, blood glucose levels were highly variable at the time of blood sampling, unlike in the Ins1+/+Ins2+/+ controls. We immunostained pancreas from mice with reduced insulin gene copy number (Ins1−/−Ins2+/+, Ins1+/+Ins2−/−, or Ins1−/−Ins2+/−) for the prototypical mature β-cell marker muscoloaponeurotic fibrosarcoma oncogene homolog A (MAFA, a basic leucine zipper transcription factor; Supplemental Fig. 1). Given previous reports of abnormal β-cell size and islet structure (29) paired with reduced nuclear immunoreactivity for MAFA in mice with reduced insulin gene copy number, we compared the Ins1−/−Ins2−/− group with wild-type Ins1+/+Ins2+/+ controls in all subsequent experiments. Histological examination of neonatal pancreata revealed normal aggregates of IAPP+βGAL+INS− clusters resembling neonatal islets of Langerhans in Ins1−/−Ins2−/− mice and IAPP+βGAL− insulin-producing (INS+) β-cells in Ins1+/+Ins2+/+ mice (Fig. 1B). Figure 1. View largeDownload slide Ins1−/−Ins2−/− mice have IAPP-expressing β-cells. (A) Upon discovery (P0 to P1), wild-type Ins1+/+Ins2+/+ and insulin knockout Ins1−/−Ins2−/− mice were weighed and their blood glucose was measured (n = 10–31). Individual animal weight or blood glucose is shown in box-and-whisker plots. Statistical analysis to compare body weight was performed with a Student t test to assess significance. ***P < 0.001. (B) Immunostaining of P1 pancreata for INS, IAPP, βGAL, and DAPI in wild-type Ins1+/+Ins2+/+ controls (upper panels) and Ins1−/−Ins2−/− mice (lower panels), revealing isletlike clusters of INS−IAPP+βGAL+ cells in Ins1−/−Ins2−/− pancreata (representative micrographs from n = 3). Scale bar, 100 µm. Figure 1. View largeDownload slide Ins1−/−Ins2−/− mice have IAPP-expressing β-cells. (A) Upon discovery (P0 to P1), wild-type Ins1+/+Ins2+/+ and insulin knockout Ins1−/−Ins2−/− mice were weighed and their blood glucose was measured (n = 10–31). Individual animal weight or blood glucose is shown in box-and-whisker plots. Statistical analysis to compare body weight was performed with a Student t test to assess significance. ***P < 0.001. (B) Immunostaining of P1 pancreata for INS, IAPP, βGAL, and DAPI in wild-type Ins1+/+Ins2+/+ controls (upper panels) and Ins1−/−Ins2−/− mice (lower panels), revealing isletlike clusters of INS−IAPP+βGAL+ cells in Ins1−/−Ins2−/− pancreata (representative micrographs from n = 3). Scale bar, 100 µm. Insulin-deficient islets are enlarged and have an abnormal endocrine cell distribution We characterized the endocrine cell distribution of neonatal islets to determine whether a lack of insulin causes a shift in endocrine cell proportions. Ins1+/+Ins2+/+ and Ins1−/−Ins2−/− islets had a similar distribution of GCG+ α-cells and SST+ δ-cells (including cells in the core of the islet) and no obvious pathological immune infiltration or fibrosis by hematoxylin and eosin and Masson’s trichrome staining (Fig. 2A). Among all hormone-expressing cells, the relative proportion of GCG- and PP-expressing cells decreased and the proportion of SST-expressing cells increased in islets of Ins1−/−Ins2−/− mice compared with Ins1+/+Ins2+/+ controls (Fig. 2B). The proportion of total pancreas section area expressing IAPP and SST was larger and PP was smaller in Ins1−/−Ins2−/− pancreata compared with Ins1+/+Ins2+/+ controls (Fig. 2C) [measurement used as a surrogate of total endocrine cell mass (30)]. We immunostained for ghrelin and found extremely rare cells, as expected, in both controls and insulin-deficient islets (data not shown). Insulin-deficient islets were fewer in number (Fig. 2D) but more than three times as large as islets in pancreata of Ins1+/+Ins2+/+ control mice (Fig. 2E). We immunostained for the proliferating cell nuclear antigen (PCNA) as a marker of replicating cells and observed a pronounced increase in proportion of IAPP+PCNA+/IAPP+ cells in Ins1−/−Ins2−/− mice compared with controls (Fig. 2F). Figure 2. View largeDownload slide Ins1−/−Ins2−/− mice have enlarged islets with abnormal islet cell number and distribution. (A) P1 pancreata of Ins1+/+Ins2+/+ (top panels) and Ins1−/−Ins2−/− (bottom panels) mice were immunostained for IAPP and GCG or GCG, SST, and PP. Hematoxylin and eosin and Masson’s trichrome staining of P1 pancreas (representative micrographs from n = 3–5). Scale bars, 100 µm. (B) Percentage of total endocrine immunoreactive area that is immunoreactive for the islet hormones IAPP, GCG, SST, and PP (n = 4 or 5 animals, 3 sections quantified per animal). (C) β-cell, α-cell, δ-cell, and pancreatic polypeptide cell area relative to total pancreas area (n = 4 or 5 animals, 3 sections quantified per animal). (D) Number of islets identified by SYN immunoreactivity relative to pancreas section area (n = 4 or 5, 3 sections quantified per animal). (E) Average islet size by SYN immunoreactive area (n = 4 or 5, 3 sections quantified per animal). (F) Quantification of percentage of IAPP+ cells that were PCNA+; representative image shown (n = 5). Scale bar, 100 µm. Individual animals are shown on box-and-whisker plots. Statistical analysis was performed via Student t test to assess significance. *P < 0.05, **P < 0.01, ***P < 0.001 vs Ins1+/+Ins2+/+ controls. Figure 2. View largeDownload slide Ins1−/−Ins2−/− mice have enlarged islets with abnormal islet cell number and distribution. (A) P1 pancreata of Ins1+/+Ins2+/+ (top panels) and Ins1−/−Ins2−/− (bottom panels) mice were immunostained for IAPP and GCG or GCG, SST, and PP. Hematoxylin and eosin and Masson’s trichrome staining of P1 pancreas (representative micrographs from n = 3–5). Scale bars, 100 µm. (B) Percentage of total endocrine immunoreactive area that is immunoreactive for the islet hormones IAPP, GCG, SST, and PP (n = 4 or 5 animals, 3 sections quantified per animal). (C) β-cell, α-cell, δ-cell, and pancreatic polypeptide cell area relative to total pancreas area (n = 4 or 5 animals, 3 sections quantified per animal). (D) Number of islets identified by SYN immunoreactivity relative to pancreas section area (n = 4 or 5, 3 sections quantified per animal). (E) Average islet size by SYN immunoreactive area (n = 4 or 5, 3 sections quantified per animal). (F) Quantification of percentage of IAPP+ cells that were PCNA+; representative image shown (n = 5). Scale bar, 100 µm. Individual animals are shown on box-and-whisker plots. Statistical analysis was performed via Student t test to assess significance. *P < 0.05, **P < 0.01, ***P < 0.001 vs Ins1+/+Ins2+/+ controls. Insulin-deficient β-cells lack many markers of mature β-cells We examined the maturity of the insulin-deficient β-cells by immunostaining for select markers of mature β-cells. Interestingly, for some proteins the immunoreactivity appeared to coincide with the feeding and glycemia status of the animals. IAPP+ cells of hypoglycemic insulin-deficient mice had normal levels of PDX1 (Fig. 3A) but completely lacked immunoreactivity for homeodomain transcription factor 6.1 (NKX6.1), in contrast to Ins1+/+Ins2+/+ controls (Fig. 3B). We also detected less frequent immunoreactivity of homeodomain transcription factor 2.2 (NKX2.2) in β-cells (IAPP+) of Ins1−/−Ins2−/− mice compared with Ins1+/+Ins2+/+ controls (Fig. 3C). Ins1−/−Ins2−/− mice had a lower proportion of IAPP+ cells that expressed paired box 6 (PAX6), and IAPP+PAX6+ cells had lower intensity of PAX6 immunoreactivity than Ins1+/+Ins2+/+ controls (Fig. 3D), as did the remainder of the islet cells. MAFA immunoreactivity was observed in a lower proportion of βGAL+ β-cells in insulin knockouts compared with INS+ cells in control mice, and the intensity of immunoreactivity trended to be reduced (Fig. 3E). Glucose transporter 2 (GLUT2) immunoreactivity was detected in fewer IAPP+ cells in Ins1−/−Ins2−/− animals, and IAPP+GLUT2+ cells had lower intensity of GLUT2 immunoreactivity in Ins1−/−Ins2−/− animals compared with Ins1+/+Ins2+/+ controls (Fig. 4A). In hypoglycemic insulin-deficient neonates, immunoreactivity of sulfonylurea receptor 1 (SUR1) (Fig. 4B), prohormone convertase 1/3 (PC1/3) (Fig. 4C), and prohormone convertase 2 (PC2) (Fig. 4D) was comparable to that of Ins1+/+Ins2+/+ controls. In hyperglycemic Ins1−/−Ins2−/− neonates, NKX6.1, NKX2.2, PAX6, MAFA, and GLUT2 appeared to be completely lacking (Supplemental Fig. 2A), and there was a lower percentage of islet area with positive immunoreactivity for NKX6.1, NKX2.2, PAX6, MAFA, GLUT2, SUR1, and PC2 relative to control Ins1+/+Ins2+/+ mice (Supplemental Fig. 2B). Figure 3. View largeDownload slide β-cells in hypoglycemic Ins1−/−Ins2−/− mice lack expression of some β-cell transcription factors. P0 to P0.5 pancreata were immunostained for (A) PDX1, (B) NKX6.1, (C) NKX2.2, (D) PAX6, and (E) MAFA. Representative micrographs shown (n = 3). Quantification of percentage of (A–D) IAPP+ cells or (E) INS/βGAL+ cells immunoreactive for the selected transcription factor and fluorescent intensities relative to Ins1+/+Ins2+/+ controls in (A) PDX1+, (B) NKX6.1+, (C) NKX2.2+, (D) PAX6+, or (E) MAFA+ cells shown to the right of representative images. Individual animal values are shown on box-and-whisker plots. A Student t test [(A–E) target+ β-cells and (B, D, E) fluorescent intensity] or Mann–Whitney U test [(A, C) fluorescent intensity] was used to assess significance. *P < 0.05, **P < 0.01 vs Ins1+/+Ins2+/+ controls. Scale bar, 100 µm and, insets are magnified ×8. Figure 3. View largeDownload slide β-cells in hypoglycemic Ins1−/−Ins2−/− mice lack expression of some β-cell transcription factors. P0 to P0.5 pancreata were immunostained for (A) PDX1, (B) NKX6.1, (C) NKX2.2, (D) PAX6, and (E) MAFA. Representative micrographs shown (n = 3). Quantification of percentage of (A–D) IAPP+ cells or (E) INS/βGAL+ cells immunoreactive for the selected transcription factor and fluorescent intensities relative to Ins1+/+Ins2+/+ controls in (A) PDX1+, (B) NKX6.1+, (C) NKX2.2+, (D) PAX6+, or (E) MAFA+ cells shown to the right of representative images. Individual animal values are shown on box-and-whisker plots. A Student t test [(A–E) target+ β-cells and (B, D, E) fluorescent intensity] or Mann–Whitney U test [(A, C) fluorescent intensity] was used to assess significance. *P < 0.05, **P < 0.01 vs Ins1+/+Ins2+/+ controls. Scale bar, 100 µm and, insets are magnified ×8. Figure 4. View largeDownload slide Hypoglycemic Ins1−/−Ins2−/−β-cells lack expression of key β-cell factors. P0 to P0.5 pancreata were immunostained for (A) GLUT2, (B) SUR1, (C) PC1/3, and (D) PC2, Representative micrographs shown (n = 3). Quantification of percentage of (A, B) INS/βGAL+ cells or (C) IAPP+ cells immunoreactive for the selected protein and fluorescent intensities relative to Ins1+/+Ins2+/+ controls in (A) GLUT2+, (B) SUR1+, or (C) PC1/3+ cells shown to the right of representative images. (D) Percentage of GCG− islet cells expressing PC2 and relative fluorescent intensity of PC2 immunoreactivity in GCG−PC2+ cells. Scale bar, 100 µm, and insets are magnified ×8. (E) Relative mRNA quantification from isolated P0 to P05 islets from Ins1+/+Ins2+/+ and Ins1−/−Ins2−/− pups. Individual animals are shown on box-and-whisker plots. A Student t test [(B, D) target+ β-cells, (A–D) fluorescent intensity, and (E) Gcg, Nkx2.2, Slc2a2, Gck, Pcsk1, Amy] or Mann–Whitney U [(A, C) target+ β-cells and (E) Sst, Ppy, Pdx1, Nkx6.1, Pax6, Abcc8, Pcsk2] was used to assess significance. *P < 0.05, **P < 0.01, ***P < 0.001 vs controls. Figure 4. View largeDownload slide Hypoglycemic Ins1−/−Ins2−/−β-cells lack expression of key β-cell factors. P0 to P0.5 pancreata were immunostained for (A) GLUT2, (B) SUR1, (C) PC1/3, and (D) PC2, Representative micrographs shown (n = 3). Quantification of percentage of (A, B) INS/βGAL+ cells or (C) IAPP+ cells immunoreactive for the selected protein and fluorescent intensities relative to Ins1+/+Ins2+/+ controls in (A) GLUT2+, (B) SUR1+, or (C) PC1/3+ cells shown to the right of representative images. (D) Percentage of GCG− islet cells expressing PC2 and relative fluorescent intensity of PC2 immunoreactivity in GCG−PC2+ cells. Scale bar, 100 µm, and insets are magnified ×8. (E) Relative mRNA quantification from isolated P0 to P05 islets from Ins1+/+Ins2+/+ and Ins1−/−Ins2−/− pups. Individual animals are shown on box-and-whisker plots. A Student t test [(B, D) target+ β-cells, (A–D) fluorescent intensity, and (E) Gcg, Nkx2.2, Slc2a2, Gck, Pcsk1, Amy] or Mann–Whitney U [(A, C) target+ β-cells and (E) Sst, Ppy, Pdx1, Nkx6.1, Pax6, Abcc8, Pcsk2] was used to assess significance. *P < 0.05, **P < 0.01, ***P < 0.001 vs controls. We examined transcript levels by qPCR and found reduced expression of Ins, Gcg, Sst, and Ppy, as well as Pdx1, Nkx6.1, Nkx2.2, Pax6, Sur1, and Pcsk2 in isolated islets from hypoglycemic Ins1−/−Ins2−/− pups compared with Ins1+/+Ins2+/+ controls, and mRNA expression levels of Slc2a2,Gck, and Pcsk1 were similar to levels in islets from control mice (Fig. 4E). We found comparable expression of Amy in samples, indicating that the differences observed in expression levels of other target genes were unlikely to be a result of variable pancreatic exocrine contamination of samples. Insulin-deficient β-cells express progenitor markers We next determined whether insulin-deficient β-cells of Ins1−/−Ins2−/− mice resembled embryonic β-cell precursors or dedifferentiated β-cells. We immunostained pancreata from three neonatal Ins1+/+Ins2+/+ and three neonatal Ins1−/−Ins2−/− animals for factors associated with a progenitor state that have been detected in dedifferentiated β-cells (31). Many βGAL+ β-cells in Ins1−/−Ins2−/− mice were immunoreactive for v-myc avian myolocytomatosis viral oncogene lung carcinoma derived (L-MYC), homeobox protein NANOG (NANOG), and aldehyde dehydrogenase 1 family member A3 (ALDH1A3; Fig. 5A–5C). This finding contrasts with that of neurogenin-3 (NGN3), which was present in the perinuclear area of SYN+ islets cells in Ins1+/+Ins2+/+ controls but absent from Ins1−/−Ins2−/− islet cells (Fig. 5D). We repeated NGN3 immunostaining by using two commercially available antibodies (R&D Systems, catalog #AF3444; and Thermo Fisher Scientific, catalog #PA5-11893) and found similar patterns of immunoreactivity (Supplemental Fig. 3). The proportion of β-cells that were L-MYC+ or ALDH1A3+ was significantly higher in Ins1−/−Ins2−/− mice relative to controls. Ins1−/−Ins2−/−β-cells had higher intensity for immunoreactive NANOG and L-MYC compared with control samples. We attempted to quantify the corresponding gene levels by qPCR but could detect sufficient transcript only of Aldh1a3, which was about eight times higher in islets of in Ins1−/−Ins2−/− mice than in controls (Fig. 5C). Figure 5. View largeDownload slide Ins1−/−Ins2−/−β-cells are not fully differentiated. P0 to P0.5 pancreata from Ins1+/+Ins2+/+ and Ins1−/−Ins2−/− mice were immunostained for (A) L-MYC, (B) NANOG, (C) ALDH1A3, and (D) NGN3. Representative micrographs shown (n = 3). Quantification of percentage of (A–D) INS/βGAL+ cells or (E) SYN+ cells immunoreactive for the selected protein and fluorescent intensity relative to Ins1+/+Ins2+/+ controls in (A) L-MYC+, (B) NANOG+, (C) ALDH1A3+, or (D) NGN3+ cells shown to the right of representative images. Relative mRNA expression of (D) Aldh1a3 by 2−ΔΔCt method in isolated P0 to P0.5 islets from Ins1+/+Ins2+/+ and Ins1−/−Ins2−/− pups. Individual animals are shown on box-and-whisker plots. A Student t test [(A, C, D, and E) target+ β-cells, and (A–E) fluorescent intensity, and qPCR relative expression] or Mann–Whitney U [(B) target+ β-cells] was used to assess significance. *P < 0.05, ***P < 0.001 vs controls. Scale bar, 50 µm, and insets are magnified ×4. Figure 5. View largeDownload slide Ins1−/−Ins2−/−β-cells are not fully differentiated. P0 to P0.5 pancreata from Ins1+/+Ins2+/+ and Ins1−/−Ins2−/− mice were immunostained for (A) L-MYC, (B) NANOG, (C) ALDH1A3, and (D) NGN3. Representative micrographs shown (n = 3). Quantification of percentage of (A–D) INS/βGAL+ cells or (E) SYN+ cells immunoreactive for the selected protein and fluorescent intensity relative to Ins1+/+Ins2+/+ controls in (A) L-MYC+, (B) NANOG+, (C) ALDH1A3+, or (D) NGN3+ cells shown to the right of representative images. Relative mRNA expression of (D) Aldh1a3 by 2−ΔΔCt method in isolated P0 to P0.5 islets from Ins1+/+Ins2+/+ and Ins1−/−Ins2−/− pups. Individual animals are shown on box-and-whisker plots. A Student t test [(A, C, D, and E) target+ β-cells, and (A–E) fluorescent intensity, and qPCR relative expression] or Mann–Whitney U [(B) target+ β-cells] was used to assess significance. *P < 0.05, ***P < 0.001 vs controls. Scale bar, 50 µm, and insets are magnified ×4. Insulin replacement by injection leads to islet fibrosis and expanded islet cell area To determine whether restoration of insulin signaling by exogenous insulin therapy was sufficient for completed β-cell maturation, we treated insulin-deficient animals with insulin injections for 2 months. Insulin-injected Ins−/−Ins2−/− mice gained weight but at a slower rate than control animals (Supplemental Fig. 4A). After insulin therapy by injection or isogenic islet transplantation from Ins1+/+Ins2+/+ donors, animals retained clusters of IAPP+ cells, but the majority of IAPP+ cells were βGAL− (Supplemental Fig. 4B–4D). Seeking to clarify this observation, we examined INS and βGAL immunoreactivity in Ins1−/−Ins2+/− and Ins1+/+Ins2−/− islets. Evidently there can be unique expression of Ins1 compared with Ins2 within β-cells, because many INS+ cells were βGAL− in Ins1+/+Ins2−/− mice. Additionally, it appears there can be unique expression of the two Ins2 alleles within β-cells, because Ins1−/−Ins2+/− islets also had abundant INS+ cells that were βGAL− (Supplemental Fig. 4E). After 2 months of insulin therapy by injections, we found islet cell hyperplasia and obvious islet fibrosis by trichrome staining in insulin-deficient islets (Fig. 6A). Although a smaller proportion of endocrine cells were IAPP+ β-cells (Fig. 6B), we observed an expansion of all endocrine cell types relative to total pancreatic area in Ins1−/−Ins2−/− mice compared with Ins1+/+Ins2+/+ controls (Fig. 6C). Furthermore, Ins1−/−Ins2−/− islets trended to being larger (Fig. 66E), and there was a trend toward a higher proportion of IAPP+PCNA+/IAPP+ islet cells compared with controls (Fig. 6F). Figure 6. View largeDownload slide Adult Ins1−/−Ins2−/− mice treated by insulin injections have fibrosis and expanded islet cell area. (A) Pancreata of 2-month-old Ins1+/+Ins2+/+ (top panels) and insulin injection–treated Ins1−/−Ins2−/− (bottom panels) mice were immunostained for IAPP and GCG or GCG, SST, and PP. Hematoxylin and eosin and Masson’s trichrome staining of pancreata (representative micrographs from n = 3–5). Scale bars, 100 µm. (B) Percentage of total endocrine (SYN+) immunoreactive area that expressed the islet hormones IAPP, GCG, SST, or PP. (C) β-cell, α-cell, δ-cell, and pancreatic polypeptide cell area relative to total pancreas area (n = 4 animals, 3 sections quantified per animal). (D) Number of islets identified by SYN immunoreactivity relative to pancreas section area. (E) Average islet size by SYN immunoreactive area. (F) Quantification of percentage of IAPP+ cells that were PCNA+; representative image shown (n = 4). Scale bar, 100 µm. (B–F) Individual animal endocrine area is shown on box-and-whisker plots. Statistical analysis was performed with a Student t test [(B) IAPP, SST, PP, (C) IAPP, GCG, PP, (F)] or Mann–Whitney U test [(B) GCG, (C) SST, (D, E)] to assess significance. *P < 0.05, **P < 0.01, ***P < 0.001 vs Ins1+/+Ins2+/+ controls. Figure 6. View largeDownload slide Adult Ins1−/−Ins2−/− mice treated by insulin injections have fibrosis and expanded islet cell area. (A) Pancreata of 2-month-old Ins1+/+Ins2+/+ (top panels) and insulin injection–treated Ins1−/−Ins2−/− (bottom panels) mice were immunostained for IAPP and GCG or GCG, SST, and PP. Hematoxylin and eosin and Masson’s trichrome staining of pancreata (representative micrographs from n = 3–5). Scale bars, 100 µm. (B) Percentage of total endocrine (SYN+) immunoreactive area that expressed the islet hormones IAPP, GCG, SST, or PP. (C) β-cell, α-cell, δ-cell, and pancreatic polypeptide cell area relative to total pancreas area (n = 4 animals, 3 sections quantified per animal). (D) Number of islets identified by SYN immunoreactivity relative to pancreas section area. (E) Average islet size by SYN immunoreactive area. (F) Quantification of percentage of IAPP+ cells that were PCNA+; representative image shown (n = 4). Scale bar, 100 µm. (B–F) Individual animal endocrine area is shown on box-and-whisker plots. Statistical analysis was performed with a Student t test [(B) IAPP, SST, PP, (C) IAPP, GCG, PP, (F)] or Mann–Whitney U test [(B) GCG, (C) SST, (D, E)] to assess significance. *P < 0.05, **P < 0.01, ***P < 0.001 vs Ins1+/+Ins2+/+ controls. Insulin replacement by injection facilitates partial maturation of Ins1−/−Ins2−/−β-cells To determine whether insulin replacement by injection was sufficient for Ins1−/−Ins2−/−β-cells to complete maturation, gain expression of NKX6.1, NKX2.2, MAFA, PAX6, and GLUT2, and lose expression of L-MYC and NANOG, we immunostained for these factors in Ins1−/−Ins2−/− mice treated with insulin injections. After 2 months of twice-daily insulin injections, Ins1−/−Ins2−/−β-cells gained immunoreactive NKX2.2 (Fig. 7A) but still had lower intensity of NKX6.1 immunoreactivity compared with INS+ Ins1+/+Ins2+/+β-cells (Fig. 7B). Furthermore, Ins1−/−Ins2−/− islet cells were deficient in PAX6, MAFA, and GLUT2 compared with control islets (Fig. 7C–7E). There was cytoplasmic NANOG immunoreactivity in Ins1−/−Ins2−/− IAPP+ cells but no L-MYC+ cells (Fig. 77G). Rare ALDH1A3+ cells were observed (Fig. 7H), and pericytoplasmic immunoreactivity for NGN3 was frequent in Ins1−/−Ins2−/− IAPP+ β-cells and neighboring cells within the islet (Fig. 7I), similar to Ins1+/+Ins2+/+ neonatal β-cells (Fig. 5D). Figure 7. View largeDownload slide Insulin therapy by injection is not sufficient for the completed maturation of Ins1−/−Ins2−/−β-cells. Pancreata of 2-month-old Ins1+/+Ins2+/+ (left panels) and insulin injection–treated Ins1−/−Ins2−/− (right panels) mice were immunostained for factors associated with (A–E) mature β-cells and (F–J)a progenitor state. Ins1−/−Ins2−/−βGAL+ cells express (A) NKX2.2 but had less intense to absent (B) NKX6.1, (C) PAX6, (D) MAFA, and (E) GLUT2 immunoreactivity compared with INS+ Ins1+/+Ins2+/+β-cells. Ins1−/−Ins2−/−βGAL+ β-cells express (G) NANOG and (I) NGN3 but not (F) L-MYC and only (H) extremely rare ALDH1A3+IAPP+ cells. Scale bars, 100 µm, and insets are magnified (A–E) ×8 or (F–J) ×4. Figure 7. View largeDownload slide Insulin therapy by injection is not sufficient for the completed maturation of Ins1−/−Ins2−/−β-cells. Pancreata of 2-month-old Ins1+/+Ins2+/+ (left panels) and insulin injection–treated Ins1−/−Ins2−/− (right panels) mice were immunostained for factors associated with (A–E) mature β-cells and (F–J)a progenitor state. Ins1−/−Ins2−/−βGAL+ cells express (A) NKX2.2 but had less intense to absent (B) NKX6.1, (C) PAX6, (D) MAFA, and (E) GLUT2 immunoreactivity compared with INS+ Ins1+/+Ins2+/+β-cells. Ins1−/−Ins2−/−βGAL+ β-cells express (G) NANOG and (I) NGN3 but not (F) L-MYC and only (H) extremely rare ALDH1A3+IAPP+ cells. Scale bars, 100 µm, and insets are magnified (A–E) ×8 or (F–J) ×4. Insulin replacement by islet transplantation facilitates expansion and completed maturation of Ins1−/−Ins2−/−β-cells We next determined whether restoration of insulin signaling by isogenic islet transplantation into the anterior chamber of the eye was sufficient for completed β-cell maturation. We attempted to have a side-by-side comparison of the endocrine pancreas of animals treated for ~1 year by insulin injections alone or islet transplantation into the anterior chamber of the eye at 2 weeks of age (Fig. 8). Because of the major technical challenges in keeping Ins1−/−Ins2−/− animals alive with insulin injections for such a prolonged time, we were able to collect samples from only one mouse at 291 days of age. The mouse treated by injections had extreme islet fibrosis (Fig. 8A), with a nearly complete loss of IAPP+ cells, and islets were >50% GCG+ (Supplemental Fig. 5A). Compared with Ins1+/+Ins2+/+ controls, mice treated with islet transplantation (n = 4) had grossly normal-appearing islets without signs of fibrosis (Fig. 8A) and did not have significantly altered islet hormone proportions (Fig. 8B), but there was a slightly expanded α-cell area (Fig. 8C) and enlarged islets (Fig. 8E). Figure 8. View largeDownload slide Insulin replacement by islet transplantation leads to islet hyperplasia and preservation of β-cell area. (A) Pancreata of Ins1+/+Ins2+/+ (top panels; n = 3, 12 months of age), insulin injection–treated Ins1−/−Ins2−/− (middle panels; n = 1, 9 months of age), and islet transplantation–treated Ins1−/−Ins2−/− (bottom panels; n = 4, 12 to 14 months of age) mice were immunostained for IAPP and GCG or for GCG, SST, and PP. Arrows point to examples of polyhormonal cells that are GCG+PP+ or SST+PP+. Hematoxylin and eosin and Masson’s trichrome staining of pancreas. Scale bars, 100 µm. (B) Percentage of total endocrine immunoreactive area that is immunoreactive for the islet hormones IAPP, GCG, SST, or PP. (C) β-cell, α-cell, δ-cell, and pancreatic polypeptide cell area relative to total pancreas area (n = 1 to 4 animals, 3 sections quantified per animal). (D) Number of islets identified by SYN immunoreactivity relative to pancreas section area. (E) Average islet size by SYN immunoreactive area. (F) Percentage of IAPP+ cells that were PCNA+. Individual animal endocrine area is shown on box-and-whisker plots. Ins1+/+Ins2+/+ and islet-treated Ins1−/−Ins2−/− groups were compared by Student t test [(B) IAPP, GCG, SST, (C) IAPP, GCG, SST, (D, E)] or Mann–Whitney U test [(B) PP, (C) PP, (F)] to assess significance. *P < 0.05 vs Ins1+/+Ins2+/+ controls. Figure 8. View largeDownload slide Insulin replacement by islet transplantation leads to islet hyperplasia and preservation of β-cell area. (A) Pancreata of Ins1+/+Ins2+/+ (top panels; n = 3, 12 months of age), insulin injection–treated Ins1−/−Ins2−/− (middle panels; n = 1, 9 months of age), and islet transplantation–treated Ins1−/−Ins2−/− (bottom panels; n = 4, 12 to 14 months of age) mice were immunostained for IAPP and GCG or for GCG, SST, and PP. Arrows point to examples of polyhormonal cells that are GCG+PP+ or SST+PP+. Hematoxylin and eosin and Masson’s trichrome staining of pancreas. Scale bars, 100 µm. (B) Percentage of total endocrine immunoreactive area that is immunoreactive for the islet hormones IAPP, GCG, SST, or PP. (C) β-cell, α-cell, δ-cell, and pancreatic polypeptide cell area relative to total pancreas area (n = 1 to 4 animals, 3 sections quantified per animal). (D) Number of islets identified by SYN immunoreactivity relative to pancreas section area. (E) Average islet size by SYN immunoreactive area. (F) Percentage of IAPP+ cells that were PCNA+. Individual animal endocrine area is shown on box-and-whisker plots. Ins1+/+Ins2+/+ and islet-treated Ins1−/−Ins2−/− groups were compared by Student t test [(B) IAPP, GCG, SST, (C) IAPP, GCG, SST, (D, E)] or Mann–Whitney U test [(B) PP, (C) PP, (F)] to assess significance. *P < 0.05 vs Ins1+/+Ins2+/+ controls. Unlike those of mice treated with insulin injections for 2 months (Fig. 7), pancreatic β-cells of Ins1−/−Ins2−/− mice with healthy islets transplanted had normal immunoreactivity for PAX6, MAFA, and GLUT2 (Supplemental Fig. 5B), but we detected the abnormal presence of NANOG+ cells (Supplemental Fig. 6). NGN3 immunoreactivity was not detected in pancreata of Ins1−/−Ins2−/− mice with transplanted islets, similar to adult Ins1+/+Ins2+/+ controls (Supplemental Fig. 6). Discussion Mice lacking IRs on β-cells have impaired glucose-stimulated insulin secretion (7), and β-cell mass expands during development (13). IR-deficient β-cells are unable to sufficiently expand in response to hepatic insulin resistance, resulting in adult-onset diabetes (8). In the current study, we characterized pancreata in neonatal Ins1−/−Ins2−/− mice and Ins1−/−Ins2−/− mice kept alive into adulthood by using insulin therapy. By using the insulin knockout mouse model as an alternative to IR-deficient mice, we provide evidence that insulin itself is necessary for β-cell maturation and insulin deficiency alters islet development. However, we cannot rule out the possibility that IGF signaling though the IR is altered by changes to expression of the IR or insulinlike growth factor 1 receptor (IGF1R) and downstream signaling components. Additionally, it is possible that other defects secondary to the genetic deletion of insulin contribute to the observed β-cell phenotype. Independent of these caveats, we posit that the insulin-deficient mouse model provides the best evidence available that signaling by insulin itself is necessary for β-cell maturation. Certainly, the Ins1−/−Ins2−/− mouse model does not have any insulin signaling via an IR-independent pathway, and unlike in the IR-deficient model, there is no direct and complete loss of IGF signaling through the IR. At birth, insulin-deficient islets were enlarged with an expanded IAPP+ area. These findings are consistent with observations of expanded β-cell mass and increased β-cell replication in the MIP-CreER/IRflox/flox (tamoxifen at E13) model (13) and reduce the likelihood that those findings were attributable to the presence of the growth hormone minigene within the transgene (32, 33). We also observed a lack of mature β-cell factors PDX1, NKX6.1, MAFA, and membranous GLUT2 in insulin-deficient β-cells. These findings contrast with normal expression of PDX1, NKX6.1, MAFA, and GLUT2 in pancreata from MIP-CreER/IRflox/flox (tamoxifen at E13) mice. This discrepancy can be explained in three ways: insulin before E13 may be essential for the normal formation of maturing β-cells; although insulin signals through the IGF1R with low affinity (34), increased expression of the IGF1R in the islets of CreER/IRflox/flox (tamoxifen at E13) mice (13) may contribute to meaningful insulin signaling through the IGF1R; and despite normal blood glucose, Ins1−/−Ins2−/− pups have hypertriglyceridemia, which could contribute to loss of membranous GLUT2 (35) and β-cell dysfunction and loss of mature β-cell factors (36). Dedifferentiated β-cells in patients with type 2 diabetes (37, 38) express progenitor markers L-MYC and NANOG (31). Insulin-deficient β-cells in Ins1−/−Ins2−/− mice at birth appeared similar, with expression of both L-MYC and NANOG. Notably, we are unaware of any evidence of L-MYC or NANOG being present in the normal developing pancreas, suggesting that insulin-deficient β-cells may not be arrested at a developmental stage. Instead, insulin-deficient β-cells appear to dedifferentiate after failed maturation because of a lack of the β-cell defining protein, insulin. Furthermore, like β-cells of patients with type 2 diabetes, an elevated proportion of the insulin-deficient β-cells expressed ALDH1A3, a marker of dysfunctional β-cells (38). NGN3 was expressed in neonatal Ins1+/+Ins2+/+ islet cells but not in insulin-deficient islet cells. Although neurogenin3 was not detectable by in situ hybridization at birth (39), NGN3 is expressed in normal postnatal β-cells (40) and in non–fully differentiated β-cells that are still dividing (41) and β-cells replicate after birth (42). A lack of NGN3 in Ins1−/−Ins2−/−β-cells is consistent with reduced expression of the post-NGN3 factor NKX2.2 (43, 44). Because NGN3 is necessary for pancreatic endocrine cell formation (39), there must be NGN3 early in development to initiate the formation of pancreatic islets, but a lack of insulin results in a secondary loss of NGN3 expression later in development. Our results suggest that insulin signaling contributes to the maturation of β-cells, and in the absence of insulin, β-cells fail normal maturation and dedifferentiate to an early replicating embryonic state lacking NGN3 and downstream factors including NKX2.2 and MAFA and expressing pluripotency markers NANOG and L-MYC. Replacing insulin contributes to the further maturation of insulin-deficient β-cells. After insulin injection therapy, insulin-deficient β-cells did not express L-MYC or ALDH1A3, but we observed islet fibrosis. Additionally, β-cells of Ins1−/−Ins2−/− adults treated with insulin injection resembled neonatal wild-type β-cells, with perinuclear immunoreactivity for NGN3. This cytoplasmic localization of NGN3 has been previously reported as marker of newly forming β-cells: cytoplasmic NGN3 has been observed during β-cell regeneration after immunological destruction of β-cells (45) and has been observed in early phases of endocrine cell neogenesis in vitro by stimulation with a growth factor cocktail (46). Additionally, unlike the nuclear localization of NANOG observed in neonatal Ins1−/−Ins2−/− islet cells that is conventionally a marker of self-renewal in stem cells (47), insulin-treated mice (by insulin injection or islet transplantation) had cytoplasmic NANOG, which has been used as a marker of an epithelial-to-mesenchymal transition in nasopharyngeal carcinoma (48), cervical cancer (49), and pancreatic cancer (50). This finding contrasts with that of endogenous β-cells in Ins1−/−Ins2−/− animals treated with insulin replacement by islet transplantation, which had normal expression of all mature β-cell factors examined, including MAFA. Insulin-deficient β-cells of islet-treated adult mice also had a normal absence of L-MYC, ALDH1A3, and NGN3. Continued presence of cells with cytoplasmic NANOG immunoreactivity provides weak evidence for ongoing epithelial-to-mesenchymal transition of nonendocrine cells as a source of new islet cells contributing to islet hyperplasia. Because the primary deficit of Ins1−/−Ins2−/− mice is a loss of insulin, it is surprising that replacing insulin by injections alone or islet transplantation resulted in dramatically divergent outcomes for the endogenous β-cells. We proposed three variables that could contribute to the differences in β-cell phenotype between Ins1−/−Ins2−/− mice treated with islet transplantation and those treated with insulin injection: glycemic control was superior in mice treated with islet transplantation relative to those treated with insulin injections, native mouse insulin produced by transplanted islets may signal in β-cells with higher bioactivity than recombinant insulin (51, 52), or insulin-deficient islets may fail to produce other essential factors that are replaced by transplanted islets. Although we are unable to conclusively discern which, if any, of these variables contribute to the differences in β-cell phenotype, the striking resemblance in fibrotic islet phenotype between injection-treated Ins1−/−Ins2−/− mice and mice with diabetes as a result of inexcitable β-cells [adenosine triphosphate (ATP)–sensitive potassium channel gain of function] (53) suggests that differences in glycemia are at least partially responsible. Hyperglycemia leading to glucotoxicity has also been shown to cause reduced expression of Pdx1, Mafa, and Slc2a2 (36), and reprogramming of exocrine cells to insulin-producing cells was more complete and abundant from a viral therapy (expressing NGN3/PDX1/MAFA) when mice were treated with islet transplantation for good control of their toxin-induced diabetes compared with crude treatment with insulin pellets (54). Similarly, in models of neonatal diabetes (expression of an activating ATP-sensitive potassium channel), animals treated with islet transplantation did not develop islet fibrosis and retained glucose-stimulated insulin secretion, unlike the untreated hyperglycemic group (55). These findings also suggest that the crude glycemic regulation of insulin injections (or pellets) compared with the ideally regulated glycemia of mice treated with islet transplantation is probably an important contributor to the differences in β-cell phenotype. Transplanted islets also secrete additional peptides beyond insulin, including C-peptide, a byproduct of proinsulin processing that may contribute to preservation of islet health (56). Finally, in this study the mice treated by islet transplantation were kept alive for ~1 year, whereas injection-treated mice were kept alive for 2 months, with the exception of a single mouse that was kept alive by injections for nearly 1 year. The extreme challenge of keeping mice alive by multiple daily injections for a long time limited our ability to have age-matched groups, and the different duration of therapy may have contributed to the differences in phenotype. Ins1−/−Ins2−/− mice develop aggregates of endocrine cells in the pancreas resembling islets, but a loss of insulin alters the cellular composition. Ins1−/−Ins2−/− islets have a lower proportion of GCG+ and PP+ endocrine cells and greater IAPP+ and SST+ cell mass. Given the known contribution of paired box 4 to the β- and δ-cell lineages and ARX to the α-cell and pancreatic polypeptide cell lineages (57, 58), reduced GCG+ and PP+ and expanded IAPP+ and SST+ cell populations align with an overall reduction in ARX and increase in paired box 4 signaling. Additionally, there was a progressive expansion of GCG+ α-cells in mice treated with insulin injections that was not observed in mice treated with islet transplantation. A loss of insulin signaling in α-cells contributes to α-cell hyperplasia (59), and poor glycemic control in insulin-injected mice also may contribute to progressive α-cell expansion (60). Additionally, we made the surprising observation that the pan-endocrine factors NKX2.2 and PAX6 are absent from not only β-cells but also the remainder of the islet cells. A reduced number of PP+ cells in the pancreas aligns with the phenotype of the NKX2.2-deficient endocrine pancreas (44), but unlike in the PAX6 knockout mouse (61), we did not observe expanded ghrelin+ cells. There is probably an undefined paracrine effect inhibiting the normal maturation of non-β islet cells in the pancreas of insulin-deficient mice. MAFA immunoreactivity was diminished in β-cells of mice with reduced insulin gene copy number (Ins1−/−, Ins2−/−, and Ins1−/−Ins2+/−). Although we did not follow up on these findings, they raise the intriguing possibility that despite not having obvious severe abnormalities in glucose homeostasis (62), reduced insulin dosed β-cells may not be functionally mature because of MAFA insufficiency (63) and perhaps other unidentified factors. Additionally, we made an unexpected observation that there can be unique regulation of the Ins2 and Ins1 loci and the two Ins2 alleles in mouse β-cells. With lacZ knocked into the Ins2 locus, the majority of β-cells in adult Ins1−/−Ins2+/− or Ins1+/+Ins2−/− animals are INS+βGAL−. In the Ins1+/+Ins2−/− animals, cells that were INS+βGAL− had an active Ins1 gene, but the Ins2 knockin of βGAL was inactive. In the Ins1−/−Ins2+/− animals, cells that were INS+βGAL− had an active wild-type Ins2 allele, but the βGAL knockin allele was inactive. Similarly, loss of βGAL in most IAPP-expressing cells happens after birth in Ins1−/−Ins2−/− animals. Although chronic hyperglycemia has been shown to suppress insulin transcription (64), loss of βGAL cannot be attributed to a diabetic state because postnatal loss of βGAL expression also occurs in euglycemic Ins1−/−Ins2−/− mice treated by islet transplantation and Ins1+/+Ins2−/− and Ins1−/−Ins2+/− animals. Because expression of insulin is arguably the most important aspect of the pancreatic β-cell identity, loss of βGAL does not align with a mature β-cell identity. However, it is important to consider the model organism because age-dependent loss of βGAL in insulin-treated Ins1−/−Ins2−/− mice is consistent with reduced lacZ expression when knocked into the globin genes in mice (65). The distinct expression pattern of βGAL compared with insulin in the insulin knockout/lacZ knockin model may be a caveat of the mouse model because there can be selective CpG methylation to silence a foreign open reading frame such as the lacZ gene (66). Nevertheless, it may be worthwhile to further explore the possibility of differential regulation of the mouse insulin alleles. There have been many reported human cases of diabetes caused by mutations in the insulin gene. Most of these patients have a heterozygous dominant negative disease caused by misfolding of the mutated insulin, but there has been a report of patients with homozygous deletion of INS (68). Although we found no published histology of the pancreas from such patients, a patient with an intronic mutation causing altered splicing of INS had undetectable C-peptide but readily detectable IAPP, suggesting that the patient had insulin-deficient β-cells in the pancreas (69). This hypothesis is supported by the abundant INS−IAPP+ β-cells in Ins1−/−Ins2−/− mouse pancreas. Relatedly, a child with severe sulfonylurea-unresponsive permanent neonatal diabetes mellitus from an activating mutation in the ATP-sensitive potassium channel had severely reduced β-cell mass on postmortem histological examination (70). Patients with mutations in the ATP-sensitive potassium channel are less likely to become insulin independent with sulfonylurea therapy the longer the patient has been insulin dependent (71). Potentially there is a major change in β-cell maturity after transfer of therapy, but prolonged insulin replacement by injection causes progressive loss of β-cell mass, thus hindering the ability of sulfonylurea therapy to induce diabetic remission. This hypothesis aligns with our findings that although a lack of insulin during development initially results in an expanded β-cell mass, insulin replacement by injection is not sufficient to maintain β-cell mass. Like mouse β-cells, adult human β-cells also contain much of the necessary machinery for insulin signaling, including IRs and insulin receptor substrate (IRS)-1 and IRS-2 (72). Functionally, inhibition of IR by short hairpin RNA impaired glucose-stimulated insulin secretion (72), and human islets with an IRS-1 polymorphism have impaired glucose-stimulated insulin secretion, elevated proinsulin secretion, and reduced insulin content (73). Furthermore, there is evidence of reduced insulin signaling in β-cells of patients with type 2 diabetes: IRS-1, IRS-2, Tyr612 IRS-1, and Tyr612 IRS-2 expression are reduced in islets from patients with type 2 diabetes compared with islets from nondiabetic donors (74). Taken together, these findings lead us to propose that though usually attributed to reduced glucolipotoxicity (75), diabetic remission after intensive insulin therapy in patients with type 2 diabetes (76) may be attributed partially to augmented β-cell insulin signaling from increased circulating insulin. Increased insulin signaling in β-cells could lead to redifferentiation of dedifferentiated β-cells, a possibility that may warrant future investigation. Collectively, we provide evidence that insulin is a necessary signaling molecule for the maturation of β-cells. Replacement of insulin contributes to β-cell maturation, but the ability of insulin therapy to complete β-cell maturation may depend on euglycemia, replacement of the native species of insulin, or other factors secreted from islets. Without insulin during development in mice, β-cells appear not fully mature. Extremely rare mutations of the INS gene may also cause altered maturation of β-cells in patients. Understanding how insulin regulates β-cell maturation is relevant to research into the generation of β-cells in vitro and therapies attempting to induce diabetic remission in patients with dedifferentiated β-cells in early type 2 diabetes. Although there has been some debate about the potential that insulin signals in an autocrine fashion on β-cells (77), our findings provide evidence that insulin itself is an essential signaling molecule on β-cells. Appendix. Table of Antibodies Used for Immunofluorescent Staining Peptide/Protein Target  Antigen Sequence  Name of Antibody  Manufacturer or Name of Individual Providing the Antibody  Catalog No.  Species Raised in  Monoclonal or Polyclonal  Dilution Used  RRID  Rabbit IgG  Whole IgG  Goat anti-rabbit AF488  Life Technologies  A11034  Goat  Polyclonal  1:1000  AB_2576217  Rabbit IgG  Whole IgG  Goat anti-rabbit AF555  Life Technologies  A21429  Goat  Polyclonal  1:1000  AB_151761  Rabbit IgG  Whole IgG  Goat anti-rabbit AF594  Life Technologies  A11037  Goat  Polyclonal  1:1000  AB_2534095  Rabbit IgG  Whole IgG  Goat anti-rabbit AF647  Life Technologies  A21245  Goat  Polyclonal  1:1000  AB_2535813  Rabbit IgG  Whole IgG  Donkey anti-rabbit AF488  Life Technologies  A21206  Donkey  Polyclonal  1:1000  AB_141708  Rabbit IgG  Whole IgG  Donkey anti-rabbit AF555  Life Technologies  A31572  Donkey  Polyclonal  1:1000  AB_162543  Rabbit IgG  Whole IgG  Donkey anti-rabbit AF594  Life Technologies  A21207  Donkey  Polyclonal  1:1000  AB_141637  Rabbit IgG  Whole IgG  Donkey anti-rabbit AF647  Life Technologies  A31573  Donkey  Polyclonal  1:1000  AB_2536183  Mouse IgG  Whole IgG  Goat anti-mouse AF488  Life Technologies  A11029  Goat  Polyclonal  1:1000  AB_2534088  Mouse IgG  Whole IgG  Goat anti-mouse AF555  Life Technologies  A21424  Goat  Polyclonal  1:1000  AB_141780  Mouse IgG  Whole IgG  Goat anti-mouse AF594  Life Technologies  A11032  Goat  Polyclonal  1:1000  AB_141672  Mouse IgG  Whole IgG  Goat anti-mouse AF647  Life Technologies  A21236  Goat  Polyclonal  1:1000  AB_2535805  Mouse IgG  Whole IgG  Donkey anti-mouse AF488  Life Technologies  A21202  Donkey  Polyclonal  1:1000  AB_2535788  Mouse IgG  Whole IgG  Donkey anti-mouse AF555  Life Technologies  A31570  Donkey  Polyclonal  1:1000  AB_2536180  Mouse IgG  Whole IgG  Donkey anti-mouse AF594  Life Technologies  A21203  Donkey  Polyclonal  1:1000  AB_141633  Mouse IgG  Whole IgG  Donkey anti-mouse AF647  Life Technologies  A31571  Donkey  Polyclonal  1:1000  AB_162542  Goat IgG  Whole IgG  Donkey anti-goat AF594  Life Technologies  A11058  Donkey  Polyclonal  1:1000  AB_142540  Sheep IgG  Whole IgG  Donkey anti-sheep AF488  Life Technologies  A11015  Donkey  Polyclonal  1:1000  AB_141362  Guinea pig IgG  Whole IgG  Goat anti–guinea pig AF488  Life Technologies  A11073  Goat  Polyclonal  1:1000  AB_2534117  Guinea pig IgG  Whole IgG  Goat anti–guinea pig AF647  Life Technologies  A21450  Goat  Polyclonal  1:1000  AB_151882  Aldehyde dehydrogenase family member 1A3  Unknown  Rabbit anti-ALDH1A3  Novus Biologicals  NBP2-15339  Rabbit  Polyclonal  1:100  AB_2665496  Islet amyloid polypeptide  Unknown  Rabbit anti-IAPP  AbCam  ab15125  Rabbit  Polyclonal  1:50  AB_2295631  Beta-galactosidase  Whole β-galactosidase  Rabbit anti-βGAL  Thermo Scientific  A11132  Rabbit  Polyclonal  1:100  AB_221539  Beta-galactosidase  Unknown  Mouse anti-βGAL  DSHB  40-1a-c  Mouse  Monoclonal  1:50  AB_528100  Glucokinase  Recombinant glucokinase  Rabbit anti-GCK  Sigma  HPA007034  Rabbit  Polyclonal  1:50  AB_888431  Glucagon  Unknown  Mouse anti-GCG  Sigma  G 2654  Mouse  Monoclonal  1:1000  AB_259852  Glucose transporter 2  First extracellular loop of Glut2  Rabbit anti-GLUT2  Millipore  07-1402  Rabbit  Polyclonal  1:500  AB_1587076  Insulin  Full-length human insulin  Guinea pig anti-INS  Thermo Fisher Scientific  PA1-26938  Guinea pig  Polyclonal  1:100  AB_794668  Insulin  Unknown  Rabbit anti-INS  Cell Signaling  C27C9  Rabbit  Monoclonal  1:200  AB_2126503  Insulin  Residues surrounding Val36 of human insulin  Mouse anti-INS  Cell Signaling  L6B10  Mouse  Monoclonal  1:250  AB_10949314  v-myc Avian myolocytomatosis viral oncogene lung carcinoma derived  AA 105-154 of human l-myc  Rabbit anti-L-MYC  AbCam  Ab28739  Rabbit  Polyclonal  1:100  AB_2148730  V-maf muscoloapo-neurotic fibrosarcoma oncogene homolog A  Unknown  Rabbit anti-MAFA  Betalogics (Johnson & Johnson)  LP9872  Rabbit  Polyclonal  1:1000  AB_2665528  V-maf muscoloaponeurotic fibrosarcoma oncogene homolog B  AA 18-140 of MAFB  Rabbit anti-MAFB  Sigma Life Sciences  HPA005653  Rabbit  Polyclonal  1:100  AB_1079293  Homeobox protein NANOG  Mouse nanog  Rabbit anti-NANOG  AbCam  Ab80892  Rabbit  Polyclonal  1:100  AB_2150114  Homeodomain transcription factor 6.1  Human Nkx6.1  Goat anti-NKX6.1  R and D Systems  AF5857  Goat  Polyclonal  1:20  AB_1857045  Homeodomain transcription factor 2.2  Nkx2.2–GST fusion protein from Escherichia. coli  Mouse anti-NKX2.2  DSHB  74.5A5  Mouse  Monoclonal  1:100  AB_531794  Neurogenin-3  Met1-Leu214 of human Ngn3  Sheep anti-NGN3  R and D Systems  AF3444  Sheep  Polyclonal  1:20  AB_2149527  Neurogenin-3  AA40-69 of human Ngn3  Sheep anti-NGN3  Thermo Fisher Scientific  PA5-11893  Rabbit  Polyclonal  1:100  AB_2149526  Pancreatic polypeptide  Ala30-Leu95 of human PP  Goat anti-PP  R and D Systems  AF6297  Goat  Polyclonal  1:200  AB_10717571  Paired box 6  C-terminus of mouse PAX6  Rabbit anti-PAX6  Covance  PRB-278P  Rabbit  Polyclonal  1:250  AB_2313780  Prohormone convertase 1/3  Unknown  Rabbit anti-PC1/3  Lakshmi Devi  Gift  Rabbit  Polyclonal  1:500  AB_2665530  Prohormone convertase 1/3  Unknown  Mouse anti-PC1/3  Gunilla Westermark  Gift  Mouse  Monoclonal  Direct  AB_2665529  Prohormone convertase 2  E622-N638 of mouse PC2  Rabbit anti-PC2  Thermo Fisher Scientific  PA1-058  Rabbit  Polyclonal  1:500  AB_2158593  Proliferating cell nuclear antigen  Rat PCNA  Mouse anti-PCNA  Abcam  ab29  Mouse  Monoclonal  1:100  AB_303394  Pancreatic and duodenal homeobox 1  N-terminus of mouse PDX1  Guinea pig anti-PDX1  Abcam  ab47308  Guinea pig  Polyclonal  1:1000  AB_777178  Somatostatin  Human somatostatin  Mouse anti-SST  Β Cell Biology Consortium  AB1985  Mouse  Polyclonal  1:500  AB_10014609  Sulfonylurea receptor 1  Aa1560-1582  Rabbit anti-SUR1  Abcam  ab32844  Rabbit  Polyclonal  1:50  AB_2273320  Synaptophysin  C-terminus of human Syn  Rabbit anti-synaptophysin  Novus Biologicals  NB120-16659  Rabbit  Monoclonal  1:50  AB_792140  Peptide/Protein Target  Antigen Sequence  Name of Antibody  Manufacturer or Name of Individual Providing the Antibody  Catalog No.  Species Raised in  Monoclonal or Polyclonal  Dilution Used  RRID  Rabbit IgG  Whole IgG  Goat anti-rabbit AF488  Life Technologies  A11034  Goat  Polyclonal  1:1000  AB_2576217  Rabbit IgG  Whole IgG  Goat anti-rabbit AF555  Life Technologies  A21429  Goat  Polyclonal  1:1000  AB_151761  Rabbit IgG  Whole IgG  Goat anti-rabbit AF594  Life Technologies  A11037  Goat  Polyclonal  1:1000  AB_2534095  Rabbit IgG  Whole IgG  Goat anti-rabbit AF647  Life Technologies  A21245  Goat  Polyclonal  1:1000  AB_2535813  Rabbit IgG  Whole IgG  Donkey anti-rabbit AF488  Life Technologies  A21206  Donkey  Polyclonal  1:1000  AB_141708  Rabbit IgG  Whole IgG  Donkey anti-rabbit AF555  Life Technologies  A31572  Donkey  Polyclonal  1:1000  AB_162543  Rabbit IgG  Whole IgG  Donkey anti-rabbit AF594  Life Technologies  A21207  Donkey  Polyclonal  1:1000  AB_141637  Rabbit IgG  Whole IgG  Donkey anti-rabbit AF647  Life Technologies  A31573  Donkey  Polyclonal  1:1000  AB_2536183  Mouse IgG  Whole IgG  Goat anti-mouse AF488  Life Technologies  A11029  Goat  Polyclonal  1:1000  AB_2534088  Mouse IgG  Whole IgG  Goat anti-mouse AF555  Life Technologies  A21424  Goat  Polyclonal  1:1000  AB_141780  Mouse IgG  Whole IgG  Goat anti-mouse AF594  Life Technologies  A11032  Goat  Polyclonal  1:1000  AB_141672  Mouse IgG  Whole IgG  Goat anti-mouse AF647  Life Technologies  A21236  Goat  Polyclonal  1:1000  AB_2535805  Mouse IgG  Whole IgG  Donkey anti-mouse AF488  Life Technologies  A21202  Donkey  Polyclonal  1:1000  AB_2535788  Mouse IgG  Whole IgG  Donkey anti-mouse AF555  Life Technologies  A31570  Donkey  Polyclonal  1:1000  AB_2536180  Mouse IgG  Whole IgG  Donkey anti-mouse AF594  Life Technologies  A21203  Donkey  Polyclonal  1:1000  AB_141633  Mouse IgG  Whole IgG  Donkey anti-mouse AF647  Life Technologies  A31571  Donkey  Polyclonal  1:1000  AB_162542  Goat IgG  Whole IgG  Donkey anti-goat AF594  Life Technologies  A11058  Donkey  Polyclonal  1:1000  AB_142540  Sheep IgG  Whole IgG  Donkey anti-sheep AF488  Life Technologies  A11015  Donkey  Polyclonal  1:1000  AB_141362  Guinea pig IgG  Whole IgG  Goat anti–guinea pig AF488  Life Technologies  A11073  Goat  Polyclonal  1:1000  AB_2534117  Guinea pig IgG  Whole IgG  Goat anti–guinea pig AF647  Life Technologies  A21450  Goat  Polyclonal  1:1000  AB_151882  Aldehyde dehydrogenase family member 1A3  Unknown  Rabbit anti-ALDH1A3  Novus Biologicals  NBP2-15339  Rabbit  Polyclonal  1:100  AB_2665496  Islet amyloid polypeptide  Unknown  Rabbit anti-IAPP  AbCam  ab15125  Rabbit  Polyclonal  1:50  AB_2295631  Beta-galactosidase  Whole β-galactosidase  Rabbit anti-βGAL  Thermo Scientific  A11132  Rabbit  Polyclonal  1:100  AB_221539  Beta-galactosidase  Unknown  Mouse anti-βGAL  DSHB  40-1a-c  Mouse  Monoclonal  1:50  AB_528100  Glucokinase  Recombinant glucokinase  Rabbit anti-GCK  Sigma  HPA007034  Rabbit  Polyclonal  1:50  AB_888431  Glucagon  Unknown  Mouse anti-GCG  Sigma  G 2654  Mouse  Monoclonal  1:1000  AB_259852  Glucose transporter 2  First extracellular loop of Glut2  Rabbit anti-GLUT2  Millipore  07-1402  Rabbit  Polyclonal  1:500  AB_1587076  Insulin  Full-length human insulin  Guinea pig anti-INS  Thermo Fisher Scientific  PA1-26938  Guinea pig  Polyclonal  1:100  AB_794668  Insulin  Unknown  Rabbit anti-INS  Cell Signaling  C27C9  Rabbit  Monoclonal  1:200  AB_2126503  Insulin  Residues surrounding Val36 of human insulin  Mouse anti-INS  Cell Signaling  L6B10  Mouse  Monoclonal  1:250  AB_10949314  v-myc Avian myolocytomatosis viral oncogene lung carcinoma derived  AA 105-154 of human l-myc  Rabbit anti-L-MYC  AbCam  Ab28739  Rabbit  Polyclonal  1:100  AB_2148730  V-maf muscoloapo-neurotic fibrosarcoma oncogene homolog A  Unknown  Rabbit anti-MAFA  Betalogics (Johnson & Johnson)  LP9872  Rabbit  Polyclonal  1:1000  AB_2665528  V-maf muscoloaponeurotic fibrosarcoma oncogene homolog B  AA 18-140 of MAFB  Rabbit anti-MAFB  Sigma Life Sciences  HPA005653  Rabbit  Polyclonal  1:100  AB_1079293  Homeobox protein NANOG  Mouse nanog  Rabbit anti-NANOG  AbCam  Ab80892  Rabbit  Polyclonal  1:100  AB_2150114  Homeodomain transcription factor 6.1  Human Nkx6.1  Goat anti-NKX6.1  R and D Systems  AF5857  Goat  Polyclonal  1:20  AB_1857045  Homeodomain transcription factor 2.2  Nkx2.2–GST fusion protein from Escherichia. coli  Mouse anti-NKX2.2  DSHB  74.5A5  Mouse  Monoclonal  1:100  AB_531794  Neurogenin-3  Met1-Leu214 of human Ngn3  Sheep anti-NGN3  R and D Systems  AF3444  Sheep  Polyclonal  1:20  AB_2149527  Neurogenin-3  AA40-69 of human Ngn3  Sheep anti-NGN3  Thermo Fisher Scientific  PA5-11893  Rabbit  Polyclonal  1:100  AB_2149526  Pancreatic polypeptide  Ala30-Leu95 of human PP  Goat anti-PP  R and D Systems  AF6297  Goat  Polyclonal  1:200  AB_10717571  Paired box 6  C-terminus of mouse PAX6  Rabbit anti-PAX6  Covance  PRB-278P  Rabbit  Polyclonal  1:250  AB_2313780  Prohormone convertase 1/3  Unknown  Rabbit anti-PC1/3  Lakshmi Devi  Gift  Rabbit  Polyclonal  1:500  AB_2665530  Prohormone convertase 1/3  Unknown  Mouse anti-PC1/3  Gunilla Westermark  Gift  Mouse  Monoclonal  Direct  AB_2665529  Prohormone convertase 2  E622-N638 of mouse PC2  Rabbit anti-PC2  Thermo Fisher Scientific  PA1-058  Rabbit  Polyclonal  1:500  AB_2158593  Proliferating cell nuclear antigen  Rat PCNA  Mouse anti-PCNA  Abcam  ab29  Mouse  Monoclonal  1:100  AB_303394  Pancreatic and duodenal homeobox 1  N-terminus of mouse PDX1  Guinea pig anti-PDX1  Abcam  ab47308  Guinea pig  Polyclonal  1:1000  AB_777178  Somatostatin  Human somatostatin  Mouse anti-SST  Β Cell Biology Consortium  AB1985  Mouse  Polyclonal  1:500  AB_10014609  Sulfonylurea receptor 1  Aa1560-1582  Rabbit anti-SUR1  Abcam  ab32844  Rabbit  Polyclonal  1:50  AB_2273320  Synaptophysin  C-terminus of human Syn  Rabbit anti-synaptophysin  Novus Biologicals  NB120-16659  Rabbit  Monoclonal  1:50  AB_792140  Abbreviations: IgG, immunoglobulin G. View Large Abbreviations: ALDH1A3 aldehyde dehydrogenase 1 family member A3 ATP adenosine triphosphate βGAL β-galactosidase DAPI 4′,6-diamidino-2-phenylindole GCG glucagon GLUT2 glucose transporter 2 IAPP islet amyloid polypeptide IGF insulinlike growth factor IGF1R insulinlike growth factor 1 receptor INS insulin IR insulin receptor IRS insulin receptor substrate L-MYC v-myc avian myolocytomatosis viral oncogene lung carcinoma derived MAFA muscoloaponeurotic fibrosarcoma oncogene homolog A mRNA messenger RNA NGN3 neurogenin-3 NKX2.2 homeodomain transcription factor 2.2 NKX6.1 homeodomain transcription factor 6.1 PAX6 paired box 6 PBS phosphate-buffered saline PC1/3 prohormone convertase 1/3 PC2 prohormone convertase 2 PCNA proliferating cell nuclear antigen PDX1 pancreatic and duodenal homeobox 1 PP pancreatic polypeptide qPCR quantitative polymerase chain reaction SST somatostatin SUR1 sulfonylurea receptor 1 SYN synaptophysin UBC University of British Columbia. Acknowledgments We express our sincere gratitude to Ali Asadi for consultation on histological procedures, Shannon O’Dwyer for expert animal handling assistance, and Nazde Edeer for technical assistance. Financial Support: This work was supported by grants from the Canadian Diabetes Association and the Canadian Institutes of Health Research (CIHR Foundation Scheme). A.R. gratefully acknowledges studentship support from the Canadian Institutes of Health Research (Vanier Canada Graduate Scholarship) and Vancouver Coastal Health (CIHR-UBC MD/PhD Studentship). Author Contributions: A.R. and T.J.K. designed the experiments. A.R. performed the experiments. M.M. performed islet transplantation, optimized insulin therapy, and generated and maintained Ins1−/−Ins2−/− animals. A.R. analyzed data, and A.R. and T.J.K wrote the manuscript. All authors were involved in the discussion and revision of the manuscript. 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