Increased SLC38A4 Amino Acid Transporter Expression in Human Pancreatic α-Cells After Glucagon Receptor Inhibition

Increased SLC38A4 Amino Acid Transporter Expression in Human Pancreatic α-Cells After Glucagon... Abstract Plasma amino acids and their transporters constitute an important part of the feedback loop between the liver and pancreatic α-cell function, and glucagon regulates hepatic amino acid turnover. Disruption of hepatic glucagon receptor action activates the loop and results in high plasma amino acids and hypersecretion of glucagon associated with α-cell hyperplasia. In the present study, we report a technique to rescue implanted human pancreatic islets from the mouse kidney capsule. Using this model, we have demonstrated that expression of the amino acid transporter SLC38A4 increases in α-cells after administration of a glucagon receptor blocking antibody. The increase in SLC38A4 expression and associated α-cell proliferation was dependent on mechanistic target of rapamycin pathway. We confirmed increased α-cell proliferation and expression of SLC38A4 in pancreas sections from patients with glucagon cell hyperplasia and neoplasia (GCHN) with loss-of-function mutations in the glucagon receptor. Collectively, using a technique to rescue implanted human islets from the kidney capsule in mice and pancreas sections from patients with GCHN, we found that expression of SLC38A4 was increased under conditions of disrupted glucagon receptor signaling. These data provide support for the existence of a liver–human α-cell endocrine feedback loop. An endocrine loop exists between the liver and the pancreatic α-cells in which the glucagon signals the α-cell → liver part and amino acids mediate the liver → α-cell part. This has been supported by findings that genetic or pharmacological inhibition of hepatic glucagon signaling results in elevated plasma amino acid levels and compensatory glucagon hypersecretion, involving expansion of the pancreatic α-cell mass (1–11). However, administration of glucagon to animals and humans will result in reductions in plasma amino acids, increased amino acid uptake by the liver, and their conversion into gluconeogenic precursors, promoting hepatic glucose production and an increase in blood glucose levels (12, 13). We, and others, have recently shown that the amino acid transporter Slc38a5 contributes to glucagon receptor inhibition-induced hyperglucagonemia and α-cell hyperplasia in mice (1, 5). The increase in α-cell proliferation, but not glucagon secretion, is mediated via an mechanistic target of rapamycin (mTOR)–dependent pathway (5). α-Cell growth was primarily triggered by glutamine and, to a lesser extent, alanine (1, 5, 9). The concentrations of these amino acids were dramatically increased in the circulation of mice when glucagon receptor signaling was blocked (5, 9, 10, 14). However, these findings did not translate into human α-cells, because SLC38A5 mRNA was not detected, and its expression was not induced in settings of glucagon receptor inhibition (5). The present study sought to examine the expression of human α-cell amino acid transporters in the setting of reduced hepatic glucagon action. Thus, we developed a technique to rescue human islets implanted under the mouse kidney capsule to allow for RNA sequencing and histological examination of the islet cells. We have confirmed our human islet implantation findings using pancreas sections from patients with glucagon cell hyperplasia and neoplasia (GCHN) with loss-of-function mutations in the glucagon receptor. Materials and Methods Human islets Human pancreatic islets were obtained from Prodo Laboratories (Aliso Viejo, CA) with appropriate consent. The islets were cultured in CMRL-1066 media supplemented with 10% (v/v) fetal bovine serum, 2 mM glutamine, 100 U/mL of penicillin, and 100 µg/mL of streptomycin (islet media). Islets were incubated in a tissue culture incubator at 37°C with 5% carbon dioxide in atmosphere air for 3 to 5 days while conducting a pathogen analysis (mouse pathogen testing; IDEXX BioResearch, Columbia, MO). The islet donors had no history of diabetes, and their information is presented in Table 1. Table 1. Islet Donor Information Donor Age, y Ethnicity Sex BMI, kg/m2 Height Weight, lb HbA1c, % Cause of Death Usage 1 29 White Male 26.7 6’3” 214 5.5 Anoxia Histological examination 2 50 White Male 28.6 5’7” 185 5.4 Stroke Histological examination 3 56 White Male 21.2 5’11” 152 5.4 Stroke Histological examination 4 46 White Female 20.1 5’5” 121 5.0 Stroke RNA sequencing 5 41 Hispanic Female 18.3 5’3” 105 5.5 Stroke RNA sequencing 6 57 White Female 29.0 5’7” 185 5.6 Stroke RNA sequencing 7 33 Black Male 30.8 5’10” 215 5.7 Head trauma RNA sequencing Donor Age, y Ethnicity Sex BMI, kg/m2 Height Weight, lb HbA1c, % Cause of Death Usage 1 29 White Male 26.7 6’3” 214 5.5 Anoxia Histological examination 2 50 White Male 28.6 5’7” 185 5.4 Stroke Histological examination 3 56 White Male 21.2 5’11” 152 5.4 Stroke Histological examination 4 46 White Female 20.1 5’5” 121 5.0 Stroke RNA sequencing 5 41 Hispanic Female 18.3 5’3” 105 5.5 Stroke RNA sequencing 6 57 White Female 29.0 5’7” 185 5.6 Stroke RNA sequencing 7 33 Black Male 30.8 5’10” 215 5.7 Head trauma RNA sequencing View Large Table 1. Islet Donor Information Donor Age, y Ethnicity Sex BMI, kg/m2 Height Weight, lb HbA1c, % Cause of Death Usage 1 29 White Male 26.7 6’3” 214 5.5 Anoxia Histological examination 2 50 White Male 28.6 5’7” 185 5.4 Stroke Histological examination 3 56 White Male 21.2 5’11” 152 5.4 Stroke Histological examination 4 46 White Female 20.1 5’5” 121 5.0 Stroke RNA sequencing 5 41 Hispanic Female 18.3 5’3” 105 5.5 Stroke RNA sequencing 6 57 White Female 29.0 5’7” 185 5.6 Stroke RNA sequencing 7 33 Black Male 30.8 5’10” 215 5.7 Head trauma RNA sequencing Donor Age, y Ethnicity Sex BMI, kg/m2 Height Weight, lb HbA1c, % Cause of Death Usage 1 29 White Male 26.7 6’3” 214 5.5 Anoxia Histological examination 2 50 White Male 28.6 5’7” 185 5.4 Stroke Histological examination 3 56 White Male 21.2 5’11” 152 5.4 Stroke Histological examination 4 46 White Female 20.1 5’5” 121 5.0 Stroke RNA sequencing 5 41 Hispanic Female 18.3 5’3” 105 5.5 Stroke RNA sequencing 6 57 White Female 29.0 5’7” 185 5.6 Stroke RNA sequencing 7 33 Black Male 30.8 5’10” 215 5.7 Head trauma RNA sequencing View Large Human islet implantation Human islets were implanted under the kidney capsule of severe combined immunodeficient (SCID) mice (males, aged 16 to 25 weeks; Taconic, Rensselaer, NY). Each mouse received 1000 islet equivalents. Starting on day 20 after islet implantation, the mice received weekly subcutaneous injections of the glucagon receptor blocking monoclonal antibody REGN1193 (7, 15) or an isotype control antibody (16) at 10 mg/kg for 3 weeks. Blood was collected from the tail for glucose measurements without fasting. Plasma samples were collected via submandibular bleeding without fasting for analysis of hormones and total amino acids. At 3 weeks after the initial antibody dosing, the mice were euthanized, and the human islet grafts were isolated for RNA sequencing and histological analysis. The detailed procedures are described in the next sections. Islet implantation Cultured human islets were centrifuged at 1300 rpm for 5 minutes at 4°C. After careful aspiration of the media, the islets were resuspended with islet media at a density of 4000 islet equivalent/mL. Next, 250 µL of resuspended islets were transferred into a gel loading tip with the narrow end bent into a “U” shape and placed in an Eppendorf tube. The islets were allowed to settle to the bottom. The media were removed from the gel-loading tip, leaving 15 to 20 µL. The gel loading tip was inserted into the nonbeveled end of the 9-in.-long PE50 tubing (BD Biosciences, San Jose, CA) to load islets. A pipette was connected to the open end of the tip, and the piston was gently depressed to push the islets to the beveled side of the PE50 tubing. The pipet tip was disconnected and subsequently connected to a Hamilton syringe. The PE50 tubing was held in an upright position to allow the islets inside the tubing to form a small pellet. The recipient SCID mice were anesthetized, shaved, and cleaned. A small incision was made on the left flank, and the kidney was exposed by gently pressing both sides of the incision. Using a 25-guage syringe needle, a small scratch was made on the membrane of the kidney to insert the PE50 tubing. After applying 0.9% sodium chloride solution to the kidney, the beveled end of the PE50 tubing with the islets near the tip was inserted under the kidney capsule and gently moved around to create a space. The islets were slowly injected by gently pressing the plunger. The PE50 tubing was removed, the nick was cauterized, the kidney was carefully replaced back into the peritoneum, and the skin was sutured and stapled. Isolation of engrafted islets To isolate the engrafted islets, a small section of the kidney membrane with the islets was peeled off and transferred into a 15-mL tube containing 5 mL of Liberase TL (0.25 mg/mL). The samples were incubated at 37°C for 45 minutes with frequent P1000 pipetting to help islet cell dissociation. Once the islet cells were well dissociated, 10 mL of islet media was added, and the cell suspension was filtered through a 70-µm cell strainer into a 15-mL centrifuge tube. Islet cells were collected by centrifugation at 1300 rpm for 5 minutes. The cell pellets were resuspended in 5 mL of PBS containing 0.5% BSA (MACS buffer) and centrifuged again at 1300 rpm for 5 minutes. The cell pellets were resuspended in 300 µL MACS buffer. Next, 100 µL of MACS Mouse Cell Depletion Cocktail (Miltenyi Biotec, Bergisch Gladbach, Germany) was added and incubated for 15 minutes at 4°C. An LS column in a MACS Separator (Miltenyi Biotec) was prepared, and the column was equilibrated with 3 mL of MACS buffer. Next, 300 µL of MACS buffer was added to the cell suspension and applied to the column. Flow-through containing unlabeled human islet cells was collected. The column was washed three times with 1 mL of MACS buffer. The flow-through was collected in the same tube and filtered through a 30-µm cell strainer. The tubes and strainer were washed with 2 mL of PBS containing 0.04% BSA, and the cells were collected by centrifugation at 1300 rpm for 5 minutes. The cell pellets were resuspended in PBS containing 0.04% BSA to a final concentration of 300 to 500 cells/µL. Cell viability was measured using the Trypan blue assay and Cellometer Auto 1000 (Nexcelom Bioscience, Lawrence, MA). RNA preparation Total RNA was purified from all samples using MagMAX™-96 for Microarrays Total RNA Isolation Kit (Ambion; Life Technologies, Waltham, MA) according to the manufacturer’s specifications. Genomic DNA was removed using MagMAX™Turbo™DNase Buffer and TURBO DNase from the MagMAX kit (Ambion; Life Technologies). mRNA was purified from total RNA using the Dynabeads mRNA Purification Kit (Invitrogen). Strand-specific RNA-sequencing libraries were prepared using the KAPA mRNA-Seq Library Preparation Kit (Kapa Biosystems, Wilmington, MA). Twelve-cycle PCR was performed to amplify the libraries. Sequencing was performed on Illumina HiSeq®2000 (Illumina, San Diego, CA) by a multiplexed single-read run with 33 cycles. RNAseq read mapping and statistical analysis of differentially expressed RNA Raw sequence data (BCL files) were converted to FASTQ format via Illumina bcl2fastq, version 2.17. The reads were decoded based on their barcodes, and the read quality was evaluated using FastQC (available at: http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The reads were mapped to the human genome (National Center for Biotechnology Information, build 37.3) using ArrayStudio® software (OmicSoft®, Cary, NC) allowing for two mismatches. Reads mapped to the exons of a gene were summed at the gene level. Differentially expressed genes were identified by the DESeq2 package, and substantially perturbed genes were defined with fold changes of not less than 1.5 in either the up or down direction and with Benjamin-Hochberg adjusted P values of ≥ 0.01. RNA fluorescence in situ hybridization Formalin-fixed paraffin-embedded (FFPE) kidney sections with engrafted human islets or pancreas sections were deparaffinized in two changes of xylene and rehydrated. The sections were hybridized with mRNA probes for human SLC38A4, KI67, and GCG, in accordance with the manufacturer’s instructions (Advanced Cell Diagnostics, Newark, CA). In brief, FFPE sections were pretreated with hydrogen peroxide, incubated in Target Retrieval solution in a steamer for 30 minutes, and permeabilized by incubating in Protease Plus solution for 40 minutes. After hybridization, a fluorescent kit (version 2) was used to amplify the mRNA signal, and TSA Plus Fluorescein, TSA Plus Cyanine 3, and TSA Plus Cyanine 5 fluorescent signals were detected using a microscope slide scanner (Axio Scan.Z1; Zeiss, Oberkochen, Germany). Representative images were prepared in ZEN image software (Zeiss). Immunofluorescence Pancreas sections from healthy individuals and patients with GCHN were deparaffinized in three changes of xylene and rehydrated. The slides were placed in IHC-Tek Epitope Retrieval Solution (IHCWorld, Woodstock, MD) and heated for 30 minutes in a steamer, followed by 15 minutes of cooling at room temperature. The slides were washed in running water for 5 minutes, washed once in PBS, and incubated in blocking solution (5% BSA, 0.3% Triton X-100 in PBS) for 1 hour at room temperature. After applying primary antibodies for GCG (17), INS (18), KI67 (19), and pS6 (20) diluted in antibody dilution buffer (1% BSA and 0.3% Triton X-100 in PBS), the slides were incubated overnight at 4°C. The slides were washed three times in PBS and incubated with appropriate secondary antibodies [AF488-donkey anti-guinea pig (21), AF594-donkey anti-human (22), AF647-donkey anti-sheep (23), and AF647-donkey anti-rabbit (24); Jackson ImmunoResearch] diluted in antibody dilution buffer for 1 hour at room temperature. After washing three times in PBS, the slides were counterstained with 4′,6-diamidino-2-phenylindole for nuclei, mounted, and scanned using a slide scanner. Blood chemistry Blood glucose was determined using ACCU-CHEK® Compact Plus (Roche Diagnostics, Basel, Switzerland). The plasma glucagon and insulin levels were determined using glucagon (25), mouse (26), and human insulin ELISA kits (27) (Mercodia AB, Uppsala, Sweden). The plasma total amino acid levels were measured using the l-Amino Acid Quantification Kit (MAK002; Sigma-Aldrich, St. Louis, MO). GCHN and normal pancreas samples FFPE pancreatic tissue blocks of three patients with GCHN and three normal donors were used for RNA sequencing and/or histological analysis. The patients with GCHN had been confirmed to have inactivating mutations in GCGR, and their cases have been previously described (8). The three normal pancreas tissue blocks were obtained from Origene. The patient and normal donor information and analyses performed on each sample are presented in Table 2. Table 2. Normal and GCHN Patient Donor Information Donor Sex Age,a y BMI, kg/m2 GCGR Mutation Increased Serum Glucagon Normal 1 Male 45 28.2 ND ND Normal 2 Male 47 NR ND ND Normal 3 Female 60 24.3 ND ND GCHN patient 1 Male 25 Normal p.W83Lfs*35 Yes GCHN patient 2 Female 43 NR p.R8*, p.Q327* Yes GCHN patient 3 Male 58 NR p.R225H, p.V368M ND Donor Sex Age,a y BMI, kg/m2 GCGR Mutation Increased Serum Glucagon Normal 1 Male 45 28.2 ND ND Normal 2 Male 47 NR ND ND Normal 3 Female 60 24.3 ND ND GCHN patient 1 Male 25 Normal p.W83Lfs*35 Yes GCHN patient 2 Female 43 NR p.R8*, p.Q327* Yes GCHN patient 3 Male 58 NR p.R225H, p.V368M ND Asterisks indicate stop codons. Abbreviations: ND, not determined; NR, not reported. a Age at which the sample was collected. View Large Table 2. Normal and GCHN Patient Donor Information Donor Sex Age,a y BMI, kg/m2 GCGR Mutation Increased Serum Glucagon Normal 1 Male 45 28.2 ND ND Normal 2 Male 47 NR ND ND Normal 3 Female 60 24.3 ND ND GCHN patient 1 Male 25 Normal p.W83Lfs*35 Yes GCHN patient 2 Female 43 NR p.R8*, p.Q327* Yes GCHN patient 3 Male 58 NR p.R225H, p.V368M ND Donor Sex Age,a y BMI, kg/m2 GCGR Mutation Increased Serum Glucagon Normal 1 Male 45 28.2 ND ND Normal 2 Male 47 NR ND ND Normal 3 Female 60 24.3 ND ND GCHN patient 1 Male 25 Normal p.W83Lfs*35 Yes GCHN patient 2 Female 43 NR p.R8*, p.Q327* Yes GCHN patient 3 Male 58 NR p.R225H, p.V368M ND Asterisks indicate stop codons. Abbreviations: ND, not determined; NR, not reported. a Age at which the sample was collected. View Large Total RNA extraction from GCHN sections for RNA sequencing FFPE tissues from three patients with GCHN were used to extract mRNA for sequencing. mRNA was isolated from each patient sample using two freshly cut 15-µm FFPE sections using the RNeasy FFPE Kit according to the manufacturer’s instructions (Qiagen, Hilden, Germany). In brief, the sections were deparaffinized by incubating in deparaffinization solution and digested with proteinase K in PKD buffer. After DNase treatment, the samples were loaded onto RNeasy MinElute spin columns. After washing twice with RPE buffer, RNA was eluted with RNase-free water. Statistical analysis Statistical analyses were performed using GraphPad software Prism 7.0. All parameters were analyzed using the Student t test, one-way ANOVA or two-way ANOVA. A threshold of P < 0.05 was considered to indicate statistical significance. If a statistically significant F ratio was obtained with one- or two-way ANOVA, post hoc analysis was conducted with Bonferroni post-tests. All data are presented as the mean ± SEM. The statistical details of the experiments are reported with the figures. Results Recovery of engrafted human islet cells The workflow for treatment and recovery of human islets implanted under the kidney capsule in SCID mice is shown in Fig. 1A. The engrafted human islets did not affect the blood glucose, plasma amino acid, insulin, or glucagon levels (Fig. 1B–1D). Isolation of the islets from the kidney capsule had little effect on cell viability (implanted and recovered cells, 83% ± 7%; corresponding nonimplanted cells, 91% ± 3%). Overall, the gene expression profile (30,373 detected genes) was similar to that of nonimplanted human islets (R = 0.96; Fig. 1G). This included expression of the major islet cell hormones (GCG, INS, SST, and PYY) and key α-cell (ARX), β-cell (NKX6.1, MAFA, and PDX1), and δ-cell (HHEX) transcription factors. Figure 1. View largeDownload slide Implanted human islets isolated through the “peel” technique expressed comparable key endocrine cell markers to nonimplanted islets. (A) Schematic diagram of human islet implantation and recovery from the mouse kidney capsule. (B) Blood glucose, (C) plasma amino acid, (D) glucagon, (E) mouse insulin, and (F) human insulin levels in SCID mice implanted with 1000 human islet equivalents under the kidney capsule and treated with weekly injections of either control or GCGR antibody (10 mg/kg, weekly) for 3 weeks. Data presented as mean ± SEM; n = 6 mice per group. All mice had received islets from donor 1. Similar changes in circulating factors were observed in SCID mice implanted with islets from donors 2 and 3. ***P < 0.001; ****P < 0.0001. (G) Scatterplot depicting correlation of expressed genes in implanted vs nonimplanted human islets. Red triangles indicate selected key endocrine markers. mAb, monoclonal antibody; TPM, transcripts per million. Figure 1. View largeDownload slide Implanted human islets isolated through the “peel” technique expressed comparable key endocrine cell markers to nonimplanted islets. (A) Schematic diagram of human islet implantation and recovery from the mouse kidney capsule. (B) Blood glucose, (C) plasma amino acid, (D) glucagon, (E) mouse insulin, and (F) human insulin levels in SCID mice implanted with 1000 human islet equivalents under the kidney capsule and treated with weekly injections of either control or GCGR antibody (10 mg/kg, weekly) for 3 weeks. Data presented as mean ± SEM; n = 6 mice per group. All mice had received islets from donor 1. Similar changes in circulating factors were observed in SCID mice implanted with islets from donors 2 and 3. ***P < 0.001; ****P < 0.0001. (G) Scatterplot depicting correlation of expressed genes in implanted vs nonimplanted human islets. Red triangles indicate selected key endocrine markers. mAb, monoclonal antibody; TPM, transcripts per million. Administration of the GCGR blocking antibody (10 mg/kg) for 21 days to human islet-engrafted SCID mice lowered the blood glucose (Fig. 1B) and increased the plasma amino acid (Fig. 1C) and glucagon (Fig. 1D) levels. No effect of GCGR inhibition was observed on mouse and human plasma insulin levels (Fig. 1E and 1F). SLC38A4 expression is increased in implanted and α-GCGR–treated human α-cells GCGR inhibition did not significantly change the relative α-, β-, and δ-cell distributions in the human islet grafts (Fig. 2A and 2B). As expected from the increase in plasma glucagon levels, we detected an increase in GCG and PCSK2 expression in the recovered human islet cells (Fig. 2C). No change in INS or PCSK1 expression was observed, consistent with the lack of change in human insulin plasma levels. SST expression was also unchanged. Overall, the expression of 485 genes (303 upregulated and 182 downregulated) were affected by α-GCGR treatment (28). Figure 2. View largeDownload slide GCGR inhibition using a monoclonal blocking antibody increased the expression of amino acid transporter SLC38A4 in human α-cells. (A) Representative RNA-FISH images for GCG (red), INS (green), and SST (white) of human islet grafts from mice treated with control or GCGR antibody (10 mg/kg, weekly) for 3 weeks. (B) Relative distribution (percentage) of GCG+, INS+, and SST+ cells in the islet grafts. All mice had received islets from donor 2; 900 to 1200 cells were analyzed for each mouse. The percentage of relative distribution was calculated for each mouse, and statistical analysis was performed for five mice per group. (C) Differential gene expression of endocrine hormones and related processing enzymes in human islets implanted in mice treated with control or GCGR antibody. (D) Amino acid transporter expression in human islet endocrine cells. The average expression of nine amino acid transporters in each islet endocrine cell type using our previously reported human islet single-cell RNA sequencing data set of ∼19,000 cells from 12 nondiabetic donors (29, 30). (E) Differential expression of α-cell–expressed amino acid transporters in human islets implanted in mice treated with control or GCGR antibody. (C and E) Islets from donors 4, 5, and 7 were transplanted to three to four mice per donor, and the islets recovered from the mice were pooled and submitted for RNA sequencing. *Adjusted P < 0.01. (F) Representative RNA-FISH images for SLC38A4 (green), GCG (red), and INS (white) in human islet grafts from control or GCGR antibody-treated mice (10 mg/kg, weekly for 3 weeks). UMI, unique molecular identifier. Figure 2. View largeDownload slide GCGR inhibition using a monoclonal blocking antibody increased the expression of amino acid transporter SLC38A4 in human α-cells. (A) Representative RNA-FISH images for GCG (red), INS (green), and SST (white) of human islet grafts from mice treated with control or GCGR antibody (10 mg/kg, weekly) for 3 weeks. (B) Relative distribution (percentage) of GCG+, INS+, and SST+ cells in the islet grafts. All mice had received islets from donor 2; 900 to 1200 cells were analyzed for each mouse. The percentage of relative distribution was calculated for each mouse, and statistical analysis was performed for five mice per group. (C) Differential gene expression of endocrine hormones and related processing enzymes in human islets implanted in mice treated with control or GCGR antibody. (D) Amino acid transporter expression in human islet endocrine cells. The average expression of nine amino acid transporters in each islet endocrine cell type using our previously reported human islet single-cell RNA sequencing data set of ∼19,000 cells from 12 nondiabetic donors (29, 30). (E) Differential expression of α-cell–expressed amino acid transporters in human islets implanted in mice treated with control or GCGR antibody. (C and E) Islets from donors 4, 5, and 7 were transplanted to three to four mice per donor, and the islets recovered from the mice were pooled and submitted for RNA sequencing. *Adjusted P < 0.01. (F) Representative RNA-FISH images for SLC38A4 (green), GCG (red), and INS (white) in human islet grafts from control or GCGR antibody-treated mice (10 mg/kg, weekly for 3 weeks). UMI, unique molecular identifier. In our previously reported single human islet cell RNA sequencing data set (29, 30), nine amino acid transporters were detected (defined by unique molecular identifier >0 in >10% of the cell population) in at least one endocrine cell type and six of the nine transporters were detected in α-cells (Fig. 2D). The most abundant α-cell transporter was SLC3A2, and the most α-cell–enriched transporter was SLC38A4 (Fig. 2D). The expression of SLC38A4 was increased in the recovered human islet cells after α-GCGR treatment (Fig. 2E). RNA fluorescence in situ hybridization (FISH) of human islet grafts in α-GCGR–treated mice for SLC38A4, GCG, and INS demonstrated predominant expression of SLC38A4 in GCG-expressing cells (Fig. 2F). Increase in SLC38A4 expression is mTOR-dependent We administered the mTOR inhibitor rapamycin in the absence or presence of α-GCGR to determine whether the GCGR inhibition-induced increase in SLC38A4 expression in α-cells was mTOR-dependent. The SLC38A4 fluorescence intensity was increased by 50% in α-cells from mice treated with α-GCGR (Fig. 3A and 3B). Figure 3B shows that rapamycin inhibited the α-GCGR–induced increase in SLC38A4 expression but did not affect the baseline levels. Rapamycin also inhibited α-GCGR–induced human α-cell proliferation (Fig. 3C). We observed a small, but statistically significant, increase in SLC38A4 expression in proliferating vs nonproliferating α-cells (Fig. 3D). Consistent with the involvement of mTOR, we detected an increase in p-6S staining in implanted GCG+ cells treated with α-GCGR (Fig. 4A and 4B). Figure 3. View largeDownload slide GCGR blockade-induced SLC38A4 expression is mTOR dependent. (A) Representative RNA-FISH images for SLC38A4 (green), GCG (red), and KI67 (white) in mouse kidney sections containing human islet grafts from mice treated with control or GCGR antibody (10 mg/kg, weekly) for 3 weeks combined with PBS or rapamycin (10 mg/kg, daily). (B–D) Relative expression of SLC38A4 fluorescent intensity in human α-cells. (B) Percentage of KI67-positive human α-cells (C) and SLC38A4 fluorescence intensities in KI67-negative or KI67-positive human α-cells (D) from mice described in (A). All the mice had received islets from donor 2. A total of 1000 to 1100 α-cells were analyzed for each mouse. The percentage or fluorescence intensity was calculated for each mouse, and statistical analysis was performed for five mice per group. Data presented as mean ± SEM. *P < 0.05; **P < 0.01; ****P < 0.0001. Figure 3. View largeDownload slide GCGR blockade-induced SLC38A4 expression is mTOR dependent. (A) Representative RNA-FISH images for SLC38A4 (green), GCG (red), and KI67 (white) in mouse kidney sections containing human islet grafts from mice treated with control or GCGR antibody (10 mg/kg, weekly) for 3 weeks combined with PBS or rapamycin (10 mg/kg, daily). (B–D) Relative expression of SLC38A4 fluorescent intensity in human α-cells. (B) Percentage of KI67-positive human α-cells (C) and SLC38A4 fluorescence intensities in KI67-negative or KI67-positive human α-cells (D) from mice described in (A). All the mice had received islets from donor 2. A total of 1000 to 1100 α-cells were analyzed for each mouse. The percentage or fluorescence intensity was calculated for each mouse, and statistical analysis was performed for five mice per group. Data presented as mean ± SEM. *P < 0.05; **P < 0.01; ****P < 0.0001. Figure 4. View largeDownload slide Increased activation of mTORC1 by GCGR inhibition. (A) Representative images of immunohistochemistry staining for GCG (red), pS6 (green), 4′,6-diamidino-2-phenylindole (DAPI; blue) in kidney sections with human islet grafts from mice treated with weekly injections of control or α-GCGR antibody (10 mg/kg) for 3 weeks. (B) Quantification of pS6 levels in glucagon-producing human α- and β-cells from mice described in (A). All the mice had received islets from donor 3; 360 to 640 α- or β-cells were analyzed for each mouse. The fluorescence intensity was calculated for each mouse, and statistical analysis was performed for eight mice per group. Data presented mean ± SEM; n = 8. *P < 0.05. Figure 4. View largeDownload slide Increased activation of mTORC1 by GCGR inhibition. (A) Representative images of immunohistochemistry staining for GCG (red), pS6 (green), 4′,6-diamidino-2-phenylindole (DAPI; blue) in kidney sections with human islet grafts from mice treated with weekly injections of control or α-GCGR antibody (10 mg/kg) for 3 weeks. (B) Quantification of pS6 levels in glucagon-producing human α- and β-cells from mice described in (A). All the mice had received islets from donor 3; 360 to 640 α- or β-cells were analyzed for each mouse. The fluorescence intensity was calculated for each mouse, and statistical analysis was performed for eight mice per group. Data presented mean ± SEM; n = 8. *P < 0.05. SLC38A4 expression is increased in human GCHN samples Insulin and glucagon staining in pancreas sections from a normal donor and three patients with GCHN caused by loss-of-function mutations in GCGR is shown in Fig. 5A (Table 2). The expression of key endocrine, acinar, and ductal genes in the GCHN patient samples indicated that the section from patient 2 was composed mostly of glucagon producing α-cells and the sections from patients 1 and 3 contained more acinar and ductal cells (Fig. 5B). The detected amino acid transporters in the three GCHN samples were comparable to those detected in the α-cells from the engrafted human islets (Fig. 2B and Fig. 5C). GCG and KI67 staining in the pancreas sections showed that the α-cells in GCHN proliferate with a greater rate than that in islets from normal donors (Fig. 6A and 6B). Very few KI67+β-cells were detected (Fig. 6B). However, SLC38A4 RNA FISH fluorescence intensity was 3.5-fold greater in the α-cells from patients with GCHN compared with those from the normal donors, and the RNA level was further increased in the proliferating α-cells (Fig. 6C and 6D). Figure 5. View largeDownload slide Islet hormone and gene expression profile in GCHN patient pancreatic tissues. (A) Representative images of immunohistochemistry staining for 4′,6-diamidino-2-phenylindole (DAPI; blue), INS (green), and GCG (red) in normal pancreas and GCHN patient samples. (B) Expression levels of key endocrine, acinar, and ductal genes in three GCHN patient samples. (C) Expression levels of amino acid transporters in GCHN patient samples. Figure 5. View largeDownload slide Islet hormone and gene expression profile in GCHN patient pancreatic tissues. (A) Representative images of immunohistochemistry staining for 4′,6-diamidino-2-phenylindole (DAPI; blue), INS (green), and GCG (red) in normal pancreas and GCHN patient samples. (B) Expression levels of key endocrine, acinar, and ductal genes in three GCHN patient samples. (C) Expression levels of amino acid transporters in GCHN patient samples. Figure 6. View largeDownload slide Glucagon receptor inactivating mutations are associated with increased α-cell proliferation and SLC38A4 expression in humans. (A) Representative images of immunohistochemistry staining for GCG (red) and KI67 (white) from normal pancreas and GCHN sections from patients with inactivating mutations in GCGR. (B) Percentage of KI67-positive α-cells and β-cells in normal and GCHN patient sections. (C) Representative images of RNA-FISH staining for SLC38A4 (green) and GCG (red) from normal pancreas and GCHN patient sections. (D) SLC38A4 fluorescence intensities in α-cells in normal pancreas, KI67− and KI67+α-cells in GCHN patient samples; (B and D) 4000 to 6700 α- or β-cells were analyzed for each donor. The percentage or fluorescence intensity was calculated for each donor, and statistical analysis was performed for three donors per group. Data presented as mean ± SEM. *P < 0.05. Figure 6. View largeDownload slide Glucagon receptor inactivating mutations are associated with increased α-cell proliferation and SLC38A4 expression in humans. (A) Representative images of immunohistochemistry staining for GCG (red) and KI67 (white) from normal pancreas and GCHN sections from patients with inactivating mutations in GCGR. (B) Percentage of KI67-positive α-cells and β-cells in normal and GCHN patient sections. (C) Representative images of RNA-FISH staining for SLC38A4 (green) and GCG (red) from normal pancreas and GCHN patient sections. (D) SLC38A4 fluorescence intensities in α-cells in normal pancreas, KI67− and KI67+α-cells in GCHN patient samples; (B and D) 4000 to 6700 α- or β-cells were analyzed for each donor. The percentage or fluorescence intensity was calculated for each donor, and statistical analysis was performed for three donors per group. Data presented as mean ± SEM. *P < 0.05. Discussion We have reported on a technique to isolate human islets engrafted under the kidney capsule in SCID mice. The rescued islet cells showed high viability, and their global gene expression profile was similar to that of their nonimplanted counterparts. Using this technique, we were able to study the effects of glucagon receptor inhibition using a monoclonal α-GCGR antibody on human islet expression. α-GCGR treatment increased expression of SLC38A4, an amino acid transporter enriched in α-cells, in the rescued human islets. RNA-FISH of the human islet grafts verified that the elevated SLC38A4 expression was specific to α-cells. We confirmed the increase in SLC38A4 expression in three pancreas samples from patients with GCHN and confirmed the loss-of-function mutations in the glucagon receptor. We found that the increase in SLC38A4 expression was secondary to activation of the amino acid sensor, mTOR, and that rapamycin inhibited the increase in SLC38A4 expression. However, rapamycin did not affect the baseline SLC38A4 expression. Rapamycin also inhibited human α-cell proliferation, similar to its reported effects in mice (1, 5, 9). We detected an increase in phosphorylation of S6, confirming the α-GCGR–induced activation of mTOR. Six patients have been described with GCHN due to loss-of-function mutations in GCGR (8, 31–34). We used pancreas sections from three of these patients to establish their gene expression profiles and confirmed an increase in SLC38A4 expression. The α-cells in GCHN proliferated at a greater rate than observed in their counterparts from normal individuals. However, the proliferation rate was lower than that in α-cells engrafted in mice treated with α-GCGR. The reason for the difference in proliferation rates is unclear. However, it could reflect a burst in cell division after implantation and α-GCGR treatment, because the proliferation rates of engrafted human α-cells is comparable to that of α-cells from patients with GCHN after implantation for 3 months (5). In conclusion, RNA sequencing data have shown that six amino acid transporters are expressed in human α-cells. We speculated that the increase in SLC38A4 expression reflected an adaptation of the α-cells to prolonged elevations in plasma amino acids after α-GCGR treatment, including an increase in proliferation capacity. Future studies should aim to understand the contribution of each amino acid transporter to the regulation of glucagon secretion and the compensatory increase in α-cell number in response to glucagon receptor inactivation. Abbreviations: Abbreviations: FFPE formalin-fixed paraffin-embedded FISH fluorescence in situ hybridization GCHN glucagon cell hyperplasia and neoplasia mTOR mechanistic target of rapamycin SCID severe combined immunodeficient Acknowledgments mRNA sequencing data can be found within the Gene Expression Omnibus database using the following accession number: GSE114297. Financial Support: The present study was funded by Regeneron Pharmaceuticals, Inc. Author Contributions: J.K., J.G., and H.O. designed the studies. J.K., K.C., B. Sung, B. Sipos, and G.K. conducted the studies. J.K., G.D.G., Y.X., J.G., and H.O. analyzed the data. J.K., G.D.G., Y.X., G.K., J.G., and H.O. wrote the manuscript. Disclosure Summary: J.K., G.D.G., Y.X., K.C., B. Sung, J.G., and H.O. are or were employees and shareholders of Regeneron Pharmaceuticals, Inc. The remaining authors have nothing to disclose. References 1. 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Increased SLC38A4 Amino Acid Transporter Expression in Human Pancreatic α-Cells After Glucagon Receptor Inhibition

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Oxford University Press
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Copyright © 2019 Endocrine Society
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0013-7227
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1945-7170
D.O.I.
10.1210/en.2019-00022
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Abstract

Abstract Plasma amino acids and their transporters constitute an important part of the feedback loop between the liver and pancreatic α-cell function, and glucagon regulates hepatic amino acid turnover. Disruption of hepatic glucagon receptor action activates the loop and results in high plasma amino acids and hypersecretion of glucagon associated with α-cell hyperplasia. In the present study, we report a technique to rescue implanted human pancreatic islets from the mouse kidney capsule. Using this model, we have demonstrated that expression of the amino acid transporter SLC38A4 increases in α-cells after administration of a glucagon receptor blocking antibody. The increase in SLC38A4 expression and associated α-cell proliferation was dependent on mechanistic target of rapamycin pathway. We confirmed increased α-cell proliferation and expression of SLC38A4 in pancreas sections from patients with glucagon cell hyperplasia and neoplasia (GCHN) with loss-of-function mutations in the glucagon receptor. Collectively, using a technique to rescue implanted human islets from the kidney capsule in mice and pancreas sections from patients with GCHN, we found that expression of SLC38A4 was increased under conditions of disrupted glucagon receptor signaling. These data provide support for the existence of a liver–human α-cell endocrine feedback loop. An endocrine loop exists between the liver and the pancreatic α-cells in which the glucagon signals the α-cell → liver part and amino acids mediate the liver → α-cell part. This has been supported by findings that genetic or pharmacological inhibition of hepatic glucagon signaling results in elevated plasma amino acid levels and compensatory glucagon hypersecretion, involving expansion of the pancreatic α-cell mass (1–11). However, administration of glucagon to animals and humans will result in reductions in plasma amino acids, increased amino acid uptake by the liver, and their conversion into gluconeogenic precursors, promoting hepatic glucose production and an increase in blood glucose levels (12, 13). We, and others, have recently shown that the amino acid transporter Slc38a5 contributes to glucagon receptor inhibition-induced hyperglucagonemia and α-cell hyperplasia in mice (1, 5). The increase in α-cell proliferation, but not glucagon secretion, is mediated via an mechanistic target of rapamycin (mTOR)–dependent pathway (5). α-Cell growth was primarily triggered by glutamine and, to a lesser extent, alanine (1, 5, 9). The concentrations of these amino acids were dramatically increased in the circulation of mice when glucagon receptor signaling was blocked (5, 9, 10, 14). However, these findings did not translate into human α-cells, because SLC38A5 mRNA was not detected, and its expression was not induced in settings of glucagon receptor inhibition (5). The present study sought to examine the expression of human α-cell amino acid transporters in the setting of reduced hepatic glucagon action. Thus, we developed a technique to rescue human islets implanted under the mouse kidney capsule to allow for RNA sequencing and histological examination of the islet cells. We have confirmed our human islet implantation findings using pancreas sections from patients with glucagon cell hyperplasia and neoplasia (GCHN) with loss-of-function mutations in the glucagon receptor. Materials and Methods Human islets Human pancreatic islets were obtained from Prodo Laboratories (Aliso Viejo, CA) with appropriate consent. The islets were cultured in CMRL-1066 media supplemented with 10% (v/v) fetal bovine serum, 2 mM glutamine, 100 U/mL of penicillin, and 100 µg/mL of streptomycin (islet media). Islets were incubated in a tissue culture incubator at 37°C with 5% carbon dioxide in atmosphere air for 3 to 5 days while conducting a pathogen analysis (mouse pathogen testing; IDEXX BioResearch, Columbia, MO). The islet donors had no history of diabetes, and their information is presented in Table 1. Table 1. Islet Donor Information Donor Age, y Ethnicity Sex BMI, kg/m2 Height Weight, lb HbA1c, % Cause of Death Usage 1 29 White Male 26.7 6’3” 214 5.5 Anoxia Histological examination 2 50 White Male 28.6 5’7” 185 5.4 Stroke Histological examination 3 56 White Male 21.2 5’11” 152 5.4 Stroke Histological examination 4 46 White Female 20.1 5’5” 121 5.0 Stroke RNA sequencing 5 41 Hispanic Female 18.3 5’3” 105 5.5 Stroke RNA sequencing 6 57 White Female 29.0 5’7” 185 5.6 Stroke RNA sequencing 7 33 Black Male 30.8 5’10” 215 5.7 Head trauma RNA sequencing Donor Age, y Ethnicity Sex BMI, kg/m2 Height Weight, lb HbA1c, % Cause of Death Usage 1 29 White Male 26.7 6’3” 214 5.5 Anoxia Histological examination 2 50 White Male 28.6 5’7” 185 5.4 Stroke Histological examination 3 56 White Male 21.2 5’11” 152 5.4 Stroke Histological examination 4 46 White Female 20.1 5’5” 121 5.0 Stroke RNA sequencing 5 41 Hispanic Female 18.3 5’3” 105 5.5 Stroke RNA sequencing 6 57 White Female 29.0 5’7” 185 5.6 Stroke RNA sequencing 7 33 Black Male 30.8 5’10” 215 5.7 Head trauma RNA sequencing View Large Table 1. Islet Donor Information Donor Age, y Ethnicity Sex BMI, kg/m2 Height Weight, lb HbA1c, % Cause of Death Usage 1 29 White Male 26.7 6’3” 214 5.5 Anoxia Histological examination 2 50 White Male 28.6 5’7” 185 5.4 Stroke Histological examination 3 56 White Male 21.2 5’11” 152 5.4 Stroke Histological examination 4 46 White Female 20.1 5’5” 121 5.0 Stroke RNA sequencing 5 41 Hispanic Female 18.3 5’3” 105 5.5 Stroke RNA sequencing 6 57 White Female 29.0 5’7” 185 5.6 Stroke RNA sequencing 7 33 Black Male 30.8 5’10” 215 5.7 Head trauma RNA sequencing Donor Age, y Ethnicity Sex BMI, kg/m2 Height Weight, lb HbA1c, % Cause of Death Usage 1 29 White Male 26.7 6’3” 214 5.5 Anoxia Histological examination 2 50 White Male 28.6 5’7” 185 5.4 Stroke Histological examination 3 56 White Male 21.2 5’11” 152 5.4 Stroke Histological examination 4 46 White Female 20.1 5’5” 121 5.0 Stroke RNA sequencing 5 41 Hispanic Female 18.3 5’3” 105 5.5 Stroke RNA sequencing 6 57 White Female 29.0 5’7” 185 5.6 Stroke RNA sequencing 7 33 Black Male 30.8 5’10” 215 5.7 Head trauma RNA sequencing View Large Human islet implantation Human islets were implanted under the kidney capsule of severe combined immunodeficient (SCID) mice (males, aged 16 to 25 weeks; Taconic, Rensselaer, NY). Each mouse received 1000 islet equivalents. Starting on day 20 after islet implantation, the mice received weekly subcutaneous injections of the glucagon receptor blocking monoclonal antibody REGN1193 (7, 15) or an isotype control antibody (16) at 10 mg/kg for 3 weeks. Blood was collected from the tail for glucose measurements without fasting. Plasma samples were collected via submandibular bleeding without fasting for analysis of hormones and total amino acids. At 3 weeks after the initial antibody dosing, the mice were euthanized, and the human islet grafts were isolated for RNA sequencing and histological analysis. The detailed procedures are described in the next sections. Islet implantation Cultured human islets were centrifuged at 1300 rpm for 5 minutes at 4°C. After careful aspiration of the media, the islets were resuspended with islet media at a density of 4000 islet equivalent/mL. Next, 250 µL of resuspended islets were transferred into a gel loading tip with the narrow end bent into a “U” shape and placed in an Eppendorf tube. The islets were allowed to settle to the bottom. The media were removed from the gel-loading tip, leaving 15 to 20 µL. The gel loading tip was inserted into the nonbeveled end of the 9-in.-long PE50 tubing (BD Biosciences, San Jose, CA) to load islets. A pipette was connected to the open end of the tip, and the piston was gently depressed to push the islets to the beveled side of the PE50 tubing. The pipet tip was disconnected and subsequently connected to a Hamilton syringe. The PE50 tubing was held in an upright position to allow the islets inside the tubing to form a small pellet. The recipient SCID mice were anesthetized, shaved, and cleaned. A small incision was made on the left flank, and the kidney was exposed by gently pressing both sides of the incision. Using a 25-guage syringe needle, a small scratch was made on the membrane of the kidney to insert the PE50 tubing. After applying 0.9% sodium chloride solution to the kidney, the beveled end of the PE50 tubing with the islets near the tip was inserted under the kidney capsule and gently moved around to create a space. The islets were slowly injected by gently pressing the plunger. The PE50 tubing was removed, the nick was cauterized, the kidney was carefully replaced back into the peritoneum, and the skin was sutured and stapled. Isolation of engrafted islets To isolate the engrafted islets, a small section of the kidney membrane with the islets was peeled off and transferred into a 15-mL tube containing 5 mL of Liberase TL (0.25 mg/mL). The samples were incubated at 37°C for 45 minutes with frequent P1000 pipetting to help islet cell dissociation. Once the islet cells were well dissociated, 10 mL of islet media was added, and the cell suspension was filtered through a 70-µm cell strainer into a 15-mL centrifuge tube. Islet cells were collected by centrifugation at 1300 rpm for 5 minutes. The cell pellets were resuspended in 5 mL of PBS containing 0.5% BSA (MACS buffer) and centrifuged again at 1300 rpm for 5 minutes. The cell pellets were resuspended in 300 µL MACS buffer. Next, 100 µL of MACS Mouse Cell Depletion Cocktail (Miltenyi Biotec, Bergisch Gladbach, Germany) was added and incubated for 15 minutes at 4°C. An LS column in a MACS Separator (Miltenyi Biotec) was prepared, and the column was equilibrated with 3 mL of MACS buffer. Next, 300 µL of MACS buffer was added to the cell suspension and applied to the column. Flow-through containing unlabeled human islet cells was collected. The column was washed three times with 1 mL of MACS buffer. The flow-through was collected in the same tube and filtered through a 30-µm cell strainer. The tubes and strainer were washed with 2 mL of PBS containing 0.04% BSA, and the cells were collected by centrifugation at 1300 rpm for 5 minutes. The cell pellets were resuspended in PBS containing 0.04% BSA to a final concentration of 300 to 500 cells/µL. Cell viability was measured using the Trypan blue assay and Cellometer Auto 1000 (Nexcelom Bioscience, Lawrence, MA). RNA preparation Total RNA was purified from all samples using MagMAX™-96 for Microarrays Total RNA Isolation Kit (Ambion; Life Technologies, Waltham, MA) according to the manufacturer’s specifications. Genomic DNA was removed using MagMAX™Turbo™DNase Buffer and TURBO DNase from the MagMAX kit (Ambion; Life Technologies). mRNA was purified from total RNA using the Dynabeads mRNA Purification Kit (Invitrogen). Strand-specific RNA-sequencing libraries were prepared using the KAPA mRNA-Seq Library Preparation Kit (Kapa Biosystems, Wilmington, MA). Twelve-cycle PCR was performed to amplify the libraries. Sequencing was performed on Illumina HiSeq®2000 (Illumina, San Diego, CA) by a multiplexed single-read run with 33 cycles. RNAseq read mapping and statistical analysis of differentially expressed RNA Raw sequence data (BCL files) were converted to FASTQ format via Illumina bcl2fastq, version 2.17. The reads were decoded based on their barcodes, and the read quality was evaluated using FastQC (available at: http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The reads were mapped to the human genome (National Center for Biotechnology Information, build 37.3) using ArrayStudio® software (OmicSoft®, Cary, NC) allowing for two mismatches. Reads mapped to the exons of a gene were summed at the gene level. Differentially expressed genes were identified by the DESeq2 package, and substantially perturbed genes were defined with fold changes of not less than 1.5 in either the up or down direction and with Benjamin-Hochberg adjusted P values of ≥ 0.01. RNA fluorescence in situ hybridization Formalin-fixed paraffin-embedded (FFPE) kidney sections with engrafted human islets or pancreas sections were deparaffinized in two changes of xylene and rehydrated. The sections were hybridized with mRNA probes for human SLC38A4, KI67, and GCG, in accordance with the manufacturer’s instructions (Advanced Cell Diagnostics, Newark, CA). In brief, FFPE sections were pretreated with hydrogen peroxide, incubated in Target Retrieval solution in a steamer for 30 minutes, and permeabilized by incubating in Protease Plus solution for 40 minutes. After hybridization, a fluorescent kit (version 2) was used to amplify the mRNA signal, and TSA Plus Fluorescein, TSA Plus Cyanine 3, and TSA Plus Cyanine 5 fluorescent signals were detected using a microscope slide scanner (Axio Scan.Z1; Zeiss, Oberkochen, Germany). Representative images were prepared in ZEN image software (Zeiss). Immunofluorescence Pancreas sections from healthy individuals and patients with GCHN were deparaffinized in three changes of xylene and rehydrated. The slides were placed in IHC-Tek Epitope Retrieval Solution (IHCWorld, Woodstock, MD) and heated for 30 minutes in a steamer, followed by 15 minutes of cooling at room temperature. The slides were washed in running water for 5 minutes, washed once in PBS, and incubated in blocking solution (5% BSA, 0.3% Triton X-100 in PBS) for 1 hour at room temperature. After applying primary antibodies for GCG (17), INS (18), KI67 (19), and pS6 (20) diluted in antibody dilution buffer (1% BSA and 0.3% Triton X-100 in PBS), the slides were incubated overnight at 4°C. The slides were washed three times in PBS and incubated with appropriate secondary antibodies [AF488-donkey anti-guinea pig (21), AF594-donkey anti-human (22), AF647-donkey anti-sheep (23), and AF647-donkey anti-rabbit (24); Jackson ImmunoResearch] diluted in antibody dilution buffer for 1 hour at room temperature. After washing three times in PBS, the slides were counterstained with 4′,6-diamidino-2-phenylindole for nuclei, mounted, and scanned using a slide scanner. Blood chemistry Blood glucose was determined using ACCU-CHEK® Compact Plus (Roche Diagnostics, Basel, Switzerland). The plasma glucagon and insulin levels were determined using glucagon (25), mouse (26), and human insulin ELISA kits (27) (Mercodia AB, Uppsala, Sweden). The plasma total amino acid levels were measured using the l-Amino Acid Quantification Kit (MAK002; Sigma-Aldrich, St. Louis, MO). GCHN and normal pancreas samples FFPE pancreatic tissue blocks of three patients with GCHN and three normal donors were used for RNA sequencing and/or histological analysis. The patients with GCHN had been confirmed to have inactivating mutations in GCGR, and their cases have been previously described (8). The three normal pancreas tissue blocks were obtained from Origene. The patient and normal donor information and analyses performed on each sample are presented in Table 2. Table 2. Normal and GCHN Patient Donor Information Donor Sex Age,a y BMI, kg/m2 GCGR Mutation Increased Serum Glucagon Normal 1 Male 45 28.2 ND ND Normal 2 Male 47 NR ND ND Normal 3 Female 60 24.3 ND ND GCHN patient 1 Male 25 Normal p.W83Lfs*35 Yes GCHN patient 2 Female 43 NR p.R8*, p.Q327* Yes GCHN patient 3 Male 58 NR p.R225H, p.V368M ND Donor Sex Age,a y BMI, kg/m2 GCGR Mutation Increased Serum Glucagon Normal 1 Male 45 28.2 ND ND Normal 2 Male 47 NR ND ND Normal 3 Female 60 24.3 ND ND GCHN patient 1 Male 25 Normal p.W83Lfs*35 Yes GCHN patient 2 Female 43 NR p.R8*, p.Q327* Yes GCHN patient 3 Male 58 NR p.R225H, p.V368M ND Asterisks indicate stop codons. Abbreviations: ND, not determined; NR, not reported. a Age at which the sample was collected. View Large Table 2. Normal and GCHN Patient Donor Information Donor Sex Age,a y BMI, kg/m2 GCGR Mutation Increased Serum Glucagon Normal 1 Male 45 28.2 ND ND Normal 2 Male 47 NR ND ND Normal 3 Female 60 24.3 ND ND GCHN patient 1 Male 25 Normal p.W83Lfs*35 Yes GCHN patient 2 Female 43 NR p.R8*, p.Q327* Yes GCHN patient 3 Male 58 NR p.R225H, p.V368M ND Donor Sex Age,a y BMI, kg/m2 GCGR Mutation Increased Serum Glucagon Normal 1 Male 45 28.2 ND ND Normal 2 Male 47 NR ND ND Normal 3 Female 60 24.3 ND ND GCHN patient 1 Male 25 Normal p.W83Lfs*35 Yes GCHN patient 2 Female 43 NR p.R8*, p.Q327* Yes GCHN patient 3 Male 58 NR p.R225H, p.V368M ND Asterisks indicate stop codons. Abbreviations: ND, not determined; NR, not reported. a Age at which the sample was collected. View Large Total RNA extraction from GCHN sections for RNA sequencing FFPE tissues from three patients with GCHN were used to extract mRNA for sequencing. mRNA was isolated from each patient sample using two freshly cut 15-µm FFPE sections using the RNeasy FFPE Kit according to the manufacturer’s instructions (Qiagen, Hilden, Germany). In brief, the sections were deparaffinized by incubating in deparaffinization solution and digested with proteinase K in PKD buffer. After DNase treatment, the samples were loaded onto RNeasy MinElute spin columns. After washing twice with RPE buffer, RNA was eluted with RNase-free water. Statistical analysis Statistical analyses were performed using GraphPad software Prism 7.0. All parameters were analyzed using the Student t test, one-way ANOVA or two-way ANOVA. A threshold of P < 0.05 was considered to indicate statistical significance. If a statistically significant F ratio was obtained with one- or two-way ANOVA, post hoc analysis was conducted with Bonferroni post-tests. All data are presented as the mean ± SEM. The statistical details of the experiments are reported with the figures. Results Recovery of engrafted human islet cells The workflow for treatment and recovery of human islets implanted under the kidney capsule in SCID mice is shown in Fig. 1A. The engrafted human islets did not affect the blood glucose, plasma amino acid, insulin, or glucagon levels (Fig. 1B–1D). Isolation of the islets from the kidney capsule had little effect on cell viability (implanted and recovered cells, 83% ± 7%; corresponding nonimplanted cells, 91% ± 3%). Overall, the gene expression profile (30,373 detected genes) was similar to that of nonimplanted human islets (R = 0.96; Fig. 1G). This included expression of the major islet cell hormones (GCG, INS, SST, and PYY) and key α-cell (ARX), β-cell (NKX6.1, MAFA, and PDX1), and δ-cell (HHEX) transcription factors. Figure 1. View largeDownload slide Implanted human islets isolated through the “peel” technique expressed comparable key endocrine cell markers to nonimplanted islets. (A) Schematic diagram of human islet implantation and recovery from the mouse kidney capsule. (B) Blood glucose, (C) plasma amino acid, (D) glucagon, (E) mouse insulin, and (F) human insulin levels in SCID mice implanted with 1000 human islet equivalents under the kidney capsule and treated with weekly injections of either control or GCGR antibody (10 mg/kg, weekly) for 3 weeks. Data presented as mean ± SEM; n = 6 mice per group. All mice had received islets from donor 1. Similar changes in circulating factors were observed in SCID mice implanted with islets from donors 2 and 3. ***P < 0.001; ****P < 0.0001. (G) Scatterplot depicting correlation of expressed genes in implanted vs nonimplanted human islets. Red triangles indicate selected key endocrine markers. mAb, monoclonal antibody; TPM, transcripts per million. Figure 1. View largeDownload slide Implanted human islets isolated through the “peel” technique expressed comparable key endocrine cell markers to nonimplanted islets. (A) Schematic diagram of human islet implantation and recovery from the mouse kidney capsule. (B) Blood glucose, (C) plasma amino acid, (D) glucagon, (E) mouse insulin, and (F) human insulin levels in SCID mice implanted with 1000 human islet equivalents under the kidney capsule and treated with weekly injections of either control or GCGR antibody (10 mg/kg, weekly) for 3 weeks. Data presented as mean ± SEM; n = 6 mice per group. All mice had received islets from donor 1. Similar changes in circulating factors were observed in SCID mice implanted with islets from donors 2 and 3. ***P < 0.001; ****P < 0.0001. (G) Scatterplot depicting correlation of expressed genes in implanted vs nonimplanted human islets. Red triangles indicate selected key endocrine markers. mAb, monoclonal antibody; TPM, transcripts per million. Administration of the GCGR blocking antibody (10 mg/kg) for 21 days to human islet-engrafted SCID mice lowered the blood glucose (Fig. 1B) and increased the plasma amino acid (Fig. 1C) and glucagon (Fig. 1D) levels. No effect of GCGR inhibition was observed on mouse and human plasma insulin levels (Fig. 1E and 1F). SLC38A4 expression is increased in implanted and α-GCGR–treated human α-cells GCGR inhibition did not significantly change the relative α-, β-, and δ-cell distributions in the human islet grafts (Fig. 2A and 2B). As expected from the increase in plasma glucagon levels, we detected an increase in GCG and PCSK2 expression in the recovered human islet cells (Fig. 2C). No change in INS or PCSK1 expression was observed, consistent with the lack of change in human insulin plasma levels. SST expression was also unchanged. Overall, the expression of 485 genes (303 upregulated and 182 downregulated) were affected by α-GCGR treatment (28). Figure 2. View largeDownload slide GCGR inhibition using a monoclonal blocking antibody increased the expression of amino acid transporter SLC38A4 in human α-cells. (A) Representative RNA-FISH images for GCG (red), INS (green), and SST (white) of human islet grafts from mice treated with control or GCGR antibody (10 mg/kg, weekly) for 3 weeks. (B) Relative distribution (percentage) of GCG+, INS+, and SST+ cells in the islet grafts. All mice had received islets from donor 2; 900 to 1200 cells were analyzed for each mouse. The percentage of relative distribution was calculated for each mouse, and statistical analysis was performed for five mice per group. (C) Differential gene expression of endocrine hormones and related processing enzymes in human islets implanted in mice treated with control or GCGR antibody. (D) Amino acid transporter expression in human islet endocrine cells. The average expression of nine amino acid transporters in each islet endocrine cell type using our previously reported human islet single-cell RNA sequencing data set of ∼19,000 cells from 12 nondiabetic donors (29, 30). (E) Differential expression of α-cell–expressed amino acid transporters in human islets implanted in mice treated with control or GCGR antibody. (C and E) Islets from donors 4, 5, and 7 were transplanted to three to four mice per donor, and the islets recovered from the mice were pooled and submitted for RNA sequencing. *Adjusted P < 0.01. (F) Representative RNA-FISH images for SLC38A4 (green), GCG (red), and INS (white) in human islet grafts from control or GCGR antibody-treated mice (10 mg/kg, weekly for 3 weeks). UMI, unique molecular identifier. Figure 2. View largeDownload slide GCGR inhibition using a monoclonal blocking antibody increased the expression of amino acid transporter SLC38A4 in human α-cells. (A) Representative RNA-FISH images for GCG (red), INS (green), and SST (white) of human islet grafts from mice treated with control or GCGR antibody (10 mg/kg, weekly) for 3 weeks. (B) Relative distribution (percentage) of GCG+, INS+, and SST+ cells in the islet grafts. All mice had received islets from donor 2; 900 to 1200 cells were analyzed for each mouse. The percentage of relative distribution was calculated for each mouse, and statistical analysis was performed for five mice per group. (C) Differential gene expression of endocrine hormones and related processing enzymes in human islets implanted in mice treated with control or GCGR antibody. (D) Amino acid transporter expression in human islet endocrine cells. The average expression of nine amino acid transporters in each islet endocrine cell type using our previously reported human islet single-cell RNA sequencing data set of ∼19,000 cells from 12 nondiabetic donors (29, 30). (E) Differential expression of α-cell–expressed amino acid transporters in human islets implanted in mice treated with control or GCGR antibody. (C and E) Islets from donors 4, 5, and 7 were transplanted to three to four mice per donor, and the islets recovered from the mice were pooled and submitted for RNA sequencing. *Adjusted P < 0.01. (F) Representative RNA-FISH images for SLC38A4 (green), GCG (red), and INS (white) in human islet grafts from control or GCGR antibody-treated mice (10 mg/kg, weekly for 3 weeks). UMI, unique molecular identifier. In our previously reported single human islet cell RNA sequencing data set (29, 30), nine amino acid transporters were detected (defined by unique molecular identifier >0 in >10% of the cell population) in at least one endocrine cell type and six of the nine transporters were detected in α-cells (Fig. 2D). The most abundant α-cell transporter was SLC3A2, and the most α-cell–enriched transporter was SLC38A4 (Fig. 2D). The expression of SLC38A4 was increased in the recovered human islet cells after α-GCGR treatment (Fig. 2E). RNA fluorescence in situ hybridization (FISH) of human islet grafts in α-GCGR–treated mice for SLC38A4, GCG, and INS demonstrated predominant expression of SLC38A4 in GCG-expressing cells (Fig. 2F). Increase in SLC38A4 expression is mTOR-dependent We administered the mTOR inhibitor rapamycin in the absence or presence of α-GCGR to determine whether the GCGR inhibition-induced increase in SLC38A4 expression in α-cells was mTOR-dependent. The SLC38A4 fluorescence intensity was increased by 50% in α-cells from mice treated with α-GCGR (Fig. 3A and 3B). Figure 3B shows that rapamycin inhibited the α-GCGR–induced increase in SLC38A4 expression but did not affect the baseline levels. Rapamycin also inhibited α-GCGR–induced human α-cell proliferation (Fig. 3C). We observed a small, but statistically significant, increase in SLC38A4 expression in proliferating vs nonproliferating α-cells (Fig. 3D). Consistent with the involvement of mTOR, we detected an increase in p-6S staining in implanted GCG+ cells treated with α-GCGR (Fig. 4A and 4B). Figure 3. View largeDownload slide GCGR blockade-induced SLC38A4 expression is mTOR dependent. (A) Representative RNA-FISH images for SLC38A4 (green), GCG (red), and KI67 (white) in mouse kidney sections containing human islet grafts from mice treated with control or GCGR antibody (10 mg/kg, weekly) for 3 weeks combined with PBS or rapamycin (10 mg/kg, daily). (B–D) Relative expression of SLC38A4 fluorescent intensity in human α-cells. (B) Percentage of KI67-positive human α-cells (C) and SLC38A4 fluorescence intensities in KI67-negative or KI67-positive human α-cells (D) from mice described in (A). All the mice had received islets from donor 2. A total of 1000 to 1100 α-cells were analyzed for each mouse. The percentage or fluorescence intensity was calculated for each mouse, and statistical analysis was performed for five mice per group. Data presented as mean ± SEM. *P < 0.05; **P < 0.01; ****P < 0.0001. Figure 3. View largeDownload slide GCGR blockade-induced SLC38A4 expression is mTOR dependent. (A) Representative RNA-FISH images for SLC38A4 (green), GCG (red), and KI67 (white) in mouse kidney sections containing human islet grafts from mice treated with control or GCGR antibody (10 mg/kg, weekly) for 3 weeks combined with PBS or rapamycin (10 mg/kg, daily). (B–D) Relative expression of SLC38A4 fluorescent intensity in human α-cells. (B) Percentage of KI67-positive human α-cells (C) and SLC38A4 fluorescence intensities in KI67-negative or KI67-positive human α-cells (D) from mice described in (A). All the mice had received islets from donor 2. A total of 1000 to 1100 α-cells were analyzed for each mouse. The percentage or fluorescence intensity was calculated for each mouse, and statistical analysis was performed for five mice per group. Data presented as mean ± SEM. *P < 0.05; **P < 0.01; ****P < 0.0001. Figure 4. View largeDownload slide Increased activation of mTORC1 by GCGR inhibition. (A) Representative images of immunohistochemistry staining for GCG (red), pS6 (green), 4′,6-diamidino-2-phenylindole (DAPI; blue) in kidney sections with human islet grafts from mice treated with weekly injections of control or α-GCGR antibody (10 mg/kg) for 3 weeks. (B) Quantification of pS6 levels in glucagon-producing human α- and β-cells from mice described in (A). All the mice had received islets from donor 3; 360 to 640 α- or β-cells were analyzed for each mouse. The fluorescence intensity was calculated for each mouse, and statistical analysis was performed for eight mice per group. Data presented mean ± SEM; n = 8. *P < 0.05. Figure 4. View largeDownload slide Increased activation of mTORC1 by GCGR inhibition. (A) Representative images of immunohistochemistry staining for GCG (red), pS6 (green), 4′,6-diamidino-2-phenylindole (DAPI; blue) in kidney sections with human islet grafts from mice treated with weekly injections of control or α-GCGR antibody (10 mg/kg) for 3 weeks. (B) Quantification of pS6 levels in glucagon-producing human α- and β-cells from mice described in (A). All the mice had received islets from donor 3; 360 to 640 α- or β-cells were analyzed for each mouse. The fluorescence intensity was calculated for each mouse, and statistical analysis was performed for eight mice per group. Data presented mean ± SEM; n = 8. *P < 0.05. SLC38A4 expression is increased in human GCHN samples Insulin and glucagon staining in pancreas sections from a normal donor and three patients with GCHN caused by loss-of-function mutations in GCGR is shown in Fig. 5A (Table 2). The expression of key endocrine, acinar, and ductal genes in the GCHN patient samples indicated that the section from patient 2 was composed mostly of glucagon producing α-cells and the sections from patients 1 and 3 contained more acinar and ductal cells (Fig. 5B). The detected amino acid transporters in the three GCHN samples were comparable to those detected in the α-cells from the engrafted human islets (Fig. 2B and Fig. 5C). GCG and KI67 staining in the pancreas sections showed that the α-cells in GCHN proliferate with a greater rate than that in islets from normal donors (Fig. 6A and 6B). Very few KI67+β-cells were detected (Fig. 6B). However, SLC38A4 RNA FISH fluorescence intensity was 3.5-fold greater in the α-cells from patients with GCHN compared with those from the normal donors, and the RNA level was further increased in the proliferating α-cells (Fig. 6C and 6D). Figure 5. View largeDownload slide Islet hormone and gene expression profile in GCHN patient pancreatic tissues. (A) Representative images of immunohistochemistry staining for 4′,6-diamidino-2-phenylindole (DAPI; blue), INS (green), and GCG (red) in normal pancreas and GCHN patient samples. (B) Expression levels of key endocrine, acinar, and ductal genes in three GCHN patient samples. (C) Expression levels of amino acid transporters in GCHN patient samples. Figure 5. View largeDownload slide Islet hormone and gene expression profile in GCHN patient pancreatic tissues. (A) Representative images of immunohistochemistry staining for 4′,6-diamidino-2-phenylindole (DAPI; blue), INS (green), and GCG (red) in normal pancreas and GCHN patient samples. (B) Expression levels of key endocrine, acinar, and ductal genes in three GCHN patient samples. (C) Expression levels of amino acid transporters in GCHN patient samples. Figure 6. View largeDownload slide Glucagon receptor inactivating mutations are associated with increased α-cell proliferation and SLC38A4 expression in humans. (A) Representative images of immunohistochemistry staining for GCG (red) and KI67 (white) from normal pancreas and GCHN sections from patients with inactivating mutations in GCGR. (B) Percentage of KI67-positive α-cells and β-cells in normal and GCHN patient sections. (C) Representative images of RNA-FISH staining for SLC38A4 (green) and GCG (red) from normal pancreas and GCHN patient sections. (D) SLC38A4 fluorescence intensities in α-cells in normal pancreas, KI67− and KI67+α-cells in GCHN patient samples; (B and D) 4000 to 6700 α- or β-cells were analyzed for each donor. The percentage or fluorescence intensity was calculated for each donor, and statistical analysis was performed for three donors per group. Data presented as mean ± SEM. *P < 0.05. Figure 6. View largeDownload slide Glucagon receptor inactivating mutations are associated with increased α-cell proliferation and SLC38A4 expression in humans. (A) Representative images of immunohistochemistry staining for GCG (red) and KI67 (white) from normal pancreas and GCHN sections from patients with inactivating mutations in GCGR. (B) Percentage of KI67-positive α-cells and β-cells in normal and GCHN patient sections. (C) Representative images of RNA-FISH staining for SLC38A4 (green) and GCG (red) from normal pancreas and GCHN patient sections. (D) SLC38A4 fluorescence intensities in α-cells in normal pancreas, KI67− and KI67+α-cells in GCHN patient samples; (B and D) 4000 to 6700 α- or β-cells were analyzed for each donor. The percentage or fluorescence intensity was calculated for each donor, and statistical analysis was performed for three donors per group. Data presented as mean ± SEM. *P < 0.05. Discussion We have reported on a technique to isolate human islets engrafted under the kidney capsule in SCID mice. The rescued islet cells showed high viability, and their global gene expression profile was similar to that of their nonimplanted counterparts. Using this technique, we were able to study the effects of glucagon receptor inhibition using a monoclonal α-GCGR antibody on human islet expression. α-GCGR treatment increased expression of SLC38A4, an amino acid transporter enriched in α-cells, in the rescued human islets. RNA-FISH of the human islet grafts verified that the elevated SLC38A4 expression was specific to α-cells. We confirmed the increase in SLC38A4 expression in three pancreas samples from patients with GCHN and confirmed the loss-of-function mutations in the glucagon receptor. We found that the increase in SLC38A4 expression was secondary to activation of the amino acid sensor, mTOR, and that rapamycin inhibited the increase in SLC38A4 expression. However, rapamycin did not affect the baseline SLC38A4 expression. Rapamycin also inhibited human α-cell proliferation, similar to its reported effects in mice (1, 5, 9). We detected an increase in phosphorylation of S6, confirming the α-GCGR–induced activation of mTOR. Six patients have been described with GCHN due to loss-of-function mutations in GCGR (8, 31–34). We used pancreas sections from three of these patients to establish their gene expression profiles and confirmed an increase in SLC38A4 expression. The α-cells in GCHN proliferated at a greater rate than observed in their counterparts from normal individuals. However, the proliferation rate was lower than that in α-cells engrafted in mice treated with α-GCGR. The reason for the difference in proliferation rates is unclear. However, it could reflect a burst in cell division after implantation and α-GCGR treatment, because the proliferation rates of engrafted human α-cells is comparable to that of α-cells from patients with GCHN after implantation for 3 months (5). In conclusion, RNA sequencing data have shown that six amino acid transporters are expressed in human α-cells. We speculated that the increase in SLC38A4 expression reflected an adaptation of the α-cells to prolonged elevations in plasma amino acids after α-GCGR treatment, including an increase in proliferation capacity. Future studies should aim to understand the contribution of each amino acid transporter to the regulation of glucagon secretion and the compensatory increase in α-cell number in response to glucagon receptor inactivation. Abbreviations: Abbreviations: FFPE formalin-fixed paraffin-embedded FISH fluorescence in situ hybridization GCHN glucagon cell hyperplasia and neoplasia mTOR mechanistic target of rapamycin SCID severe combined immunodeficient Acknowledgments mRNA sequencing data can be found within the Gene Expression Omnibus database using the following accession number: GSE114297. Financial Support: The present study was funded by Regeneron Pharmaceuticals, Inc. Author Contributions: J.K., J.G., and H.O. designed the studies. J.K., K.C., B. Sung, B. Sipos, and G.K. conducted the studies. J.K., G.D.G., Y.X., J.G., and H.O. analyzed the data. J.K., G.D.G., Y.X., G.K., J.G., and H.O. wrote the manuscript. Disclosure Summary: J.K., G.D.G., Y.X., K.C., B. Sung, J.G., and H.O. are or were employees and shareholders of Regeneron Pharmaceuticals, Inc. The remaining authors have nothing to disclose. References 1. 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EndocrinologyOxford University Press

Published: May 1, 2019

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