AJP - Gastrointestinal and Liver Physiology

Mayer, Fahima; Gunawan, Amanda L.; Tso, Patrick; Aponte, Gregory W.

doi: 10.1152/ajpgi.00189.2019pmid: 32421358

Glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are released from enteroendocrine cells (EEC) in response to nutrient ingestion and lowers blood glucose levels by stimulation of insulin secretion, and thus are defined as incretins. GLP-1 receptor (GLP-1R) expression has been identified on enteric neurons that include intrinsic afferent neurons, extrinsic spinal and vagal sensory afferents, but has not been shown to have an incretin effect through these nerves. GLP-1 and GIP enter the mesenteric lymphatic fluid (MLF) after a meal via the interstitial fluid (IF) from local tissue secretion and/or blood capillaries. We tested if MLF could induce diet dependent intransient increases in intracellular calcium ( [Ca2+]i) in cultured sensory neurons. Postprandial rat MLF, collected from the superior mesenteric lymphatic duct, induced a significant 2 - fold higher intransient increase in [Ca2+]iin primary-cultured sensory neurons than MLF from fasted rats. Inhibition of transient receptor potential vanilloid 1 (TRPV1) and TRPV1 and ankyrin 1 cation channels (TRPA1) with ruthenium red eliminated the difference. Substance P (SP) (a peptide that stimulates insulin secretion) sensor cells co-cultured with sensory neurons showed both GLP-1R agonist exendin-4 (Ex-4) and GIP induced transient increases in [Ca2+]idirectly coupled to SP secretion in the sensory nerves. Ex-4-induced release of SP required expression of either TRPA1 or TRPV1. These data identify unrecognized actions of GLP-1 and GIP as incretins by acting as neurolymphocrines, and suggest a mechanism for sensory nerves to respond to the postprandial state through MLF.

Fujita-Yoshigaki, Junko; Yokoyama, Megumi; Katsumata-Kato, Osamu

doi: 10.1152/ajpgi.00103.2020pmid: 32538138

The mechanism for segregation of cargo proteins into the regulated and constitutive secretory pathways in exocrine cells remains to be elucidated. We examined the transport of HaloTag proteins fused with full-length cystatin D (fCst5-Halo) or only its signal peptide (ssCst5-Halo) in parotid acinar cells. Although both fusion proteins were observed to be colocalized with amylase in the secretory granules, the coefficients for overlapping and correlation of fCst5-Halo with amylase were higher than those of ssCst5-Halo. The secretion of both the proteins was enhanced by the addition of the β-adrenergic receptor agonist isoproterenol, as well as endogenous amylase. In contrast, unstimulated secretion of ssCst5-Halo without isoproterenol was significantly higher than that of fCst5-Halo and amylase. Simulation analysis using a mathematical model revealed that a large proportion of ssCst5-Halo was secreted through the constitutive pathway, while fCst5-Halo was transported into the secretory granules more efficiently. Precipitation of fCst5-Halo from cell lysates was increased at a low pH, which may mimic the milieu of the trans-Golgi network. These data suggest that the addition of a full-length sequence of cystatin D facilitates efficient selective transport into the regulated pathway by aggregation at low pH in the trans-Golgi network.

Riehl, Terrence E.; Alvarado, David; Ee, Xueping; Ciorba, Matthew A.; Stenson, William F.

doi: 10.1152/ajpgi.00242.2019pmid: 32538139

Background and Aims: Hyaluronic acid (HA), a glycosaminoglycan in the extracellular matrix, binds to CD44 and TLR4. We previously demonstrated that TLR4 predominately mediated HA stimulation of Lgr5+ stem cell proliferation, crypt fission and intestinal growth in postnatal mice. Here we address which cell type expresses the relevant TLR4 in driving intestinal growth and the downstream events from TLR4 activation. Methods: Studies were done in 14-day-old mice: wild type (WT), mice deficient in either cyclooxygenase2 (COX2), myeloid cell TLR4 or epithelial cell EGFR. Biologic end points included crypt fission and Lgr5 cell proliferation. Results: In WT mice treatment with NS398 ( COX2 inhibitor), clodronate (macrophage depleting agent), or tyrphostin (EGFR inhibitor) resulted in 30% reductions in crypt fission and Lgr5+ stem cell proliferation. Mice deficient in COX2 or myeloid TLR4 or epithelial cell EGFR all had 30% reductions in crypt fission and Lgr5+ stem cell proliferation . Dimethyl PGE2, a stable PGE2 analog, increased crypt fission and Lgr5+ stem cell proliferation. Administration of dimethyl PGE2 reversed the effects of NS398, clodronate, COX2 deficiency, and myeloid TLR4 deficiency, but had no effect on mice treated with tyrphostin or mice deficient in epithelial cell EGFR. Conclusions: In postnatal mice about 30% of intestinal growth as manifested by crypt fission and Lgr5+ stem cell proliferation is driven by a novel pathway: Extracellular HA binds TLR4 on pericryptal macrophages inducing the production of PGE2 through COX2. PGE2 transactivates EGFR in Lgr5+ epithelial stem cells resulting in Lgr5+ stem cell proliferation and crypt fission.

Zhang, Xin; Xu, Yuyin; Bai, Qianming; Li, Xiaobo; Han, Jing; Hou, Yingyong; Ji, Yuan; Zhang, Zhigang

doi: 10.1152/ajpgi.00380.2019pmid: 32475129

Hydroxysteroid sulfotransferase 2B1b (SULT2B1b) plays a critical role in hepatic energy homeostasis. Liver X receptors (LXRs) are implicated in multiple physiological functions, including the inhibition of hepatocyte proliferation and regulation of fatty acid and cholesterol metabolism. We have previously reported that SULT2B1b promotes hepatocyte proliferation by inactivating LXR signaling in vivo and in vitro, leading to our hypothesis that SULT2B1b promotes fatty liver regeneration. In the present study, female C57BL/6 and S129 mice were fed a high-fat diet for 8 weeks to establish a nonalcoholic fatty liver disease (NAFLD) mouse model. 70% partial hepatectomy (PH) was performed to induce liver regeneration. Our experiments revealed that the SULT2B1b overexpression significantly promotes the regeneration of hepatocytes in NAFLD C57BL/6 mice after PH, increasing liver regrowth by 11% within 1 day, and then by 21%, 33%, and 24% by 2, 3, and 5 days post-PH, respectively. Compared with the wild-type NAFLD S129 mice, SULT2B1 deletion NAFLD S129 mice presented reduced hepatocyte regeneration at postoperative day 2, as verified by decreased liver regrowth (37.4% vs. 46.1%, p <0.05) and the results of immunohistochemical staining, quantitative real-time polymerase chain reaction, and Western blotting. Moreover, LXRα signaling and SULT2B1b expression are highly correlated in the regeneration of NAFLD mouse liver; SULT2B1b overexpression suppresses LXRα signaling, while the LXRα-signaling agonist T0901317 blocks SULT2B1b-induced hepatocyte regeneration in NAFLD mouse liver. Thus, the upregulation of SULT2B1b may promote hepatocyte regeneration via the suppression of LXRα activation in NAFLD mice, providing a potential strategy for improving hepatic-steatosis-related liver regeneration disorders.

Chin, LiKang; Theise, Neil D.; Loneker, Abigail E.; Janmey, Paul A.; Wells, Rebecca G.

doi: 10.1152/ajpgi.00098.2020pmid: 32463334

Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer death in the world. Although most cases occur in stiff, cirrhotic livers, and stiffness is a significant risk factor, HCC can also arise in non-cirrhotic livers in the setting of non-alcoholic fatty liver disease (NAFLD). We hypothesized that lipid droplets in NAFLD might apply mechanical forces to the nucleus, functioning as mechanical stressors akin to stiffness. We investigated the effect of lipid droplets on cellular mechanosensing and found that primary human hepatocytes loaded with the fatty acids oleate and linoleate exhibited decreased stiffness-induced cell spreading and disrupted focal adhesions and stress fibers. The presence of large lipid droplets in hepatocytes resulted in increased nuclear localization of the mechano-sensor Yes-associated protein (YAP). In cirrhotic livers from patients with NAFLD, hepatocytes filled with large lipid droplets showed significantly higher nuclear localization of YAP as compared to cells with small lipid droplets. This work suggests that lipid droplets induce a mechanical signal that disrupts the ability of the hepatocyte to sense its underlying matrix stiffness, and that the presence of lipid droplets can induce intracellular mechanical stresses.

Liu, Cambrian Y.; Polk, D. Brent

doi: 10.1152/ajpgi.00075.2020pmid: 32421359

The development of modern methods to induce optical transparency ("clearing") in biological tissues has enabled the 3d reconstruction of intact organs at cellular resolution. New capabilities in visualization of rare cellular events, long-range interactions, and irregular structures will facilitate novel studies in the alimentary tract and gastrointestinal systems. The tubular geometry of the alimentary tract facilitates large-scale cellular reconstruction of cleared tissue without specialized microscopy setups. However, with the rapid pace of development of clearing agents and current relative paucity of research groups in the gastrointestinal field using these techniques, it can be daunting to incorporate tissue clearing into experimental workflows. Here, we give some advice and describe our own experience bringing tissue clearing and whole-mount reconstruction into our lab's investigations. We present a brief overview of the chemical concepts that underpin tissue clearing, what sorts of questions whole-mount imaging can answer, how to choose a clearing agent, an example of how to clear and image alimentary tissue, and what to do after obtaining the image. This short review will encourage other gastrointestinal researchers to consider how utilizing tissue clearing and creating 3d "maps" of tissue might deepen the impact of their studies.

Thorne, Adam M.; Ubbink, Rinse; Brüggenwirth, Isabel M. A.; Nijsten, Maarten W.; Porte, Robert J.; de Meijer, Vincent E.

doi: 10.1152/ajpgi.00101.2020pmid: 32508156

Liver transplantation is the standard treatment for end-stage liver disease. However, due to the ongoing disparity between supply and demand for optimal donor organs, there is increasing usage of extended criteria donor organs, including steatotic liver grafts. To mitigate the increased risks associated with extended criteria donor livers, ex-situ oxygenated machine perfusion (MP) has received increasing attention in recent years as an emerging platform for dynamic preservation, reconditioning and viability assessment to increase organ utilisation. MP can be applied at different temperatures. During hypothermic MP (4-12°C), liver metabolism is reduced, while oxygenation restores the intracellular levels of adenosine triphosphate. The liver is quickly 'recharged' to support metabolism when at normothermia (35-37°C), and to ameliorate the detrimental effects of ischemia/reperfusion injury during transplantation. During normothermia, MP can be applied to assess hepatocellular and cholangiocellular viability. MP at hyperthermic (>38°C) temperatures (HyMP), however, remains relatively understudied. The liver is an important component in the regulation of core body temperature and, as such, displays significant physiological and metabolic changes in response to different temperatures. Hyperthermia may promote vasodilation, increase aerobic metabolism and induce production of protective molecules such as heat shock proteins. Therefore, HyMP could provide an attractive reconditioning strategy for steatotic livers. In this review, we describe current literature on the physiological and metabolic effects of the liver at hyperthermia for human, rodents and pigs, and provide a rationale for using therapeutic HyMP during isolated liver machine perfusion to recondition extended criteria donor livers, including steatotic livers, prior to transplantation.

Courtney, Cathleen M.; Shyr, Zeenat A.; Yan, Zihan; Onufer, Emily Jean; Steinberger, Allie E.; Tecos, Maria E.; Barron, Lauren K.; Guo, Jun; Remedi, Maria S.; Warner, Brad W.

doi: 10.1152/ajpgi.00282.2019pmid: 32463335

Background - After massive small bowel resection (SBR), mice exhibit hepatic steatosis, impaired glucose metabolism without insulin resistance, and increased pancreatic islet area. We sought to determine the consequences of SBR on pancreatic b-cell morphology, proliferation, and expression of a key regulatory hormone, glucagon like peptide-1 (GLP-1). Methods - C57Bl/6 mice underwent 50% SBR or sham operation. At 10 weeks, pancreatic insulin content and secretion was measured by ELISA. Immunohistochemistry was performed to determine alterations in pancreatic a- and b-cells. Western blot analysis was used to measure GLP-1R expression and immunoassay was used to measure plasma insulin and GLP-1. Results - After SBR, we identified pancreatic islet hypertrophy and impaired glucose tolerance. The proportion of a- and b-cells was not grossly altered. Whole pancreas and pancreatic islet insulin content was not significantly different; however, SBR pancreas demonstrated decreased insulin secretion in both static incubation and islet perfusion experiments. The expression of pancreatic GLP-1R was decreased approximately 2-fold after SBR and serum GLP-1 levels were decreased. Administration of a GLP-1 agonist mitigated the metabolic derangements. Conclusion Following massive SBR, there is significant hypertrophy of pancreatic islet cells with morphologically intact a- and b-cells. Significantly reduced pancreatic insulin release in both static and dynamic conditions demonstrate perturbed intrinsic b-cell function. GLP-1 is a key mediator of this amplification pathway. Decreased expression of serum GLP-1 and pancreatic GLP-1R in face of no change in insulin content offer a novel pathway for enteropancreatic glucose regulation following SBR.

Liu, Julia Yuen Hang; Du, Peng; Rudd, John Anthony

doi: 10.1152/ajpgi.00003.2020pmid: 32475128

The effect of acetylcholine (ACh) on pacemaking and spontaneous contractions in the gastrointestinal tract is not well-characterized. The current study aims to profile the effect of several muscarinic and nicotinic receptor agonists and antagonists on pacemaker potentials in the ICR mouse ileum. Pacemaker potentials of whole-thickness mouse ileal segments were recorded extracellularly using a 60-channel microelectrode array (MEA) platform. A spatiotemporal analysis integrated the frequency, amplitude and velocity measurements of pacemaker currents. Comparative data were obtained by recording spontaneous smooth muscle tone in a conventional organ bath. On the MEA, ACh (0.3-300 μM) and bethanechol (0.3-300 μM) reduced ileal pacemaker potentials, significantly. The inhibitory effect of ACh was mimicked by donepezil (300 μM), but not nicotine (0.3-7 mM). Atropine (300 μM), but not hexamethonium (300 μM), reversed the inhibitory actions of ACh and bethanechol and revealed excitatory properties manifested as increases in pacemaker frequency. A spatial analysis also revealed that atropine, but not hexamethonium, reversed the ACh-induced distortion of pacemaker propagation activity. Atropine (0.001-3 mM) and hexamethonium (0.3-7 mM) alone were inactive. In the organ bath, ACh (300 nM) and bethanechol (30 μM) induced ileal tonic contractions, while inhibiting basal spontaneous contractions at 300 μM. Atropine (1 μM), but not hexamethonium (1-300 μM), reversed both the tonic contractions and the inhibition of the spontaneous contractions of ACh and bethanechol, and revealed an excitatory effect manifested as an increasing in frequency of contractions. Muscarinic, but not nicotinic, receptors appear to mediate the inhibitory actions of ACh on mouse ileal pacemaker potentials.

Lee, Dustin M.; Ecton, Kayl E.; Trikha, S. Raj J.; Wrigley, Scott D.; Thomas, Keely N.; Battson, Micah L.; Wei, Yuren; Johnson, Sarah A.; Weir, Tiffany L.; Gentile, Christopher L.

doi: 10.1152/ajpgi.00375.2019pmid: 32421360

Emerging evidence suggests that intestinal microbes regulate host physiology and cardiometabolic health, although the mechanism(s) by which they do so are unclear. Indoles are a group of compounds produced from bacterial metabolism of the amino acid tryptophan. In light of recent data suggesting broad physiological effects of indoles on host physiology, we examined whether indole-3-propionic acid (IPA) would protect mice from the cardiometabolic consequences of a western diet. Male C57BL/6J mice were fed either a standard diet (SD) or western diet (WD) for 5mo and received normal autoclaved drinking water or water supplemented with IPA (0.1mg/ml) (SD+IPA and WD+IPA). WD-feeding led to increased liver triglycerides and makers of inflammation with no effect of IPA. At 5mo, arterial stiffness was significantly higher in WD and WD+IPA compared to SD (WD: 485.7±6.7 & WD+IPA: 492.8±8.6 vs SD: 436.9±7.0 cm/s, p<0.05), but not SD+IPA (SD+IPA: 468.1±6.6 vs WD groups, p>0.05). Supplementation with IPA in the SD+IPA group significantly increased glucose AUC compared to SD mice (SD+IPA: 1763.3±92.0 vs SD: 1397.6±64.0, p<0.05) and no significant differences were observed among either WD or WD+IPA groups (WD: 1623.5±77.3 & WD+IPA: 1658.4±88.4, p>0.05). Gut microbiota changes were driven by WD-feeding, whereas IPA supplementation drove differences in SD-fed mice. In conclusion, supplementation with IPA did not improve cardiometabolic outcomes in WD-fed mice and may have worsened some parameters in SD-fed mice, suggesting that IPA is not a critical signal mediating WD-induced cardiometabolic dysfunction downstream of the gut microbiota.