Yin Nam Kwok

Glucose-Dependent Insulinotropic Polypeptide Is Expressed in Pancreatic Islet α-Cells and Promotes Insulin Secretion

BACKGROUND & AIMS Glucose-dependent insulinotropic polypeptide (GIP) and the proglucagon product glucagon-like peptide-1 (GLP-1) are gastrointestinal hormones that are released in response to nutrient intake and promote insulin secretion. Interestingly, a subset of enteroendocrine cells express both GIP and GLP-1. We sought to determine whether GIP also might be co-expressed with proglucagon in pancreatic alpha-cells. METHODS We assessed GIP expression via reverse-transcription polymerase chain reaction, in situ hybridization, and immunohistochemistry. We developed a novel bioassay to measure GIP release from isolated islets, compared the biological activities of full-length and truncated GIP, and assessed the impact of immunoneutralization of islet GIP on glucose-stimulated insulin secretion in isolated islets. RESULTS GIP messenger RNA was present in mouse islets; GIP protein localized to islet alpha-cells of mouse, human, and snake pancreas, based on immunohistochemical analyses. However, using a C-terminal GIP antibody, immunoreactivity was detected in islets from prohormone convertase (PC) 2 knockout but not wild-type mice. Bioactive GIP was secreted from mouse and human islets after arginine stimulation. In the perfused mouse pancreas, GIP(1-42) and amidated GIP(1-30) had equipotent insulinotropic actions. Finally, immunoneutralization of GIP secreted by isolated islets decreased glucose-stimulated insulin secretion. CONCLUSIONS GIP is expressed in and secreted from pancreatic islets; in alpha-cells, PC2 processes proGIP to yield a truncated but bioactive form of GIP that differs from the PC1/3-derived form from K-cells. Islet-derived GIP promotes islet glucose competence and also could support islet development and/or survival.

A GIP Receptor Agonist Exhibits β-Cell Anti-Apoptotic Actions in Rat Models of Diabetes Resulting in Improved β-Cell Function and Glycemic Control

Aims The gastrointestinal hormone GIP promotes pancreatic islet function and exerts pro-survival actions on cultured β-cells. However, GIP also promotes lipogenesis, thus potentially restricting its therapeutic use. The current studies evaluated the effects of a truncated GIP analog, D-Ala2-GIP1–30 (D-GIP1–30), on glucose homeostasis and β-cell mass in rat models of diabetes. Materials and Methods The insulinotropic and pro-survival potency of D-GIP1–30 was evaluated in perfused pancreas preparations and cultured INS-1 β-cells, respectively, and receptor selectivity evaluated using wild type and GIP receptor knockout mice. Effects of D-GIP1–30 on β-cell function and glucose homeostasis, in vivo, were determined using Lean Zucker rats, obese Vancouver diabetic fatty rats, streptozotocin treated rats, and obese Zucker diabetic fatty rats, with effects on β-cell mass determined in histological studies of pancreatic tissue. Lipogenic effects of D-GIP1–30 were evaluated on cultured 3T3-L1 adipocytes. Results Acutely, D-GIP1–30 improved glucose tolerance and insulin secretion. Chronic treatment with D-GIP1–30 reduced levels of islet pro-apoptotic proteins in Vancouver diabetic fatty rats and preserved β-cell mass in streptozotocin treated rats and Zucker diabetic fatty rats, resulting in improved insulin responses and glycemic control in each animal model, with no change in body weight. In in vitro studies, D-GIP1–30 exhibited equivalent potency to GIP1–42 on β-cell function and survival, but greatly reduced action on lipoprotein lipase activity in 3T3-L1 adipocytes. Conclusions These findings demonstrate that truncated forms of GIP exhibit potent anti-diabetic actions, without pro-obesity effects, and that the C-terminus contributes to the lipogenic actions of GIP.

Gastrin secretion from human antral G cells in culture

Receptor-dependent and -independent regulation of gastrin secretion from cultured human antral G cells was investigated. Human antral mucosal cell preparations that were enriched for G cells were obtained by sequential incubations with collagenase and ethylenediaminetetraacetic acid, centrifugal elutriation, and short-term culture. After a 2-day incubation period, gastrin- and somatostatin-containing cells accounted for 15% and 5%, respectively, of the total adhered-cell population. Forskolin, A23187, and beta-phorbol 12 myristate 13-acetate stimulated basal gastrin secretion from cultured human G cells in a concentration-dependent fashion. These results indicate that gastrin release could be mediated by elevations in cytosolic cyclic adenosine monophosphate levels, calcium influx, or activation of protein kinase C. A direct stimulatory role for bombesin- and gastrin-releasing peptide was supported by experiments showing concentration-dependent enhancement of gastrin release by bombesin from 0.01 fmol/L to 10 nmol/L. The putative bombesin antagonist [Leu13-psi-CH2NH-Leu14] bombesin augmented basal gastrin levels by itself and produced weak inhibition of bombesin-induced gastrin secretion from human antral G cells. Somatostatin potently suppressed forskolin- and bombesin-mediated gastrin release but did not significantly alter basal gastrin levels. These results suggest that bombesin and somatostatin directly activate and inhibit G-cell function via specific and sensitive receptors. Furthermore, the adenylate cyclase and phosphatidyl inositide second messenger systems seem to be intracellular mediators of gastrin secretion from human antral G cells.

Effect of galanin on endocrine secretions from the isolated perfused rat stomach and pancreas

Nerves containing galanin immunoreactivity have been shown to be present in the stomach and pancreas. The present experiments were designed to test the effect of galanin on the release of gastric somatostatin-like immunoreactivity (SLI) and immunoreactive gastrin (IR-G), pancreatic SLI and immunoreactive insulin (IR-I) from the isolated perfused rat stomach and pancreas respectively. Galanin (2 X 10(-10)-5 X 10(-8) M) inhibited gastric SLI and IR-G release dose dependently. At a concentration of 10(-8) M, galanin also suppressed IR-I release stimulated by gastric inhibitory polypeptide (2 X 10(-10) M) in the presence of 8.9 X 10(-3) M glucose. Pancreatic SLI release under this condition was not altered. Thus the present study suggests that galanin might be involved in the neural regulation of gastric and pancreatic endocrine secretions.

Release of substance P-like immunoreactivity from the vascularly perfused rat stomach

The release of gastric substance P-like immunoreactivity (SP-LI) has been studied in the vascularly perfused rat stomach. In the presence of 20 microM bacitracin and captopril, basal release of SP-LI was sustained throughout the experiments. Gastric SP-LI release was stimulated in a concentration-dependent manner by increasing the concentration of KCl in the perfusion medium. This stimulated release was reduced by the omission of Ca2+, indicating that a Ca2(+)-dependent mechanism was involved. Naloxone did not alter basal SP-LI secretion. [Met5]Enkephalin also had no significant effect on K(+)-stimulated secretion suggesting that enkephalinergic mechanisms are not involved. Gastric SP-LI release was also increased by capsaicin perfusion but this was not sustained. In conclusion, the present results provide the first evidence for the release of SP-LI into the rat stomach vasculature.

Synthesis of Acetylcholine from Acetate in a Sympathetic Ganglion

Abstract: The present experiments tested whether acetate plays a role in the provision of acetyl‐CoA for acetylcholine synthesis in the cats superior cervical ganglion. Labeled acetylcholine was identified in extracts of ganglia that had been perfused for 20 min with Krebs solution containing choline (10−5M) and [3H], [1‐4C], or [2‐14C]acetate (103M); perfusion for 60 min or with [3H]acetate (10−2M) increased the labeling. The acetylcholine synthesized from acetate was available for release by a Ca2+‐dependent mechanism during subsequent periods of preganglionic nerve stimulation. When ganglia were stimulated via their preganglionic nerves or by exposure to 46 mM K+, the labeling of acetylcholine from [3H]acetate was reduced when compared with resting ganglia. The reduced synthesis of acetylcholine from acetate during stimulation was not due to acetate recapture, shunting of acetate into lipid synthesis, or the transmitter release process itself. In ganglia perfused with [2‐14C]glucose, the amount of labeled acetylcholine formed was clearly enhanced during stimulation. An increase in acetylcholine labeling from [3H]acetate was shown during a 15‐min resting period following a 60‐min period of preganglionic nerve stimulation (20 Hz). It is concluded that acetate is not the main physiological acetyl precursor for acetylcholine synthesis in this sympathetic ganglion, and that during preganglionic nerve stimulation there is enhanced delivery of acetyl‐CoA to choline acetyltransferase from a source other than acetate.

Differential processing of pro-glucose-dependent insulinotropic polypeptide in gut

Glucose-dependent insulinotropic polypeptide (GIP) is a hormone released from enteroendocrine K cells in response to meals. Posttranslational processing of the precursor protein pro-GIP at residue 65 by proprotein convertase subtilisin/kexin type 1 (PC1/3) in gut K cells gives rise to the established 42-amino-acid form of GIP (GIP(1-42)). However, the pro-GIP peptide sequence contains a consensus cleavage site for PC2 at residues 52-55 and we identified PC2 immunoreactivity in a subset of K cells, suggesting the potential existence of a COOH-terminal truncated GIP isoform, GIP(1-30). Indeed a subset of mouse and human K cells display GIP immunoreactivity with GIP antibodies directed to the mid portion of the peptide, but not with a COOH-terminal-directed GIP antibody, indicative of the presence of a truncated form of GIP. This population of cells represents approximately 5-15% of the total GIP-immunoreactive cells in mice, depending on the region of intestine, and is virtually absent in mice lacking PC2. Amidated GIP(1-30) and GIP(1-42) have comparable potency at stimulating somatostatin release in the perfused mouse stomach. Therefore, GIP(1-30) represents a naturally occurring, biologically active form of GIP.

Increase of p75 immunoreactivity in rat urinary bladder following inflammation

PREVIOUS studies suggest that NGF may function as a mediator of inflammatory pain. Here, we examined the effect of inflammation on expression of the low affinity neurotrophin receptor p75, using the model of cyclophosphamide-induced cystitis in rats. In control rats, p75-positive thick fibre bundles were scattered in the muscle layer. At 2 and 3 days after injection of cyclophosphamide, numbers of p75-positive fine fibres in the muscle layer were dramatically increased. Electron microscopy revealed that p75 immunoreactivity was localized on the surface of Schwann cells and at the sites where they were apposed to axons. Results show that p75 is up-regulated in inflamed tissues, suggesting that p75 may bind to and take up nerve growth factor (NGF), thus participating in NGF-induced hyperalgesia.

Increased Acetylcholine Synthesis and Release Following Presynaptic Activity in a Sympathetic Ganglion

Abstract: The acetylcholine (ACh) content of sympathetic ganglia increases above its normal level following a period of preganglionic nerve stimulation. In the present experiments, this extra ACh that accumulates following activity was labeled radioactively from [3H]choline and its specific activity was compared with that of ACh subsequently released during preganglionic nerve stimulation. The specific activity of the released ACh was similar to that of the total tissue ACh, suggesting that the extra ACh mixes fully with endogenous stores. The present experiments also show that transmitter release during neuronal stimulation is necessary for the poststimulation increase in transmitter store. However, the increase was not evident when transmitter release was induced by K+. It is concluded that both transmitter release and impulse invasion of the nerve terminals are necessary for the adaptive phenomenon to manifest itself. The role of choline delivery and choline acetyltransferase activity in generating the poststimulation increase in transmitter store was tested. When choline transport activity measured as choline analogue (homocholine) accumulation increased, ACh synthesis was increased and when transport activity was not increased, neither was ACh synthesis. There was no poststimulation increase in measured choline acetyltransferase activity.

Muscarinic Regulation of Somatostatin Release from Primary Cultures of Human Antral Epithelial Cells

A newly developed, primary culture of human antral epithelial cells has been utilized to examine the effect of parasympathomimetics on somatostatin release. The cholinergic agonists, carbachol and methacholine, stimulated somatostatin secretion in a concentration-dependent manner. Maximal release in response to carbachol was observed at 0.1 mmol/l. Methacholine was 10 times more potent with a significant release being observed at 1 mumol/l, maximal secretion was observed at 10 mumol/l. Somatostatin release, stimulated by the mixed nicotinic and muscarinic agonist, carbachol, was attenuated by the addition of atropine at 0.1 mumol/l but was unaffected by the same concentration of pirenzepine. Methacholine-stimulated release was attenuated by addition of 0.1 mumol/l atropine and unaffected by the same concentration of pirenzepine. The response to methacholine was reversed by the addition of 0.1 mumol/l 4-diphenylacetoxy-n-methylpiperidine methiodide (4-DAMP) and attenuated by 1 nmol/l 4-DAMP indicating that the effect was mediated by an M3 receptor. In conclusion, human antral D cells are stimulated by parasympathomimetics acting at an M3 receptor.