Yihong Wang

Glucagon-like peptide-1 can reverse the age-related decline in glucose tolerance in rats.

Wistar rats develop glucose intolerance and have a diminished insulin response to glucose with age. The aim of this study was to investigate if these changes were reversible with glucagon-like peptide-1 (GLP-1), a peptide that we have previously shown could increase insulin mRNA and total insulin content in insulinoma cells. We infused 1.5 pmol/ kg-1.min-1 GLP-1 subcutaneously using ALZET microosmotic pumps into 22-mo-old Wistar rats for 48 h. Rat infused with either GLP-1 or saline were then subjected to an intraperitoneal glucose (1 g/kg body weight) tolerance test, 2 h after removing the pump. 15 min after the intraperitoneal glucose, GLP-1-treated animals had lower plasma glucose levels (9.04+/-0.92 mmol/liter, P < 0.01) than saline-treated animals (11.61+/-0.23 mmol/liter). At 30 min the plasma glucose was still lower in the GLP-1-treated animals (8.61+/-0.39 mmol/liter, P < 0.05) than saline-treated animals (10.36+/-0.43 mmol/liter). This decrease in glucose levels was reflected in the higher insulin levels attained in the GLP-1-treated animals (936+/-163 pmol/liter vs. 395+/-51 pmol/liter, GLP-1 vs. saline, respectively, P < 0.01), detected 15 min after glucose injection. GLP-1 treatment also increased pancreatic insulin, GLUT2, and glucokinase mRNA in the old rats. The effects of GLP-1 were abolished by simultaneous infusion of exendin [9-39], a specific antagonist of GLP-1. GLP-1 is therefore able to reverse some of the known defects that arise in the beta cell of the pancreas of Wistar rats, not only by increasing insulin secretion but also by inducing significant changes at the molecular level.

GLP-1 action in L6 myotubes is via a receptor different from the pancreatic GLP-1 receptor

The incretin hormone glucagon-like peptide-1 (GLP-1)-(7-36) amide is best known for its antidiabetogenic actions mediated via a GLP-1 receptor present on pancreatic endocrine cells. To investigate the molecular mechanisms of GLP-1 action in muscle, we used cultured L6 myotubes. In L6 myotubes, GLP-1 enhanced insulin-stimulated glycogen synthesis by 140% while stimulating CO2 production and lactate formation by 150%. In the presence of IBMX, GLP-1 diminished cAMP levels to 83% of IBMX alone. In L6 myotubes transfected with pancreatic GLP-1 receptor, GLP-1 increased cAMP levels and inhibited glycogen synthesis by 60%. An antagonist of pancreatic GLP-1 receptor, exendin-4-(9-39), inhibited GLP-1-mediated glycogen synthesis in GLP-1 receptor-transfected L6 myotubes. However, in parental L6 myotubes, exendin-4-(9-39) and GLP-1-(1-36) amide, an inactive peptide on pancreatic GLP-1 receptor, displaced 125I-labeled GLP-1 binding and stimulated glycogen synthesis by 186 and 130%, respectively. These results suggest that the insulinomimetic effects of GLP-1 in L6 cells are likely to be mediated by a receptor that is different from the GLP-1 receptor found in the pancreas.The incretin hormone glucagon-like peptide-1 (GLP-1)-(7-36) amide is best known for its antidiabetogenic actions mediated via a GLP-1 receptor present on pancreatic endocrine cells. To investigate the molecular mechanisms of GLP-1 action in muscle, we used cultured L6 myotubes. In L6 myotubes, GLP-1 enhanced insulin-stimulated glycogen synthesis by 140% while stimulating CO2 production and lactate formation by 150%. In the presence of IBMX, GLP-1 diminished cAMP levels to 83% of IBMX alone. In L6 myotubes transfected with pancreatic GLP-1 receptor, GLP-1 increased cAMP levels and inhibited glycogen synthesis by 60%. An antagonist of pancreatic GLP-1 receptor, exendin-4-(9-39), inhibited GLP-1-mediated glycogen synthesis in GLP-1 receptor-transfected L6 myotubes. However, in parental L6 myotubes, exendin-4-(9-39) and GLP-1-(1-36) amide, an inactive peptide on pancreatic GLP-1 receptor, displaced125I-labeled GLP-1 binding and stimulated glycogen synthesis by 186 and 130%, respectively. These results suggest that the insulinomimetic effects of GLP-1 in L6 cells are likely to be mediated by a receptor that is different from the GLP-1 receptor found in the pancreas.

Novel signal transduction and peptide specificity of glucagon-like peptide receptor in 3T3-L1 adipocytes

Glucagon‐like peptide‐1 (7–36) amide (GLP‐1), in addition to its well known effect of enhancing glucose‐mediated insulin release, has been shown to have insulinomimetic effects and to enhance insulin‐mediated glucose uptake and lipid synthesis in 3T3‐L1 adipocytes. To elucidate the mechanisms of GLP‐1 action in these cells, we studied the signal transduction and peptide specificity of the GLP‐1 response. In 3T3‐L1 adipocytes, GLP‐1 caused a decrease in intracellular cAMP levels which is the opposite to the response observed in pancreatic beta cells in response to the same peptide. In 3T3‐L1 adipocytes, free intracellular calcium was not modified by GLP‐1. Peptide specificity was examined to help determine if a different GLP receptor isoform was expressed in 3T3‐L1 adipocytes vs. beta cells. Peptides with partial homology to GLP‐1 such as GLP‐2, GLP‐1 (1–36), and glucagon all lowered cAMP levels in 3T3‐L1 adipocytes. In addition, an antagonist of pancreatic GLP‐1 receptor, exendin‐4 (9–39), acted as an agonist to decrease cAMP levels in 3T3‐L1 adipocytes as did exendin‐4 (1–39), a known agonist for the pancreatic GLP‐1 receptor. Binding studies using 125I‐GLP‐1 also suggest that pancreatic GLP‐1 receptor isoform is not responsible for the effect of GLP‐1 and related peptides in 3T3‐L1 adipocytes. Based on these results, we propose that the major form of the GLP receptor in 3T3‐L1 adipocytes is functionally different from the pancreatic GLP‐1 receptor. J. Cell. Physiol. 172:275–283, 1997. Published 1997 Wiley‐Liss, Inc. This article was prepared by a group of United States government employees and non‐United States government employees, and as such is subject to 17 U.S.C. Sec. 105.

GIP REGULATES GLUCOSE TRANSPORTERS, HEXOKINASES, AND GLUCOSE-INDUCED INSULIN SECRETION IN RIN 1046-38 CELLS

Acute studies of glucose-dependent insulinotropic peptide (GIP) have shown that GIP can synergize with glucose in stimulating insulin secretion both in vivo and in vitro. Here we studied the effects of extended exposure of RIN 1046-38 cells, an insulin-secreting cell line, to GIP and the mechanisms by which GIP synergizes with glucose in stimulating insulin secretion. Incubation of the cells with 100 nM GIP in the presence of glucose for 12 h significantly increased insulin release (287 +/- 31.7 vs. 102 +/- 9.7 ng/mg protein; n = 3), intracellular insulin content (12.8 +/- 0.83 vs. 8.2 +/- 0.52 ng/mg protein; n = 3), and insulin mRNA (approximately 2.7-fold; 24 h incubation) when compared to cells cultured with glucose alone. The insulinotropic effects of GIP on RIN 1046-38 cells were accompanied by an up-regulation of GLUT-1 and hexokinase I mRNA (1.75-fold) compared to non-GIP-treated cells; mRNA levels of GLUT-2 and glucokinase were unchanged by GIP, in the presence or absence of glucose. Our study suggests that the mechanism by which extended exposure of RIN 1046-38 cells to GIP increases glucose-stimulated insulin secretion includes up-regulation of glucose sensing elements.

Overexpression of glucagon-like peptide-1 receptor in an insulin-secreting cell line enhances glucose responsiveness.

Glucagon-like peptide-1 (GLP-1), secreted from intestine in response to food intake, enhances insulin secretion from pancreatic beta-cells. In this study, we evaluated the effects of stably transfecting the GLP-1 receptor into an insulinoma cell line, RIN 1046-38, on basal and glucose-mediated insulin secretion and on second messenger pathways involved in insulin secretion. The GLP-1 receptor transfected cells had similar insulin mRNA levels but higher insulin content compared with parental cells. In GLP-1 receptor transfected cells, glucose (0.5 mM)-mediated insulin release was increased compared with parental cells (4.52 +/- 0.79 pmol insulin/l per mg protein x h vs. 2.21 +/- 0.36 pmol insulin/l per mg protein x h; mean +/- S.E., n = 6, P = 0.015, in transfected vs. parental cells, respectively). By hemolytic plaque assay measuring single cell insulin secretion, we observed that in the GLP-1 receptor transfected cells versus parental cells the increased insulin secretion was due to the presence of more glucose-responsive cells as well as more insulin released in response to glucose per cell. Resting intracellular cAMP was higher in the GLP-1 transfected cells (35.96 +/- 3.88 vs. 18.6 +/- 2.01 nmol/l per mg protein x h; mean +/- S.E., n = 4, P = 0.039, in transfected vs. parental cells, respectively). In response to GLP-1, both GLP-1 receptor transfected cells and parental cells showed increased cAMP levels independent of glucose. Resting intracellular calcium was the same in both parental and GLP-1 receptor transfected cells. However, more cells were responsive to glucose in the GLP-1 receptor transfected cells and the calcium transients attained in the presence of glucose developed at a faster rate and reached a higher amplitude than in parental cells. We conclude that having an excess of GLP-1 receptors renders beta-cells more sensitive to glucose.

Insulin release and insulin mRNA levels in rat islets of Langerhans cultured on extracellular matrix.

Primary culture of rat islets of Langerhans lose glucose responsiveness and eventually die when cultured for a long period of time. In this study we evaluated the effect of matrigel, a basement membrane extract, on (i) islet cell survival, (ii) cell responsiveness following a glucose challenge, and (iii) mRNA levels for insulin, glu-cagon, and somatostatin. Pancreatic islets were isolated by collagenase digestion and plated in culture dishes either coated or not with a matrigel layer. Using the reverse hemolytic plaque assay, we determined the total number of insulin-secreting cells and the amount of insulin secreted by individual beta cells. After 1 h of exposure to 5 mM glucose, β cells from 6-month-old rat islets cultured for 6 weeks on matrigel showed an equal number of insulin-secreting cells compared to freshly isolated islets cultured for only 3 days in the absence of matrigel (39.5 ± 2.5 vs. 37.1 ± 9.6%). Furthermore, the release of insulin by cells cultured on matrigel for 6 weeks increased in a glucose-dependent manner (p < 0.001) and showed an ED50 of 7 mM. However, the amount of insulin released per single β cell was reduced by 40–60% (p < 0.02) compared to that released from isolated β cells derived from a 3-day culture of islets. Finally, there was a 35–55% increase (p < 0.05) in the levels of insulin, glucagon, and somatostatin mRNAs in cells cultured for 6 weeks on matrigel. These data suggest a trophic effect of matrigel on the maintenance of normal β-cell activity and function and may lead the way to the development of a new model for the study of pancreatic islets in long-term culture.

GLUCAGON-LIKE PEPTIDE-1 DOES NOT MEDIATE AMYLASE RELEASE FROM AR42J CELLS

In this study, AR42J pancreatic acinar cells were used to investigate if glucagon‐like peptide‐1 (GLP‐1) or glucagon might influence amylase release and acinar cell function. We first confirmed the presence of GLP‐1 receptors on AR42J cells by reverse trasncriptase‐polymerase chain reaction (RT‐PCR), Western blotting, and partial sequencing analysis. While cholecystokinin (CCK) increased amylase release from AR42J cells, GLP‐1, alone or in the presence of CCK, had no effect on amylase release but both CCK and GLP‐1 increased intracellular calcium. Similar to GLP‐1, glucagon increased both cyclic adenosine monophosphate (cAMP) and intracellular calcium in AR42J cells but it actually decreased CCK‐mediated amylase release (n = 20, P < 0.01). CCK stimulation resulted in an increase in tyrosine phosphorylation of several cellular proteins, unlike GLP‐1 treatment, where no such increased phosphorylation was seen. Instead, GLP‐1 decreased such protein phosphorylations. Genestein blocked CCK‐induced phosphorylation events and amylase secretion while vanadate increased amylase secretion. These results provide evidence that tyrosine phosphorylation is necessary for amylase release and that signaling through GLP‐1 receptors does not mediate amylase release in AR42J cells. J. Cell. Physiol. 181:470–478, 1999. Published 1999 Wiley‐Liss, Inc.

Modulation of CCAAT/Enhancer-Binding Protein-α Gene Expression by Metabolic Signals in Rodent Adipocytes

The transcription factor CCAAT/enhancer-binding protein-α (C/EBPα) is a positive modulator of transcription for several adipocyte-specific genes that play a role in energy metabolism. However, there is little information available regarding the regulation of its expression by metabolic signals. Exposure to insulin for 5–24 h attenuated C/EBPα expression when 3T3-L1 adipocytes were incubated in 24 mm glucose, but not in 5.7 mm glucose. Nuclear run-on transcription assays indicated a transcriptional repression of C/EBPα gene, but not that of C/EBPβ. Glucosamine, a product of the hexosamine pathway, in the presence of low glucose mimicked high glucose’s ability to reduce C/EBPα messenger RNA expression in insulin-treated cells. Similar results were obtained with xylitol, an activator of the pentose phosphate pathway. There was no correlation between the accumulation of hexosamine pathway metabolites (e.g. UDP-N-acetylhexosamines) and/or changes in intracellular protein glycosylation with the ability of high ...