Chahrzad Montrose-Rafizadeh

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.

Benzopyrans are selective estrogen receptor Beta agonists with novel activity in models of benign prostatic hyperplasia.

Benzopyran selective estrogen receptor beta agonist-1 (SERBA-1) shows potent, selective binding and agonist function in estrogen receptor beta (ERbeta) in vitro assays. X-ray crystal structures of SERBA-1 in ERalpha and beta help explain observed beta-selectivity of this ligand. SERBA-1 in vivo demonstrates involution of the ventral prostate in CD-1 mice (ERbeta effect), while having no effect on gonadal hormone levels (ERalpha effect) at 10x the efficacious dose, consistent with in vitro properties of this molecule.

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.

Novel 3-aryl indoles as progesterone receptor antagonists for uterine fibroids.

We report the synthesis and characterization of novel 3-aryl indoles as potent and efficacious progesterone receptor (PR) antagonists with potential for the treatment of uterine fibroids. These compounds demonstrated excellent selectivity over other steroid nuclear hormone receptors such as the mineralocorticoid receptor (MR). They were prepared from 2-bromo-6-nitro indole in four to six steps using a Suzuki cross-coupling as the key step. Compound 8f was orally active in the complement 3 model of progesterone antagonism in the rat uterus and demonstrated partial antagonism in the McPhail model of progesterone activity.

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.

A Synthetic Peptide Derived from a COOH-terminal Domain of the Insulin Receptor Specifically Enhances Insulin Receptor Signaling

The role of the insulin receptor COOH-terminal domain in the regulation of insulin signal transduction was explored with a variety of synthetic peptides. One of the peptides, termed peptide HC, whose structure corresponds to residues 1293-1307 of the insulin proreceptor sequence, enhanced insulin-stimulated autophosphorylation of the insulin receptor in cell-free systems and in semipermeabilized Chinese hamster ovary (CHO) cells that had been transfected with an expression plasmid encoding the human insulin receptor (CHO/HIRc) at concentrations where there was no detectable effect on basal autophosphorylation levels or on receptor dephosphorylation. A lipophilic analogue of peptide HC, stearyl peptide HC, added to intact CHO/HIRc cells enhanced significantly insulin-stimulated insulin receptor autophosphorylation while having no effect on ligand-stimulated receptor phosphorylation in CHO cells overexpressing either the IGF-1 receptor or epidermal growth factor receptor. Addition of stearyl peptide HC to CHO/HIRc cells resulted in a 2.4 ± 0.3-fold increase in the amount of insulin-stimulated phosphatidylinositol 3-kinase detected in anti-IRS-1 immunoprecipitates and a 2.1 ± 0.6-fold increase in the levels of tyrosine phosphorylation of mitogen-activated protein kinase in response to insulin. Finally, a derivative of peptide HC coupled to a biotin moiety was prepared and showed to bind with the β-subunit of the wild-type insulin receptor and a truncated receptor that lacks 43 amino acids from its carboxyl terminus. However, there was little binding, if any, of the peptide with the IGF-1 receptors or the epidermal growth factor receptors. Taken together, our data demonstrate that a pentadecapeptide related to the carboxyl terminus of the insulin receptor binds to the insulin receptor β-subunit and that this interaction may contribute to the increased receptors intrinsic activity and signal transduction.

A nonsecosteroidal vitamin D receptor ligand with improved therapeutic window of bone efficacy over hypercalcemia

Vitamin D3 analogues were shown to be beneficial for osteoporosis and other indications, but their narrow therapeutic window between efficacy and hypercalcemia has limited their clinical utility. A nonsecosteroidal, tissue‐selective, orally bioavailable, vitamin D receptor (VDR) ligand was ascertained to be efficacious in bone while having modest calcemic effects in vivo. This compound (VDRM2) potently induced Retinoid X Receptor alpha (RXR)‐VDR heterodimerization (EC50 = 7.1 ± 1.6 nM) and induced osteocalcin promoter activity (EC50 = 1.9 ± 1.6 nM). VDRM2 was less potent in inducing Ca2+ channel transient receptor potential cation channel, subfamily V, member 6 (TRPV6) expression (EC50 = 37 ± 12 nM). VDRM2 then was evaluated in osteopenic ovariectomized (OVX) rats and shown to dose‐dependently restore vertebral bone mineral density (BMD) from OVX to sham levels at 0.08 µg/kg per day. Hypercalcemia was observed at a dose of 4.6 µg/kg per day of VDRM2, suggesting a safety margin of 57 [90% confidence interval (CI) 35–91]. 1α,25‐dihydroxyvitamin D3 [1α,25(OH)2D], ED71, and alfacalcidol restored BMD at 0.030, 0.0055, and 0.046 µg/kg per day, respectively, whereas hypercalcemia was observed at 0.22, 0.027, and 0.23 µg/kg per day, indicating a safety margin of 7.3, 4.9, and 5.0, respectively (90% CIs 4.1–13, 3.2–7.7, and 3.5–6.7, respectively). Histomorphometry showed that VDRM2 increased cortical bone area and stimulated the periosteal bone‐formation rate relative to OVX at doses below the hypercalcemic dose. By contrast, ED71 increased the periosteal bone‐formation rate only above the hypercalcemic dose. VDRM2 suppressed eroded surface on trabecular bone surfaces at normal serum calcium dosage levels, suggesting dual anabolic and antiresorptive activity. In summary, vitamin D analogues were more potent than VDRM2, but VDRM2 had a greater safety margin, suggesting possible therapeutic potential.