Pax6 and Cdx2/3 form a functional complex on the rat glucagon gene promoter G1-element.
(17/3774)
Alpha-cell specific transcription of the glucagon gene is mainly conferred by the glucagon promoter G1-element, while additional elements G2, G3, and G4 have broad islet cell specificity. Transcription of the glucagon gene has been shown to be stimulated by Pax6 through binding to the glucagon gene promoter G3-element. In this report, we show that Pax6 additionally binds the glucagon gene promoter G1-element and forms a transcriptionally active complex with another homeodomain protein, Cdx2/3. Two distinct mutations in the G1-element, that both reduce promoter activity by 85-90%, is shown to eliminate binding of either Pax6 or Cdx2/3. Additionally, Pax6 enhanced Cdx2/3 mediated activation of a glucagon reporter in heterologous cells. We discuss how Pax6 may contribute to cell-type specific transcription in the pancreatic islets by complex formation with different transcription factors. (+info)
Decreases in cAMP phosphodiesterase activity in hepatocytes cultured with herbimycin A due to cellular microtubule polymerization related to inhibition of tyrosine phosphorylation of alpha-tubulin.
(18/3774)
The increase in cellular cAMP concentration during 10-min incubation of rat hepatocytes with glucagon or forskolin was enhanced markedly when the hepatocytes had been cultured for several hours with herbimycin A. This effect of herbimycin was accompanied by inhibition of tyrosine-phosphorylation of cellular proteins including alpha-tubulin, antagonized by coaddition of Na3VO4 plus H2O2, which also antagonized the herbimycin-induced tyrosine phosphorylation, and overcome by the addition to the 10-min incubation medium of a certain inhibitor of cAMP phosphodiesterase (PDE), which caused a huge accumulation of cAMP. The effective PDE inhibitors were 4-[3-(cyclopentyloxy)-4-methoxyphenyl]-2-pyrrolidinone (rolipram) and 4-(3-butyloxy-4-methoxyphenyl)-2-imidazolidinone (Ro-20-1724, a PDE4 inhibitor), in addition to 3-isobutyl-1-methylxanthine (a nonselective inhibitor). Rapid breakdown of the once-accumulated cAMP in cultured hepatocytes during the subsequent incubation without PDE inhibitors was progressively prevented when the concentration of herbimycin was increased from 0.3 to 10 microM during prior culture. This effect of herbimycin to inhibit PDE activity in intact cells was abolished by coaddition of a microtubule-disrupting agent, either colchicine or vinblastine, into the culture, but remained unchanged if the vinblastine-containing medium was further supplemented with taxol, a microtubule-stabilizing agent, which by itself mimicked the effect of herbimycin. None of these agents, which thus affected PDE activity in intact cells, inhibited the PDE activity assayable in the cell lysates. The taxol-like and vinblastine-suppressible action of herbimycin to stimulate microtubular assembly was antagonized by Na3VO4/H2O2, as confirmed by confocal microscopic images of the cells stained with fluorescein-bound anti-(alpha-tubulin). Thus, 4-h culture of hepatocytes with herbimycin inhibits phosphorylation of the C-terminal tyrosine residue of alpha-tubulin, thereby stimulating formation of a microtubular network which is responsible for the inhibition of PDE4 in the intact cells by an unknown mechanism. (+info)
Glucose-dependent stimulatory effect of glucagon-like peptide 1(7-36) amide on the electrical activity of pancreatic beta-cells recorded in vivo.
(19/3774)
The stimulatory effect of the glucagon-like peptide (GLP)-1(7-36) amide on electrical activity in pancreatic b-cells recorded in vivo was studied. The injection of GLP-1 produces a lengthening of the active phase with respect to the silent phase, leading to a stimulation of insulin release, which produces a secondary decrease in blood glucose concentration and eventually, to the hyperpolarization of the membrane at a blood glucose level of approximately 5 mmol/l. The injection of GLP-1 at a glycemic level <5 mmol/l does not stimulate electrical activity. This is in contrast to the effect of tolbutamide, which stimulates electrical activity at low glucose concentrations. These results demonstrate that in vivo, the stimulatory effect of GLP-1 on insulin secretion is at least partially mediated by its effect on beta-cell electrical activity. Furthermore, the glucose dependence of the effect confers to GLP-1, a security factor that supports its potential use in the treatment of type 2 diabetes. (+info)
Within- and between-subject variation in commonly measured anthropometric and biochemical variables.
(20/3774)
BACKGROUND: The biological variation of some commonly assessed metabolic variables in healthy subjects has not been studied extensively. The aim of the study was to assess, in 12 healthy subjects (6 male and 6 female; mean (SD) age; 22.7 (1.5) years) following an overnight fast, the day-to-day variation of body fat (impedance method), triglycerides, nonesterified fatty acid (NEFAs), glycerol, 3-hydroxybutyrate (3-OHB), lactate, glucose, insulin (RIA), C-peptide, and glucagon on 12 consecutive days. METHODS: Between- and within-subject coefficients of variation (CVG and CVW) were estimated using a random effects analysis of variance, and assay variation was subtracted to give the coefficient of within-subject biological variation (CVI). Individuality indices were calculated as CVW/CVG. RESULTS: The overall means, CVI, and individuality indices were as follows: for body fat, 24.2%, 10%, and 0.3; for triglycerides, 0.61 mmol/L, 21%, and 1.1; for NEFAs, 376 micromol/L, 45%, and 1.4; for glycerol, 48 micromol/L, 36%, and 0.8; for 3-OHB, 43 micromol/L, 61%, and 1.5; for lactate, 0.88 mmol/L, 31%, and 1.1; for glucose, 4.9 mmol/L, 4.8%, and 0.7; for insulin, 52 pmol/L, 26%, and 1.0; for C-peptide, 0.39 nmol/L, 24%, and 0.9; and for glucagon, 53 ng/L, 19%, and 0.8. CONCLUSIONS: The data presented here are necessary for the evaluation of several important metabolic variables in individual and group studies. The biological variation of some metabolites makes it difficult to characterize the status of healthy subjects with a single measurement. (+info)
Intact proinsulin and beta-cell function in lean and obese subjects with and without type 2 diabetes.
(21/3774)
OBJECTIVE: Type 2 diabetes is a heterogeneous disease in which both beta-cell dysfunction and insulin resistance are pathogenetic factors. Disproportionate hyperproinsulinemia (elevated proinsulin/insulin) is another abnormality in type 2 diabetes whose mechanism is unknown. Increased demand due to obesity and/or insulin resistance may result in secretion of immature beta-cell granules with a higher content of intact proinsulin. RESEARCH DESIGN AND METHODS: We investigated the impact of obesity on beta-cell secretion in normal subjects and in type 2 diabetic patients by measuring intact proinsulin, total proinsulin immunoreactivity (PIM), intact insulin, and C-peptide (by radioimmunoassay) by specific enzyme-linked immunosorbent assays in the fasting state and during a 120-min glucagon (1 mg i.v.) stimulation test. Lean (BMI 23.5 +/- 0.3 kg/m2) (LD) and obese (30.1 +/- 0.4 kg/m2) (OD) type 2 diabetic patients matched for fasting glucose (10.2 +/- 0.6 vs. 10.3 +/- 0.4 mmol/l) were compared with age- and BMI-matched lean (22.4 +/- 0.6 kg/m2) (LC) and obese (30.8 +/- 0.9 kg/m2) (OC) normal control subjects. RESULTS: Diabetic patients (LD vs. LC and OD vs. OC) had elevated fasting levels of intact proinsulin 6.6 +/- 1.0 vs. 1.6 +/- 0.3 pmol/l and 7.7 +/- 2.0 vs. 1.2 +/- 0.2 pmol/l; PIM: 19.9 +/- 2.5 vs. 5.4 +/- 1.0 pmol/l and 29.6 +/- 6.1 vs. 6.1 +/- 0.9 pmol/l; and total PIM/intact insulin: 39 +/- 4 vs. 15 +/- 2% and 35 +/- 5 vs. 13 +/- 2%, all P < 0.01. After glucagon stimulation, PIM levels were disproportionately elevated (PIM/intact insulin based on area under the curve analysis) in diabetic patients (LD vs. LC and OD vs. OC): 32.6 +/- 6.7 vs. 9.2 +/- 1.1% and 22.7 +/- 5.2 vs. 9.1 +/- 1.1%, both P < 0.05. Intact insulin and C-peptide net responses were significantly reduced in type 2 diabetic patients, most pronounced in the lean group. The ratio of intact proinsulin to PIM was higher in diabetic patients after stimulation in both LD versus LC: 32 +/- 3 vs. 23 +/- 2%, and OD versus OC: 28 +/- 4 vs. 16 +/- 2%, both P < 0.01. In obese normal subjects, intact proinsulin/PIM was lower both in the fasting state and after glucagon stimulation: OC versus LC: 22 +/- 3 vs. 33 +/- 3% (fasting) and 16 +/- 2 vs. 23 +/- 2% (stimulated), both P < 0.05. CONCLUSIONS: Increased secretory demand from obesity-associated insulin resistance cannot explain elevated intact proinsulin and disproportionate hyperproinsulinemia in type 2 diabetes. This abnormality may be an integrated part of pancreatic beta-cell dysfunction in this disease. (+info)
Cell-specific coupling of PGE2 to different transduction pathways in arginine vasopressin- and glucagon-sensitive segments of the rat renal tubule.
(22/3774)
1. The aim of the present study was to investigate the transduction pathways elicited by prostaglandin E2 (PGE2) to inhibit hormone-stimulated adenosine 3':5'-cyclic monophosphate (cyclic AMP) accumulation in the outer medullary collecting duct (OMCD) and medullary thick ascending limb (MTAL) microdissected from the rat nephron. 2. In the OMCD, 0.3 microM PGE2 and low concentrations of Ca2+ ionophores (10 nM ionomycin or 50 nM A23187) inhibited by about 50% a same pool of arginine vasopressin (AVP)-stimulated cyclic AMP content through a same process insensitive to Bordetella pertussis toxin (PTX). 3. Sulprostone, an agonist of the EP1/EP3 subtypes of the PGE2 receptor, decreased AVP-dependent cyclic AMP accumulation in OMCD and MTAL samples. The concentration eliciting half-maximal inhibition was of about 50 nM in OMCD and 0.1 nM in MTAL. 4. In MTAL, 1 nM sulprostone and PGE2 inhibited by about 90% a same pool of AVP-dependent cyclic AMP content through a PTX-sensitive, Ca2+ -independent pathway. 5. In the OMCD, PGE2 decreased by about 50% glucagon-dependent cyclic AMP synthesis by a process sensitive to PTX and Ca2+ -independent. Sulprostone 1 nM induced the same level of inhibition. 6. These results demonstrate that PGE2 decrease hormone-dependent cyclic AMP accumulation through a G(alpha)i-mediated inhibition of adenylyl cyclase activity in MTAL cells and glucagon-sensitive cells of the OMCD or through a PTX-insensitive increase of intracellular Ca2+ concentration in AVP-sensitive cells of the OMCD. (+info)
The influence of the colon on postprandial glucagon-like peptide 1 (7-36) amide concentration in man.
(23/3774)
Glucagon-like peptide (7-36) amide (GLP-1) is an incretin hormone of the enteroinsular axis released rapidly after meals despite the fact that GLP-1 secreting cells (L-cells) occur predominantly in the distal gut. The importance of these colonic L-cells for postprandial GLP-1 was determined in healthy control subjects and in ileostomy patients with minimal small bowel resection (<5 cm). Subjects were fed a high complex carbohydrate test meal (15.3 g starch) followed by two carbohydrate-free, high fat test meals (25 g and 48.7 g fat respectively). Circulating levels of glucose, insulin, glucagon, glucose insulinotrophic peptide (GIP) and GLP-1 were measured over a 9-h postprandial period. For both subject groups the complex carbohydrate test meal failed to elicit a rise in either GIP or GLP-1. However, both hormones were elevated after the fat load although the GLP-1 concentration was significantly reduced in the ileostomist group when compared with controls (P=0.02). Associated with this reduction in circulating GLP-1 was an elevation in glucagon concentration (P=0.012) and a secondary rise in the plasma glucose concentration (P=0.006). These results suggest that the loss of colonic endocrine tissue is an important determinant in the postprandial GLP-1 concentration. Ileostomists should not be assumed to have normal enteroinsular function as the colon appears to have an important role in postprandial metabolism. (+info)
Miniglucagon (glucagon 19-29), a potent and efficient inhibitor of secretagogue-induced insulin release through a Ca2+ pathway.
(24/3774)
Using the MIN6 B-cell line, we investigated the hypothesis that miniglucagon, the C-terminal () fragment processed from glucagon and present in pancreatic A cells, modulates insulin release, and we analyzed its cellular mode of action. We show that, at concentrations ranging from 0.01 to 1000 pM, miniglucagon dose-dependently (ID50 = 1 pM) inhibited by 80-100% the insulin release triggered by glucose, glucagon, glucagon-like peptide-1-(7-36) amide (tGLP-1), or glibenclamide, but not that induced by carbachol. Miniglucagon had no significant effects on cellular cAMP levels. The increase in 45Ca2+ uptake induced by depolarizing agents (glucose or extracellular K+), by glucagon, or by the Ca2+channel agonist Bay K-8644 was blocked by miniglucagon at the doses active on insulin release. Electrophysiological experiments indicated that miniglucagon induces membrane hyperpolarization, probably by opening potassium channels, which terminated glucose-induced electrical activity. Pretreatment with pertussis toxin abolished the effects of miniglucagon on insulin release. It is concluded that miniglucagon is a highly potent and efficient inhibitor of insulin release by closing, via hyperpolarization, voltage-dependent Ca2+ channels linked to a pathway involving a pertussis toxin-sensitive G protein. (+info)