Liver cirrhosis is often accompanied by a disturbed carbohydrate metabolism similar to type 2 diabetes. To investigate the severity of the defect in insulin secretion in this form of diabetes, we measured insulin release from isolated pancreatic islets of rats with CCl(4)-phenobarbital-induced liver cirrhosis. Cirrhosis was confirmed by clinical signs, elevated liver enzymes and histology. Fasting venous plasma glucose concentrations were equal in rats with liver cirrhosis and in controls. Plasma insulin and glucagon concentrations were significantly greater (P<0.01) in cirrhotic rats than in control animals. Glucose (16.7 mM)-induced stimulation of insulin release from pancreatic islets revealed a twofold increase in control and cirrhotic rats. Basal and stimulated insulin secretion, however, were significantly lower in cirrhotic animals. The incretin hormone, glucagon-like peptide-1 (GLP-1), has therapeutic potential for the treatment of type 2 diabetes. Therefore, islets from control and cirrhotic animals were incubated with GLP-1 in concentrations from 10(-)(11) to 10(-)(6) M. GLP-1 stimulated insulin release in a concentration-dependent manner. In islets from cirrhotic rats, basal and stimulated insulin secretion was blunted compared with controls. These data show that the hyperinsulinemia observed in liver cirrhosis is not due to an increase of insulin secretion from islets, but could be explained by decreased hepatic clearance of insulin. GLP-1 may ameliorate diabetes in patients with liver cirrhosis.
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- Abstract: Diabetes x
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EG Siegel, A Seidenstucker, B Gallwitz, F Schmitz, A Reinecke-Luthge, G Kloppel, UR Folsch and WE Schmidt
JA Shaw, MI Delday, AW Hart, HM Docherty, CA Maltin and K Docherty
The objective of these studies was to evaluate human insulin gene expression following intramuscular plasmid injection in non-diabetic rats as a potential approach to gene therapy for diabetes mellitus avoiding the need for immunosuppression. A wild-type human preproinsulin construct and a mutant construct in which PC2/PC3 sites were engineered to form furin consensus sites were evaluated in in vitro transfections of hepatocyte (HepG2) and myoblast (C2C12/L6) cell lines, primary rat myoblasts, and dermal fibroblasts. In vivo gene transfer by percutaneous plasmid injection of soleus muscle +/- prior notexin-induced myolysis was assessed in rats. In vitro transfection of non-neuroendocrine cell lines and primary cultures with wild-type human preproinsulin resulted in secretion of predominantly unprocessed proinsulin. Employing the mutant construct, there was significant processing to mature insulin (HepG2, 95%; C2C12, 75%; L6, 65%; primary myoblasts, 48%; neonatal fibroblasts, 56%; adult fibroblasts, 87%). In rats aged 5 weeks, circulating human (pro)insulin was detected from 1 to 37 days following plasmid injection and the potential of augmenting transfection efficiency by prior notexin injection was demonstrated (wild-type processing, 87%; mutant, 90%). Relative hypoglycaemia was confirmed by HbA1C (saline, 5.5%; wild type, 5.1%; mutant, 5.1% (P<0.05)). Human (pro)insulin levels and processing (wild-type, 8%; mutant, 53%) were lower in rats aged 9 months but relative hypoglycaemia was confirmed by serum glucose at 10 days (saline, 6.4 mmol/l; wild-type, 6.0 mmol/l; mutant, 5.4 mmol/l). In conclusion, prolonged constitutive systemic secretion of bioactive human (pro)insulin has been attained in non-neuroendocrine cells in vitro and in growing and mature rats following intramuscular plasmid injection.
ME Guibbolini, PM Pierson and B Lahlou
Neurohypophysial hormone receptors and second messengers were studied in trout (Oncorhynchus mykiss) hepatocytes. Arginine vasotocin (AVT) and isotocin (IT) elicited a concentration-dependent inhibition of cAMP accumulation in the presence of 5x10(-8) M glucagon (maximal effect for 4.5x10(-7) M and 1.4x10(-7) M, half-maximal effect for 2.1x10(-8) M and 0.7x10(-8) M, AVT and IT respectively). The effect of glucagon was inhibited up to 90% by AVT and 80% by IT. While AVT inhibited (up to 50%) the basal cAMP production, IT had no such action. Specific V(1) or V(2) analogues (with reference to vasopressin in mammals) were used for pharmacological characterization of the type of neurohypophysial hormone receptor involved in this inhibition. The V(1) agonist [Phe(2), Orn(8)]-oxytocin inhibited the glucagon-stimulated cAMP production with a maximal effect for 6x10(-7) M and a half-maximal effect for 0.9x10(-8) M concentrations of the analogue. While the V(1) agonist reduced the glucagon-stimulated cAMP level by 70%, it showed only a tendency to reduce the basal level. The V(2) agonist [deamino(1), Val(4),d -Arg(8)]-vasopressin had no effect either on basal or on glucagon-stimulated cAMP production. The V(1) antagonist [d(CH(2))(5)(1), O-Me-Tyr(2), Arg(8)]-vasopressin totally reversed the 10(-8) M AVT-induced inhibition of 5x10(-8) M glucagon-stimulated cAMP production, whereas the V(2) antagonist [d(CH(2))(5)(1),d -Ile(2), Ile(4), Arg(8), Ala(9)]-vasopressin had no such effect. In this particular case, maximal and half-maximal effects of the V(1) antagonist were obtained for 2.3x10(-6) M and 1. 2x10(-6 )M respectively. Changes in intracellular calcium content were measured using the fluorescent probe FURA-2/AM. AVT and IT elicited a concentration-dependent increase in Ca(2+) accumulation. The comparison of the effect of 10(-8) M agonists versus AVT showed the following order of potency: AVT=IT>V(1) agonist>V(2) agonist. The V(1) antagonist reversed the AVT-induced Ca(2+) accumulation whereas the V(2) antagonist had no such effect. These results are taken as evidence for the presence in trout hepatocytes of neurohypophysial hormone receptors functionally close to the V(1a)-type linked to cAMP production and Ca(2+) mobilization.
Joshua A Kulas, Kendra L Puig and Colin K Combs
The amyloid precursor protein (APP) has been extensively investigated for its role in the production of amyloid beta (Aβ), a plaque-forming peptide in Alzheimer’s disease (AD). Epidemiological evidence suggests type 2 diabetes is a risk factor for AD. The pancreas is an essential regulator of blood glucose levels through the secretion of the hormones insulin and glucagon. Pancreatic dysfunction is a well-characterized consequence of type 1 and type 2 diabetes. In this study, we have examined the expression and processing of pancreatic APP to test the hypothesis that APP may play a role in pancreatic function and the pathophysiology of diabetes. Our data demonstrate the presence of APP within the pancreas, including pancreatic islets in both mouse and human samples. Additionally, we report that the APP/PS1 mouse model of AD overexpresses APP within pancreatic islets, although this did not result in detectable levels of Aβ. We compared whole pancreas and islet culture lysates by Western blot from C57BL/6 (WT), APP−/− and APP/PS1 mice and observed APP-dependent differences in the total protein levels of GLUT4, IDE and BACE2. Immunohistochemistry for BACE2 detected high levels in pancreatic α cells. Additionally, both mouse and human islets processed APP to release sAPP into cell culture media. Moreover, sAPP stimulated insulin but not glucagon secretion from islet cultures. We conclude that APP and its metabolites are capable of influencing the basic physiology of the pancreas, possibly through the release of sAPP acting in an autocrine or paracrine manner.
Jun-ichi Eiki, Kaori Saeki, Norihiro Nagano, Tomoharu Iino, Mari Yonemoto, Yoko Takayenoki-Iino, Satoru Ito, Teruyuki Nishimura, Yoshiyuki Sato, Makoto Bamba, Hitomi Watanabe, Kaori Sasaki, Sumika Ohyama, Akio Kanatani, Toshio Nagase and Toshihiko Yada
Glucagon-like peptide-1 (GLP-1) is an incretin hormone that potentiates insulin secretion in a glucose-dependent manner. Selective GLP-1 secretagogue would be one of the potential therapeutic targets for type 2 diabetes. Here, we describe a newly identified small molecule compound (compound A) that stimulates secretion of GLP-1 in murine enteroendocrine cell lines, STC-1 and GLUTag cells, and in primary cultured fetal rat intestinal cells (FRIC). The underlying mechanism by which compound A stimulated GLP-1 secretion was also examined. Compound A stimulated GLP-1 secretion from STC-1 cells in a concentration-dependent manner, and also from GLUTag cells and FRIC. The action of compound A was selective against other tested endocrine functions such as secretion of insulin from rat islets, growth hormone from rat pituitary gland cells, and norepinephrine from rat PC-12 cells. In STC-1 cells, the compound A-stimulated GLP-1 secretion was neither due to cyclic AMP production nor to Ca2+ release from intracellular stores, but to extracellular Ca2+ influx. The response was inhibited by the presence of either L-type Ca2+ channel blockers or K+ ionophore. Perforated-patch clamp study revealed that compound A induces membrane depolarization. These results suggest that neither Gαs- nor Gαq-coupled signaling account for the mechanism of action, but depolarization-coupled Ca2+ influx from extracellular space is the primary cause for the GLP-1 secretion stimulated by compound A. Identifying a specific target molecule for compound A will reveal a selective regulatory pathway that leads to depolarization-mediated GLP-1 secretion.
C Y Shan, J H Yang, Y Kong, X Y Wang, M Y Zheng, Y G Xu, Y Wang, H Z Ren, B C Chang and L M Chen
For centuries, Berberine has been used in the treatment of enteritis in China, and it is also known to have anti-hyperglycemic effects in type 2 diabetic patients. However, as Berberine is insoluble and rarely absorbed in gastrointestinal tract, the mechanism by which it works is unclear. We hypothesized that it may act locally by ameliorating intestinal barrier abnormalities and endotoxemia. A high-fat diet combined with low-dose streptozotocin was used to induce type 2 diabetes in male Sprague Dawley rats. Berberine (100 mg/kg) was administered by lavage to diabetic rats for 2 weeks and saline was given to controls. Hyperinsulinemia and insulin resistance improved in the Berberine group, although there was no significant decrease in blood glucose. Berberine treatment also led to a notable restoration of intestinal villi/mucosa structure and less infiltration of inflammatory cells, along with a decrease in plasma lipopolysaccharide (LPS) level. Tight junction protein zonula occludens 1 (ZO1) was also decreased in diabetic rats but was restored by Berberine treatment. Glutamine-induced glucagon-like peptide 2 (GLP2) secretion from ileal tissue decreased dramatically in the diabetic group but was restored by Berberine treatment. Fasting insulin, insulin resistance index, plasma LPS level, and ZO1 expression were significantly correlated with GLP2 level. In type 2 diabetic rats, Berberine treatment not only augments GLP2 secretion and improves diabetes but is also effective in repairing the damaged intestinal mucosa, restoring intestinal permeability, and improving endotoxemia. Whether these effects are mechanistically related will require further studies, but they certainly support the hypothesis that Berberine acts via modulation of intestinal function.
GE Rice, MH Wong, W Farrugia and KF Scott
Although phospholipase A2 (PLA2) enzymatic activities have been implicated in the regulation of phospholipid metabolism and eicosanoid formation in human gestational tissues, the role and contribution made by individual PLA2 isozymes has not been established. The aim of this study, therefore, was to determine the contribution made by Type II PLA2 to PLA2 enzymatic activity present in human term placenta. The experimental paradigm used to establish the contribution made by Type II PLA2 to total in vitro PLA2 enzymatic activity present in placental extracts was to remove Type II PLA2 by immunoaffinity extraction and then to quantify residual PLA2 enzymatic activity. Before immunoaffinity extraction, Type II PLA2 immunoactivity and total PLA2 enzymatic activity present in placental extracts averaged 28.0 +/- 10.0 ng/mg protein and 1040 +/- 367 pmol/h per mg protein (n = 3) respectively. After solid-phase immunoaffinity batch extraction of placental extracts, immunoreactive Type II PLA2 was not detectable by ELISA, and PLA2 enzymatic activity was decreased by 82 +/- 1% (P < 0.001). Residual (i.e. non-Type II) PLA2 enzymatic activity was further characterised by Western blot analysis and enzyme activity assay. The data obtained are consistent with a contribution by both cytosolic PLA2 and other secretory PLA2 isozymes (i.e. non-Type II) to residual PLA2 enzymatic activity. The results obtained in this study support the conclusion that Type II PLA2 is quantitatively the primary PLA2 isozyme that contributes to in vitro PLA2 enzymatic activity present in extracts of human term placenta, accounting for at least 80% of total activity. These data further support the involvement of this extracellularly active isozyme in the regulation of placental phospholipid metabolism and eicosanoid formation during late gestation.
SJ Fisher, ZQ Shi, HL Lickley, S Efendic, M Vranic and A Giacca
At supraphysiological levels, IGF-I bypasses some forms of insulin resistance and has been proposed as a therapeutic agent in the treatment of diabetes. Unfortunately, side effects of high-dose IGF-I (100-250 microg/kg) have precluded its clinical use. Low-dose IGF-I (40-80 microg/kg), however, shows minimal side effects but has not been systematically evaluated. In our previous study under conditions of declining glucose, low-dose IGF-I infusion was more effective in stimulating glucose utilization, but less effective in suppressing glucose production and lipolysis than low-dose insulin. However, under conditions of hyperglycemia, we could not observe any differential effects between high-dose infusions of IGF-I and insulin. To determine whether the differential effects of IGF-I and insulin are dose-related or related to the prevailing glucose level, 3 h glucose clamps were performed in the same animal model as in the previous studies, i.e. the moderately hyperglycemic (175 mg/dl) insulin-infused depancreatized dog, with additional infusions of low-dose IGF-I (67.8 microg/kg, i.e. 29.1 microg/kg bolus plus 0.215 microg/kg( )per min infusion; n=5) or insulin 49.5 mU/kg (9 mU/kg bolus plus 0.45 mU/kg per min; n=7). As in the previous study under conditions of declining glucose, low-dose IGF-I had significant metabolic effects in vivo, in our model of complete absence of endogenous insulin secretion. Glucose production was similarly suppressed with both IGF-I and insulin, by 54+/-3 and 56+/-2% s.e. (P=NS) respectively. Glucose utilization was stimulated to the same extent (IGF-I 5.2+/-0.2, insulin 5.5+/-0.3 mg/kg per min, P=NS). Glucagon, free fatty acid, glycerol, alanine and beta-hydroxybutyrate, were suppressed, while lactate and pyruvate levels were raised, similarly with IGF-I and insulin. We conclude that: (i) differential effects of IGF-I and insulin may be masked under hyperglycemic conditions, independent of the hormone dose; (ii) low-dose IGF-I has no selective advantage over additional insulin in suppressing glucose production and lipolysis, nor in stimulating glucose utilization during hyperglycemia and subbasal insulin infusion when insulin secretion is absent, as in type 1 diabetes mellitus.
G Üçkaya, P Delagrange, A Chavanieu, G Grassy, M-F Berthault, A Ktorza, E Cerasi, G Leibowitz and N Kaiser
Glucagon-like peptide 1 (GLP-1) analogues are considered potential drugs for type 2 diabetes. We studied the effect of a novel GLP-1 analogue, S 23521 ([a8-des R36] GLP-1-[7–37]-NH2), on the metabolic state and β-cell function, proliferation and survival in the Psammomys obesus model of diet-induced type 2 diabetes. Animals with marked hyperglycaemia after 6 days of high-energy diet were given twice-daily s.c. injection of 100 μg/kg S 23521 for 15 days. Food intake was significantly decreased in S 23251-treated P. obesus; however, there was no significant difference in body weight from controls. Progressive worsening of hyperglycaemia was noted in controls, as opposed to maintenance of pre-treatment glucose levels in the S 23521 group. Prevention of diabetes progression was associated with reduced mortality. In addition, the treated group had higher serum insulin, insulinogenic index and leptin, whereas plasma triglyceride and non-esterified fatty acid levels were decreased. S 23521 had pronounced effect on pancreatic insulin, which was 5-fold higher than the markedly depleted insulin reserve of control animals. Immunohistochemical analysis showed islet degranulation with disrupted morphology in untreated animals, whereas islets from S 23521-treated animals appeared intact and filled with insulin; β-cell apoptosis was approximately 70% reduced, without a change in β-cell proliferation. S 23521 treatment resulted in a 2-fold increase in relative β-cell volume. Overall, S 23521 prevented the progression of diabetes in P. obesus with marked improvement of the metabolic profile, including increased pancreatic insulin reserve, β-cell viability and mass. These effects are probably due to actions of S 23521 both directly on islets and via reduced food intake, and emphasize the feasibility of preventing blood glucose deterioration over time in type 2 diabetes.
Maaike M Roefs, Françoise Carlotti, Katherine Jones, Hannah Wills, Alexander Hamilton, Michael Verschoor, Joanna M Williams Durkin, Laura Garcia-Perez, Melissa F Brereton, Laura McCulloch, Marten A Engelse, Paul R V Johnson, Barbara C Hansen, Kevin Docherty, Eelco J P de Koning and Anne Clark
Type 2 diabetes (T2DM) is associated with pancreatic islet dysfunction. Loss of β-cell identity has been implicated via dedifferentiation or conversion to other pancreatic endocrine cell types. How these transitions contribute to the onset and progression of T2DM in vivo is unknown. The aims of this study were to determine the degree of epithelial-to-mesenchymal transition occurring in α and β cells in vivo and to relate this to diabetes-associated (patho)physiological conditions. The proportion of islet cells expressing the mesenchymal marker vimentin was determined by immunohistochemistry and quantitative morphometry in specimens of pancreas from human donors with T2DM (n = 28) and without diabetes (ND, n = 38) and in non-human primates at different stages of the diabetic syndrome: normoglycaemic (ND, n = 4), obese, hyperinsulinaemic (HI, n = 4) and hyperglycaemic (DM, n = 8). Vimentin co-localised more frequently with glucagon (α-cells) than with insulin (β-cells) in the human ND group (1.43% total α-cells, 0.98% total β-cells, median; P < 0.05); these proportions were higher in T2DM than ND (median 4.53% α-, 2.53% β-cells; P < 0.05). Vimentin-positive β-cells were not apoptotic, had reduced expression of Nkx6.1 and Pdx1, and were not associated with islet amyloidosis or with bihormonal expression (insulin + glucagon). In non-human primates, vimentin-positive β-cell proportion was larger in the diabetic than the ND group (6.85 vs 0.50%, medians respectively, P < 0.05), but was similar in ND and HI groups. In conclusion, islet cell expression of vimentin indicates a degree of plasticity and dedifferentiation with potential loss of cellular identity in diabetes. This could contribute to α- and β-cell dysfunction in T2DM.