Enzymatic,metabolic and secretory perturbations in pancreatic islets of thyroidectomized rats

In thyroidectomized rats, the activity of FAD-linked glycerophosphate dehydrogenase was severely diminished in liver homogenates but not affected significantly in pancreatic islet homogenates, whilst the activity of 2-ketoglutarate dehydrogenase was decreased modestly in both liver and islet homogenates. Likewise, in intact islets of thyroidectomized rats, the generation of³HOH from [2-³H]glycerol was not decreased, and the ratio between oxidative and total glycolysis not significantly lower than in islets from sham-operated rats, at least in the presence of a high concentration of D-glucose. Nevertheless impaired oxidation of both D-[3,4-¹⁴C]glucose and D-[6-¹⁴C]glucose was observed in islets of thyroidectomized rats, the relative magnitude of such a decrease being more pronounced at a low than at a high D-glucose concentration. Such metabolic anomalies coincided with a lower level of plasma insulin and a decreased output of insulin by islets incubated at low (2·8 mM ), but not higher, concentrations of D-glucose. It is concluded that hypothyroidism does not mimic the deficiency in islet FAD-linked glycerophosphate dehydrogenase activity found in rats with inherited or acquired non-insulin-dependent diabetes.     

Reduced expression of SIRT1 is associated with diminished glucose-induced insulin secretion in islets from calorie-restricted rats

Alterations in food intake such as caloric restriction modulate the expression of SIRT1 and SIRT4 proteins that are involved in pancreatic β-cell function. Here, we search for a possible relationship between insulin secretion and the expression of SIRT1, SIRT4, PKC and PKA in islets from adult rats submitted to CR for 21 days. Rats were fed with an isocaloric diet (CTL) or received 60% (CR) of the food ingested by CTL. The dose-response curve of insulin secretion to glucose was shifted to the right in the CR compared with CTL islets (EC₅₀ of 15.1±0.17 and 10.5±0.11 mmol/L glucose). Insulin release by the depolarizing agents arginine and KCl was reduced in CR compared with CTL islets. Total islet insulin content and glucose oxidation were also reduced in CR islets. Leucine-stimulated secretion was similar in both groups, slightly reduced in CR islets stimulated by leucine plus glutamine but higher in CR islets stimulated by ketoisocaproate (KIC). Insulin secretion was also higher in CR islets stimulated by carbachol, compared with CTL islets. No differences in the rise of cytosolic Ca²⁺ concentrations stimulated by either glucose or KCl were observed between groups of islets. Finally, SIRT1, but not SIRT4, protein expression was lower in CR compared with CTL islets, whereas no differences in the expression of PKC and PKA proteins were observed. In conclusion, the lower insulin secretion in islets from CR rats was, at least in part, due to an imbalance between the expression of SIRT1 and SIRT4.     

Effect of insulin on exocrine pancreatic secretion in healthy and diabetic anaesthetised rats

This investigation characterised the effects of exogenous insulin on exocrine pancreatic secretion in anaesthetised healthy and diabetic rats. Animals were rendered diabetic by a single injection of streptozotocin (STZ, 60 mg kg^–1 I.P.). Age-matched controls were injected citrate buffer. Rats were tested for hyperglycaemia 4 days after STZ injection and 7–8 weeks later when they were used for the experiments. Following anaesthesia (1 g kg^–1 urethane I.P.), laparotomy was performed and the pancreatic duct cannulated for collection of pure pancreatic juice. Basal pancreatic juice flow rate in diabetic rats was significantly (p < 0.001) increased whereas protein and amylase outputs were significantly (p < 0.001) decreased compared to control rats. Insulin (1 IU, I.P.) produced in healthy rats significant increases in pancreatic flow rate, amylase secretion and protein output compared to basal (p < 0.05). Insulin action also included a reduction in blood glucose (152.7 ± 16.9 mg dl^–1, n= 6, prior to insulin and 42.0 ± 8.4 mg dl^–1, n= 4, 100 min later). In fact, flow rate and glycaemia showed a strong negative correlation (p < 0.01, Pearson). Pretreatment with atropine (0.2 mg kg^–1, I.V.) abolished the effects of insulin on secretory parameters despite a similar reduction in glycaemia; in this series of experiments the correlation between flow rate and blood glucose was lost. In diabetic rats, insulin (4 IU, I.P.) did not modify exocrine pancreatic secretion. There was a fall in blood glucose (467.6 ± 14.0 mg dl^–1, n= 10, prior to insulin and 386.6 ± 43.6 mg dl^–1, n= 7, 120 min later). Rats, however, did not become hypoglycaemic. Similar results were observed in diabetic atropinized rats. The results of this study indicate that the effects of insulin on exocrine pancreatic secretion in anaesthetised healthy rats are mediated by hypoglycaemia-evoked vagal cholinergic activation. (Mol Cell Biochem 261: 105–110, 2004)     

Cyproheptadine metabolites inhibit proinsulin and insulin biosynthesis and insulin release in isolated rat pancreatic islets

The contribution of drug metabolites to cyproheptadine (CPH)-induced alterations in endocrine pancreatic -cells was investigated by examining the inhibitory activity of CPH and its biotransformation products, desmethylcyproheptadine (DMCPH), CPH-epoxide and DMCPH-epoxide, on hormone biosynthesis and secretion in pancreatic islets isolated from 50-day-old rats. Measurement of (pro)insulin (proinsulin and insulin) synthesis using incorporation of ³H-leucine showed that DMCPH-epoxide, DMCPH and CPH-epoxide were 22, 10 and 4 times, respectively, more potent than CPH in inhibiting hormone synthesis. The biosynthesis of (pro)insulin was also inhibited by CPH and DMCPH-epoxide in islets isolated from 21-day-old rat fetuses. The inhibitory action of CPH and its metabolites was apparently specific for (pro)insulin, and the synthesis of other islet proteins was not affected. Other experiments showed the metabolites of CPH were active in inhibiting glucose-stimulated insulin secretion but were less potent than the parent drug in producing this effect. CPH and its structurally related metabolites, therefore, have differential inhibitory activities on insulin synthesis and release. The observation that CPH metabolites have higher potency than CPH to inhibit (pro)insulin synthesis, when considered with published reports on the disposition of the drug in rats, indicate that CPH metabolites, particularly DMCPH-epoxide, are primarily responsible for the insulin depletion observed when the parent compound is given to fetal and adult animals.Abbreviations CPH cyproheptadine - CPH-epoxide cyproheptadine-10-11-epoxide - DMCPH desmethylcyproheptadine - DMCPH-epoxide desmethylcyproheptadine-10,11-epoxide - HPLC high-performance liquid chromatography - KBB Krebs biocarbonate buffer Recipient of a Society of Toxicology Predoctoral Research Fellowship.Present address: Department of Biochemistry, The University of Hong Kong, Hong Kong.     

The zinc transporter Slc39a5 controls glucose sensing and insulin secretion in pancreatic β-cells via Sirt1- and Pgc-1α-mediated regulation of Glut2

Zinc levels are high in pancreatic β-cells, and zinc is involved in the synthesis, processing and secretion of insulin in these cells. However, precisely how cellular zinc homeostasis is regulated in pancreatic β-cells is poorly understood. By screening the expression of 14 Slc39a metal importer family member genes, we found that the zinc transporter Slc39a5 is significantly downregulated in pancreatic β-cells in diabetic db/db mice, obese ob/ob mice and high-fat diet-fed mice. Moreover,β-cell-specific Slc39a5 knockout mice have impaired insulin secretion. In addition, Slc39a5-deficient pancreatic islets have reduced glucose tolerance accompanied by reduced expression of Pgc-1α and its downstream target gene Glut2. The down-regulation of Glut2 in Slc39a5-deficient islets was rescued using agonists of Sirt1, Pgc-1α and Ppar-γ. At the mechanistic level, we found that Slc39a5-mediated zinc influx induces Glut2 expression via Sirt1-mediated Pgc-1α activation. These findings suggest that Slc39a5 may serve as a possible therapeutic target for diabetes-related conditions.     

Increased resistance to hydrogen peroxide‐induced cardiac contracture is associated with decreased myocardial oxidative stress in hypothyroid rats

The purpose of this study was to determine whether decreased oxidative stress would increase the resistance to cardiac contracture induced by H₂O₂ in hypothyroid rats. Male Wistar rats were divided into two groups: control and hypothyroid. Hypothyroidism was induced via thyroidectomy. Four weeks post surgery, blood samples were collected to perform thyroid hormone assessments, and excised hearts were perfused at a constant flow with or without H₂O₂ (1 mmol/L), being divided into two sub‐groups: control, hypothyroid, control + H₂O₂, hypothyroid + H₂O₂. Lipid peroxidation (LPO) was evaluated by chemiluminescence (CL) and thiobarbituric acid reactive substances (TBARS) methods, and protein oxidation by carbonyls assay in heart homogenates. Cardiac tissue was also screened for superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) activities, and for total radical‐trapping antioxidant potential (TRAP). Analyses of SOD and glutathione‐S‐transferase (GST) protein expression were also performed in heart homogenates. Hypothyroid hearts were found to be more resistant to H₂O₂‐induced contracture (60% elevation in LVEDP) as compared to control. CL, TBARS, carbonyl, as well as SOD, CAT, GPx activities and TRAP levels were reduced (35, 30, 40, 30, 16, 25, and 33%, respectively) in the cardiac homogenates of the hypothyroid group as compared to controls. A decrease in SOD and GST protein levels by 20 and 16%, respectively, was also observed in the hypothyroid group. These results suggest that a hypometabolic state caused by thyroid hormone deficiency can lead to an improved response to H₂O₂ challenge and is associated with decreased oxidative myocardial damage. Copyright © 2009 John Wiley & Sons, Ltd.     

Pyrroloquinoline quinone ameliorates l‐thyroxine‐induced hyperthyroidism and associated problems in rats

Pyrroloquinoline quinone (PQQ) is believed to be a strong antioxidant. In this study, we have evaluated its hitherto unknown role in l ‐thyroxin (L‐T₄)‐induced hyperthyroidism considering laboratory rat as a model. Alterations in the serum concentration of thyroxin (T₄) and triiodothyronine (T₃); lipid peroxidation (LPO) of liver, kidney, heart, muscles and brain; in the endogenous antioxidants such as superoxide dismutase, catalase and glutathione and in serum total cholesterol, high‐density lipoprotien, triglycerides, serum glutamate pyruvate transaminase (SGPT), serum glutamate oxaloacetate transaminase (SGOT) and urea were evaluated. Administration of l ‐T₄ (500‐µg kg^?1 body weight) enhanced not only the serum T₃ and T₄ levels but also the tissue LPO, serum SGOT, SGPT and urea with a parallel decrease in the levels of antioxidants and serum lipids. However, on simultaneous administration of PQQ (5 mg kg^?1 for 6 days), all these adverse effects were ameliorated, indicating the potential of PQQ in the amelioration of hyperthyroidism and associated problems. Possibly, the curative effects were mediated through inhibition of oxidative stress. We suggest that PQQ may be considered for therapeutic use for hyperthyroidism after dose standardization. Copyright © 2014 John Wiley & Sons, Ltd.     

The nucleosome binding protein HMGN3 is expressed in pancreatic α‐cells and affects plasma glucagon levels in mice

Glucose homeostasis requires the coordinated actions of various organs and is critically dependent on the proper functioning of the various cell types present in the pancreatic Langerhans islets. Here we report that chromatin architectural protein HMGN3 is highly expressed in all pancreatic endocrine islet cells, and that Hmgn3?/? mice which have a mild diabetic phenotype, have reduced glucagon levels in their blood. To elucidate the mechanism leading to altered glucagon secretion of Hmgn3?/? mice, we tested whether HMGN3 affect glucagon synthesis and secretion in αTC1‐9 cells, a glucagon secreting cell line that is used to study pancreatic α‐cell function. We find that in these cells deletion of either HMGN3 or other HMGN variants, does not significantly affect glucagon gene expression or glucagon secretion. Our studies demonstrate a link between HMGN3 and glucagon blood levels that is not directly dependent of the function of pancreatic α‐cells. J. Cell. Biochem. 109: 49–57, 2010. Published 2009 Wiley‐Liss, Inc.     

Comparative study on the effects of a hypoglycemic 2-substituted-2-imidazoline derivative (DG-5128) and tolbutamide on insulin secretion from and insulin synthesis in the isolated rat pancreatic islets

2[2-(4,5-Dihydro-1H-imidazol-2-yl)-1-phenylethyl]pyridine dihydrochloride sesquihydrate (DG-5128) was found to stimulate the glucose-primed insulin secretion from the isolated rat pancreatic islets throughout the incubation period, unlike tolbutamide which stimulated it only in the initial phase of incubation. The effect of DG-5128 was more pronounced at a higher glucose concentration (5 mg/ml). In the islet perifusion study, DG-5128 was also found to stimulate the glucose-induced insulin secretion in both the first and the second phases of the reaction, in contrast to tolbutamide which stimulated only the first phase of insulin secretion from the perifused islets. DG-5128 gave no significant effect on the glucose-stimulated increase in incorporation of [³H]leucine into the pro-insulin and insulin fractions, while tolbutamide significantly inhibited the incorporation especially at a low glucose concentration (1 mg/ml). These and the previous findings indicate that DG-5128 is a new class of hypoglycemic agent with a unique mode of action different from the known hypoglycemics ever reported.     

SREBP‐1c,Pdx‐1, and GLP‐1R involved in palmitate–EPA regulated glucose‐stimulated insulin secretion in INS‐1 cells

Impairment of glucose‐stimulated insulin secretion (GSIS) caused by glucolipotoxicity is an essential feature in type 2 diabetes mellitus (T2DM). Palmitate and eicosapentaenoate (EPA), because of their lipotoxicity and protection effect, were found to impair or restore the GSIS in beta cells. Furthermore, palmitate was found to up‐regulate the expression level of sterol regulatory element‐binding protein (SREBP)‐1c and down‐regulate the levels of pancreatic and duodenal homeobox (Pdx)‐1 and glucagon‐like peptide (GLP)‐1 receptor (GLP‐1R) in INS‐1 cells. To investigate the underlying mechanism, the lentiviral system was used to knock‐down or over‐express SREBP‐1c and Pdx‐1, respectively. It was found that palmitate failed to suppress the expression of Pdx‐1 and GLP‐1R in SREBP‐1c‐deficient INS‐1 cells. Moreover, down‐regulation of Pdx‐1 could cause the low expression of GLP‐1R with/without palmitate treatment. Additionally, either SREBP‐1c down‐regulation or Pdx‐1 over‐expression could partially alleviate palmitate‐induced GSIS impairment. These results suggested that sequent SREBP‐1c‐Pdx‐1‐GLP‐1R signal pathway was involved in the palmitate‐caused GSIS impairment in beta cells. J. Cell. Biochem. 111: 634–642, 2010. © 2010 Wiley‐Liss, Inc.     

Reduced insulin secretion in response to nutrients in islets from malnourished young rats is associated with a diminished calcium uptake

Changes in (45)Ca uptake and insulin secretion in response to glucose, leucine, and arginine were measured in isolated islets derived from 4-week-old rats born of mothers maintained with normal protein (NP, 17%) or low protein (LP, 6%) diet during pregnancy and lactation. Glucose provoked a dose-dependent stimulation of insulin secretion in both groups of islets, with basal (2.8 mmol/L glucose) and maximal release (27.7 mmol/L glucose) significantly reduced in LP compared with NP islets. In the LP group the concentration-response curve to glucose was shifted to the right compared with the NP group, with the half-maximal response occurring at 16.9 and 13.3 mmol/L glucose, respectively. In LP islets, glucose-induced first and second phases of insulin secretions were drastically reduced. In addition, insulin response to individual amino acids, or in association with glucose, was also significantly reduced in the LP group compared with NP islets. Finally, in LP islets the (45)Ca uptake after 5 minutes or 90 minutes of incubation (which reflect mainly the entry and retention, respectively, of Ca(2+)), was lower than in NP islets. These data indicate that in malnourished rats both initial and sustained phases of insulin secretion in response to glucose were reduced. This poor secretory response to nutrients seems to be the consequence of an altered Ca(2+) handling by malnourished islet cells.     

Overexpression of Nmnat3 efficiently increases NAD and NGD levels and ameliorates age‐associated insulin resistance

Nicotinamide adenine dinucleotide (NAD) is an important cofactor that regulates various biological processes, including metabolism and gene expression. As a coenzyme, NAD controls mitochondrial respiration through enzymes of the tricarboxylic acid (TCA) cycle, β‐oxidation, and oxidative phosphorylation and also serves as a substrate for posttranslational protein modifications, such as deacetylation and ADP‐ribosylation by sirtuins and poly(ADP‐ribose) polymerase (PARP), respectively. Many studies have demonstrated that NAD levels decrease with aging and that these declines cause various aging‐associated diseases. In contrast, activation of NAD metabolism prevents declines in NAD levels during aging. In particular, dietary supplementation with NAD precursors has been associated with protection against age‐associated insulin resistance. However, it remains unclear which NAD synthesis pathway is important and/or efficient at increasing NAD levels in vivo. In this study, Nmnat3 overexpression in mice efficiently increased NAD levels in various tissues and prevented aging‐related declines in NAD levels. We also demonstrated that Nmnat3‐overexpressing (Nmnat3 Tg) mice were protected against diet‐induced and aging‐associated insulin resistance. Moreover, in skeletal muscles of Nmnat3 Tg mice, TCA cycle activity was significantly enhanced, and the energy source for oxidative phosphorylation was shifted toward fatty acid oxidation. Furthermore, reactive oxygen species (ROS) generation was significantly suppressed in aged Nmnat3 Tg mice. Interestingly, we also found that concentrations of the NAD analog nicotinamide guanine dinucleotide (NGD) were dramatically increased in Nmnat3 Tg mice. These results suggest that Nmnat3 overexpression improves metabolic health and that Nmnat3 is an attractive therapeutic target for metabolic disorders that are caused by aging.     

Zingerone induced caspase‐dependent apoptosis in MCF‐7 cells and prevents 7,12‐dimethylbenz(a)anthracene‐induced mammary carcinogenesis in experimental rats

Breast cancer is a prevalent of tumoregenesis in women and reports for the maximum mortality and morbidity in the global. Ginger (Zingiber officinale) is the mainly widespread spice and herbal remedies used in the world. Since antique periods, ginger has been used in Greece, India and China for the curing of upset stomach, nausea, diarrhea, colds, and headaches. The current work was planned to explore the anticancer properties of zingerone (ZO) toward 7,12‐dimethylbenz(a)anthracene (DMBA)‐treated mammary carcinogenesis in Sprague‐Dawley (SD) rats and MCF‐7 mammary cancer cells. The mammary carcinogenesis was produced through a single dosage of DMBA (20 mg/kg bwt) mixed in soya oil (1 mL) administrated intragastrically with a gavage. We found improved concentrations of lipid peroxidation (LOOH and TBARS), carcinoembryonic antigen, lowered levels of enzymatic (CAT, GPx, and SOD), and nonenzymatic (vitamin E, GSH, and vitamin C) antioxidant in mammary tissues and plasma of DMBA‐induced cancer bearing animals. Moreover, augmented concentrations of phase I (Cyt‐b₅ and CYP₄₅₀) and reduced levels of phase II (GR and GST) detoxification microsomal proteins in mammary tissues were noticed. ZO administrations significantly reverted back to all these parameters in this way, showing efficient of anticancer effect. Furthermore, our in vitro study also supported the anticancer effect of the treatment of ZO were noticed loss of cell viability, improved reactive oxygen species formation, and reduced MMP. Furthermore, the status of apoptosis proteins such as Bcl‐2, Bax, and Bid expressions was determined by using Western blot analysis techniques. Overall, these results proposed the anticancer effect of ZO toward DMBA‐induced mammary cancer in SD animals and Michigan cancer foundation‐7 mammary cancer cells.