Hexosamines stimulate leptin production in transgenic mice

Hexosamine flux has been shown to mediate aspects of nutrient sensing in insulin sensitive tissues and has been hypothesized to represent a satiety signal that results in shunting of fuel toward storage as fat. It has been recently reported that in vitro treatment of fat and muscle cells with hexosamines and acute glucosamine infusion in intact rats stimulate leptin secretion. In order to investigate the effects of chronic, physiologic increases in hexosamine flux on leptin we have examined leptin mRNA and serum leptin in mice overexpressing the rate-limiting enzyme for hexosamine synthesis, GFA, in muscle and fat. Increased levels of UDP-N-acetylglucosamine, the principal end-product of the hexosamine pathway were seen in transgenic fat, consistent with the overexpression of GFA. After overnight fasting, the transgenic mice were hyperleptinemic compared to littermate controls (4.5+/-0.5 ng/ml in transgenic, 2.8+/-0.2 in control, p = 0.005) despite equal body weights. In the random-fed state, the leptin levels of control mice increased to 4.1+/-0.5 ng/ml (p = 0.01) whereas the leptin levels in the transgenics did not increase any further (3.7+/-0.4 ng/ml). Leptin mRNA levels were also increased in transgenic fat (2.7+/-0.6 in transgenic compared to 0.8+/-0.2 in control, arbitrary units normalized to actin, p < 0.007). Despite increased leptin, the transgenic animals did not have lower body fat content. We conclude that hexosamine flux in fat regulates leptin synthesis and secretion.     

Hexosamines regulate leptin production in human subcutaneous adipocytes

The hexosamine biosynthetic pathway has recently been proposed as a mechanism through which cells "sense" nutrient flux to regulate leptin release. This study was undertaken to examine the regulation of leptin production by hexosamines in human adipocytes. Adipose tissue UDP-N-acetylglucosamine, an end product of hexosamine biosynthesis, was elevated 3.2-fold, and ob messenger ribonucleic acid was elevated 2-fold in the sc adipose tissue of 17 obese [body mass index (BMI), 41.3+/-12.0 kg/m2; age, 31+/-5 yr] subjects compared to 14 lean (BMI, 23.4+/-1.6 kg/m2; age, 33+/-11 yr) subjects. Serum leptin was increased 2.7-fold in the obese subjects. A significant positive relationship was found between adipose tissue UDP-N-acetylglucosamine and BMI (Spearman correlation = 0.576; P = 0.0007) and between UDP-N-acetylglucosamine and serum leptin (Spearman correlation = 0.4650; P = 0.0145). Treatment of isolated sc adipocytes with 1 mmol/L glucosamine, an intermediate product in UDP-N-acetylglucosamine biosynthesis, increased leptin release 21.4+/-17.6% (mean +/- SD) over control (P = 0.0365) and 74.5+/-82.8% over control (P = 0.0271) in adipocytes from lean (BMI, 23.2+/-1.6 kg/m2; n = 6) and obese (BMI, 55.4+/-13.0 kg/m2,; n = 9) subjects, respectively, by 48 h of culture. Inhibition of UDP-N-acetylglucosamine biosynthesis with 6-diazo-5-oxo-norleucine reduced glucose-stimulated leptin release from cultured adipocytes 21.8+/-32.4% (P = 0.0395; n = 12) and ob gene expression 19.9+/-18.9% (P = 0.0208; n = 8) by 48 h of treatment. These findings suggest that hexosamine biosynthesis regulates leptin production in human adipose tissue.     

Ocimum sanctum leaf extracts stimulate insulin secretion from perfused pancreas, isolated islets and clonal pancreatic beta-cells

Ocimum sanctum leaves have previously been reported to reduce blood glucose when administered to rats and humans with diabetes. In the present study, the effects of ethanol extract and five partition fractions of O. sanctum leaves were studied on insulin secretion together with an evaluation of their mechanisms of action. The ethanol extract and each of the aqueous, butanol and ethylacetate fractions stimulated insulin secretion from perfused rat pancreas, isolated rat islets and a clonal rat beta-cell line in a concentration-dependent manner. The stimulatory effects of ethanol extract and each of these partition fractions were potentiated by glucose, isobutylmethylxanthine, tolbutamide and a depolarizing concentration of KCl. Inhibition of the secretory effect was observed with diazoxide, verapamil and Ca2+ removal. In contrast, the stimulatory effects of the chloroform and hexane partition fractions were associated with decreased cell viability and were unaltered by diazoxide and verapamil. The ethanol extract and the five fractions increased intracellular Ca2+ in clonal BRIN-BD11 cells, being partly attenuated by the addition of verapamil. These findings indicated that constituents of O. sanctum leaf extracts have stimulatory effects on physiological pathways of insulin secretion which may underlie its reported antidiabetic action.     

Insulin secretory actions of extracts of Asparagus racemosus root in perfused pancreas, isolated islets and clonal pancreatic beta-cells

Asparagus racemosus root has previously been reported to reduce blood glucose in rats and rabbits. In the present study, the effects of the ethanol extract and five partition fractions of the root of A. racemosus were evaluated on insulin secretion together with exploration of their mechanisms of action. The ethanol extract and each of the hexane, chloroform and ethyl acetate partition fractions concentration-dependently stimulated insulin secretion in isolated perfused rat pancreas, isolated rat islet cells and clonal beta-cells. The stimulatory effects of the ethanol extract, hexane, chloroform and ethyl acetate partition fractions were potentiated by glucose, 3-isobutyl-1-methyl xanthine IBMX, tolbutamide and depolarizing concentration of KCl. Inhibition of A. racemosus-induced insulin release was observed with diazoxide and verapamil. Ethanol extract and five fractions increased intracellular Ca(2+), consistent with the observed abolition of insulin secretory effects under Ca(2+) -free conditions. These findings reveal that constituents of A. racemosus root extracts have wide-ranging stimulatory effects on physiological insulinotropic pathways. Future work assessing the use of this plant as a source of active components may provide new opportunities for diabetes therapy.     

Uncoupling protein-2 increases nitric oxide production and TNFAIP3 pathway activation in pancreatic islets

Mutations in the uncoupling protein 2 (Ucp2) gene are linked to type-2 diabetes. Here, a potential mechanism by which lack of UCP2 is cytoprotective in pancreatic β-cells was investigated. Nitric oxide (NO) production was elevated in Ucp2(-/-) islets. Proliferation (cyclin D2, Ccnd2) and anti-apoptosis (Tnfaip3) genes had increased expression in Ucp2(-/-) islets, whereas the mRNA of pro-apoptosis genes (Jun, Myc) was reduced. TNFAIP3 cellular localization was detected in both α- and β-cells of Ucp2(-/-) islets but in neither α- nor β-cells of UCP2(+)(/)(+) islets, where it was detected in pancreatic polypeptide-expressing cells. TNFAIP3 distribution was not markedly altered 14 days after streptozotocin treatment. Basal apoptosis was attenuated in Ucp2(-/-) β-cells, while the nuclear factor κB (NF-κB) pathway was transactivated after islet isolation. Ucp2(+/+) and Ucp2(-/-) islets were treated with cytokines for 24?h. Cytokines did not increase NF-κB transactivation or apoptosis in Ucp2(-/-) islets and TNFAIP3 was more strongly induced in Ucp2(-/-) islets. Inhibition of NO production strongly reduced NF-κB activation and apoptosis. These data show that null expression of Ucp2 induces transactivation of NF-κB in isolated islets, possibly due to NO-dependent up-regulation of inhibitor of κB kinase β activity. NF-κB transactivation appears to result in altered expression of genes that enhance a pro-survival phenotype basally and when β-cells are exposed to cytokines. TNFAIP3 is of particular interest because of its ability to regulate NF-κB signaling pathways.     

Proteasomal activation mediates down-regulation of inositol 1,4,5-trisphosphate receptor and calcium mobilization in rat pancreatic islets

Inositol 1,4,5-trisphosphate receptor (IP3R) protein levels in isolated rat pancreatic islets were investigated in response to carbachol (CCh) and sulfated cholecystokinin 26-33 amide stimulation. Within 2 h, CCh reduced IP3R-I protein levels by 22% and IP3R-II and -III levels to 65% or more below basal. Sulfated cholecystokinin 26-33 amide decreased the levels of IP3R-I, -II, and -III by 34%, 60%, and 66% below basal, respectively. The effect of CCh was concentration- and time-dependent, with a persistent decline in IP3R levels for up to 6 h after the onset of stimulation. CCh-pretreated islets also showed an inhibition of glucose-stimulated insulin secretion. Proteasome inhibition completely blocked the down-regulatory effects of CCh on IP3Rs and significantly increased the insulin secretory response to glucose stimulation in the presence of CCH: Islet stimulation by glucose, alpha-ketoisocaproic acid, and tolbutamide completely protected IP3Rs against the down-regulatory effects of CCH: 2-deoxyglucose and 3-O-methyl glucose failed to affect CCh-induced IP3R down-regulation. The protective effects of glucose on IP3R down-regulation were completely inhibited by the Ca(2+) channel-blocking agent nimodipine. Intracellular Ca(2+) ([Ca(2+)](i)) levels in Fura-2 (fluorescent Ca(2+) indicator)-loaded islets, in the absence of extracellular Ca(2+), increased in response to glucose stimulation; but in islets pretreated with CCh, glucose did not increase [Ca(2+)](i) above basal levels. However, in islets pretreated with CCh and the proteasomal inhibitor MG-132 (carbobenzoxyl-leucinyl-leucinyl-leucinyl-H), the glucose-stimulated increase in [Ca(2+)](i) was significantly higher than the change observed for glucose-stimulated [Ca(2+)](i) in the absence of MG-132. The results suggest that muscarinic receptor stimulation modulates IP3R protein levels in islets through a proteasomal activation pathway, and that down-regulation of IP3Rs has a profound effect on Ca(2+) mobilization in islets that may relate to insulin secretory responsiveness.     

Nutrient and hormone-neurotransmitter stimuli induce hydrolysis of polyphosphoinositides in rat pancreatic islets

Preincubation of rat pancreatic islets with 3H-inositol, and subsequent exposure, in the presence of LiCl, to either glucose or carbamylcholine resulted in a rapid stimulation of 3H-inositol 1,4,5-triphosphate and 3H-myo-inositol 1,4-bisphosphate formation, the level of which reached a plateau after about 5 min of stimulation. Both stimuli also caused an approximately linear accumulation of 3H-myo-inositol 1-phosphate. The amounts of 3H-inositol phosphates formed were dependent on the concentration of LiCl. Studies of 32P-labeling of islet ATP, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), and phosphatidylinositol 4-phosphate revealed that these approached isotopic equilibrium after about 240-min incubation, whereas 32P-labeling of phosphatidylinositol, phosphatidic acid, phosphatidylcholine, and phosphatidylethanolamine proceeded at a lower rate. Carbamylcholine provoked an immediate fall in 32P-PtdIns(4,5)P2 and, to a lesser extent, 32P-phosphatidylinositol 4-phosphate. Glucose caused a similar response although, in this case, the most marked decline was in a more polar 32P-labeled lipid. Cholecystokinin-pancreozymin was also found to induce 32P-PtdIns(4,5)P2 hydrolysis, although the ionophore A23187 was without effect. Both carbamylcholine and glucose induced an increase in 32P-phosphatidic acid. The results provide two independent pieces of evidence suggesting that phospholipase C-mediated hydrolysis of polyphosphoinositides occurs as an early response in rat islets to either nutrient or neurotransmitter secretagogues.     

Leptin decreases apoptosis and alters BCL-2 : Bax ratio in clonal rodent pancreatic beta-cells

AIMS/HYPOTHESIS: The adipocyte derived peptide hormone leptin is known to regulate apoptosis and cell viability in several cells and tissues, as well as having several pancreatic islet beta-cell specific effects such as inhibition of glucose-stimulated insulin secretion. This study investigated the effects of leptin upon apoptosis induced by serum depletion and on expression of the apoptotic regulators B-cell leukaemia 2 gene product (BCL-2) and BCL2-associated X protein (Bax) in the glucose-responsive BRIN-BD11 beta-cell line. METHODS: BRIN-BD11 cells were cultured in RPMI 1640 and subsequently serum depleted +/- leptin (10 and 50 ng/mL) for 24 h. Cell viability and apoptosis were measured using a modified MTS assay and TUNEL/YO-PRO-1 assays, respectively. BCL-2 and Bax expression were measured by real-time PCR and Western blotting. RESULTS: Leptin caused a reduction in serum-depleted apoptosis, although it failed to have any effect on the overall cell viability, causing a 68% shift from apoptosis to necrosis. Leptin significantly increased the level of BCL-2 mRNA expression (150% compared to serum depletion alone), without altering Bax mRNA expression. At the protein level, leptin increased BCL-2 and decreased Bax, altering the BCL-2 : Bax ratio. CONCLUSIONS: We conclude that leptin reduces apoptosis in beta-cells at physiological concentrations, possibly via its ability to up-regulate BCL-2 and Bax expression.     

Hexosamines as mediators of nutrient sensing and regulation in diabetes

High concentrations of glucose induce insulin resistance, impair insulin secretion, and affect hepatic glucose production in a manner that mirrors Type 2 diabetes, and hexosamines mimic many of these effects. This has led to the hypothesis that cells use hexosamine flux as a glucose- and satiety-sensing pathway. The hexosamine hypothesis for glucose sensing has been validated by overexpressing the rate-limiting enzyme for hexosamine synthesis, glutamine: fructose-6-phosphate amidotransferase (GFA) in several tissues including muscle, liver, fat, and beta cells. With overexpression of GFA in transgenic animals, skeletal muscle becomes insulin resistant, the liver synthesizes excess fatty acid, and the beta cell secretes excess insulin leading to hyperinsulinemia. Thus, excess hexosamine flux leads to a coordinated response whereby fuel is shunted toward long-term storage, mirroring the "thrifty phenotype." Chronically, however, these same adaptive changes result ultimately in obesity, hyperlipidemia, beta cell failure, and Type 2 diabetes. These results suggest a mechanism by which chronic overnutrition leads to the phenotype of Type 2 diabetes.     

CD40 activation in human pancreatic islets and ductal cells

AIMS/HYPOTHESIS: CD40 expression on non-haematopoietic cells is linked to inflammation. We previously reported that CD40 is expressed on isolated human and non-human primate islets and its activation results in secretion of IL-8, macrophage inflammatory protein 1-beta (MIP-1beta) and monocyte chemoattractant protein-1 (MCP-1) through nuclear factor-kappaB and extracellularly regulated kinases 1/2 pathways. The objective of this study was to identify the pattern of gene expression, and to study viability and functionality affected by CD40-CD40 ligand (CD40L) interaction in human islets. Furthermore, we have studied the CD40-mediated cytokine/chemokine profile in pancreatic ductal cells, as they are always present in human islet transplant preparations and express CD40 constitutively. METHODS: CD40-CD40L gene expression modulation was studied by microarray on islet cells depleted of ductal cells. Selected genes were validated by quantitative RT-PCR. The cytokine profile in purified ductal cells was evaluated by Luminex technology, based on the use of fluorescent-coated beads, known as microspheres, and capable of multiplex detection of proteins from a single sample. Glucose-stimulated insulin secretion and islet viability were assessed by perifusion and 7-aminoactinomycin D membrane exclusion, respectively. RESULTS: Statistical analysis of microarrays identified 30 genes exhibiting at least a 2.5-fold increase across all replicate arrays. The majority of them were related to oxidative stress/inflammation. Prominently upregulated were chemokine C-X-C motif ligand 1 (CXCL1), CXCL2 and CXCL3 belonging to the CXC family of chemokines related to IL-8. CD40-mediated CXCL1 secretion was confirmed by ELISA. The viability or in vitro function was not affected by CD40 activation. In addition to previously reported IL-8, MIP-1beta and MCP-1, CD40 stimulation in ductal cells produced IL-1beta, IFN-gamma, TNF-alpha, granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor. CONCLUSIONS/INTERPRETATION: CD40 activation in islets and ductal cells produces cytokines/chemokines with a broad-spectrum range of biological functions.     

Effects of nutrient secretagogues upon phospholipid metabolism in rat pancreatic islets

Rat pancreatic islets labelled with [32P]Pi were used to investigate the effects of nutrient secretagogues upon phospholipid metabolism. Stimulatory concentrations of glucose, 4-methyl-2-oxopentanoate, 3-phenylpyruvate and 2-endo-aminonorbornane-2-carboxylic acid caused a marked and specific stimulation of labelling with [32P]Pi of phosphatidylinositol, phosphatidic acid and the polyphosphoinositides. The effects of glucose were concentration-related and were inhibited by mannoheptulose and menadione. Enhanced phospholipid labelling persisted in the absence of added calcium ions but was abolished by excess EDTA. The time-course of labelling of these phospholipids in response to glucose contrasted with that previously observed using carbamylcholine in that the accumulation of radioactivity in phosphatidic acid and in phosphatidylinositol occurred in parallel. We conclude that glucose, in common with other nutrient secretagogues or those which promote the metabolism of endogenous nutrients, causes a marked stimulation of turnover in the phosphatidylinositol cycle. This effect requires the integrity of nutrient catabolism and is probably not dependent upon the stimulation of Ca2+ uptake from the extracellular medium.     

Adenine nucleotide pattern in rat pancreatic islets exposed to nutrient secretagogues

The effects of d-glucose, d-mannose, d-galactose, d-glyceraldehyde, pyruvate, l-lactate, 2-ketoisocaproate, l-leucine, and/or l-glutamine on the ATP and ADP content of rat isolated pancreatic islets were reevaluated in order to compare changes evoked by these nutrient secretagogues in the islet ATP content and ATP/ADP ratio to their effects upon insulin release. Although being compatible with the fuel concept for nutrient-stimulated insulin secretion, the results of this study also argue against the monolithic view that the adenine nucleotide pattern in islet cells represents the sole coupling factor between metabolic and more distal events in the process of nutrient-stimulated insulin release.     

Forskolin-induced activation of adenylate cyclase, cyclic adenosine monophosphate production and insulin release in rat pancreatic islets

Forskolin activated adenylate cyclase in rat islet homogenates and stimulated cAMP production in intact islets incubated in the absence or presence of either D-glucose or Ca2+. Forskolin failed to affect D-[U-14C]glucose oxidation, glucose-stimulated net 45Ca uptake, or basal insulin release, but enhanced insulin secretion evoked by either nutrients (D-glucose, 2-ketoisocaproate, L-leucine alone or in combination with L-glutamine), or nonnutrient secretagogues (12-O-tetradecanoylphorbol-13-acetate, Ba2+ alone or in combination with theophylline). Forskolin stimulated insulin release from islets incubated in the presence of glucose but in the absence of Ca2+. These findings confirm that a marked increase in cAMP production is not sufficient to cause sustained insulin release. They also suggest that the enhancing action of endogenous cAMP upon insulin release does not depend on a facilitation of Ca2+ influx into islet cells.     

Induction of monocyte chemoattractant protein-1 expression by angiotensin II in the pancreatic islets and beta-cells

Angiotensin II (AngII), the principal hormone of the renin-angiotensin system, is actively generated in the pancreas and has been suggested as a key mediator of inflammation. Monocyte chemoattractant protein-1 (MCP-1) is a chemokine that plays an important role in the recruitment of mononuclear cells into the pancreatic islets. In this study, we investigated the potential molecular basis for the role of AngII in islet inflammation through studying its effect on MCP-1. AngII significantly increased the expression of MCP-1 mRNA and protein in the RINm5F beta-cell line and activated MCP-1 promoter. AngII-MCP-1 mRNA induction was inhibited by an AngII type 1 receptor antagonist but was unchanged by an AngII type 2 receptor antagonist. AngII-MCP-1 induction was inhibited by the tyrosine kinase inhibitor genistein, suggesting a MAPK signaling mechanism. AngII activated the phosphorylation of ERK1/2 but not p38 or c-Jun NH(2)-terminal MAPKs. Inhibition of ERK1/2 activation reduced the AngII-induced MCP-1 synthesis. In nonobese diabetic mice pancreata, the temporal pattern of angiotensin-converting enzyme expression correlated well with progression of insulitis and beta-cell destruction. Immunostaining of pancreatic serial sections show colocalization of angiotensin-converting enzyme with MCP-1 in beta-cells in the islets. In freshly isolated islets from normoglycemic mice, AngII alone and in combination with IL-1beta elicited an inflammatory response by stimulation of MCP-1. Our data suggest a positive autocrine/paracrine action for the local pancreatic AngII-generating system during insulitis and provide the first insight into an AngII-initiated signal transduction pathway that regulates MCP-1 as a possible inflammatory mechanism in the islets.