L-type channel antagonists are of therapeutic benefit in the treatment of hyperlipidaemia and insulin resistance. Our aim was to identify L-type voltage-gated Ca2+ channels in white fat adipocytes, and determine if they affect intracellular Ca2+, lipolysis and lipogenesis. We used a multidisciplinary approach of molecular biology, confocal microscopy, Ca2+ imaging and metabolic assays to explore this problem using adipocytes isolated from adult rat epididymal fat pads. CaV1.2, CaV1.3 and CaV1.1 alpha1, beta and alpha2delta subunits were detected at the gene expression level. The CaV1.2 and CaV1.3 alpha1 subunits were identified in the plasma membrane at the protein level. Confocal microscopy with fluorescent antibodies labelled CaV1.2 in the plasma membrane. Ca2+ imaging revealed that the intracellular Ca2+ concentration, [Ca2 +]i was reversibly decreased by removal of extracellular Ca2+, an effect mimicked by verapamil, nifedipine and Co2+, all blockers of L-type channels, whereas the Ca2+ channel agonist BAY-K8644 increased [Ca2+]i. The finding that the magnitude of these effects correlated with basal [Ca2+]i suggests that adipocyte [Ca2+]i is controlled by L-type Ca2+ channels that are constitutively active at the adipocyte depolarized membrane potential. Pharmacological manipulation of L-type channel activity modulated both basal and catecholamine-stimulated lipolysis but not insulin-induced glucose uptake or lipogenesis. We conclude that white adipocytes have constitutively active L-type Ca2+ channels which explains their sensitivity of lipolysis to Ca2+ channel modulators. Our data suggest CaV1.2 as a potential novel therapeutic target in the treatment of obesity.
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- Abstract: Diabetes x
- Abstract: Islets x
- Abstract: Insulin x
- Abstract: BetaCells x
- Abstract: Pancreas x
- Abstract: Obesity x
- Abstract: Glucose x
- Abstract: Hyperglycemia x
- Abstract: Hypoglycemia x
- Abstract: Insulinoma x
- Abstract: Glucagon x
- Abstract: IGF* x
- Abstract: Type 2 x
Olena A Fedorenko, Pawitra Pulbutr, Elin Banke, Nneoma E Akaniro-Ejim, Donna C Bentley, Charlotta S Olofsson, Sue Chan and Paul A Smith
Linda Ahlkvist, Bilal Omar, Anders Valeur, Keld Fosgerau and Bo Ahrén
Stimulation of insulin secretion by short-term glucagon receptor (GCGR) activation is well characterized; however, the effect of long-term GCGR activation on β-cell function is not known, but of interest, since hyperglucagonemia occurs early during development of type 2 diabetes. Therefore, we examined whether chronic GCGR activation affects insulin secretion in glucose intolerant mice. To induce chronic GCGR activation, high-fat diet fed mice were continuously (2 weeks) infused with the stable glucagon analog ZP-GA-1 and challenged with oral glucose and intravenous glucose±glucagon-like peptide 1 (GLP1). Islets were isolated to evaluate the insulin secretory response to glucose±GLP1 and their pancreas were collected for immunohistochemical analysis. Two weeks of ZP-GA-1 infusion reduced insulin secretion both after oral and intravenous glucose challenges in vivo and in isolated islets. These inhibitory effects were corrected for by GLP1. Also, we observed increased β-cell area and islet size. We conclude that induction of chronic ZP-GA-1 levels in glucose intolerant mice markedly reduces insulin secretion, and thus, we suggest that chronic activation of the GCGR may contribute to the failure of β-cell function during development of type 2 diabetes.
Paige V Bauer and Frank A Duca
The rising global rates of type 2 diabetes and obesity present a significant economic and social burden, underscoring the importance for effective and safe therapeutic options. The success of glucagon-like-peptide-1 receptor agonists in the treatment of type 2 diabetes, along with the potent glucose-lowering effects of bariatric surgery, highlight the gastrointestinal tract as a potential target for diabetes treatment. Furthermore, recent evidence suggests that the gut plays a prominent role in the ability of metformin to lower glucose levels. As such, the current review highlights some of the current and potential pathways in the gut that could be targeted to improve glucose homeostasis, such as changes in nutrient sensing, gut peptides, gut microbiota and bile acids. A better understanding of these pathways will lay the groundwork for novel gut-targeted antidiabetic therapies, some of which have already shown initial promise.
The proglucagon gene (gcg) encodes a number of peptide hormones that are of cell-type specifically expressed in the pancreatic islets, the distal ileum and the large intestine, as well as certain brain neuronal cells. These hormones are important in controlling blood glucose homeostasis, intestinal cell proliferation, and satiety. More importantly, the major hormone generated in the pancreas (i.e. glucagon) exerts opposite effects to the ones that are produced in the intestines (i.e. glucagon-like peptide-1 (GLP-1) and GLP-2). To understand the mechanisms underlying cell-type-specific gcg expression may lead to the identification of novel drug targets to control endogenous hormone production for therapeutic purposes. Extensive in vitro examinations have shown that more than a half dozen of homeodomain (HD) proteins are able to interact with the gcg gene promoter and activate its expression. In vivo ‘knock-out’ mouse studies, however, cannot demonstrate the role of some of them (i.e. Cdx-2, Brn-4, and Nkx6.2) in the development of pancreatic islet α-cells, suggesting that these HD proteins may exert some redundant functions in the genesis of gcg-producing cells. Investigations have also revealed that gcg expression is controlled by both protein kinase A and Epac signaling pathways in response to cAMP elevation, and cell-type specifically controlled by insulin and the effectors of the Wnt signaling pathway. This review summarizes our current understanding on the mechanisms underlying gcg transcription and presented my interpretations on how the interactions between different signaling networks regulate gcg expression.
S. J. Winder, S. D. Wheatley and I. A. Forsyth
Sucrose density centrifugation was used to prepare a partially purified membrane fraction from the mammary glands of non-pregnant, pregnant and lactating sheep. The binding of125 I-labelled insulin-like growth factor-I (IGF-I) was dependent on membrane protein concentration, pH, time and temperature. The binding showed the characteristics of a type-1 IGF receptor, being displaced by IGF-I (median effective dose (ED50) 0·55 nmol/l), less effectively by IGF-II (ED50 8·8 nmol/l) and least effectively by insulin. Glucagon, ovine prolactin and ovine placental lactogen could not displace binding. A molecular weight of 135 000 was determined by affinity cross-linking using disuccinimidyl suberate; this was consistent with the reported size of the type-1 receptor α-subunit. Scatchard analysis was used to determine binding affinity and numbers of IGF-I-binding sites. A single class of high-affinity binding sites was found in all physiological states. In non-pregnant sheep and sheep at days 40, 75 and 110–120 of pregnancy and at term, the binding affinity was similar (apparent dissociation constant (K d) 2·73 ±0·31 nmol/l, n = 22). In lactating sheep (weeks 1, 4 and 10), the binding affinity was significantly (P = 0·02) higher (K d 0·77± 0·06 nmol/l n = 9). Binding capacity was similar in non-pregnant and pregnant sheep (1005 ± 113 fmol/mg, n = 19), but fell by parturition and remained low in lactation (570±52 fmol/mg membrane protein, n = 12). The results suggest that the mammary growth of pregnancy is not regulated at the level of the type-1 IGF receptor.
Journal of Endocrinology (1993) 136, 297–304
ST Dheen, K Rajkumar and LJ Murphy
Transgenic mice which overexpress insulin-like growth factor binding protein-1 (IGFPB-1) demonstrate fasting hyperglycemia, hyperinsulinemia and glucose intolerance in adult life. Here we have examined the ontogeny of pancreatic endocrine dysfunction and investigated islet cell proliferation and apoptosis in this mouse model. In addition we have examined pancreatic insulin content in transgenic mice derived from blastocyst transfer into non-transgenic mice. Transgenic mice were normoglycemic at birth but had markedly elevated plasma insulin levels, 56.2 +/- 4.5 versus 25.4 +/- 1.5 pmol/l, p < 0.001, and pancreatic insulin concentration, 60.5 +/- 2.5 versus 49.0 +/- 2.6 ng/mg of tissue, P < 0.01, compared with wild-type mice. Transgenic mice derived from blastocyst transfer to wild-type foster mothers had an elevated pancreatic insulin content similar to that seen in pups from transgenic mice. There was an age-related decline in pancreatic insulin content and plasma insulin levels and an increase in fasting blood glucose concentrations, such that adult transgenic mice had significantly less pancreatic insulin than wild-type mice. Pancreatic islet number and the size of mature islets were increased in transgenic animals at birth compared with wild-type mice. Both islet cell proliferation, measured by 5-bromo-2'-deoxyuridine labeling, and apoptosis, assessed by the in situ terminal deoxynucleotidyl transferase and nick translation assay, were increased in islets of newborn transgenic mice compared with wild-type mice. In adult mice both islet cell proliferation and apoptosis were low and similar in transgenic and wild-type mice. Islets remained significantly larger and more numerous in adult transgenic mice despite a reduction in pancreatic insulin content. These data suggest that overexpression of IGFBP-1, either directly or indirectly via local or systemic mechanisms, has a positive trophic effect on islet development.
Malin Fex, Lisa M Nicholas, Neelanjan Vishnu, Anya Medina, Vladimir V Sharoyko, David G Nicholls, Peter Spégel and Hindrik Mulder
Mitochondrial metabolism is a major determinant of insulin secretion from pancreatic β-cells. Type 2 diabetes evolves when β-cells fail to release appropriate amounts of insulin in response to glucose. This results in hyperglycemia and metabolic dysregulation. Evidence has recently been mounting that mitochondrial dysfunction plays an important role in these processes. Monogenic dysfunction of mitochondria is a rare condition but causes a type 2 diabetes-like syndrome owing to β-cell failure. Here, we describe novel advances in research on mitochondrial dysfunction in the β-cell in type 2 diabetes, with a focus on human studies. Relevant studies in animal and cell models of the disease are described. Transcriptional and translational regulation in mitochondria are particularly emphasized. The role of metabolic enzymes and pathways and their impact on β-cell function in type 2 diabetes pathophysiology are discussed. The role of genetic variation in mitochondrial function leading to type 2 diabetes is highlighted. We argue that alterations in mitochondria may be a culprit in the pathogenetic processes culminating in type 2 diabetes.
Yoko Yagishita, Akira Uruno, Dionysios V Chartoumpekis, Thomas W Kensler and Masayuki Yamamoto
The transcription factor Nrf2 (NF-E2-related factor 2) plays a critical role in oxidative stress responses. Although activation of Nrf2 signaling is known to exert anti-inflammatory effects, the function of Nrf2 in inflammation-mediated autoimmune disorders, such as type 1 diabetes, is not well established. To address the roles of Nrf2 in protection against autoreactive T-cell-induced type 1 diabetes, we used non-obese diabetic (NOD) mice, which are a polygenic model of human type 1 diabetes, to generate a genetic model for assessment of the contribution of Nrf2 activation to prevention and/or treatment of type 1 diabetes. Because Keap1 (Kelch-like ECH-associated protein 1) negatively regulates Nrf2, we used Keap1 gene knockdown driven by either hypomorphic or knockout Keap1 alleles, which enhanced Nrf2 signaling to moderate or excess levels, respectively. Nrf2 activation in the NOD::Keap1 FA/ – mice inhibited T-cell infiltration within or near the islets, ameliorated impairment of insulin secretion and prevented the development of diabetes mellitus. Notably, Nrf2 activation decreased both the plasma interferon-γ (IFN-γ) levels and the IFN-γ-positive cell numbers in the pancreatic islets. The amelioration of diabetes was also observed in the NOD mice with two hypomorphic Keap1 alleles (Keap1 FA/FA) by intermediate activation of Nrf2. Both NOD::Keap1 FA/ – and NOD::Keap1 FA/FA mice had a decreased incidence of diabetes mellitus, demonstrating that activation of Nrf2 signaling prevented the onset of type 1 diabetes mellitus in NOD mice. Thus, Nrf2 appears to be a potential target for the prevention and treatment of type 1 diabetes.
K Fosgerau, P Galle, T Hansen, A Albrechtsen, C de Lemos Rieper, B Klarlund Pedersen, L Kongskov Larsen, A Randrup Thomsen, O Pedersen, M Bagge Hansen and A Steensberg
Interleukin-6 (IL6) is critically involved in inflammation and metabolism. About 1% of people produce IL6 autoantibodies (aAb-IL6) that impair IL6 signaling in vivo. We tested the hypothesis that the prevalence of such aAb-IL6 is increased in type 2 diabetic patients and that aAb-IL6 plays a direct role in causing hyperglycemia. In humans, the prevalence of circulating high-affinity neutralizing aAb-IL6 was 2.5% in the type 2 diabetic patients and 1% in the controls (odds ratio 2.5, 95% confidence interval 1.2–4.9, P=0.01). To test for the role of aAb-IL6 in causing hyperglycemia, such aAb-IL6 were induced in mice by a validated vaccination procedure. Mice with plasma levels of aAb-IL6 similar to the 2.5% type 2 diabetic patients developed obesity and impaired glucose tolerance (area under the curve (AUC) glucose, 2056±62 vs 1793±62, P=0.05) as compared with sham-vaccinated mice, when challenged with a high-fat diet. Mice with very high plasma levels of aAb-IL6 developed elevated fasting plasma glucose (mM, 4.8±0.4 vs 3.3±0.1, P<0.001) and impaired glucose tolerance (AUC glucose, 1340±38 vs 916±25, P<0.001) as compared with sham-control mice on normal chow. In conclusion, the prevalence of plasma aAb-IL6 at levels known to impair IL6 signaling in vivo is increased 2.5-fold in people with type 2 diabetes. In mice, matching levels of aAb-IL6 cause obesity and hyperglycemia. These data suggest that a small subset of type 2 diabetes may in part evolve from an autoimmune attack against IL6.
Xue Jiang, Jia Xiao, Mulan He, Ani Ma and Anderson O L Wong
Type II suppressor of cytokine signaling (SOCS) serve as feedback repressors for cytokines and are known to inhibit growth hormone (GH) actions. However, direct evidence for SOCS modulation of GH-induced insulin-like growth factor 1 (Igf1) expression is lacking, and the post-receptor signaling for SOCS expression at the hepatic level is still unclear. To shed light on the comparative aspects of SOCS in GH functions, grass carp was used as a model to study the role of type II SOCS in GH-induced Igf1 expression. Structural identity of type II SOCS, Socs1–3 and cytokine-inducible SH2-containing protein (Cish), was established in grass carp by 5’/3’-RACE, and their expression at both transcript and protein levels were confirmed in the liver by RT-PCR and LC/MS/MS respectively. In carp hepatocytes, GH treatment induced rapid phosphorylation of JAK2, STATs, MAPK, PI3K, and protein kinase B (Akt) with parallel rises in socs1–3 and cish mRNA levels, and these stimulatory effects on type II SOCS were shown to occur before the gradual loss of igf1 gene expression caused by prolonged exposure of GH. Furthermore, GH-induced type II SOCS gene expression could be negated by inhibiting JAK2, STATs, MEK1/2, P38 MAPK, PI3K, and/or Akt respectively. In CHO cells transfected with carp GH receptor, over-expression of these newly cloned type II SOCS not only suppressed JAK2/STAT5 signaling with GH treatment but also inhibited GH-induced grass carp Igf1 promoter activity. These results, taken together, suggest that type II SOCS could be induced by GH in the carp liver via JAK2/STATs, MAPK, and PI3K/Akt cascades and serve as feedback repressors for GH signaling and induction of igf1 gene expression.