Heather C. Denroche

Circulating miR-375 as a Biomarker of β-Cell Death and Diabetes in Mice

Type 1 diabetes is a progressive autoimmune disease that is largely silent in its initial stages. Yet, sensitive methods for detection of β-cell death and prediction and prevention of diabetes are lacking. Micro-RNAs (miRNAs) have been found at high concentrations in body fluids. Here in this study we sought to determine whether an islet enriched miRNA, miR-375, is a suitable blood marker to detect β-cell death and predict diabetes in mice. We measured miR-375 levels by quantitative RT-PCR in plasma samples of streptozotocin (STZ)-treated C57BL/6 mice and nonobese diabetic (NOD) mice. We also measured miR-375 levels in media samples of cytokine- or STZ-treated islets in the presence or absence of cell-death inhibitors. High-dose STZ administration dramatically increased circulating miR-375 levels, prior to the onset of hyperglycemia. Similarly, in the NOD mouse model of autoimmune diabetes, circulating miR-375 levels were significantly increased 2 weeks before diabetes onset. Moreover, cytokine- and STZ-induced cell death in isolated mouse islets produced a striking increase in extracellular miR-375 levels, which was reduced by cell death inhibitors. These data suggest that circulating miR-375 can be used as a marker of β-cell death and potential predictor of diabetes.

Leptin Therapy Reverses Hyperglycemia in Mice With Streptozotocin-Induced Diabetes, Independent of Hepatic Leptin Signaling

OBJECTIVE Leptin therapy has been found to reverse hyperglycemia and prevent mortality in several rodent models of type 1 diabetes. Yet the mechanism of leptin-mediated reversal of hyperglycemia has not been fully defined. The liver is a key organ regulating glucose metabolism and is also a target of leptin action. Thus we hypothesized that exogenous leptin administered to mice with streptozotocin (STZ)-induced diabetes reverses hyperglycemia through direct action on hepatocytes. RESEARCH DESIGN AND METHODS After the induction of diabetes in mice with a high dose of STZ, recombinant mouse leptin was delivered at a supraphysiological dose for 14 days by an osmotic pump implant. We characterized the effect of leptin administration in C57Bl/6J mice with STZ-induced diabetes and then examined whether leptin therapy could reverse STZ-induced hyperglycemia in mice in which hepatic leptin signaling was specifically disrupted. RESULTS Hyperleptinemia reversed hyperglycemia and hyperketonemia in diabetic C57Bl/6J mice and dramatically improved glucose tolerance. These effects were associated with reduced plasma glucagon and growth hormone levels and dramatically enhanced insulin sensitivity, without changes in glucose uptake by skeletal muscle. Leptin therapy also ameliorated STZ-induced hyperglycemia and hyperketonemia in mice with disrupted hepatic leptin signaling to a similar extent as observed in wild-type littermates with STZ-induced diabetes. CONCLUSIONS These observations reveal that hyperleptinemia reverses the symptoms of STZ-induced diabetes in mice and that this action does not require direct leptin signaling in the liver.

IL-33 Reverses an Obesity-Induced Deficit in Visceral Adipose Tissue ST2+ T Regulatory Cells and Ameliorates Adipose Tissue Inflammation and Insulin Resistance

Obesity is associated with insulin resistance and inflammation thought to be caused by a visceral adipose tissue (VAT)–localized reduction in immunoregulatory cells and increase in proinflammatory immune cells. We previously found that VAT regulatory T cells (Tregs) normally express high levels of IL-10 and that expression of this cytokine in VAT Tregs is specifically reduced in mice fed a high-fat diet. In this study, we further investigated the phenotype of VAT Tregs and found that the majority of IL-10–expressing Tregs in the VAT of lean mice also expressed the ST2 chain of the IL-33R. In addition to high expression of IL-10, ST2+ Tregs in lean VAT expressed higher proportions of Th2-associated proteins, including GATA3 and CCR4, and Neuropillin-1 compared with ST2− Tregs. The proportion of ST2+ Tregs in VAT was severely diminished in obese mice that had been fed a high-fat/sucrose diet, and this effect could be completely reversed by treatment with IL-33. IL-33 treatment also reversed VAT inflammation in obese mice and resulted in a reduction of hyperinsulinemia and insulin resistance. These data suggest that IL-33 contributes to the maintenance of the normal pool of ST2+ Tregs in the VAT, and that therapeutic administration of IL-33 results in multiple anti-obesity effects, including the reversal of VAT inflammation and alleviation of insulin resistance.

Hepatic leptin signalling and subdiaphragmatic vagal efferents are not required for leptin-induced increases of plasma IGF binding protein-2 (IGFBP-2) in ob/ob mice

Aims/hypothesisThe fat-derived hormone leptin plays a crucial role in the maintenance of normal body weight and energy expenditure as well as in glucose homeostasis. Recently, it was reported that the liver-derived protein, insulin-like growth factor binding protein-2 (IGFBP-2), is responsible for at least some of the glucose-normalising effects of leptin. However, the exact mechanism by which leptin upregulates IGFBP-2 production is unknown. Since it is believed that circulating IGFBP-2 is predominantly derived from the liver and leptin has been shown to have both direct and indirect actions on the liver, we hypothesised that leptin signalling in hepatocytes or via brain–liver vagal efferents may mediate leptin control of IGFBP-2 production.MethodsTo address our hypothesis, we assessed leptin action on glucose homeostasis and plasma IGFBP-2 levels in both leptin-deficient ob/ob mice with a liver-specific loss of leptin signalling and ob/ob mice with a subdiaphragmatic vagotomy. We also examined whether restoring hepatic leptin signalling in leptin receptor-deficient db/db mice could increase plasma IGFBP-2 levels.ResultsContinuous leptin administration increased plasma IGFBP-2 levels in a dose-dependent manner, in association with reduced plasma glucose and insulin levels. Interestingly, leptin was still able to increase plasma IGFBP-2 levels and improve glucose homeostasis in both ob/ob mouse models to the same extent as their littermate controls. Further, restoration of hepatic leptin signalling in db/db mice did not increase either hepatic or plasma IGFBP-2 levels.Conclusions/interpretationTaken together, these data indicate that hepatic leptin signalling and subdiaphragmatic vagal inputs are not required for leptin upregulation of plasma IGFBP-2 nor blood glucose lowering in ob/ob mice.

Differential Activation of Innate Immune Pathways by Distinct Islet Amyloid Polypeptide (IAPP) Aggregates.

Aggregation of islet amyloid polypeptide (IAPP) contributes to beta cell dysfunction in type 2 diabetes and islet transplantation. Like other amyloidogenic peptides, human IAPP induces macrophage IL-1β secretion by stimulating both the synthesis and processing of proIL-1β, a pro-inflammatory cytokine that (when chronically elevated) impairs beta cell insulin secretion. We sought to determine the specific mechanism of IAPP-induced proIL-1β synthesis. Soluble IAPP species produced early during IAPP aggregation provided a Toll-like-receptor-2- (TLR2-) dependent stimulus for NF-κB activation in HEK 293 cells and bone marrow-derived macrophages (BMDMs). Non-amyloidogenic rodent IAPP and thioflavin-T-positive fibrillar amyloid produced by human IAPP aggregation failed to activate TLR2. Blockade of TLR6 but not TLR1 prevented hIAPP-induced TLR2 activation, consistent with stimulation of a TLR2/6 heterodimer. TLR2 and its downstream adaptor protein MyD88 were required for IAPP-induced cytokine production by BMDMs, a process that is partially dependent on autoinduction by IL-1. BMDMs treated with soluble but not fibrillar IAPP provided a TLR2-dependent priming stimulus for ATP-induced IL-1β secretion, whereas late IAPP aggregates induced NLRP3-dependent IL-1β secretion by LPS-primed macrophages. Moreover, inhibition of TLR2 and depletion of islet macrophages prevented up-regulation of Il1b and Tnf expression in human IAPP-expressing transgenic mouse islets. These data suggest participation by both soluble and fibrillar aggregates in IAPP-induced islet inflammation. IAPP-induced activation of TLR2 and secretion of IL-1 may be important therapeutic targets to prevent amyloid-associated beta cell dysfunction.

Partial ablation of leptin signaling in mouse pancreatic α-cells does not alter either glucose or lipid homeostasis.

The role of glucagon in the pathological condition of diabetes is gaining interest, and it has been recently reported that its action is essential for hyperglycemia to occur. Glucagon levels, which are elevated in some diabetic models, are reduced following leptin therapy. Likewise, hyperglycemia is corrected in type 1 diabetic mice treated with leptin, although the mechanisms have not been fully determined. A direct inhibitory effect of leptin on mouse and human α-cells has been demonstrated at the levels of electrical activity, calcium signaling, and glucagon secretion. In the present study we employed the Cre-loxP strategy to generate Lepr(flox/flox) Gcg-cre mice, which specifically lack leptin receptors in glucagon-secreting α-cells, to determine whether leptin resistance in α-cells contributes to hyperglucagonemia, and also whether leptin action in α-cells is required to improve glycemia in type 1 diabetes with leptin therapy. Immunohistochemical analysis of pancreas sections revealed Cre-mediated recombination in ∼ 43% of the α-cells. We observed that in vivo Lepr(flox/flox) Gcg-cre mice display normal glucose and lipid homeostasis. In addition, leptin administration in streptozotocin-induced diabetic Lepr(flox/flox) Gcg-cre mice restored euglycemia similarly to control mice. These findings suggest that loss of leptin receptor signaling in close to one-half of α-cells does not alter glucose metabolism in vivo, nor is it sufficient to prevent the therapeutic action of leptin in type 1 diabetes.

Leptin Administration Enhances Islet Transplant Performance in Diabetic Mice

Islet transplantation is an effective method to obtain long-term glycemic control for patients with type 1 diabetes, yet its widespread use is limited by an inadequate supply of donor islets. The hormone leptin has profound glucose-lowering and insulin-sensitizing action in type 1 diabetic rodent models. We hypothesized that leptin administration could reduce the dose of transplanted islets required to achieve metabolic control in a mouse model of type 1 diabetes. We first performed a leptin dose-response study in C57Bl/6 mice with streptozotocin (STZ)-induced diabetes to determine a leptin dose insufficient to reverse hyperglycemia. Subsequently, we compared the ability of suboptimal islet transplants of 50 or 125 syngeneic islets to achieve glycemic control in STZ-induced diabetic C57Bl/6 mice treated with or without this dose of leptin. The dose-response study revealed that leptin reverses STZ-induced diabetes in a dose-dependent manner. Supraphysiological leptin levels were necessary to restore euglycemia but simultaneously increased risk of hypoglycemia, and also lost efficacy after 12 days of administration. In contrast, 1 µg/day leptin only modestly reduced blood glucose but maintained efficacy throughout the study duration. We then administered 1 µg/day leptin to diabetic mice that underwent transplantation of 50 or 125 islets. Although these islet doses were insufficient to ameliorate hyperglycemia alone, coadministration of leptin with islet transplantation robustly improved control of glucose and lipid metabolism, without increasing circulating insulin levels. This study reveals that low-dose leptin administration can reduce the number of transplanted islets required to achieve metabolic control in STZ-induced diabetic mice.

Insulin Knockout Mice Have Extended Survival but Volatile Blood Glucose Levels on Leptin Therapy.

Leptin can reverse hyperglycemia in rodent models of type 1 diabetes. However, these models have used chemical or immune mediated β-cell destruction where insulin depletion is incomplete. Thus it is unknown which actions of leptin are entirely insulin independent, versus those which require insulin. To directly assess this we maximized blockage of insulin action using an insulin receptor antagonist in combination with streptozotocin-diabetic mice; leptin treatment was still able to reduce blood glucose. Next, we leptin-treated adult insulin knockout (InsKO) mice. Remarkably, leptin-treated InsKO mice were viable for up to 3 weeks without insulin therapy. Leptin treatment reduced plasma corticosterone, glucagon, β-hydroxybutyrate, triglycerides, cholesterol, fatty acids and glycerol. However, leptin-treated InsKO mice exhibited overt fed hyperglycemia and severe fasting hypoglycemia. Therefore, leptin can normalize many metabolic parameters in the complete absence of insulin, but blood glucose levels are volatile and the length of survival finite.

Impaired Ca2+ Signaling in β-Cells Lacking Leptin Receptors by Cre-loxP Recombination

Obesity is a major risk factor for diabetes and is typically associated with hyperleptinemia and a state of leptin resistance. The impact of chronically elevated leptin levels on the function of insulin-secreting β-cells has not been elucidated. We previously generated mice lacking leptin signaling in β-cells by using the Cre-loxP strategy and showed that these animals develop increased body weight and adiposity, hyperinsulinemia, impaired glucose-stimulated insulin secretion and insulin resistance. Here, we performed several in vitro studies and observed that β-cells lacking leptin signaling in this model are capable of properly metabolizing glucose, but show impaired intracellular Ca2+ oscillations and lack of synchrony within the islets in response to glucose, display reduced response to tolbutamide and exhibit morphological abnormalities including increased autophagy. Defects in intracellular Ca2+ signaling were observed even in neonatal islets, ruling out the possible contribution of obesity to the β-cell irregularities observed in adults. In parallel, we also detected a disrupted intracellular Ca2+ pattern in response to glucose and tolbutamide in control islets from adult transgenic mice expressing Cre recombinase under the rat insulin promoter, despite these animals being glucose tolerant and secreting normal levels of insulin in response to glucose. This unexpected observation impeded us from discerning the consequences of impaired leptin signaling as opposed to long-term Cre expression in the function of insulin-secreting cells. These findings highlight the need to generate improved Cre-driver mouse models or new tools to induce Cre recombination in β-cells.

The role of autonomic efferents and uncoupling protein 1 in the glucose-lowering effect of leptin therapy

Objective Leptin reverses hyperglycemia in rodent models of type 1 diabetes (T1D). Direct application of leptin to the brain can lower blood glucose in diabetic rodents, and can activate autonomic efferents and non-shivering thermogenesis in brown adipose tissue (BAT). We investigated whether leptin reverses hyperglycemia through a mechanism that requires autonomic innervation, or uncoupling protein 1 (UCP1)-mediated thermogenesis. Methods To examine the role of parasympathetic and sympathetic efferents in the glucose-lowering action of leptin, mice with a subdiaphragmatic vagotomy or 6-hydroxydopamine induced chemical sympathectomy were injected with streptozotocin (STZ) to induce hyperglycemia, and subsequently leptin treated. To test whether the glucose-lowering action of leptin requires activation of UCP1-mediated thermogenesis in BAT, we administered leptin in STZ-diabetic Ucp1 knockout (Ucp1−/−) mice and wildtype controls. Results Leptin ameliorated STZ-induced hyperglycemia in both intact and vagotomised mice. Similarly, mice with a partial chemical sympathectomy did not have an attenuated response to leptin-mediated glucose lowering relative to sham controls, and showed intact leptin-induced Ucp1 expression in BAT. Although leptin activated BAT thermogenesis in STZ-diabetic mice, the anti-diabetic effect of leptin was not blunted in Ucp1−/− mice. Conclusions These results suggest that leptin lowers blood glucose in insulin-deficient diabetes through a manner that does not require parasympathetic or sympathetic innervation, and thus imply that leptin lowers blood glucose through an alternative CNS-mediated mechanism or redundant target tissues. Furthermore, we conclude that the glucose lowering action of leptin is independent of UCP1-dependent thermogenesis.