Emerielle C. Vanzela

Altered Glucose Homeostasis and Hepatic Function in Obese Mice Deficient for Both Kinin Receptor Genes

The Kallikrein-Kinin System (KKS) has been implicated in several aspects of metabolism, including the regulation of glucose homeostasis and adiposity. Kinins and des-Arg-kinins are the major effectors of this system and promote their effects by binding to two different receptors, the kinin B2 and B1 receptors, respectively. To understand the influence of the KKS on the pathophysiology of obesity and type 2 diabetes (T2DM), we generated an animal model deficient for both kinin receptor genes and leptin (obB1B2KO). Six-month-old obB1B2KO mice showed increased blood glucose levels. Isolated islets of the transgenic animals were more responsive to glucose stimulation releasing greater amounts of insulin, mainly in 3-month-old mice, which was corroborated by elevated serum C-peptide concentrations. Furthermore, they presented hepatomegaly, pronounced steatosis, and increased levels of circulating transaminases. This mouse also demonstrated exacerbated gluconeogenesis during the pyruvate challenge test. The hepatic abnormalities were accompanied by changes in the gene expression of factors linked to glucose and lipid metabolisms in the liver. Thus, we conclude that kinin receptors are important for modulation of insulin secretion and for the preservation of normal glucose levels and hepatic functions in obese mice, suggesting a protective role of the KKS regarding complications associated with obesity and T2DM.

Taurine supplementation enhances nutrient-induced insulin secretion in pancreatic mice islets.

Taurine (TAU), a naturally occurring sulfur‐containing amino acid, is found at high concentrations in plasma and mammalian tissues and regulates osmolarity, ion channel activity, and glucose homeostasis. Several reports have shown that physiological plasma TAU levels seem to be important for adequate beta (β)‐cell function and insulin action, since low concentrations of TAU in the plasma have been reported in the pre‐diabetic and diabetic states.

Taurine supplementation: involvement of cholinergic/phospholipase C and protein kinase A pathways in potentiation of insulin secretion and Ca2+ handling in mouse pancreatic islets.

Taurine (TAU) supplementation increases insulin secretion in response to high glucose concentrations in rodent islets. This effect is probably due to an increase in Ca2+ handling by the islet cells. Here, we investigated the possible involvement of the cholinergic/phospholipase C (PLC) and protein kinase (PK) A pathways in this process. Adult mice were fed with 2% TAU in drinking water for 30 d. The mice were killed and pancreatic islets isolated by the collagenase method. Islets from TAU-supplemented mice showed higher insulin secretion in the presence of 8.3 mm-glucose, 100 μm-carbachol (Cch) and 1 mm-3-isobutyl-1-methyl-xanthine (IBMX), respectively. The increase in insulin secretion in response to Cch in TAU islets was accompanied by a higher intracellular Ca2+ mobilisation and PLCβ2 protein expression. The Ca2+ uptake was higher in TAU islets in the presence of 8.3 mm-glucose, but similar when the islets were challenged by glucose plus IBMX. TAU islets also showed an increase in the expression of PKAα protein. This protein may play a role in cation accumulation, since the amount of Ca2+ in these islets was significantly reduced by the PKA inhibitors: N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline sulfonamide (H89) and PK inhibitor-(6-22)-amide (PKI). In conclusion, TAU supplementation increases insulin secretion in response to glucose, favouring both influx and internal mobilisation of Ca2+, and these effects seem to involve the activation of both PLC-inositol-1,4,5-trisphosphate and cAMP-PKA pathways.

Primary hypercholesterolaemia impairs glucose homeostasis and insulin secretion in low-density lipoprotein receptor knockout mice independently of high-fat diet and obesity.

We investigated whether primary hypercholesterolaemia per se affects glucose homeostasis and insulin secretion in low-density lipoprotein receptor knockout mice (LDLR(-/-)). Glucose plasma levels were increased and insulin decreased in LDLR(-/-) compared to the wild-type mice. LDLR(-/-) mice presented impaired glucose tolerance, but normal whole body insulin sensitivity. The dose-response curve of glucose-stimulated insulin secretion was shifted to the right in LDLR(-/-) islets. Significant reductions in insulin secretion in response to l-leucine or 2-ketoisocaproic acid were also observed in LDLR(-/-). Islet morphometric parameters, total insulin and DNA content were similar in both groups. Glucose uptake and oxidation were reduced in LDLR(-/-) islets. Removal of cholesterol from LDLR(-/-) islets corrected glucose-stimulated insulin secretion. These results indicate that enhanced membrane cholesterol content due to hypercholesterolaemia leads to a lower insulin secretion and glucose intolerance without affecting body insulin sensitivity. This represents an additional risk factor for diabetes and atherosclerosis in primary hypercholesterolaemia.

Early onset of obesity induces reproductive deficits in female rats

The incidence of obesity is increasing rapidly all over the world and results in numerous health detriments, including disruptions in reproduction. However, the mechanisms by which excess body fat interferes with reproductive functions are still not fully understood. After weaning, female rats were treated with a cafeteria diet or a chow diet (control group). Biometric and metabolic parameters were evaluated in adulthood. Reproductive parameters, including estradiol, progesterone, LH and prolactin during the proestrus afternoon, sexual behavior, ovulation rates and histological analysis of ovaries were also evaluated. Cafeteria diet was able to induce obesity in female rats by increasing body and fat pad weight, which resulted in increased levels of triglycerides, total cholesterol, LDL and induced insulin resistance. The cafeteria diet also negatively affected female reproduction by reducing the number of oocytes and preantral follicles, as well as the thickness of the follicular layer. Obese females did not show preovulatory progesterone and LH surges, though plasma estradiol and prolactin showed preovulatory surges similar to control rats. Nevertheless, sexual receptiveness was not altered by cafeteria diet. Taken together, our results suggest that the cafeteria diet administered from weaning age was able to induce obesity and reduce the reproductive capability in adult female rats, indicating that this obesity model can be used to better understand the mechanisms underlying reproductive dysfunction in obese subjects.

Exercise increases pancreatic β-cell viability in a model of type 1 diabetes through IL-6 signaling

Type 1 diabetes (T1D) is provoked by an autoimmune assault against pancreatic β cells. Exercise training enhances β‐cell mass in T1D. Here, we investigated how exercise signals β cells in T1D condition. For this, we used several approaches. Wild‐type and IL‐6 knockout (KO) C57BL/6 mice were exercised. Afterward, islets from control and trained mice were exposed to inflammatory cytokines (IL‐1β plus IFN‐γ). Islets from control mice and β‐cell lines (INS‐1E and MIN6) were incubated with serum from control or trained mice or medium obtained from 5‐aminoimidazole‐4 carboxamide 1‐β‐d‐ribofuranoside (AICAR)‐treated C2C12 skeletal muscle cells. Subsequently, islets and β cells were exposed to IL‐1β plus IFN‐γ. Proteins were assessed by immunoblotting, apoptosis was determined by DNA‐binding dye propidium iodide fluorescence, and NO* was estimated by nitrite. Exercise reduced 25, 75, and 50% of the IL‐1β plus IFN‐γ‐induced iNOS, nitrite, and cleaved caspase‐3 content, respectively, in pancreatic islets. Serum from trained mice and medium from AICAR‐treated C2C12 cells reduced β‐cell death, induced by IL‐1β plus IFN‐γ treatment, in 15 and 38%, respectively. This effect was lost in samples treated with IL‐6 inhibitor or with serum from exercised IL‐6 KO mice. In conclusion, muscle contraction signals β‐cell survival in T1D through IL‐6.—Paula, F.M.M., Leite, N. C., Vanzela, E. C., Kurauti, M. A., Freitas‐Dias, R., Carneiro, E. M., Boschero, A. C., Zoppi, C. C. Exercise increases pancreatic β‐cell viability in a model of type 1 diabetes through IL‐6 signaling. FASEB J. 29, 1805‐1816 (2015). www.fasebj.org

Short-term calorie restriction improves glucose homeostasis in old rats: involvement of AMPK

The occurrence of metabolic disorders, such as diabetes, obesity, atherosclerosis, and hypertension, increases with age. Inappropriate food intake, when combined with genetic and hormonal factors, can trigger the occurrence of these diseases in aged organisms. This study investigated whether short-term calorie restriction (CR; 40% of the intake of control animals (CTL) for 21 days) benefits 1-year-old (CR1yr) and 2-year-old (CR2yr) Wistar rats, with regard to insulin secretion and action. Plasma insulin and the insulin secreted by isolated islets were measured with radioimmunoassay, and the insulin sensitivity of peripheral tissues was assessed with the intraperitoneal glucose tolerance test (IPGTT), intraperitoneal insulin tolerance test, and hepatic and muscle adenosine monophosphate-activated protein kinase (AMPK) phosphorylation measurements. Body weight, epididymal fat pad, epididymal fat pad/body weight index, plasma glucose, and insulin were lower in the CR1yr than in the control (CTL1yr) rats. Serum cholesterol, triglycerides, and protein, as well as hepatic and muscle glycogen content, were similar between the CR and CTL groups. The IPGTT was higher in CR2yr and CTL2yr rats than in CR1yr and CTL1yr rats, and insulin sensitivity was higher in CR1yr and CR2yr rats than in their respective CTLs. This was associated with an increase in hepatic and muscle AMPK phosphorylation. No differences in glucose-induced insulin secretion in the isolated islets were observed between CRs and their respective CTL rats. In conclusion, short-term calorie restriction provoked more severe alterations in CR1yr than CR2yr rats. The normoglycemia observed in both CR groups seems to be due to an increase in insulin sensitivity, with the involvement of liver and muscle AMPK.

Pregnancy restores insulin secretion from pancreatic islets in cafeteria diet-induced obese rats

Insulin resistance during pregnancy is counteracted by enhanced insulin secretion. This condition is aggravated by obesity, which increases the risk of gestational diabetes. Therefore, pancreatic islet functionality was investigated in control nonpregnant (C) and pregnant (CP), and cafeteria diet-fed nonpregnant (Caf), and pregnant (CafP) obese rats. Isolated islets were used for measurements of insulin secretion (RIA), NAD(P)H production (MTS), glucose oxidation ((14)CO(2) production), intracellular Ca(2+) levels (fura-2 AM), and gene expression (real-time PCR). Impaired glucose tolerance was clearly established in Caf and CafP rats at the 14th wk on a diet. Insulin secretion induced by direct depolarizing agents such as KCl and tolbutamide and increasing concentrations of glucose was significantly reduced in Caf, compared with C islets. This reduction was not observed in islets from CP and CafP rats. Accordingly, the glucose oxidation and production of reduced equivalents were increased in CafP islets. The glucose-induced Ca(2+) increase was significantly lower in Caf and higher in CafP, compared with all other groups. CP and CafP islets demonstrated an increased Ca(2+) oscillation frequency, compared with both C and Caf islets, and the amplitude of oscillations was augmented in CafP, compared with Caf islets. In addition, Ca(v)alpha1.2 and SERCA2a mRNA levels were reduced in Caf islets. Ca(v)alpha1.2, but not SERCA2a, mRNA was normalized in CafP islets. In conclusion, cafeteria diet-induced obesity impairs insulin secretion. This alteration is related to the impairment of Ca(2+) handling in pancreatic islets, in especial Ca(2+) influx, a defect that is reversed during pregnancy allowing normalization of insulin secretion.

Cholesterol reduction ameliorates glucose-induced calcium handling and insulin secretion in islets from low-density lipoprotein receptor knockout mice

AIMS/HYPOTHESIS Changes in cellular cholesterol level may contribute to beta cell dysfunction. Islets from low density lipoprotein receptor knockout (LDLR(-/-)) mice have higher cholesterol content and secrete less insulin than wild-type (WT) mice. Here, we investigated the association between cholesterol content, insulin secretion and Ca(2+) handling in these islets. METHODS Isolated islets from both LDLR(-/-) and WT mice were used for measurements of insulin secretion (radioimmunoassay), cholesterol content (fluorimetric assay), cytosolic Ca(2+) level (fura-2AM) and SNARE protein expression (VAMP-2, SNAP-25 and syntaxin-1A). Cholesterol was depleted by incubating the islets with increasing concentrations (0-10mmol/l) of methyl-beta-cyclodextrin (MβCD). RESULTS The first and second phases of glucose-stimulated insulin secretion (GSIS) were lower in LDLR(-/-) than in WT islets, paralleled by an impairment of Ca(2+) handling in the former. SNAP-25 and VAMP-2, but not syntaxin-1A, were reduced in LDLR(-/-) compared with WT islets. Removal of excess cholesterol from LDLR(-/-) islets normalized glucose- and tolbutamide-induced insulin release. Glucose-stimulated Ca(2+) handling was also normalized in cholesterol-depleted LDLR(-/-) islets. Cholesterol removal from WT islets by 0.1 and 1.0mmol/l MβCD impaired both GSIS and Ca(2+) handling. In addition, at 10mmol/l MβCD WT islet showed a loss of membrane integrity and higher DNA fragmentation. CONCLUSION Abnormally high (LDLR(-/-) islets) or low cholesterol content (WT islets treated with MβCD) alters both GSIS and Ca(2+) handling. Normalization of cholesterol improves Ca(2+) handling and insulin secretion in LDLR(-/-) islets.

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 (EC(50) 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 Ca(2+) 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.