Jean Franciesco Vettorazzi

The bile acid TUDCA increases glucose-induced insulin secretion via the cAMP/PKA pathway in pancreatic beta cells.

OBJECTIVE While bile acids are important for the digestion process, they also act as signaling molecules in many tissues, including the endocrine pancreas, which expresses specific bile acid receptors that regulate several cell functions. In this study, we investigated the effects of the conjugated bile acid TUDCA on glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells. METHODS Pancreatic islets were isolated from 90-day-old male mice. Insulin secretion was measured by radioimmunoassay, protein phosphorylation by western blot, Ca(2+) signals by fluorescence microscopy and ATP-dependent K(+) (KATP) channels by electrophysiology. RESULTS TUDCA dose-dependently increased GSIS in fresh islets at stimulatory glucose concentrations but remained without effect at low glucose levels. This effect was not associated with changes in glucose metabolism, Ca(2+) signals or KATP channel activity; however, it was lost in the presence of a cAMP competitor or a PKA inhibitor. Additionally, PKA and CREB phosphorylation were observed after 1-hour incubation with TUDCA. The potentiation of GSIS was blunted by the Gα stimulatory, G protein subunit-specific inhibitor NF449 and mimicked by the specific TGR5 agonist INT-777, pointing to the involvement of the bile acid G protein-coupled receptor TGR5. CONCLUSION Our data indicate that TUDCA potentiates GSIS through the cAMP/PKA pathway.

Vagotomy ameliorates islet morphofunction and body metabolic homeostasis in MSG-obese rats

The parasympathetic nervous system is important for β-cell secretion and mass regulation. Here, we characterized involvement of the vagus nerve in pancreatic β-cell morphofunctional regulation and body nutrient homeostasis in 90-day-old monosodium glutamate (MSG)-obese rats. Male newborn Wistar rats received MSG (4 g/kg body weight) or saline [control (CTL) group] during the first 5 days of life. At 30 days of age, both groups of rats were submitted to sham-surgery (CTL and MSG groups) or subdiaphragmatic vagotomy (Cvag and Mvag groups). The 90-day-old MSG rats presented obesity, hyperinsulinemia, insulin resistance, and hypertriglyceridemia. Their pancreatic islets hypersecreted insulin in response to glucose but did not increase insulin release upon carbachol (Cch) stimulus, despite a higher intracellular Ca2+ mobilization. Furthermore, while the pancreas weight was 34% lower in MSG rats, no alteration in islet and β-cell mass was observed. However, in the MSG pancreas, increases of 51% and 55% were observed in the total islet and β-cell area/pancreas section, respectively. Also, the β-cell number per β-cell area was 19% higher in MSG rat pancreas than in CTL pancreas. Vagotomy prevented obesity, reducing 25% of body fat stores and ameliorated glucose homeostasis in Mvag rats. Mvag islets demonstrated partially reduced insulin secretion in response to 11.1 mM glucose and presented normalization of Cch-induced Ca2+ mobilization and insulin release. All morphometric parameters were similar among Mvag and CTL rat pancreases. Therefore, the higher insulin release in MSG rats was associated with greater β-cell/islet numbers and not due to hypertrophy. Vagotomy improved whole body nutrient homeostasis and endocrine pancreatic morphofunction in Mvag rats.

Protein malnutrition potentiates the amplifying pathway of insulin secretion in adult obese mice

Pancreatic beta cell (β) dysfunction is an outcome of malnutrition. We assessed the role of the amplifying pathway (AMP PATH) in β cells in malnourished obese mice. C57Bl-6 mice were fed a control (C) or a low-protein diet (R). The groups were then fed a high-fat diet (CH and RH). AMP PATH contribution to insulin secretion was assessed upon incubating islets with diazoxide and KCl. CH and RH displayed increased glucose intolerance, insulin resistance and glucose-stimulated insulin secretion. Only RH showed a higher contribution of the AMP PATH. The mitochondrial membrane potential of RH was decreased, and ATP flux was unaltered. In RH islets, glutamate dehydrogenase (GDH) protein content and activity increased, and the AMP PATH contribution was reestablished when GDH was blunted. Thus, protein malnutrition induces mitochondrial dysfunction in β cells, leading to an increased contribution of the AMP PATH to insulin secretion through the enhancement of GDH content and activity.

Taurine Supplementation Enhances Insulin Secretion Without Altering Islet Morphology in Non-obese Diabetic Mice

Taurine (TAU) is a sulfated amino acid that improves pancreatic islet function and regulates β-cell mass in pre- and diabetic states. We herein analyzed glucose homeostasis and islet morphofunction in non-obese diabetic (NOD) mice supplemented with 2 % TAU in their drinking water from birth until 90-days of age. TAU-supplemented female NOD mice (TAU group) showed a better glucose tolerance without modification in insulinemia, when compared to non-supplemented NOD mice (CTL). Glucose-induced insulin secretion was higher in islets isolated from female and male TAU groups. In addition, a better insulin release was observed at 30 mM K+ in islets from female TAU mice. These effects were accompanied by a higher total intracellular Ca2+ concentration in islets from female and male TAU mice. TAU-treated mice did not show any alteration in β-cell and islet areas, compared with CTL mice. Islets from TAU female mice presented a higher ratio of phosphorylated Akt and ERK (extracellular signal-regulated kinase) related to Akt and ERK protein content, respectively, in comparison with CTL islets. Additional experiments using isolated islets from Swiss mice showed that 3 mM TAU prevented the reduction in insulin secretion induced by 12 h incubation with IL1-β or IL1-β + IFN-γ. In conclusion, TAU supplementation improved NOD islet function without altering endocrine pancreatic morphometry, an effect that may be associated with a protective TAU effect upon cytokine-induced islet dysfunction, together with an improved protein expression of Akt and ERK.

Protein malnutrition blunts the increment of taurine transporter expression by a high-fat diet and impairs taurine reestablishment of insulin secretion

Taurine (Tau) restores β‐cell function in obesity; however, its action is lost in malnourished obese rodents. Here, we investigated the mechanisms involved in the lack of effects of Tau in this model. C57BL/6 mice were fed a control diet (CD)(14% protein) oraprotein‐restricted diet (RD) (6% protein) for 6 wk. Afterward, mice received a high‐fat diet (HFD) for 8 wk [CD + HFD (CH) and RD + HFD (RH)] with or without 5% Tau supplementation after weaning on their drinking water [CH + Tau (CHT) and RH + Tau (RHT)]. The HFD increased insulin secretion through mitochondrial metabolism in CH and RH. Tau prevented all those alterations in CHT only. The expression of the taurine transporter (Tau‐T), as well as Tau content in pancreatic islets, was increased in CH but had no effect on RH. Protein malnutrition programs β cells and impairs Tau‐induced restoration of mitochondrial metabolism and biogenesis. This may be associated with modulation of the expression of Tau‐T in pancreatic islets, which may be responsible for the absence of effect of Tau in protein‐malnourished obese mice.—Branco, R.C. S., Camargo, R. L., Batista, T. M., Vettorazzi, J. F., Borck, P. C., dos Santos‐Silva, J. C. R., Boschero, A. C., Zoppi, C. C., Carneiro, E. M. Protein malnutrition blunts the increment of taurine transporter expression by a high‐fat diet and impairs taurine reestablishment of insulin secretion. FASEB J. 31, 4078–4087 (2017). www.fasebj.org—Branco, Renato Chaves Souto, Camargo, Rafael Ludemann, Batista, Thiago Martins, Vettorazzi, Jean Franciesco, Borck, Patrícia Cristine, dos Santos‐Silva, Junia Carolina Rebelo, Boschero, Antonio Carlos, Zoppi, Cláudio Cesar, Carneiro, Everardo Magalhães Protein malnutrition blunts the increment of taurine transporter expression by a high‐fat diet and impairs taurine reestablishment of insulin secretion. FASEB J. 31, 4078–4087 (2017)

Bile acid TUDCA improves insulin clearance by increasing the expression of insulin-degrading enzyme in the liver of obese mice

Disruption of insulin secretion and clearance both contribute to obesity-induced hyperinsulinemia, though reduced insulin clearance seems to be the main factor. The liver is the major site for insulin degradation, a process mainly coordinated by the insulin-degrading enzyme (IDE). The beneficial effects of taurine conjugated bile acid (TUDCA) on insulin secretion as well as insulin sensitivity have been recently described. However, the possible role of TUDCA in insulin clearance had not yet been explored. Here, we demonstrated that 15 days treatment with TUDCA reestablished plasma insulin to physiological concentrations in high fat diet (HFD) mice, a phenomenon associated with increased insulin clearance and liver IDE expression. TUDCA also increased IDE expression in human hepatic cell line HepG2. This effect was not observed in the presence of an inhibitor of the hepatic membrane bile acid receptor, S1PR2, nor when its downstream proteins were inhibited, including IR, PI3K and Akt. These results indicate that treatment with TUDCA may be helpful to counteract obesity-induced hyperinsulinemia through increasing insulin clearance, likely through enhanced liver IDE expression in a mechanism dependent on S1PR2-Insulin pathway activation.

Vagotomy Reduces Insulin Clearance in Obese Mice Programmed by Low-Protein Diet in the Adolescence

The aim of this study was to investigate the effect of subdiaphragmatic vagotomy on insulin sensitivity, secretion, and degradation in metabolic programmed mice, induced by a low-protein diet early in life, followed by exposure to a high-fat diet in adulthood. Weaned 30-day-old C57Bl/6 mice were submitted to a low-protein diet (6% protein). After 4 weeks, the mice were distributed into three groups: LP group, which continued receiving a low-protein diet; LP + HF group, which started to receive a high-fat diet; and LP + HFvag group, which underwent vagotomy and also was kept at a high-fat diet. Glucose-stimulated insulin secretion (GSIS) in isolated islets, ipGTT, ipITT, in vivo insulin clearance, and liver expression of the insulin-degrading enzyme (IDE) was accessed. Vagotomy improved glucose tolerance and reduced insulin secretion but did not alter adiposity and insulin sensitivity in the LP + HFvag, compared with the LP + HF group. Improvement in glucose tolerance was accompanied by increased insulinemia, probably due to a diminished insulin clearance, as judged by the lower C-peptide : insulin ratio, during the ipGTT. Finally, vagotomy also reduced liver IDE expression in this group. In conclusion, when submitted to vagotomy, the metabolic programmed mice showed improved glucose tolerance, associated with an increase of plasma insulin concentration as a result of insulin clearance reduction, a phenomenon probably due to diminished liver IDE expression.

Protein malnutrition mitigates the effects of a high-fat diet on glucose homeostasis in mice: BRANCO et al.

Nutrient malnutrition, during the early stages of development, may facilitate the onset of metabolic diseases later in life. However, the consequences of nutritional insults, such as a high‐fat diet (HFD) after protein restriction, are still controversial. We assessed overall glucose homeostasis and molecular markers of mitochondrial function in the gastrocnemius muscle of protein‐restricted mice fed an HFD until early adulthood. Male C57BL/6 mice were fed a control (14% protein‐control diet) or a protein‐restricted (6% protein‐restricted diet) diet for 6 weeks. Afterward, mice received an HFD or not for 8 weeks (mice fed a control diet and HFD [CH] and mice fed a protein‐restricted diet and HFD [RH]). RH mice showed lower weight gain and fat accumulation and did not show an increase in fasting plasma glucose and insulin levels compared with CH mice. RH mice showed higher energy expenditure, increased citrate synthase, peroxisome‐proliferator‐activated receptor gamma coactivator 1‐alpha protein content, and higher levels of malate and α‐ketoglutarate compared with CH mice. Moreover, RH mice showed increased AMPc‐dependent kinase and acetyl coenzyme‐A (CoA) carboxylase phosphorylation, lower intramuscular triacylglycerol content, and similar malonyl‐CoA levels. In conclusion, protein undernourishment after weaning does not potentiate fat accumulation and insulin resistance in adult young mice fed an HFD. This outcome seems to be associated with increased skeletal muscle mitochondrial oxidative capacity and reduced lipids accumulation.

Protein malnutrition after weaning disrupts peripheral clock and daily insulin secretion in mice

Changes in nutritional state may alter circadian rhythms through alterations in expression of clock genes. Protein deficiency has a profound effect on body metabolism, but the effect of this nutrient restriction after weaning on biological clock has not been explored. Thus, this study aims to investigate whether the protein restriction affects the daily oscillation in the behavior and metabolic rhythms, as well as expression of clock genes in peripheral tissues. Male C57BL/6 J mice, after weaning, were fed a normal-protein (NP) diet or a low-protein (LP) diet for 8 weeks. Mice fed an LP diet did not show difference in locomotor activity and energy expenditure, but the food intake was increased, with parallel increased expression of the orexigenic neuropeptide Npy and disruption of the anorexigenic Pomc oscillatory pattern in the hypothalamus. LP mice showed disruption in the daily rhythmic patterns of plasma glucose, triglycerides and insulin. Also, the rhythmic expression of clock genes in peripheral tissues and pancreatic islets was altered in LP mice. In pancreatic islets, the disruption of clock genes was followed by impairment of daily glucose-stimulated insulin secretion and the expression of genes involved in exocytosis. Pharmacological activation of REV-ERBα could not restore the insulin secretion in LP mice. The present study demonstrates that protein restriction, leading to development of malnutrition, alters the peripheral clock and metabolic outputs, suggesting that this nutrient provides important entraining cues to regulate the daily fluctuation of biological clock.

Effects of paternal hypothalamic obesity and taurine supplementation on adiposity and vascular reactivity in rat offspring.

Obesity predisposes to the development of cardiovascular diseases and type 2 diabetes. Detrimental impacts of maternal obesity on adiposity and cardiovascular function in offspring are well known, whereas the contribution of obese fathers to these processes is poorly described. Taurine (Tau) regulates glucose homeostasis and cardiovascular function. Here, we analyzed whether paternal obesity may influence adiposity, glucose tolerance and vascular reactivity in their offspring. We also determined whether Tau supplementation in fathers modulates these effects. Male rats received subcutaneous injections of MSG [4 g/kg body weight (BW)] or saline [1.25 g/kg BW, control (CTL)] during the first 5 days of life. At 21 days, rats were distributed into four groups: CTL, MSG, and CTL and MSG supplemented with 2.5 % Tau (CTAU and MTAU). At 90 days of age, all groups were mated with female CTL rats to obtain offspring that were designated according to their fathers’ treatment. MSG-treated rats were obese, hyperinsulinemic and hypertriglyceridemic. Tau decreased fat stores and plasma triglyceride levels in MTAU. At 5 months of age, female offspring from MSG rats demonstrated higher retroperitoneal and perigonadal fat stores. While male offspring did not exhibit any alteration in adiposity or glucose homeostasis, the aortas of these rats had a lower maximal vasodilatory response to acetylcholine (Ach). In contrast, Ach-induced aorta relaxation was normal in MTAU offspring. In conclusion, data indicate that MSG-hypothalamic obesity in fathers may contribute to obesity, in female offspring, and vascular dysfunction in male offspring, and that supplementation of Tau to founders may preserve only vascular function