Renato Chaves Souto Branco

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.

Reduced insulin clearance and lower insulin-degrading enzyme expression in the liver might contribute to the thrifty phenotype of protein-restricted mice

Nutrient restriction during the early stages of life usually leads to alterations in glucose homeostasis, mainly insulin secretion and sensitivity, increasing the risk of metabolic disorders in adulthood. Despite growing evidence regarding the importance of insulin clearance during glucose homeostasis in health and disease, no information exists about this process in malnourished animals. Thus, in the present study, we aimed to determine the effect of a nutrient-restricted diet on insulin clearance using a model in which 30-d-old C57BL/6 mice were exposed to a protein-restricted diet for 14 weeks. After this period, we evaluated many metabolic variables and extracted pancreatic islet, liver, gastrocnemius muscle (GCK) and white adipose tissue samples from the control (normal-protein diet) and restricted (low-protein diet, LP) mice. Insulin concentrations were determined using RIA and protein expression and phosphorylation by Western blot analysis. The LP mice exhibited lower body weight, glycaemia, and insulinaemia, increased glucose tolerance and altered insulin dynamics after the glucose challenge. The improved glucose tolerance could partially be explained by an increase in insulin sensitivity through the phosphorylation of the insulin receptor/protein kinase B and AMP-activated protein kinase/acetyl-CoA carboxylase in the liver, whereas the changes in insulin dynamics could be attributed to reduced insulin secretion coupled with reduced insulin clearance and lower insulin-degrading enzyme (IDE) expression in the liver and GCK. In summary, protein-restricted mice not only produce and secrete less insulin, but also remove and degrade less insulin. This phenomenon has the double benefit of sparing insulin while prolonging and potentiating its effects, probably due to the lower expression of IDE in the liver, possibly with long-term consequences.

Low-protein diet disrupts the crosstalk between the PKA and PKC signaling pathways in isolated pancreatic islets

Protein restriction in the early stages of life can result in several changes in pancreatic function. These alterations include documented reductions in insulin secretion and in cytoplasmic calcium concentration [Ca(2+)]i. However, the mechanisms underlying these changes have not been completely elucidated and may result, in part, from alterations in signaling pathways that potentiate insulin secretion in the presence of glucose. Our findings suggest that protein restriction disrupts the insulin secretory synergism between Cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) and Ca(2+)-dependent protein kinase C (PKC) in isolated islets. Western blot analysis demonstrated reduced levels of both phospho-cAMP response element-binding protein (phospho-CREB) at Ser-133 and substrates phosphorylated by PKCs (Phospho-(Ser) PKC substrate), suggesting that PKA and PKC activity was impaired in islets from rats fed a low-protein diet (LP). cAMP levels and global Ca(2+) entry were also reduced in LP islets. In summary, our findings showed that protein restriction altered the crosstalk between PKA and PKC signaling pathways, resulting in the alteration of secretory synergism in isolated islets.

Long-Term Taurine Supplementation Leads to Enhanced Hepatic Steatosis, Renal Dysfunction and Hyperglycemia in Mice Fed on a High-Fat Diet

There is growing interest in the nutraceutical applications of taurine (TAU) for the prevention and treatment of obesity, diabetes and cardiovascular diseases. The lack of long-term clinical and animal studies available makes it difficult to address the safety of supraphysiological TAU exposure over prolonged periods. Here, we assessed growth parameters, renal function and glucose homeostasis in mice fed on a high-fat diet (HFD) and supplemented with 5 % TAU for 12 months. Body weight and fat depots were increased by the HFD and unaltered by TAU supplementation. TAU enhanced diet-induced hepatomegaly and liver steatosis. TAU-supplemented mice developed renal dysfunction as judged by increased urinary proteins and albumin, kidney weight and accumulation of lipid vacuoles in renal tubule. Long-term TAU enhanced the deleterious effects of the HFD upon glucose control, as indicated by fasting hyperglycemia, insulin hypersecretion, lower hepatic Akt activation and peripheral insulin resistance. In conclusion, long-term TAU supplementation enhanced the HFD-induced ectopic lipid accumulation in the liver and kidney and disrupted body glucose control and renal function. TAU-based interventions for obese and diabetic subjects should be carefully planned to avoid extended treatments over prolonged periods.

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.

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.

The effect of taurine supplementation on glucose homeostasis: the role of insulin-degrading enzyme.

Taurine is a semi-essential sulfur-containing amino acid derived from the metabolism of methionine and cysteine. Taurine controls several biological processes, including glucose homeostasis. The effects of taurine supplementation on insulin secretion and its effects on peripheral organs have been previously described. However, there are no data describing the effects of this amino acid on insulin clearance and the expression of insulin-degrading enzyme (IDE).

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.