Vanessa Cristina Arantes

Impaired insulin secretion and decreased expression of the nutritionally responsive ribosomal kinase protein S6K-1 in pancreatic islets from malnourished rats

Low protein diet has been shown to affect the levels and activities of several enzymes from pancreatic islets. To further extend the knowledge on how malnutrition affects insulin secretion pathway, we investigated in this work the insulin release induced by glucose or leucine, an insulin secretagogue, and the expression of insulin receptor (IR), insulin receptor substrate 1 (IRS1), phosphatidylinositol 3-kinase (PI3K), and p70S6K1 (S6K-1) proteins from pancreatic islets of rats fed a normal (17%; NP) or a low (6%; LP) protein diet for 8 weeks. Isolated islets were incubated for 1 h in Krebs-bicarbonate solution containing 16.7 mmol/L of glucose, or 2.8 mmol/L of glucose in the presence or absence of 20 mmol/L of leucine. Glucose- and leucine-induced insulin secretions were higher in NP than in LP islets. Western blotting analysis showed an increase in the expression of IR and PI3K protein levels whereas IRS1 and S6K-1 protein expression were lower in LP compared to NP islets. In addition, S6K-1 mRNA expression was also reduced in islets from LP rats. Our data indicate that a low protein diet modulates the levels of several proteins involved in the insulin secretion pathway. Particularly, the decrease in S6K-1 expression might be an important factor affecting either glucose- or leucine-induced insulin secretion.

Soybean diet improves insulin secretion through activation of cAMP/PKA pathway in rats.

Maternal malnutrition leads to permanent alterations in insulin secretion of offspring and the soybean diet contributes to improve insulin release. At least a soy component, genistein, seems to increase the insulin secretion by activating the cAMP/PKA and PLC/PKC pathways. Here, we investigated the effect of the soybean diet on the expression of PKAalpha and PKCalpha, and insulin secretion in response to glucose and activators of adenylate cyclase and PKC in adult pancreatic rat islets. Rats from mothers fed with 17% or 6% protein (casein) during pregnancy and lactation were maintained with 17% casein (CC and CR groups) or soybean (SC and SR groups) diet until 90 days of life. The soybean diet improved the insulin response to a physiological concentration of glucose in control islets, but only in the presence of supra-physiological concentrations of glucose in islets from CR and SR groups. PMA also improved the insulin response in islets of SC and SR groups. The expression of PKCalpha was similar in all groups. Forskolin increased the insulin secretion; however, the magnitude of the increment was lower in islets from CR and SR groups than in control animals and in those from rats maintained with soybean diet than in rats fed with casein diet. The PKAalpha expression was similar between SR and CR groups and lower in SC than in CC islets. Thus, soybean diet improved the secretory pattern of beta cells, at least in part, by activating the cAMP/PKA-signaling cascade.

Soybean diet alters the insulin-signaling pathway in the liver of rats recovering from early-life malnutrition.

OBJECTIVE We investigated if alterations in the insulin-signaling pathway could contribute to reduced hepatic glycogen levels in adult rats subjected to a protein deficiency during intrauterine life and lactation and reared through to recovery on a soybean diet. METHODS Rats from mothers fed with 17% or 6% protein (casein) during pregnancy and lactation were maintained with a 17% casein diet (offspring born to and suckled by mothers fed a control diet and subsequently fed the same diet after weaning [CC group] and offspring born to and suckled by mothers fed a control diet and subsequently fed a soybean flour diet with 17% protein after weaning [CS group]), a soybean diet (offspring of mothers fed a low-protein diet and a control diet after weaning [LC group] and offspring of mothers fed a low-protein diet and fed a soybean flour diet containing 17% protein after weaning [LS group]), or a 6% casein diet (offspring of mothers fed a low-protein diet and subsequently fed the same diet after weaning [LL group]) from weaning until 90 d of life. RESULTS A soybean diet did not modify basal serum glucose and glucagon concentrations, but raised basal serum insulin and consequently increased the serum insulin/glucose ratio. Insulin receptor and insulin receptor substrate-1 levels were lower in rats fed a soybean diet compared with those maintained with a casein diet. In the LS group, the p85 levels were higher than in the LC group, whereas in CS rats its expression was lower than in CC rats. The expression of p110 was lower in the CS group compared with the CC group and similar in the LS and LC groups. Insulin receptor substrate-1 phosphorylation was similar in the LS, LC, and CS groups and lower compared with the CC group. The insulin receptor substrate-1-p85/phosphatidylinositol 3-kinase association was lower in LS than in LC rats and in CS than in CC rats. Akt phosphorylation was lower in the CS and LS groups than in the CC and LC groups. CONCLUSION Adult rats maintained with a soybean diet exhibited insulin resistance due, at least in part, to alterations in the early steps of the insulin signal transduction pathway.

Serum leptin and insulin levels in lactating protein-restricted rats: implications for energy balance

The present study analysed the effect of protein restriction on serum insulin and leptin levels and their relationship with energy balance during lactation. Four groups of rats received isocaloric diets containing 170 g protein/kg or 60 g protein/kg from pregnancy until the 14th day of lactation: control non-lactating, control lactating (both fed a control diet), low-protein non-lactating and low-protein lactating. Energy intake, body composition, energy balance, serum insulin and leptin concentrations and the relationship between these hormones and several factors related to obesity were analysed. Low-protein-intake lactating rats exhibited hypoinsulinaemia, hyperleptinaemia, hypophagia and decreased energy expenditure compared with control lactating rats. The protein level in the carcasses was lower in the low-protein lactating group than in the control lactating group, resulting in a higher fat content in the first group compared with the latter. Body fat correlated inversely with serum insulin and positively with serum leptin level. There was a significant negative correlation between serum leptin and energy intake, and a positive relationship between energy intake and serum insulin level in lactating rats and in the combined data from both groups. Energy expenditure was correlated positively with serum insulin and negatively with serum leptin in lactating rats and when data from control non-lactating and lactating rats were pooled. Lactating rats submitted to protein restriction, compared with lactating control rats, showed that maternal reserves were preserved owing to less severe negative energy balance. This metabolic adaptation was obtained, at least in part, by hypoinsulinaemia that resulted in increased insulin sensitivity favouring enhanced fat deposition, hyperleptinaemia and hypophagia.

A low-protein diet during pregnancy alters glucose metabolism and insulin secretion.

In pancreatic islets, glucose metabolism is a key process for insulin secretion, and pregnancy requires an increase in insulin secretion to compensate for the typical insulin resistance at the end of this period. Because a low‐protein diet decreases insulin secretion, this type of diet could impair glucose homeostasis, leading to gestational diabetes. In pancreatic islets, we investigated GLUT2, glucokinase and hexokinase expression patterns as well as glucose uptake, utilization and oxidation rates. Adult control non‐pregnant (CNP) and control pregnant (CP) rats were fed a normal protein diet (17%), whereas low‐protein non‐pregnant (LPNP) and low‐protein pregnant (LPP) rats were fed a low‐protein diet (6%) from days 1 to 15 of pregnancy. The insulin secretion in 2.8 mmol l−1 of glucose was higher in islets from LPP rats than that in islets from CP, CNP and LPNP rats. Maximal insulin release was obtained at 8.3 and 16.7 mmol l−1 of glucose in LPP and CP groups, respectively. The glucose dose–response curve from LPNP group was shifted to the right in relation to the CNP group. In the CP group, the concentration–response curve to glucose was shifted to the left compared with the CNP group. The LPP groups exhibited an “inverted U‐shape” dose–response curve. The alterations in the GLUT2, glucokinase and hexokinase expression patterns neither impaired glucose metabolism nor correlated with glucose islet sensitivity, suggesting that β‐cell sensitivity to glucose requires secondary events other than the observed metabolic/molecular events. Copyright

Effect of nutritional recovery with soybean flour diet on body composition, energy balance and serum leptin concentration in adult rats.

BackgroundMalnutrition in early life is associated with obesity in adulthood and soybean products may have a beneficial effect on its prevention and treatment. This study evaluated body composition, serum leptin and energy balance in adult rats subjected to protein restriction during the intrauterine stage and lactation and recovering on a soybean flour diet.MethodsFive groups of the Wistar strain of albino rats were used: CC, offspring born to and suckled by mothers fed a control diet and fed the same diet after weaning; CS, offspring born to and suckled by mothers fed a control diet and fed a soybean diet with 17% protein after weaning; LL, offspring of mothers fed a low protein diet and fed the same diet after weaning; LC, offspring of mothers fed a low protein diet, but fed a control diet after weaning; LS, offspring of mothers fed a low protein diet, but fed a soybean diet with 17% protein after weaning. Food intake, body, perirenal and retroperitoneal adipose tissue were measured in grams. Leptin was quantified using the Enzyme Linked Immuno Sorbent Assay (ELISA) and insulin by radioimmunoassay (RIA). Carcass composition was determined by chemical methods and energy expenditure was calculated by the difference between energy intake and carcass energy gain. Data were tested by analysis of variance (ANOVA).ResultsThe LC and LS groups had higher energetic intake concerning body weight, lower energy expenditure, proportion of fat carcass and fat pads than CC and CS groups. The LS group showed reduced body weight gain and lower energy efficiency, which was reflected in less energy gain as protein and the proportion of carcass protein, and lower energy gain as lipid than in the LC groups, although both groups had eaten the same amount of diet and showed equal energy expenditure. Serum leptin did not differ among groups and was unrelated to food or energy intake and energy expenditure. Serum insulin was higher in the LS than in the LC group.ConclusionProtein restriction during intrauterine life and lactation periods did not provoke obesity in adulthood. Nutritional recovery with soybean diet decreased the body weight at the expense of lower energy efficiency with repercussion on lean mass.

Soybean diet modulates acetyl-coenzyme A carboxylase expression in livers of rats recovering from early-life malnutrition.

OBJECTIVE The present study evaluated the effect of nutritional recovery with a soybean diet on the gene and protein expressions and protein phosphorylation of several enzymes and transcription factors involved in hepatic lipid metabolism. METHODS Rats from mothers fed with 17% or 6% protein (casein) during pregnancy and lactation were maintained with a 17% casein (CC and LC groups) or soybean (CS and LS groups) diet and with a 6% casein (LL group) diet until 90 d of life. RESULTS The soybean diet enhanced serum insulin levels but decreased body and liver weights and hepatic lipid and glycogen concentrations. Liver peroxisome proliferator receptor-alpha mRNA abundance was higher in the LS and CS groups than in the LC and CC groups, but the protein content was similar in all groups. Hepatic acetyl-coenzyme A carboxylase (ACC)-alpha and ACCbeta mRNA expression was markedly lower in the LS and CS rats than in the LC and CC rats. ACC protein expression was lower in the CS group than in the CC, LC, and LS groups. Phospho-[Ser(79)]2-ACC content was similar in the CS, LC, and LS groups and lower than the CC group. In the CS rats this reduction paralleled the decrease in total ACC protein. Messenger RNA and protein expression of sterol regulatory element-binding protein 1c, adenosine monophosphate-activated protein kinase, and phospho-[Thr(172)]-adenosine monophosphate-activated protein kinase was not modified by the soybean diet. CONCLUSION Thus, the soybean diet reduced the liver lipid concentration through downregulation of the ACC gene and protein expressions rather than by phosphorylation status, which possibly resulted in decreased lipogenesis and increased beta-oxidation.

Correlation of serum leptin and insulin levels of pregnant protein-restricted rats with predictive obesity variables

During pregnancy and protein restriction, changes in serum insulin and leptin levels, food intake and several metabolic parameters normally result in enhanced adiposity. We evaluated serum leptin and insulin levels and their correlations with some predictive obesity variables in Wistar rats (90 days), up to the 14th day of pregnancy: control non-pregnant (N = 5) and pregnant (N = 7) groups (control diet: 17% protein), and low-protein non-pregnant (N = 5) and pregnant (N = 6) groups (low-protein diet: 6%). Independent of the protein content of the diet, pregnancy increased total (F1,19 = 22.28, P < 0.001) and relative (F1,19 = 5.57, P < 0.03) food intake, the variation of weight (F1,19 = 49.79, P < 0.000) and final body weight (F1,19 = 19.52, P < 0.001), but glycemia (F1,19 = 9.02, P = 0.01) and the relative weight of gonadal adipose tissue (F1,19 = 17.11, P < 0.001) were decreased. Pregnancy (F1,19 = 18.13, P < 0.001) and low-protein diet (F1,19 = 20.35, P < 0.001) increased the absolute weight of brown adipose tissue. However, the relative weight of this tissue was increased only by protein restriction (F1,19 = 15.20, P < 0.001) and the relative lipid in carcass was decreased in low-protein groups (F1,19 = 4.34, P = 0.05). Serum insulin and leptin levels were similar among groups and did not correlate with food intake. However, there was a positive relationship between serum insulin levels and carcass fat depots in low-protein groups (r = 0.37, P < 0.05), while in pregnancy serum leptin correlated with weight of gonadal (r = 0.39, P < 0.02) and retroperitoneal (r = 0.41, P < 0.01) adipose tissues. Unexpectedly, protein restriction during 14 days of pregnancy did not alter the serum profile of adiposity signals and their effects on food intake and adiposity, probably due to the short term of exposure to low-protein diet.

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.

Nutritional Recovery Promotes Hypothalamic Inflammation in Rats during Adulthood

We evaluated whether protein restriction in fetal life alters food intake and glucose homeostasis in adulthood by interfering with insulin signal transduction through proinflammatory mechanisms in the hypothalamus and peripheral tissues. Rats were divided into the following: a control group (C); a recovered group (R); and a low protein (LP) group. Relative food intake was greater and serum leptin was diminished in LP and R compared to C rats. Proinflammatory genes and POMC mRNA were upregulated in the hypothalamus of R group. Hypothalamic NPY mRNA expression was greater but AKT phosphorylation was diminished in the LP than in the C rats. In muscle, AKT phosphorylation was higher in restricted than in control animals. The HOMA-IR was decreased in R and C compared to the LP group. In contrast, the K itt in R was similar to that in C and both were lower than LP rats. Thus, nutritional recovery did not alter glucose homeostasis but produced middle hyperphagia, possibly due to increased anorexigenic neuropeptide expression that counteracted the hypothalamic inflammatory process. In long term protein deprived rats, hyperphagia most likely resulted from increased orexigenic neuropeptide expression, and glucose homeostasis was maintained, at least in part, at the expense of increased muscle insulin sensitivity.