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Purpose Observational studies show that longer breastfeeding periods reduce maternal risk of type 2 diabetes mellitus. However, it is currently unknown if the long-term benefits of breastfeeding for maternal glucose homeostasis are linked to changes in the endocrine pancreas. Methods We presently evaluated functional, morphological and molecular aspects of the endocrine pancreas of mice subjected to two sequential cycles of pregnancy and lactation (L21). Age-matched mice not allowed to breastfeed (L0) and virgin mice were used as controls. Results L21 mice exhibited increased tolerance and increased glucose-stimulated insulin secretion (GSIS) by isolated islets. Pancreatic islets of L21 mice did not present evident morphological changes to justify the increased GSIS. On the other hand, islets of L21 mice exhibited a reduction in Cavb3 and Kir6.2 expression with concordant increased intracellular Ca2+ levels after challenge with glucose. Conclusion Altogether, the present findings show the breastfeeding exerts long-term benefits for maternal endocrine pancreas by increasing intracellular Ca2+ levels and GSIS.

In the present study we investigated the participation of hepatic peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) in the metabolic programming of newborn rats exposed in utero to dexamethasone (DEX). On the 21st day of life, fasted offspring born to DEX-treated mothers displayed increased conversion of pyruvate into glucose with simultaneous upregulation of PEPCK (phosphoenolpyruvate carboxykinase) and G6Pase (glucose-6-phosphatase). Increased oxidative phosphorylation, higher ATP/ADP ratio and mitochondrial biogenesis and lower pyruvate levels were also found in the progeny of DEX-treated mothers. On the other hand, the 21-day-old progeny of DEX-treated mothers had increased hepatic triglycerides (TAG) and lower CPT-1 activity when subjected to short-term fasting. At the mechanistic level, rats exposed in utero to DEX exhibited increased hepatic PGC-1α protein content with lower miR-29a-c expression. Increased PGC-1α content was concurrent with increased association to HNF-4α and NRF1 and reduced PPARα expression. The data presented herein reveal that changes in the transcription machinery in neonatal liver of rats born to DEX-treated mothers leads to an inflexible metabolic response to fasting. Such programming is hallmarked by increased oxidative phosphorylation of pyruvate with impaired FFA oxidation and hepatic TAG accumulation.

Antenatal corticosteroid therapy is used to reduce neonatal mortality in preterm infants but it is currently unknown whether this intervention affects lipid metabolism at the peripartum. This study aimed to evaluate if antenatal corticosteroid therapy in pregnant rats and women affects lipid metabolism during early lactation. We evaluated women at risk of preterm delivery that received corticosteroid therapy (CASE) and women that were not exposed to corticosteroid and were not at risk of preterm delivery (CONTROL). Samples were collected to measure serum and milk triacylglycerol (TAG) three days after delivery. Rats were treated with dexamethasone (DEX) between the 15th and the 20th days of pregnancy. Samples were collected at different days after delivery (L3, L8 and L14). TAG was measured in serum, liver and mammary gland (MG). TAG appearance rates were measured after tyloxapol injection and gavage with olive oil. We also evaluated the expression of key genes related to lipid metabolism in the liver and in the MG and hepatic phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC). CASE volunteers delivered earlier than CONTROL but presented unaltered milk and serum TAG concentrations. Early lactating DEX rats exhibited increased TAG in serum, MG and milk. No changes in CD36 and LPL were detected in the MG and liver. Early lactating DEX rats displayed increased TAG appearance rate and reduced hepatic AMPK/ACC phosphorylation. Our data revealed that antenatal corticosteroid therapy reduces hepatic AMPK/ACC phosphorylation during early lactation that reflects in increased TAG concentration in serum, MG and milk.

Pancreatic islets from pregnant rats develop a transitory increase in the pancreatic β-cell proliferation rate and mass. Increased apoptosis during early lactation contributes to the rapid reversal of those morphological changes. Exposure to synthetic glucocorticoids during pregnancy has been previously reported to impair insulin secretion, but its impacts on pancreatic islet morphological changes during pregnancy and lactation have not been described. To address this issue, we assessed the morphological and molecular characteristics of pancreatic islets from rats that underwent undisturbed pregnancy (CTL) or were treated with dexamethasone between the 14th and 19th days of pregnancy (DEX). Pancreatic islets were analyzed on the 20th day of pregnancy (P20) and on the 3rd, 8th, 14th and 21st days of lactation (L3, L8, L14 and L21, respectively). Pancreatic islets from CTL rats exhibited transitory increases in cellular proliferation and pancreatic β-cell mass at P20, which were reversed at L3, when a transitory increase in apoptosis was observed. This was followed by the appearance of morphological features of pancreatic islet neogenesis at L8. Islets from DEX rats did not demonstrate an increase in apoptosis at L3, which coincided with an increase in the expression of M2 macrophage markers relative to M1 macrophage and T lymphocyte markers. Islets from DEX rats also did not exhibit the morphological characteristics of pancreatic islet neogenesis at L8. Our data demonstrate that maternal pancreatic islets undergo a renewal process during lactation that is impaired by exposure to DEX during pregnancy.

Aims The present investigation evaluated whether pregnancy followed by lactation exerts long-term impacts on maternal hepatic lipid metabolism. Main methods Female mice were subjected to two pregnancies, after which they were either allowed to breastfeed their pups for 21 days (L21) or had their litter removed (L0). Age-matched virgin mice were used as controls (CTL). Three months after the second delivery, serum was collected for lipid profiling, and fragments of liver were used to assess lipid content and to evaluate the key steps of de novo non-esterified fatty acid (NEFA) synthesis, esterification and β-oxidation, very low density lipoprotein (VLDL) assembly and secretion and autophagy. Key findings L0 exhibited a significant increase in hepatic TG and reduced apolipoprotein B-100 (ApoB-100) expression. L21 mice had increased ATP citrate lyase (ACLY) activity and reduced acetyl-CoA carboxylase (ACC) phosphorylation but no increased hepatic TG. On the other hand, L21 mice had reduced hepatic sequestosome 1 (SQSTM1/p62) levels. Increased high density lipoprotein (HDL) cholesterol and hepatic apolipoprotein A-1 (ApoA-1) expression were found exclusively in L21. Significance The present study reveals that long-term hepatic lipid accumulation is induced by the history of pregnancy without lactation. On the other hand, reduced SQSTM1/p62 levels indicate that increased autophagic flux during life may prevent hepatic fat in dams subjected to lactation. Lactation after pregnancy is also obligatory for a long-term increase in maternal HDL. The present data may contribute to the understanding of the mechanisms leading to elevated cardiometabolic risk in women limited to short periods of lactation.

The progeny of rats born and breastfed by mothers receiving dexamethasone (DEX) during pregnancy exhibits permanent reduction in body weight and adiposity but the precise mechanisms related to this programming are not fully understood. In order to clarify this issue, the present study investigated key aspects of lipoprotein production and lipid metabolism by the liver and the intestine that would explain the reduced adiposity seen in the adult offspring exposed to DEX in utero. Female Wistar rats were treated with DEX (0.1 mg/kg/day) between the 15th and the 21st days of pregnancy, while control mothers were treated with vehicle. Male offspring born to control mothers were nursed by either adoptive control mothers (CTL/CTL) or DEX-treated mothers (CTL/DEX). Male offspring born to DEX-treated mothers were nursed by either control mothers (DEX/CTL) or adoptive DEX-treated mothers (DEX/DEX). We found that only the male DEX/DEX offspring had reduced adiposity. Additionally, male DEX/DEX progeny had lower circulating triacylglycerol (TAG) levels only in fed-state. The four groups of offspring presented similar energy expenditure, respiratory quotient and very low-density lipoprotein (VLDL) production. On the other hand, DEX/DEX rats displayed reduced TAG levels after gavage with olive oil and reduced expression of fatty acid translocase Cd36 (Fat/Cd36) and peroxisome proliferator-activated receptor γ (Pparg) in the jejunum. Altogether, our study supports the notion that reduced fat absorption by the jejunum may contribute to the lower adiposity of the adult offspring born and breastfed by mothers treated with DEX during pregnancy.

Fructose consumption by rodents modulates both hepatic and intestinal lipid metabolism and gluconeogenesis. We have previously demonstrated that in utero exposure to dexamethasone (DEX) interacts with fructose consumption during adult life to exacerbate hepatic steatosis in rats. The aim of this study was to clarify if adult rats born to DEX-treated mothers would display differences in intestinal gluconeogenesis after excessive fructose intake. To address this issue, female Wistar rats were treated with DEX during pregnancy and control (CTL) mothers were kept untreated. Adult offspring born to CTL and DEX-treated mothers were assigned to receive either tap water (Control-Standard Chow (CTL-SC) and Dexamethasone-Standard Chow (DEX-SC)) or 10% fructose in the drinking water (CTL-fructose and DEX-fructose). Fructose consumption lasted for 80 days. All rats were subjected to a 40 h fasting before sample collection. We found that DEX-fructose rats have increased glucose and reduced lactate in the portal blood. Jejunum samples of DEX-fructose rats have enhanced phosphoenolpyruvate carboxykinase (PEPCK) expression and activity, higher facilitated glucose transporter member 2 (GLUT2) and facilitated glucose transporter member 5 (GLUT5) content, and increased villous height, crypt depth, and proliferating cell nuclear antigen (PCNA) staining. The current data reveal that rats born to DEX-treated mothers that consume fructose during adult life have increased intestinal gluconeogenesis while recapitulating metabolic and morphological features of the neonatal jejunum phenotype.

The expression levels of some reference genes and proteins are used for data normalization and quantification. However, these levels can change in response to experimental conditions or treatments. Aim. The aim of this work was to evaluate reference gene and protein expression in models of nonalcoholic fatty liver disease, using mice fed with a high-fat diet (HFD) and mice that are genetically obese (ob/ob). Main Methods. Histological staining techniques were used to verify the morphology and quantify the amount of lipid droplets present in the liver. Real-time polymerase chain reaction and immunoblotting were employed for monitoring protein expression and gene expression levels, respectively. Key Finding. The results showed that there was a substantial increase in the amount of lipid droplets in the livers of HFD and ob/ob animals when compared to the standard diet (SD) group. There was an observed reduction in the expression of β-actin (10%), α-tubulin (6%), GAPDH (19%), and RPL3 (15%) genes when comparing the ob/ob group to the HFD group. Additionally, the ob/ob mice displayed GAPDH protein levels that were substantially, but not significantly, reduced when compared to SD. Significance. It was concluded that there are slight differences in the expression levels of reference genes and proteins in these two NAFLD animal models, and researchers should consider these alterations when working with these models.

Aims The aim of the present study was to clarify if in utero exposure to DEX would affect the development of different types of pancreatic endocrine cells during postnatal life. Main methods We investigated morphological and transcriptional features of both pancreatic β- and α-cell populations within the pancreatic islets during the early postnatal life of rats born to mothers treated with DEX (0.1 mg/kg) from day 14 to 19 of pregnancy. Untreated pregnant Wistar rats of the same age (12-week-old) were used as control (CTL). Pups were euthanized on the 1st, 3rd and 21st (PND1, PND3 and PND21, respectively) days of life, regardless of sex. Serum insulin and glucagon levels were also evaluated. Key findings Rats born to DEX-treated mothers exhibited increased pancreatic α-cell mass, circulating glucagon levels and Gcg, Pax6, MafB and Nkx2.2 expression. Rats born to DEX-treated mothers also presented a rise in serum insulin levels on the PND3 that was paralleled by reduced β-cell mass. Such increase in serum insulin levels, instead, was associated with increased expression of genes associated to insulin secretion such as Gck and Slc2a2. Significance Altogether, the present data reveals yet unknown changes in endocrine pancreas during early postnatal life of rats exposed to DEX in utero. Such data may contribute to the understanding of the metabolic features of rats born to DEX-treated mothers.

Agomelatine (AGO) is an antidepressant drug with agonistic activity at melatonin receptor 1 (MT1) and MT2 and with neutral antagonistic activity at serotonin receptor 5-HT2C. Although experimental studies show that melatonin reduces hypertriglyceridemia and hepatic steatosis induced by excessive fructose intake, no studies have tested if AGO exerts similar actions. To address this issue we have treated male Wistar rats with fructose (15% in the drinking water) and/or AGO (40 mg/kg/day) for two weeks. AGO reduced body weight gain, feeding efficiency and hepatic lipid levels without affecting caloric intake in fructose-treated rats. AGO has also decreased very low-density lipoprotein (VLDL) production and circulating TAG levels after an oral load with olive oil. Accordingly, treatment with AGO reduced the hepatic expression of fatty acid synthase (Fasn), a limiting step for hepatic de novo lipogenesis (DNLG). The expression of apolipoprotein B (Apob) and microsomal triglyceride transfer protein (Mttp) in the ileum, two crucial proteins for intestinal lipoprotein production, were also downregulated by treatment with AGO. Altogether, the present data show that AGO mimics the metabolic benefits of melatonin when used in fructose-treated rats. This study also suggests that it is relevant to evaluate the potential of AGO to treat metabolic disorders in future clinical trials.

To evaluate the role of miR-124a in the regulation of genes involved in insulin exocytosis and its effects on the kinetics of insulin secretion in pancreatic islets from pregnant rats submitted to a low-protein diet. 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. Kinetics of the glucose-induced insulin release and measurement of [Ca2+]i in pancreatic islets were assessed by standard protocols. The miR-124a expression and gene transcriptions from pancreatic islets were determined by real-time polymerase chain reaction. In islets from LPP rats, the first phase of insulin release was abrogated. The AUC [Ca2+]i from the LPP group was lower compared with the other groups. miR-124a expression was reduced by a low-protein diet. SNAP-25 mRNA, protein expression, and Rab3A protein content were lower in the LPP rats than in CP rats. Syntaxin 1A and Kir6.2 mRNA levels were decreased in islets from low-protein rats compared with control rats, whereas their protein content was reduced in islets from pregnant rats. Loss of biphasic insulin secretion in islets from LPP rats appears to have resulted from reduced [Ca2+]i due, at least in part, to Kir6.2 underexpression and from the changes in exocytotic elements that are influenced either directly or indirectly by miR-124a.

Distinct environmental insults might interact with fructose consumption and contribute to the development of metabolic disorders. To address whether in utero glucocorticoid exposure and fructose intake modulate metabolic responses, adult female Wistar rats were exposed to dexamethasone (DEX) during pregnancy, and the offspring were administered fructose at a later time. Briefly, dams received DEX during the third period of pregnancy, while control dams remained untreated. Offspring born to control and DEX-treated mothers were defined as CTL-off and DEX-off, respectively, while untreated animals were designated CTL-off-CTL and DEX-off-CTL. CLT-off and DEX-off treated with 10% fructose in the drinking water for 8 weeks are referred to as CTL-off-FRU and DEX-off-FRU. We found that fructose promoted glucose intolerance and whole-body gluconeogenesis in both CTL-off-FRU and DEX-off-FRU animals. On the other hand, hepatic lipid accumulation was significantly stimulated in DEX-off-FRU rats when compared to the CTL-off-FRU group. The DEX-off-FRU group also displayed impaired very-low-density lipoprotein (VLDL) production and reduced hepatic expression of apoB, mttp, and sec22b. DEX-off-FRU has lower hepatic levels of autophagy markers. Taken together, our results support the unprecedented notion that in utero glucocorticoid exposure exacerbates hepatic steatosis caused by fructose consumption later in life.

A rise in plasma triacylglycerol levels is a common physiological occurrence during late gestation and excess of glucocorticoids (GCs) has been shown to impair lipid metabolism. Based on those observations, we investigated whether the administration of dexamethasone during the late gestational period could exacerbate this pregnancy associated hypertriacylglycerolemia in rats. For this, female Wistar rats were treated with dexamethasone (0.2 mg/kg of body mass in the drinking water on days 14-19 of pregnancy; DP group) or equivalent days in the virgin rats (DV group). Untreated pregnant rats (control pregnant group) and age-matched virgin rats (control virgin group) were used as controls. Functional, biochemical, and molecular analyses were carried out after treatment with GC and in the control groups. Euthanasia was performed on day 20 of pregnancy. The metabolic parameters of the mothers (dams) at the time of weaning and 6 months later, as well as newborn survival, were evaluated. We observed that neither dexamethasone nor pregnancy affected blood glucose or glucose tolerance. Hypertriacylglycerolemia associated with lipid intolerance or reduced hepatic triacylglycerol clearance was observed during the late gestational period. GC treatment caused a further increase in basal plasma triacylglycerol levels, but did not have a significant effect on lipid tolerance and hepatic triacylglycerol clearance in pregnant rats. GC, but not pregnancy, caused few significant changes in mRNA expression of proteins involved in lipid metabolism. Dexamethasone during pregnancy had no impact on lipid metabolism later in the dams' life; however, it led to intra-uterine growth restriction and reduced pup survival rate. In conclusion, GC exposure during the late gestational period in rats has no major impact on maternal lipid homeostasis, soon after parturition at weaning, or later in the dams' life, but GC exposure is deleterious to the newborn when high doses are administered at late gestation. These data highlight the importance of performing an individualized and rigorous control of a GC treatment during late pregnancy considering its harmful impact on the fetuses' health.

We investigated the effect of the crude herbal extract from Uncaria tomentosa (UT) on non-alcoholic fatty liver disease (NAFLD) in two models of obesity: high fat diet (HFD) and genetically obese (ob/ob) mice. Both obese mouse models were insulin resistant and exhibited an abundance of lipid droplets in the hepatocytes and inflammatory cell infiltration in the liver, while only the HFD group had collagen deposition in the perivascular space of the liver. UT treatment significantly reduced liver steatosis and inflammation in both obese mouse models. Furthermore, serine phosphorylation of IRS-1 was reduced by 25% in the HFD mice treated with UT. Overall, UT treated animals exhibited higher insulin sensitivity as compared to vehicle administration. In conclusion, Uncaria tomentosa extract improved glucose homeostasis and reverted NAFLD to a benign hepatic steatosis condition and these effects were associated with the attenuation of liver inflammation in obese mice.

We evaluate the mRNA expression of monocarboxylate transporters 1 and 4 (MCT1 and MCT4) in skeletal muscle (soleus, red and white gastrocnemius), heart and liver tissues in mice submitted to a single bout of swimming exercise at the maximal lactate steady state workload (MLSSw). After 72 h of MLSS test, the animals were submitted to a swimming exercise session for 25 min at individual MLSSw. Tissues and muscle samples were obtained at rest (control, n=5), immediately (n=5), 5 h (n=5) and 10 h (n=5) after exercise for determination of the MCT1 and MCT4 mRNA expression (RT-PCR). The MCT1 mRNA expression in liver increased after 10 h in relation to the control, immediate and 5 h groups, but the MCT4 remained unchanged. The MCT1 mRNA expression in heart increased by 31 % after 10 h when compared to immediate, but no differences were observed in relation to the control group. No significant differences were observed for red gastrocnemius in MCT1 and MCT4 mRNA expression. However, white gastrocnemius increased MCT1 mRNA expression immediately when compared to rest, 5 and 10 h test groups. In soleus muscle, the MCT1 mRNA expression increased immediately, 5 and 10 h after exercise when compared to the control. In relation to MCT4 mRNA expression, the soleus increased immediately and 10 h after acute exercise when compared to the control group. The soleus, liver and heart were the main tissues that showed improved the MCT1 mRNA expression, indicating its important role in controlling MLSS concentration in mice.

We investigated the effect of dexamethasone during the last week of pregnancy on glucose and lipid metabolism in male offspring. Twelve-week old offspring were evaluated after fasting for 12-hours (physiological) and 60-hours (prolonged). Physiological fasting resulted in glucose intolerance, decreased glucose clearance after pyruvate load and increased PEPCK expression in rats born to dexamethasone-treated mothers (DEX). Prolonged fasting resulted in increased glucose tolerance and increased glucose clearance after pyruvate load in DEX. These modulations were accompanied by accumulation of hepatic triglycerides (TG). Sixty-hour fasted DEX also showed increased citrate synthase (CS) activity, ATP citrate lyase (ACLY) content, and pyruvate kinase 2 (pkm2), glucose transporter 1 (slc2a1) and lactate dehydrogenase-a (ldha) expressions. Hepatic AKT2 was increased in 60-hour fasted DEX, in parallel with reduced miRNAs targeting the AKT2 gene. Altogether, we show that metabolic programming by prenatal dexamethasone is characterized by an unexpected hepatic TG accumulation during prolonged fasting. The underlying mechanism may depend on increased hepatic glycolytic flux due to increased pkm2 expression and consequent conversion of pyruvate to non-esterified fatty acid synthesis due to increased CS activity and ACLY levels. Upregulation of AKT2 due to reduced miRNAs may serve as a permanent mechanism leading to increased pkm2 expression.

Recent studies show that the metabolic effects of fructose may vary depending on the phase of its consumption along with the light/dark cycle. Here, we investigated the metabolic outcomes of fructose consumption by rats during either the light (LPF) or the dark (DPF) phases of the light/dark cycle. This experimental approach was combined with other interventions, including restriction of chow availability to the dark phase, melatonin administration or intracerebroventricular inhibition of adenosine monophosphate-activated protein kinase (AMPK) with Compound C. LPF, but not DPF rats, exhibited increased hypothalamic AMPK phosphorylation, glucose intolerance, reduced urinary 6-sulfatoxymelatonin (6-S-Mel) (a metabolite of melatonin) and increased corticosterone levels. LPF, but not DPF rats, also exhibited increased chow ingestion during the light phase. The mentioned changes were blunted by Compound C. LPF rats subjected to dark phase-restricted feeding still exhibited increased hypothalamic AMPK phosphorylation but failed to develop the endocrine and metabolic changes. Moreover, melatonin administration to LPF rats reduced corticosterone and prevented glucose intolerance. Altogether, the present data suggests that consumption of fructose during the light phase results in out-of-phase feeding due to increased hypothalamic AMPK phosphorylation. This shift in spontaneous chow ingestion is responsible for the reduction of 6-S-Mel and glucose intolerance.

The liver plays an essential role in maternal metabolic adaptation during late pregnancy. With regard to lipid metabolism, increased secretion of very low-density lipoprotein (VLDL) is characteristic of late pregnancy. Despite this well-described metabolic plasticity, the molecular changes underlying the hepatic adaptation to pregnancy remain unclear. As AMPK is a key intracellular energy sensor, we investigated whether this protein assumes a causal role in the hepatic adaptation to pregnancy. Pregnant Wistar rats were treated with vehicle or AICAR (5-aminoimidazole-4-carboxamide ribonucleotide) for 5 days starting at gestational day 14. At the end of treatment, the rats were subjected to an intraperitoneal pyruvate tolerance test and in situ liver perfusion with pyruvate. The livers were processed for Western blot analysis, quantitative PCR, thin-layer chromatography, enzymatic activity, and glycogen content measurements. Blood biochemical profiles were also assessed. We found that AMPK and ACC phosphorylation were reduced in the livers of pregnant rats in parallel with a reduced level of hepatic gluconeogenesis of pyruvate. This effect was accompanied by both a reduction in the levels of hepatic triglycerides (TG) and an increase in circulating levels of TG. Treatment with AICAR restored hepatic levels of TG to those observed in nonpregnant rats. Additionally, AMPK activation reduced the upregulation of genes related to VLDL synthesis and secretion observed in the livers of pregnant rats. We conclude that the increased secretion of hepatic TG in late pregnancy is concurrent with a transcriptional profile that favors VLDL production. This transcriptional profile results from the reduction in hepatic AMPK activity. ispartof: American Journal of Physiology. Regulatory, Integrative and Comparative Physiology vol:307 issue:9 pages:R1146-R1156 ispartof: location:United States status: published

Inflammation plays a key role in the development of insulin resistance and atherosclerosis. Fatty acids and fiber intake can selectively alter gene expression by modifying inflammation. We compared the postprandial expression of inflammatory genes after 2 distinct high-fat breakfast meals, before and after 1-month dietary interventions. This crossover clinical trial included 18 individuals at low-to-moderate cardiometabolic risk participating in evaluations before and after two 4-week breakfast interventions—one rich in saturated fatty acids (SFA) and the other in unsaturated fatty acids (unSFA) and fiber. Participants underwent meal tests with similar compositions to the breakfasts. Variables were compared by Student t test. The expression of inflammatory genes in leukocytes was analyzed using RT-PCR. Before and after the intervention with the SFA-enriched breakfast, this meal test induced a higher relative postprandial IL-1β expression compared to the responses to the unSFA and fiber-enriched meal (p = 0.02). On the other hand, following the intervention with the unSFA-fiber-enriched breakfast, postprandial IL-6 expression showed a reduction tendency comparing to the pre-intervention value (p = 0.08). Although fasting IL-1β, IL-6, MCP-1 and IFN-γ expressions had not changed after interventions, their circulating levels increased after the SFA-enriched meal test but not after the unSFA meal (p value between changes

SUMMARY Tight control of blood glucose reduces cardiovascular events and total mortality is conflicting. To summarize clinical effects of tight versus conventional glucose control in patients with type 2 diabetes. We systematically searched MEDLINE, EMBASE, Cochrane Library, and ISI Web of Knowledge with no limits of language and time. Further trials were searched from the reference lists of identified studies. We included randomized controlled comparing different levels of blood glucose control intensity in type 2 diabetic patients. Two independent reviewers extracted data of eligible studies using standard case report forms. We investigated total mortality, cardiovascular and microvascular events, and hypoglycemia in patients with type 2 diabetes. We used random-effects models to obtain relative risks (RR) with 95% confidence intervals (CI). We included 6 trials involving 27,654 patients. There was no significant effect of tight blood glucose control on all-cause mortality (RR 1.03; 95% CI 0.90‐1.17) or cardiovascular mortality (RR 1.04; 95% CI 0.83‐1.29). Tight glucose control reduced the risk for nonfatal MI (RR 0.85; 95% CI 0.76‐0.95), although had no effect on the incidence of nonfatal stroke (RR 1.02; 95% CI 0.88‐1.17). For microvascular events, tight glucose control reduced the risk progression of retinopathy (RR 0.80; 95% CI 0.71‐0.91), incidence of peripheral neuropathy (RR 0.94; 95% CI 0.89‐0.99), and progression of nephropathy (RR 0.55; 95% CI 0.37‐0.80), but had not significant effect on the incidence of nephropathy (RR 0.69; 95% CI 0.42‐1.14). The risk of severe hypoglycemia increased with tight glucose control (RR 2.39; 95% CI 1.79‐3.18). Tight blood glucose control reduces the risk for some macrovascular and microvascular events, without effect on all-cause mortality and cardiovascular mortality. Tight glucose control increases the risk of severe hypoglycemia.

It is well known that melatonin participates in the regulation of many important physiological functions such as sleep-wakefulness cycle, motor coordination and neural plasticity, and cognition. However, as there are contradictory results regarding the melatonin production diurnal profile under alcohol consumption, the aim of this paper was to study the phenomenology and mechanisms of the putative modifications on the daily profile of melatonin production in rats submitted to chronic alcohol intake. The present results show that rats receiving 10% ethanol in drinking water for 35 days display an altered daily profile of melatonin production, with a phase delay and a reduction in the nocturnal peak. This can be partially explained by a loss of the daily rhythm and the 25% reduction in tryptophan hydroxylase activity and, mainly, by a phase delay in arylalkylamine N-acetyltransferase gene expression and a 70% reduction in its peak activity. Upstream in the melatonin synthesis pathway, the results showed that noradrenergic signaling is impaired as well, with a decrease in β1 and α1 adrenergic receptors' mRNA contents and in vitro sustained loss of noradrenergic-stimulated melatonin production by glands from alcohol-treated rats. Together, these results confirm the alterations in the daily melatonin profile of alcoholic rats and suggest the possible mechanisms for the observed melatonin synthesis modification.

It is known that the circadian rhythm in hepatic phosphoenolpyruvate carboxykinase expression (a limiting catalytic step of gluconeogenesis) and hepatic glucose production is maintained by both daily oscillation in autonomic inputs to the liver and night feeding behavior. However, increased glycemia and reduced melatonin (Mel) levels have been recently shown to coexist in diabetic patients at the end of the night period. In parallel, pinealectomy (PINX) is known to cause glucose intolerance with increased basal glycemia exclusively at the end of the night. The mechanisms that underlie this metabolic feature are not completely understood. Here, we demonstrate that PINX rats show night-time hepatic insulin resistance characterized by reduced insulin-stimulated RAC-α serine/threonine-protein kinase phosphorylation and increased phosphoenolpyruvate carboxykinase expression. In addition, PINX rats display increased conversion of pyruvate into glucose at the end of the night. The regulatory mechanism suggests the participation of unfolded protein response (UPR), because PINX induces night-time increase in activating transcription factor 6 expression and prompts a circadian fashion of immunoglobulin heavy chain-binding protein, activating transcription factor 4, and CCAAT/enhancer-binding protein-homologous protein expression with Zenith values at the dark period. PINX also caused a night-time increase in Tribble 3 and regulatory-associated protein of mammalian target of rapamycin; both were reduced in liver of PINX rats treated with Mel. Treatment of PINX rats with 4-phenyl butyric acid, an inhibitor of UPR, restored night-time hepatic insulin sensitivity and abrogated gluconeogenesis in PINX rats. Altogether, the present data show that a circadian oscillation of UPR occurs in the liver due to the absence of Mel. The nocturnal UPR activation is related with night-time hepatic insulin resistance and increased gluconeogenesis in PINX rats. ispartof: Endocrinology vol:152 issue:4 pages:1253-63 ispartof: location:United States status: published

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Pesquisa Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Maternal pancreatic islets undergo a robust increase of mass and proliferation during pregnancy, which allows a compensation of gestational insulin resistance. Studies have described that this adaptation switches to a low proliferative status after the delivery. the mechanisms underlying this reversal are unknown, but the action of glucocorticoids (GCs) is believed to play an important role because GCs counteract the pregnancy-like effects of PRL on isolated pancreatic islets maintained in cell culture. Here, we demonstrate that ERK1/2 phosphorylation (phospho-ERK1/2) is increased in maternal rat islets isolated on the 19th day of pregnancy. Phospho-ERK1/2 status on the 3rd day after delivery (L3) rapidly turns to values lower than that found in virgin control rats (CTL). MKP-1, a protein phosphatase able to dephosphorylate ERK1/2, is increased in islets from L3 rats. Chromatin immunoprecipitation assay revealed that binding of glucocorticoid receptor (GR) to MKP-1 promoter is also increased in islets from L3 rats. in addition, dexamethasone (DEX) reduced phospho-ERK1/2 and increased MKP-1 expression in RINm5F and MIN-6 cells. Inhibition of transduction with cycloheximide and inhibition of phosphatases with orthovanadate efficiently blocked DEX-induced downregulation of phospho-ERK1/2. in addition, specific knockdown of MKP-1 with siRNA suppressed the downregulation of phosphoERK1/2 and the reduction of proliferation induced by DEX. Altogether, our results indicate that downregulation of phospho-ERK1/2 is associated with reduction in proliferation found in islets of early lactating mothers. This mechanism is probably mediated by GC-induced MKP-1 expression. Univ São Paulo, Inst Biomed Sci, Dept Physiol & Biophys, BR-05508 São Paulo, Brazil Universidade Federal de São Paulo, Dept Biol Sci, Diadema, SP, Brazil Univ Estadual Campinas, Fac Med Sci, Dept Pharmacol, São Paulo, Brazil Universidade Federal de São Paulo, Dept Biol Sci, Diadema, SP, Brazil Web of Science

Insulin-induced glucose uptake by skeletal muscle results from Akt2 activation and is severely impaired during insulin resistance. Recently, we and others have demonstrated that BMP9 improves glucose homeostasis in diabetic and non-diabetic rodents. However, the mechanism by which BMP9 modulates insulin action remains unknown. Here we demonstrate that Smad5, a transcription factor activated by BMP9, and Akt2, are upregulated in differentiated L6 myotubes. Smad5, rather than Smad1/8, is downregulated "in vivo" and "in vitro" by dexamethasone. Smad5 knockdown decreased Akt2 expression and serine phosphorylation and insulin-induced glucose uptake, and increased the expression of the lipid phosphatase Ship2. Additionally, binding of Smad5 to Akt2 gene is decreased in dexamethasone-treated rats and increased in L6 myotubes compared to myoblasts. The present study indicates that Smad5 regulates glucose uptake in skeletal muscle by controlling Akt2 expression and phosphorylation. These finding reveals Smad5 as a potential target for the therapeutic of type 2 diabetes.

Interleukin-10 (IL-10) is an endogenous factor that restrains hepatic insulin resistance in diet-induced steatosis. Reducing IL-10 expression increases proinflammatory activity in the steatotic liver and worsens insulin resistance. As the transcriptional coactivator proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha) plays a central role in dysfunctional hepatocytic activity in diet-induced steatosis, we hypothesized that at least part of the action of PGC-1alpha could be mediated by reducing the transcription of the IL-10 gene. Here, we used immunoblotting, real-time polymerase chain reaction, immunocytochemistry, and chromatin immunoprecipitation assay to investigate the role of PGC-1alpha in the control of IL-10 expression in hepatic cells. First, we show that, in the intact steatotic liver, the expressions of IL-10 and PGC-1alpha are increased. Inhibiting PGC-1alpha expression by antisense oligonucleotide increases IL-10 expression and reduces the steatotic phenotype. In cultured hepatocytes, the treatment with saturated and unsaturated fatty acids increased IL-10 expression. This was accompanied by increased association of PGC-1alpha with c-Maf and p50-nuclear factor (NF) kappaB, 2 transcription factors known to modulate IL-10 expression. In addition, after fatty acid treatment, PGC-1alpha, c-Maf, and p50-NFkappaB migrate from the cytosol to the nuclei of hepatocytes and bind to the IL-10 promoter region. Inhibiting NFkappaB activation with salicylate reduces IL-10 expression and the association of PGC-1alpha with p50-NFkappaB. Thus, PGC-1alpha emerges as a potential transcriptional regulator of the inflammatory phenomenon taking place in the steatotic liver.

In animal models of diet-induced obesity, the activation of an inflammatory response in the hypothalamus produces molecular and functional resistance to the anorexigenic hormones insulin and leptin. The primary events triggered by dietary fats that ultimately lead to hypothalamic cytokine expression and inflammatory signaling are unknown. Here, we test the hypothesis that dietary fats act through the activation of toll-like receptors 2/4 and endoplasmic reticulum stress to induce cytokine expression in the hypothalamus of rodents. According to our results, long-chain saturated fatty acids activate predominantly toll-like receptor 4 signaling, which determines not only the induction of local cytokine expression but also promotes endoplasmic reticulum stress. Rats fed on a monounsaturated fat-rich diet do not develop hypothalamic leptin resistance, whereas toll-like receptor 4 loss-of-function mutation and immunopharmacological inhibition of toll-like receptor 4 protects mice from diet-induced obesity. Thus, toll-like receptor 4 acts as a predominant molecular target for saturated fatty acids in the hypothalamus, triggering the intracellular signaling network that induces an inflammatory response, and determines the resistance to anorexigenic signals.

We have shown that rats chronically treated with Arginine (Arg), although normoglycemic, exhibit hyperinsulinemia and decreased blood glucose disappearance rate after an insulin challenge. Attempting to investigate the processes underlying these alterations, male Wistar rats were treated with Arg (35 mg/d), in drinking water, for 4 wk. Rats were then acutely stimulated with insulin, and the soleus and extensorum digitalis longus muscles, white adipose tissue (WAT), and liver were excised for total and/or phosphorylated insulin receptor (IR), IR substrate 1/2, Akt, Janus kinase 2, signal transducer and activator of transcription (STAT) 1/3/5, and p85α/55α determination. Muscles and WAT were also used for plasma membrane (PM) and microsome evaluation of glucose transporter (GLUT) 4 content. Pituitary GH mRNA, GH, and liver IGF-I mRNA expression were estimated. It was shown that Arg treatment: 1) did not affect phosphotyrosine-IR, whereas it decreased phosphotyrosine-IR substrate 1/2 and phosphoserine-Akt content in all tissues studied, indicating that insulin signaling is impaired at post-receptor level; 2) decreased PM GLUT4 content in both muscles and WAT; 3) increased the pituitary GH mRNA, GH, and liver IGF-I mRNA expression, the levels of phosphotyrosine-STAT5 in both muscles, phosphotyrosine-Janus kinase 2 in extensorum digitalis longus, phosphotyrosine-STAT3 in liver, and WAT as well as total p85α in soleus, indicating that GH signaling is enhanced in these tissues; and 4) increased p55α total content in muscles, WAT, and liver. The present findings provide the molecular mechanisms by which insulin resistance and, by extension, reduced GLUT4 content in PM of muscles and WAT take place after chronic administration of Arg, and further suggest a putative role for GH in its genesis, considering its diabetogenic effect.

Our aim was to evaluate the effects of granulocyte colony-stimulating factor (G-CSF) on early cardiac arrhythmias after myocardial infarction (MI) and the impact on survival. Male Wistar rats received repeated doses of 50 μg/kg G-CSF (MI-GCSF group) or vehicle (MI group) at 7, 3, and 1 days before surgery. MI was induced by permanent occlusion of left coronary artery. The electrocardiogram was obtained before occlusion and then for 30 minutes after surgery. Events and duration of ventricular arrhythmias were analyzed. The levels of connexin43 (Cx43) were measured by Western blot immediately before MI production. Survival was significantly increased in MI-GCSF pretreated group (74% versus 52.9% MI, P < 0.05). G-CSF pretreatment also significantly reduced the ventricular premature beats when compared with the untreated-MI group (201 ± 47 versus 679 ± 117, P < 0.05). The number and the duration of ventricular tachycardia were smaller in the MI-G-CSF group, as well as the number of ventricular fibrillation episodes (10% versus 69% in MI, P < 0.05). Cx43 levels were significantly increased by G-CSF treatment (1.27 ± 0.13 versus 0.86 ± 0.11; P < 0.05). The MI size 24 hours after occlusion was reduced by G-CSF pretreatment (36 ± 3% versus 44 ± 2% of left ventricle in MI group; P < 0.05). The increase of Cx43 expression in the heart may explain the reduced incidence in ventricular arrhythmias in the early phases after coronary artery occlusion in rats, thus increasing survival after MI.

Mutations in Na(+)-glucose transporters (SGLT)-2 and hepatocyte nuclear factor (HNF)-1alpha genes have been related to renal glycosuria and maturity-onset diabetes of the young 3, respectively. However, the expression of these genes have not been investigated in type 1 and type 2 diabetes. Here in kidney of diabetic rats, we tested the hypotheses that SGLT2 mRNA expression is altered; HNF-1alpha is involved in this regulation; and glycemic homeostasis is a related mechanism. The in vivo binding of HNF-1alpha into the SGLT2 promoter region in renal cortex was confirmed by chromatin immunoprecipitation assay. SGLT2 and HNF-1alpha mRNA expression (by Northern and RT-PCR analysis) and HNF-1 binding activity of nuclear proteins (by EMSA) were investigated in diabetic rats and treated or not with insulin or phlorizin (an inhibitor of SGLT2). Results showed that diabetes increases SGLT2 and HNF-1alpha mRNA expression (~50%) and binding of nuclear proteins to a HNF-1 consensus motif (~100%). Six days of insulin or phlorizin treatment restores these parameters to nondiabetic-rat levels. Moreover, both treatments similarly reduced glycemia, despite the differences in plasma insulin and urinary glucose concentrations, highlighting the plasma glucose levels as involved in the observed modulations. This study shows that SGLT2 mRNA expression and HNF-1alpha expression and activity correlate positively in kidney of diabetic rats. It also shows that diabetes-induced changes are reversed by lowering glycemia, independently of insulinemia. Our demonstration that HNF-1alpha binds DNA that encodes SGLT2 supports the hypothesis that HNF-1alpha, as a modulator of SGLT2 expression, may be involved in diabetic kidney disease.

It is known that at the moment of delivery immediate lost of conceptus (main site of glucose disposal in late pregnancy) is not able to disturb glucose homeostasis in early lactating mothers. However, the mechanism by which this adaptation takes place in early lactation is still unknown. Most studies concerning insulin sensitivity in lactating rats were carried out at 11–13 days postpartum and did not describe functional changes in insulin response in early lactation. Here we show that lactation hypersensitivity to insulin is observed as early as 3 days after delivery (L3). We show that the oxidative soleus muscle displays a transient increased maximal insulin-induced glucose uptake and CO2production, which is temporally limited to L3. Response of soleus muscle was accompanied by an increase in glucose transporter 4 (GLUT4) content at L3. This adaptive response was not detected in the glycolytic plantaris muscle, which displayed lower content of GLUT4. We also found that soleus muscle from early lactating rats have higher insulin receptor expression and tyrosine phosphorylation. Downstream steps of insulin signaling pathway; e.g., insulin receptor substrate 2 tyrosine phosphorylation and its association with phosphatidylinositol 3-kinase were also upregulated in soleus muscle. In parallel, protein tyrosine phosphatase 1B expression, a negative regulator of insulin signal, was reduced. Importantly, all of these molecular alterations were time limited to L3 and were not observed in plantaris muscle. These results suggest that improved insulin action in oxidative, but not in glycolytic muscle might contribute to achievement of glucose homeostasis postpartum.

Glucocorticoid (GC) hormone exerts numerous physiological roles that include modulation of immune, nervous, cardiovascular and metabolic systems. Synthetic GCs such as dexamethasone and prednisone/prednisolone are widely prescribed in the clinical context to the treatment of inflammatory-related diseases. In spite of its positive therapeutic effect, GC-based therapies may cause several adverse effects including glucose intolerance and peripheral insulin resistance. Reduction of insulin sensitivity in the adipocytes and adipose tissue caused by GC treatment is associated with increased lipolysis and abnormal Ser phosphorylation of insulin substrate receptor (IRS)-1 and protein kinase B (PKB). However, there is no consensus about the precise mechanisms whereby GC treatment promotes such attenuation in the insulin signaling pathway. In this paper, we will briefly discuss and present some molecular evidences that might be involved with this negative impact of GC in the insulin signaling in the adipose tissue.

Acute or chronic exposure of beta cells to glucose, palmitic acid or pro-inflammatory cytokines will result in increased production of the p47 phox component of the NADPH oxidase and subsequent production of reactive oxygen species (ROS). Rat pancreatic islets or clonal rat BRIN BD11 beta cells were incubated in the presence of glucose, palmitic acid or pro-inflammatory cytokines for periods between 1 and 24 h. p47 phox production was determined by western blotting. ROS production was determined by spectrophotometric nitroblue tetrazolium or fluorescence-based hydroethidine assays. Incubation for 24 h in 0.1 mmol/l palmitic acid or a pro-inflammatory cytokine cocktail increased p47 phox protein production by 1.5-fold or by 1.75-fold, respectively, in the BRIN BD11 beta cell line. In the presence of 16.7 mmol/l glucose protein production of p47 phox was increased by 1.7-fold in isolated rat islets after 1 h, while in the presence of 0.1 mmol/l palmitic acid or 5 ng/ml IL-1β it was increased by 1.4-fold or 1.8-fold, respectively. However, palmitic acid or IL-1β-dependent production was reduced after 24 h. Islet ROS production was significantly increased after incubation in elevated glucose for 1 h and was completely abolished by addition of diphenylene iodonium, an inhibitor of NADPH oxidase or by the oligonucleotide anti-p47 phox . Addition of 0.1 mmol/l palmitic acid or 5 ng/ml IL-1β plus 5.6 mmol/l glucose also resulted in a significant increase in islet ROS production after 1 h, which was partially attenuated by diphenylene iodonium or the protein kinase C inhibitor GF109203X. However, ROS production was reduced after 24 h incubation. NADPH oxidase may play a key role in normal beta cell physiology, but under specific conditions may also contribute to beta cell demise.

The Islet Neogenesis Associated Protein (INGAP) increases pancreatic beta-cell mass and potentiates glucose-induced insulin secretion. We currently studied the effects of a pentadecapeptide having the 104-118 amino acid sequence of INGAP (INGAP-PP) on insulin secretion and on transcript profile expression in 4-day-cultured normal pancreatic neonatal rat islets. Islets cultured with INGAP-PP released significantly more insulin in response to 2.8 and 16.7 mM glucose than those cultured without the peptide. The macroarray analysis showed that 210 out of 2352 genes spotted in the nylon membranes were up-regulated while only 4 were down-regulated by INGAP-PP-treatment. The main categories of genes modified by INGAP-PP included several related with islet metabolism, insulin secretion mechanism, beta-cell mass and islet neogenesis. RT-PCR confirmed the macroarray results for ten selected genes involved in growing, maturation, maintenance of pancreatic islet-cells, and exocytosis, i.e., Hepatocyte nuclear factor 3beta (HNF3beta), Upstream stimulatory factor 1 (USF1), K(+)-channel proteins (SUR1 and Kir6.2), PHAS-I protein, Insulin 1 gene, Glucagon gene, Mitogen-activated protein kinase 1 (MAP3K1), Amylin (IAPP), and SNAP-25. INGAP-PP also stimulated PDX-1 expression. The expression of three transcripts (HNF3beta, SUR1, and SNAP-25) was confirmed by Western blotting for the corresponding proteins. In conclusion, our results show that INGAP-PP enhances specifically the secretion of insulin and the transcription of several islet genes, many of them directly or indirectly involved in the control of islet metabolism, beta-cell mass and islet neogenesis. These results, together with other previously reported, strongly indicate an important role of INGAP-PP, and possibly of INGAP, in the regulation of islet function and development.

In the present study, we investigated the protein levels and phosphorylation status of the insulin receptor and insulin receptor substrates (IRS-1, IRS-2, and IRS-3) as well as their association with PI(3)-kinase in the rat adipose tissue of two models of insulin resistance: dexamethasone treatment and aging. AKT and atypical PKC phosphorylation detection were also performed. Both models showed decreased insulin-induced IRS-1 and IRS-2 tyrosine phosphorylation, accompanied by reduced protein levels of IRS-1 and IRS-2. Nevertheless, IRS-3 protein level was unchanged in aging but increased in dexamethasone-treated rats. PI(3)-kinase association with IRS-1 was reduced in aged rats, whereas dexamethasone-treated rats showed a reduced IRS-2/ PI(3)-kinase association. However, IRS-3 association with PI(3)-kinase was reduced in both models, as well as insulin-induced AKT and PKC phosphorylation. The alterations described in the present study show that the action of insulin is differently impaired depending on the origin of insulin resistance. These differences might be directly linked to the singular metabolic features of the models we tested.

The GLUT4 gene transcriptional activity has a profound impact on the insulin-mediated glucose disposal and it is, therefore, important to understand the mechanisms underlying it. Insulin and exercise modulate GLUT4 expression in vivo, but the net control and involved mechanisms of each one have not been established yet. This paper sought to discriminate, in soleus muscle, the effects of insulin and muscle contraction on GLUT4 gene expression, and the involvement of transcriptional factors: myocite enhancer factor 2 (MEF2 A/C/D), hypoxia inducible factor 1-a (HIF1-a) and nuclear factor-kappa B (NF-kappaB). The GLUT4 mRNA was reduced by fasting (40%), and increased by in vitro incubation with insulin (25%) or insulin plus glucose (40%), which was accompanied by opposite regulations of NF-kappaB mRNA. Differently, in vitro, muscle contraction led to a rapid increase (35-80%) in GLUT4, MEF2A, MEF2D and HIF1-a mRNAs. Additionally, electrophoretic mobility shift assay confirmed changes in the binding activity of nuclear proteins to consensus NF-kappaB, GLUT4-Ebox and GLUT4-AT-rich element probes, parallel to the mRNA changes of their respective transcriptional factors NF-kappaB, HIF1-a and MEF2s. Concluding, insulin- and contraction-induced regulation of GLUT4 expression involves distinct transcriptional factors.

Melatonin is a neurohormone that works as a nighttime signal for circadian integrity and health maintenance. It is crucial for energy metabolism regulation, and the diabetes effects on its synthesis are unresolved. Using diverse techniques that included pineal microdialysis and ultrahigh-performance liquid chromatography, the present data show a clear acute and sustained melatonin synthesis reduction in diabetic rats as a result of pineal metabolism impairment that is unrelated to cell death. Hyperglycemia is the main cause of several diabetic complications, and its consequences in terms of melatonin production were assessed. Here, we show that local high glucose (HG) concentration is acutely detrimental to pineal melatonin synthesis in rats both in vivo and in vitro. The clinically depressive action of high blood glucose concentration in melatonin levels was also observed in type 1 diabetes patients who presented a negative correlation between hyperglycemia and 6-sulfatoxymelatonin excretion. Additionally, high-mean-glycemia type 1 diabetes patients presented lower 6-sulfatoxymelatonin levels when compared to control subjects. Although further studies are needed to fully clarify the mechanisms, the present results provide evidence that high circulating glucose levels interfere with pineal melatonin production. Given the essential role played by melatonin as a powerful antioxidant and in the control of energy homeostasis, sleep and biological rhythms and knowing that optimal glycemic control is usually an issue for patients with diabetes, melatonin supplementation may be considered as an additional tool to the current treatment.

Excess of glucocorticoids (GCs) during pregnancy is strongly associated with the programming of glucose intolerance in the offspring. However, the impact of high GC levels on maternal metabolism is not clearly documented. This study aimed to test the hypothesis that mothers exposed to elevated levels of GCs might also display long-term disturbances in glucose homeostasis. Dexamethasone (DEX) was administered noninvasively to the mothers via drinking water between the 14th and the 19th days of pregnancy. Mothers were subjected to glucose and insulin tolerance tests at 1, 2, 3, 6, and 12 mo postweaning. Pregnant rats not treated with DEX and age-matched virgin rats were used as controls. Pancreatic islets were isolated at the 20th day of pregnancy and 12 mo postweaning in order to evaluate glucose-stimulated insulin secretion. The expression of the miR-29 family was also studied due to its responsiveness to GCs and its well-documented role in the regulation of pancreatic β-cell function. Rats treated with DEX during pregnancy presented long-term glucose intolerance and impaired insulin secretion. These changes correlated with 1) increased expression of miR-29 and its regulator p53, 2) reduced expression of syntaxin-1a, a direct target of miR-29, and 3) altered expression of genes related to cellular senescence. Our data demonstrate that the use of DEX during pregnancy results in deleterious outcomes to the maternal metabolism, hallmarked by reduced insulin secretion and glucose intolerance. This maternal metabolic programming might be a consequence of time-sustained upregulation of miR-29s in maternal pancreatic islets.

The effect of tetracaine on 45Ca efflux, cytoplasmic Ca2+ concentration [Ca2+]i, and insulin secretion in isolated pancreatic islets and beta-cells was studied. In the absence of external Ca2+, tetracaine (0.1-2.0 mM) increased the 45Ca efflux from isolated islets in a dose-dependentOFF efflux caused by 50 mM K+ or by the association of carbachol (0.2 mM) and 50 mM K+. Tetracaine permanently increased the [Ca2+]i in isolated beta-cells in Ca2+-free medium enriched with 2.8 mM glucose and 25 microM D-600 (methoxiverapamil). This effect was also observed in the presence of 10 mM caffeine or 1 microM thapsigargin. In the presence of 16.7 mM glucose, tetracaine transiently increased the insulin secretion from islets perfused in the absence and presence of external Ca2+. These data indicate that tetracaine mobilises Ca2+ from a thapsigargin-insensitive store and stimulates insulin secretion in the absence of extracellular Ca2+. The increase in 45Ca efflux caused by high concentrations of K+ and by carbachol indicates that tetracaine did not interfere with a cation or inositol triphosphate sensitive Ca2+ pool in beta-cells.

The effects of the muscarinic agonist oxotremorine-m (Oxo-m) on 45Ca and 86Rb fluxes, insulin secretion, cytoplasmic Ca2+ concentration ([Ca2+]i) and membrane potential in pancreatic B-cells were studied. Oxo-m (40-200 microM) increased the [Ca2+]i by about 250 nM, irrespective of the glucose concentration present in the medium (2.8-22 mM). This effect was reduced by 50% upon the addition of EGTA. Oxo-m (50 microM) increased the 45Ca efflux from islets perifused in the absence or presence of [Ca2+]o, although under the former condition this efflux was transient. The difference between effluxes measured in the absence and presence of [Ca2+]o represents the sustained second component, which presumably reflects Ca2+ influx. In both the absence and presence of 11.2 mM glucose. Oxo-m (50 microM) transiently increased 86Rb efflux. In the presence of glucose, Oxo-m provoked a transient polarization of the B-cell membrane associated with an increase in the K+ permeability values. K+ permeability returned to basal values (no Oxo-m) after 1-2 min. These results indicate that the initial phase of Oxo-m-induced insulin secretion depends partially on intracellular Ca2+ release, and that the sustained enhancement of release depends on Ca2+ influx. The participation of a calcium release-activated current (ICRAC) is proposed to explain the sustained small changes in membrane potential.

The effect of the local anesthetic, tetracaine, on 45 Ca efflux, cytoplasmic Ca 2+ concentration [Ca 2+ ] i and insulin secretion in pancreatic B-cells was studied. At a physiological level of [Ca 2+ ] o , tetracaine (0.1–5 mM) dose-dependently inhibited insulin secretion induced by 22 mM glucose. Paradoxically, at the same glucose concentration but in the absence of external Ca 2+ , tetracaine dose-dependently increased insulin secretion. At a low glucose level (2.8 mM) tetracaine failed to affect secretion, either in the presence or absence of external Ca 2+ . At high (22 mM) or low (2.8 mM) glucose, [Ca 2+ ] i was increased by tetracaine in a dose-dependent manner. Tetracaine (2 mM) also increased the 45 Ca efflux from isolated islets. This effect was of the same magnitude at both low and high glucose concentrations, and was independent of the presence of extracellular Ca 2+ . Finally, tetracaine increased 45 Ca efflux from islets perifused in the presence of thapsigargin. In conclusion, our data indicate that tetracaine releases Ca 2+ from a thapsigargin-insensitive store in pancreatic B-cells. Under suitable experimental conditions, insulin release can be elicited by a [Ca 2+ ] o -independent pathway. The existence of a ryanodine-like Ca 2+ channel in pancreatic B-cells is proposed.

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) A positive relationship between obesity and asthma has been well documented. the AMP-activated protein kinase (AMPK) activator metformin reverses obesity-associated insulin resistance (IR) and inhibits different types of inflammatory responses. This study aimed to evaluate the effects of metformin on the exacerbation of allergic eosinophilic inflammation in obese mice. Male C57BL6/J mice were fed for 10 weeks with high-fat diet (HFD) to induce obesity. the cell infiltration and inflammatory markers in bronchoalveolar lavage (BAL) fluid and lung tissue were evaluated at 48 h after ovalbumin (OVA) challenge. HFD obese mice displayed peripheral IR that was fully reversed by metformin (300 mg/kg/day, two weeks). OVA-challenge resulted in higher influx of total cell and eosinophils in lung tissue of obese mice compared with lean group. As opposed, the cell number in BAL fluid of obese mice was reduced compared with lean group. Metformin significantly reduced the tissue eosinophil infiltration and prevented the reduction of cell counts in BAL fluid. in obese mice, greater levels of eotaxin, TNF-alpha and NOx, together with increased iNOS protein expression were observed, all of which were normalized by metformin. in addition, metformin nearly abrogated the binding of NF-kappa B subunit p65 to the iNOS promoter gene in lung tissue of obese mice. Lower levels of phosphorylated AMPK and its downstream target acetyl CoA carboxylase (ACC) were found in lung tissue of obese mice, which were restored by metformin. in separate experiments, the selective iNOS inhibitor aminoguanidine (20 mg/kg, 3 weeks) and the anti-TNF-alpha mAb (2 mg/kg) significantly attenuated the aggravation of eosinophilic inflammation in obese mice. in conclusion, metformin inhibits the TNF-alpha-induced inflammatory signaling and NF-kappa B-mediated iNOS expression in lung tissue of obese mice. Metformin may be a good pharmacological strategy to control the asthma exacerbation in obese individuals. State Univ Campinas UNICAMP, Fac Med Sci, Dept Pharmacol, Campinas, SP, Brazil Univ São Paulo, Inst Biomed Sci, Dept Physiol & Biophys, São Paulo, Brazil Universidade Federal de São Paulo, Dept Biol Sci, São Paulo, Brazil Universidade Federal de São Paulo, Dept Biol Sci, São Paulo, Brazil FAPESP: 2012/14225-1 Web of Science

Melatonin can contribute to glucose homeostasis either by decreasing gluconeogenesis or by counteracting insulin resistance in distinct models of obesity. However, the precise mechanism through which melatonin controls glucose homeostasis is not completely understood. Male Wistar rats were administered an intracerebroventricular (icv) injection of melatonin and one of following: an icv injection of a phosphatidylinositol 3-kinase (PI3K) inhibitor, an icv injection of a melatonin receptor (MT) antagonist, or an intraperitoneal (ip) injection of a muscarinic receptor antagonist. Anesthetized rats were subjected to pyruvate tolerance test to estimate in vivo glucose clearance after pyruvate load and in situ liver perfusion to assess hepatic gluconeogenesis. The hypothalamus was removed to determine Akt phosphorylation. Melatonin injections in the central nervous system suppressed hepatic gluconeogenesis and increased hypothalamic Akt phosphorylation. These effects of melatonin were suppressed either by icv injections of PI3K inhibitors and MT antagonists and by ip injection of a muscarinic receptor antagonist. We conclude that melatonin activates hypothalamus-liver communication that may contribute to circadian adjustments of gluconeogenesis. These data further suggest a physiopathological relationship between the circadian disruptions in metabolism and reduced levels of melatonin found in type 2 diabetes patients.

Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-27T11:28:16Z No. of bitstreams: 0 Made available in DSpace on 2014-05-27T11:28:16Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-01-30 High systolic blood pressure caused by endothelial dysfunction is a comorbidity of metabolic syndrome that is mediated by local inflammatory signals. Insulin-induced vasorelaxation due to endothelial nitric oxide synthase (eNOS) activation is highly dependent on the activation of the upstream insulin-stimulated serine/threonine kinase (AKT) and is severely impaired in obese, hypertensive rodents and humans. Neutralisation of circulating tumor necrosis factor-α (TNFα) with infliximab improves glucose homeostasis, but the consequences of this pharmacological strategy on systolic blood pressure and eNOS activation are unknown. To address this issue, we assessed the temporal changes in the systolic pressure of spontaneously hypertensive rats (SHR) treated with infliximab. We also assessed the activation of critical proteins that mediate insulin activity and TNFα-mediated insulin resistance in the aorta and cardiac left ventricle. Our data demonstrate that infliximab prevents the upregulation of both systolic pressure and left ventricle hypertrophy in SHR. These effects paralleled an increase in AKT/eNOS phosphorylation and a reduction in the phosphorylation of inhibitor of nuclear factor-κB (Iκβ) and c-Jun N-terminal kinase (JNK) in the aorta. Overall, our study revealed the cardiovascular benefits of infliximab in SHR. In addition, the present findings further suggested that the reduction of systolic pressure and left ventricle hypertrophy by infliximab are secondary effects to the reduction of endothelial inflammation and the recovery of AKT/eNOS pathway activation. © 2012 Elsevier B.V. Department of Pharmacology Faculty of Medical Sciences State University of Campinas, Campinas, SP Department of Internal Medicine Faculty of Medical Sciences State University of Campinas, Campinas, SP Department of Physiology and Biophysics Institute of Biomedical Sciences State University of Sao Paulo, Sao Paulo, SP

In aged rats, insulin signaling pathway (ISP) is impaired in tissues that play a pivotal role in glucose homeostasis, such as liver, skeletal muscle, and adipose tissue. Moreover, the aging process is also associated with obesity and reduction in melatonin synthesis from the pineal gland and other organs. The aim of the present work was to evaluate, in male old obese Wistar rats, the effect of melatonin supplementation in the ISP, analyzing the total protein amount and the phosphorylated status (immunoprecipitation and immunoblotting) of the insulin cascade components in the rat hypothalamus, liver, skeletal muscle, and periepididymal adipose tissue. Melatonin was administered in the drinking water for 8- and 12 wk during the night period. Food and water intake and fasting blood glucose remained unchanged. The insulin sensitivity presented a 2.1-fold increase both after 8- and 12 wk of melatonin supplementation. Animals supplemented with melatonin for 12 wk also presented a reduction in body mass. The acute insulin-induced phosphorylation of the analyzed ISP proteins increased 1.3- and 2.3-fold after 8- and 12 wk of melatonin supplementation. The total protein content of the insulin receptor (IR) and the IR substrates (IRS-1, 2) remained unchanged in all investigated tissues, except for the 2-fold increase in the total amount of IRS-1 in the periepididymal adipose tissue. Therefore, the known age-related melatonin synthesis reduction may also be involved in the development of insulin resistance and the adequate supplementation could be an important alternative for the prevention of insulin signaling impairment in aged organisms.

The transition from gestation to lactation is characterized by a robust adaptation of maternal pancreatic β-cells. Consistent with the loss of β-cell mass, glucose-induced insulin secretion is down-regulated in the islets of early lactating dams. Extensive experimental evidence has demonstrated that the surge of prolactin is responsible for the morphofunctional remodeling of the maternal endocrine pancreas during pregnancy, but the precise molecular mechanisms by which this phenotype is rapidly reversed after delivery are not completely understood. This study investigated whether glucocorticoid-regulated expression of Rasd1/Dexras, a small inhibitory G protein, is involved in this physiological plasticity. Immunofluorescent staining demonstrated that Rasd1 is localized within pancreatic β-cells. Rasd1 expression in insulin-secreting cells was increased by dexamethasone and decreased by prolactin. In vivo data confirmed that Rasd1 expression is decreased in islets from pregnant rats and increased in islets from lactating mothers. Knockdown of Rasd1 abolished the inhibitory effects of dexamethasone on insulin secretion and the protein kinase A, protein kinase C, and ERK1/2 pathways. Chromatin immunoprecipitation experiments revealed that glucocorticoid receptor (GR) and signal transducer and activator of transcription 5b (STAT5b) cooperatively mediate glucocorticoid-induced Rasd1 expression in islets. Prolactin inhibited the stimulatory effect of GR/STAT5b complex on Rasd1 transcription. Overall, our data indicate that the stimulation of Rasd1 expression by glucocorticoid at the end of pregnancy reverses the increased insulin secretion that occurs during pregnancy. Prolactin negatively regulates this pathway by inhibiting GR/STAT5b transcriptional activity on the Rasd1 gene. ispartof: Endocrinology vol:153 issue:8 pages:3668-78 ispartof: location:United States status: published

Fructose consumption causes insulin resistance and favors hepatic gluconeogenesis through mechanisms that are not completely understood. Recent studies demonstrated that the activation of hypothalamic 5′-AMP-activated protein kinase (AMPK) controls dynamic fluctuations in hepatic glucose production. Thus, the present study was designed to investigate whether hypothalamic AMPK activation by fructose would mediate increased gluconeogenesis. Both ip and intracerebroventricular (icv) fructose treatment stimulated hypothalamic AMPK and acetyl-CoA carboxylase phosphorylation, in parallel with increased hepatic phosphoenolpyruvate carboxy kinase (PEPCK) and gluconeogenesis. An increase in AMPK phosphorylation by icv fructose was observed in the lateral hypothalamus as well as in the paraventricular nucleus and the arcuate nucleus. These effects were mimicked by icv 5-amino-imidazole-4-carboxamide-1-β-d-ribofuranoside treatment. Hypothalamic AMPK inhibition with icv injection of compound C or with injection of a small interfering RNA targeted to AMPKα2 in the mediobasal hypothalamus (MBH) suppressed the hepatic effects of ip fructose. We also found that fructose increased corticosterone levels through a mechanism that is dependent on hypothalamic AMPK activation. Concomitantly, fructose-stimulated gluconeogenesis, hepatic PEPCK expression, and glucocorticoid receptor binding to the PEPCK gene were suppressed by pharmacological glucocorticoid receptor blockage. Altogether the data presented herein support the hypothesis that fructose-induced hypothalamic AMPK activation stimulates hepatic gluconeogenesis by increasing corticosterone levels.

BackgroundShift work was recently described as a factor that increases the risk of Type 2 diabetes mellitus. In addition, rats born to mothers subjected to a phase shift throughout pregnancy are glucose intolerant. However, the mechanism by which a phase shift transmits metabolic information to the offspring has not been determined. Among several endocrine secretions, phase shifts in the light/dark cycle were described as altering the circadian profile of melatonin production by the pineal gland. The present study addresses the importance of maternal melatonin for the metabolic programming of the offspring.Methodology/principal findingsFemale Wistar rats were submitted to SHAM surgery or pinealectomy (PINX). The PINX rats were divided into two groups and received either melatonin (PM) or vehicle. The SHAM, the PINX vehicle and the PM females were housed with male Wistar rats. Rats were allowed to mate and after weaning, the male and female offspring were subjected to a glucose tolerance test (GTT), a pyruvate tolerance test (PTT) and an insulin tolerance test (ITT). Pancreatic islets were isolated for insulin secretion, and insulin signaling was assessed in the liver and in the skeletal muscle by western blots. We found that male and female rats born to PINX mothers display glucose intolerance at the end of the light phase of the light/dark cycle, but not at the beginning. We further demonstrate that impaired glucose-stimulated insulin secretion and hepatic insulin resistance are mechanisms that may contribute to glucose intolerance in the offspring of PINX mothers. The metabolic programming described here occurs due to an absence of maternal melatonin because the offspring born to PINX mothers treated with melatonin were not glucose intolerant.Conclusions/significanceThe present results support the novel concept that maternal melatonin is responsible for the programming of the daily pattern of energy metabolism in their offspring.