Unveiling the presence of epigenetic mark by Lactobacillus supplementation in high-fat diet-induced metabolic disorder in Sprague-Dawley rats.

Gut dysbiosis, particularly bacteria from Firmicutes and Bacteroidetes phyla, plays a fundamental role in the progression of metabolic disorders. Probiotics have shown to restore the gut microbiota composition in metabolic disorders with subsequent beneficial effects. Recent studies have reported that several species of Lactobacillus as probiotic supplementation improve insulin sensitivity and glucose metabolism. Nonetheless, whether Lactobacillus could influence the epigenetic modifications that underlie insulin-resistant conditions is still unexplored. Therefore, the current study examined the therapeutic effects and underlying epigenetic mechanisms of three different species of Lactobacillus in the high-fat diet (HFD)-induced insulin-resistant rats. Three different species of Lactobacillus; Lactobacillus casei, Lactobacillus gasseri, and Lactobacillus rhamnosus were individually supplemented orally (109 CFU/mL) to insulin-resistant SD rats for 12 weeks. Lactobacillus supplementation led to a significant reduction in the hyperglycemia, hyperinsulinemia, and hyperlipidemia associated with HFD-induced insulin resistance. Histopathological examination also indicated the protective effects of Lactobacillus supplementation against the hepatic and intestinal damage caused by the high-fat diet. Lactobacillus supplementation also down-regulated the expression of FOXO1, a major transcription factor of insulin signaling. In addition, at the epigenetic level, Lactobacillus supplementation predominantly prevented methylation and demethylation of H3K79me2 and H3K27me3, respectively. Chromatin Immunoprecipitation (ChIP) coupled with quantitative PCR (ChIP-qPCR) assay revealed the presence of cross-talk between these two histone modifications at the promoter region of FOXO1. Taken together, this is the first report to observe that the effects of Lactobacillus supplementation involve alteration in FOXO1 expression via cross-talking between H3K79me2 and H3K27me3 histone modifications.

Effect of Ursolic Acid on Insulin Resistance and Hyperinsulinemia in Rats with Diet-Induced Obesity: Role of Adipokines Expression.

Excess of visceral adipose tissue (VAT) characteristic of obesity leads to a proinflammatory state disrupting the insulin signaling pathway, triggering insulin resistance (IR) and inflammation, the main processes contributing to obesity comorbidities. Ursolic acid (UA), a pentacyclic triterpenoid occurring in a variety of plant foods, exhibits anti-inflammatory properties. The aim of this study was to evaluate UA effects on IR, hyperinsulinemia, and inflammation in experimental diet-induced obesity. Forty male Wistar rats were randomly assigned to eight groups (n = 5). One group was used for time 0. Three groups were labeled as OBE (control): receiving high-fat diet (HFD; fat content 45.24% of energy) during 3, 6, or 9 weeks; three groups UA-PREV: exposed to simultaneous HFD and UA during 3, 6, or 9 weeks to evaluate UA preventive effects; one group UA-REV: receiving HFD for 6 weeks, followed by simultaneous HFD and UA for three additional weeks to analyze UA reversal effects. Measurements were performed after 3, 6, or 9 weeks of treatment. Adiposity was calculated by weighing VAT after sacrifice. Serum markers were quantified through colorimetric and enzyme-linked immunosorbent assay methods. VAT adipokines RNAm expression was evaluated by quantitative reverse transcriptase-polymerase chain reaction. Data were analyzed by Kruskal-Wallis and Mann-Whitney U tests. UA significantly decreased adiposity, IR, hyperinsulinemia, triacylglycerides, and cholesterol levels, and also VAT mRNA expression of MCP-1 (monocyte chemoattractant protein-1), IL (interleukin)-1ß and IL-6, concomitantly increasing adiponectin levels. UA metabolic effects demonstrated in this study support its potential therapeutic utility to improve IR, hyperinsulinemia, and inflammation observed in obesity and diabetes.

Olanzapine Induced Dysmetabolic Changes Involving Tissue Chromium Mobilization in Female Rats.

Atypical antipsychotics, such as olanzapine, are commonly prescribed to patients with schizophrenic symptoms and other psychiatric disorders. However, weight gain and metabolic disturbance cause adverse effects, impair patient compliance and limit clinical utility. Thus, a better understanding of treatment-acquired adverse effects and identification of targets for therapeutic intervention are believed to offer more clinical benefits for patients with schizophrenia. Beyond its nutritional effects, studies have indicated that supplementation of chromium brings about beneficial outcomes against numerous metabolic disorders. In this study, we investigated whether olanzapine-induced weight gain and metabolic disturbance involved chromium dynamic mobilization in a female Sprague-Dawley rat model, and whether a dietary supplement of chromium improved olanzapine-acquired adverse effects. Olanzapine medicated rats experienced weight gain and adiposity, as well as the development of hyperglycemia, hyperinsulinemia, insulin resistance, hyperlipidemia, and inflammation. The olanzapine-induced metabolic disturbance was accompanied by a decrease in hepatic Akt and AMP-activated Protein Kinase (AMPK) actions, as well as an increase in serum interleukin-6 (IL-6), along with tissue chromium depletion. A daily intake of chromium supplements increased tissue chromium levels and thermogenic uncoupling protein-1 (UCP-1) expression in white adipose tissues, as well as improved both post-olanzapine weight gain and metabolic disturbance. Our findings suggest that olanzapine medicated rats showed a disturbance of tissue chromium homeostasis by inducing tissue depletion and urinary excretion. This loss may be an alternative mechanism responsible for olanzapine-induced weight gain and metabolic disturbance.

The Obesity-Linked Gene Nudt3 Drosophila Homolog Aps Is Associated With Insulin Signaling.

Several genome-wide association studies have linked the Nudix hydrolase family member nucleoside diphosphate-linked moiety X motif 3 (NUDT3) to obesity. However, the manner of NUDT3 involvement in obesity is unknown, and NUDT3 expression, regulation, and signaling in the central nervous system has not been studied. We performed an extensive expression analysis in mice, as well as knocked down the Drosophila NUDT3 homolog Aps in the nervous system, to determine its effect on metabolism. Detailed in situ hybridization studies in the mouse brain revealed abundant Nudt3 mRNA and protein expression throughout the brain, including reward- and feeding-related regions of the hypothalamus and amygdala, whereas Nudt3 mRNA expression was significantly up-regulated in the hypothalamus and brainstem of food-deprived mice. Knocking down Aps in the Drosophila central nervous system, or a subset of median neurosecretory cells, known as the insulin-producing cells (IPCs), induces hyperinsulinemia-like phenotypes, including a decrease in circulating trehalose levels as well as significantly decreasing all carbohydrate levels under starvation conditions. Moreover, lowering Aps IPC expression leads to a decreased ability to recruit these lipids during starvation. Also, loss of neuronal Aps expression caused a starvation susceptibility phenotype while inducing hyperphagia. Finally, the loss of IPC Aps lowered the expression of Akh, Ilp6, and Ilp3, genes known to be inhibited by insulin signaling. These results point toward a role for this gene in the regulation of insulin signaling, which could explain the robust association with obesity in humans.

Long-term supranutritional supplementation with selenate decreases hyperglycemia and promotes fatty liver degeneration by inducing hyperinsulinemia in diabetic db/db mice.

There are conflicting reports on the link between the micronutrient selenium and the prevalence of diabetes. To investigate the possibility that selenium acts as a "double-edged sword" in diabetes, cDNA microarray profiling and two-dimensional differential gel electrophoresis coupled with mass spectrometry were used to determine changes in mRNA and protein expression in pancreatic and liver tissues of diabetic db/db mice in response to dietary selenate supplementation. Fasting blood glucose levels increased continuously in db/db mice administered placebo (DMCtrl), but decreased gradually in selenate-supplemented db/db mice (DMSe) and approached normal levels after termination of the experiment. Pancreatic islet size was increased in DMSe mice compared with DMCtrl mice, resulting in a clear increase in insulin production and a doubling of plasma insulin concentration. Genes that encode proteins involved in key pancreatic ß-cell functions, including regulation of ß-cell proliferation and differentiation and insulin synthesis, were found to be specifically upregulated in DMSe mice. In contrast, apoptosis-associated genes were downregulated, indicating that islet function was protected by selenate treatment. Conversely, liver fat accumulation increased in DMSe mice together with significant upregulation of lipogenic and inflammatory genes. Genes related to detoxification were downregulated and antioxidant enzymatic activity was reduced, indicating an unexpected reduction in antioxidant defense capacity and exacerbation of fatty liver degeneration. Moreover, proteomic analysis of the liver showed differential expression of proteins involved in glucolipid metabolism and the endoplasmic reticulum assembly pathway. Taken together, these results suggest that dietary selenate supplementation in db/db mice decreased hyperglycemia by increasing insulin production and secretion; however, long-term hyperinsulinemia eventually led to reduced antioxidant defense capacity, which exacerbated fatty liver degeneration.

The structure and allosteric regulation of mammalian glutamate dehydrogenase.

Glutamate dehydrogenase (GDH) is a homohexameric enzyme that catalyzes the reversible oxidative deamination of l-glutamate to 2-oxoglutarate. Only in the animal kingdom is this enzyme heavily allosterically regulated by a wide array of metabolites. The major activators are ADP and leucine, while the most important inhibitors include GTP, palmitoyl CoA, and ATP. Recently, spontaneous mutations in the GTP inhibitory site that lead to the hyperinsulinism/hyperammonemia (HHS) syndrome have shed light as to why mammalian GDH is so tightly regulated. Patients with HHS exhibit hypersecretion of insulin upon consumption of protein and concomitantly extremely high levels of ammonium in the serum. The atomic structures of four new inhibitors complexed with GDH complexes have identified three different allosteric binding sites. Using a transgenic mouse model expressing the human HHS form of GDH, at least three of these compounds were found to block the dysregulated form of GDH in pancreatic tissue. EGCG from green tea prevented the hyper-response to amino acids in whole animals and improved basal serum glucose levels. The atomic structure of the ECG-GDH complex and mutagenesis studies is directing structure-based drug design using these polyphenols as a base scaffold. In addition, all of these allosteric inhibitors are elucidating the atomic mechanisms of allostery in this complex enzyme.

Carcinoembryonic antigen-related cell adhesion molecule 2 controls energy balance and peripheral insulin action in mice.

BACKGROUND & AIMS: The carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is a transmembrane glycoprotein with pleotropic functions, including clearance of hepatic insulin. We investigated the functions of the related protein CEACAM2, which has tissue-specific distribution (kidney, uterus, and crypt epithelia of intestinal tissues), in genetically modified mice. METHODS: Ceacam2-null mice (Cc2-/-) were generated from a 129/SvxC57BL/6J background. Female mice were assessed by hyperinsulinemic-euglycemic clamp analysis and indirect calorimetry and body fat composition was measured. Cc2-/- mice and controls were fed as pairs, given insulin tolerance tests, and phenotypically characterized. RESULTS: Female, but not male Cc2-/- mice exhibited obesity that resulted from hyperphagia and reduced energy expenditure. Pair feeding experiments showed that hyperphagia led to peripheral insulin resistance. Insulin action was normal in liver but compromised in skeletal muscle of female Cc2-/- mice; the mice had incomplete fatty acid oxidation and impaired glucose uptake and disposal. The mechanism of hyperphagia in Cc2-/- mice is not clear, but appears to result partly from increased hyperinsulinemia-induced hypothalamic fatty acid synthase levels and activity. Hyperinsulinemia was caused by increased insulin secretion. CONCLUSIONS: In mice, CEACAM2 is expressed by the hypothalamus. Cc2-/- mice develop obesity from hyperphagia and reduced energy expenditure, indicating its role in regulating energy balance and insulin sensitivity.

Leptin receptor polymorphisms interact with polyunsaturated fatty acids to augment risk of insulin resistance and metabolic syndrome in adults.

The leptin receptor (LEPR) is associated with insulin resistance, a key feature of metabolic syndrome (MetS). Gene-fatty acid interactions may affect MetS risk. The objective was to investigate the relationship among LEPR polymorphisms, insulin resistance, and MetS risk and whether plasma fatty acids, a biomarker of dietary fatty acids, modulate this. LEPR polymorphisms (rs10493380, rs1137100, rs1137101, rs12067936, rs1805096, rs2025805, rs3790419, rs3790433, rs6673324, and rs8179183), biochemical measurements, and plasma fatty acid profiles were determined in the LIPGENE-SU.VI.MAX study of MetS cases and matched controls (n = 1754). LEPR rs3790433 GG homozygotes had increased MetS risk compared with the minor A allele carriers [odds ratio (OR) = 1.65; 95% CI: 1.05-2.57; P = 0.028], which may be accounted for by their increased risk of elevated insulin concentrations (OR 2.40; 95% CI: 1.28-4.50; P = 0.006) and insulin resistance (OR = 2.15; 95% CI: 1.18-3.90; P = 0.012). Low (less than median) plasma (n-3) and high (n-6) PUFA status exacerbated the genetic risk conferred by GG homozygosity to hyperinsulinemia (OR 2.92-2.94) and insulin resistance (OR 3.40-3.47). Interestingly, these associations were abolished against a high (n-3) or low (n-6) PUFA background. Importantly, we replicated some of these findings in an independent cohort. Homozygosity for the LEPR rs3790433 G allele was associated with insulin resistance, which may predispose to increased MetS risk. Novel gene-nutrient interactions between LEPR rs3790433 and PUFA suggest that these genetic influences were more evident in individuals with low plasma (n-3) or high plasma (n-6) PUFA.

Estudio PET-TC con 18F-fluoro-L-DOPA combinado con el análisis genético para la optimización de la clasificación y tratamiento de un niño con hiperinsulinismo congénito grave / 18F-fluoro-L-DOPA PET-CT imaging combined with genetic analysis for optimal classification and treatment in a child with severe congenital hyperinsulinism

Fundamento: La distinción entre el hiperinsulinismo congénito (CHI) focal y difuso es esencial de cara al tratamiento y pronóstico de la enfermedad. El objetivo es presentar el primer caso de CHI focal diagnosticado en España combinando los estudios genético y PET-TC. Métodos: Paciente de 13 meses con CHI y pruebas de imagen convencionales normales, tratado con diazóxido, control dietético y alimentación por gastrostomía. Se analizó la secuencia de los genes ABCC8 y KCNJ11, y realizó una PET-TC con 18F-fluoro-L-DOPA. Resultados: Se detectó una mutación patogénica (G111R) en el alelo paterno de ABCC8. La PET-TC demostró un foco hipercaptante en el cuerpo del páncreas compatible con un adenoma confirmado histopatológicamente. Tras la cirugía el paciente continúa asintomático sin tratamiento farmacológico ni medidas dietéticas. Conclusiones: La combinación del análisis genético y la PET-TC con 18F-fluoro-L-DOPA muestra un gran potencial para la identificación, localización y guía de la cirugía del CHI (AU)

Background: Congenital hyperinsulinism (CHI) is the most common cause of persistent hypoglycaemia in infancy. The differential diagnosis between focal and diffuse forms of CHI is of great importance when planning surgery. The aim of this article is to show the first case of focal CHI diagnosed in Spain using PET-CT imaging combined with genetic analysis. Methods: A 13 month child with CHI and normal conventional radiological investigations treated with diazoxide, diet control and feeding by gastrostomy is presented. Genetic analysis of ABCC8 and KCNJ11 genes and PET-TAC using 18F-fluoro-L-DOPA were performed. Results: A pathological mutation (G111R) in the paternal allele of ABCC8 was detected. PET-CT scanning using 18F-fluoro-L-DOPA showed a focus of high uptake in the body of the pancreas compatible with adenoma that was hystopathologically confirmed. After surgical resection the patient is asymptomatic without needing either pharmacological treatment or dietetic control. Conclusions: The combination of genetic analysis and 18F-fluoro-L-DOPA PET-TAC shows a great potential for the identification, location and guideline for surgery in CHI (AU)

Reduced expression of the KATP channel subunit, Kir6.2, is associated with decreased expression of neuropeptide Y and agouti-related protein in the hypothalami of Zucker diabetic fatty rats.

The link between obesity and diabetes is not fully understood but there is evidence to suggest that hypothalamic signalling pathways may be involved. The hypothalamic neuropeptides, pro-opiomelanocortin (POMC), neuropeptide Y (NPY) and agouti-related protein (AGRP) are central to the regulation of food intake and have been implicated in glucose homeostasis. Therefore, the expression of these genes was quantified in hypothalami from diabetic Zucker fatty (ZDF) rats and nondiabetic Zucker fatty (ZF) rats at 6, 8, 10 and 14 weeks of age. Although both strains are obese, only ZDF rats develop pancreatic degeneration and diabetes over this time period. In both ZF and ZDF rats, POMC gene expression was decreased in obese versus lean rats at all ages. By contrast, although there was the expected increase in both NPY and AGRP expression in obese 14-week-old ZF rats, the expression of NPY and AGRP was decreased in 6-week-old obese ZDF rats with hyperinsulinaemia and in 14-week-old rats with the additional hyperglycaemia. Therefore, candidate genes involved in glucose, and insulin signalling pathways were examined in obese ZDF rats over this age range. We found that expression of the ATP-sensitive potassium (K(ATP)) channel component, Kir6.2, was decreased in obese ZDF rats and was lower compared to ZF rats in each age group tested. Furthermore, immunofluorescence analysis showed that Kir6.2 protein expression was reduced in the dorsomedial and ventromedial hypothalamic nuclei of 6-week-old prediabetic ZDF rats compared to ZF rats. The Kir6.2 immunofluorescence colocalised with NPY throughout the hypothalamus. The differences in Kir6.2 expression in ZF and ZDF rats mimic those of NPY and AGRP, which could infer that the changes occur in the same neurones. Overall, these data suggest that chronic changes in hypothalamic Kir6.2 expression may be associated with the development of hyperinsulinaemia and hyperglycaemia in ZDF rats.

Hyperglycemia- and hyperinsulinemia-induced alteration of adiponectin receptor expression and adiponectin effects in L6 myoblasts.

Adiponectin has been shown to regulate glucose and fatty acid uptake and metabolism in skeletal muscle. Here we investigated the role of the recently cloned adiponectin receptor (AdipoR) isoforms in mediating effects of both globular (gAd) and full-length (fAd) adiponectin, and their regulation by hyperglycemia (25 mM, 20 h) and hyperinsulinemia (100 nM, 20 h). We used L6 rat skeletal muscle cells, which were found to express both AdipoR1 and AdipoR2 mRNA in a ratio of over 6:1 respectively. Hyperglycemia and hyperinsulinemia both decreased AdipoR1 receptor expression by approximately 50%, while the latter induced an increase of approximately threefold in AdipoR2 expression. The ability of gAd to increase GLUT4 myc translocation, glucose uptake, fatty acid uptake and oxidation, as well as AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) phosphorylation, was decreased by both hyperglycemia and hyperinsulinemia. Interestingly, hyperinsulinemia induced the ability of fAd to elicit fatty acid uptake and enhanced fatty acid oxidation in response to fAd. In summary, our results suggest that both hyperglycemia and hyperinsulinemia cause gAd resistance in rat skeletal muscle cells. However, hyperinsulinemia induces a switch toward increased fAd sensitivity in these cells.

SOX6 attenuates glucose-stimulated insulin secretion by repressing PDX1 transcriptional activity and is down-regulated in hyperinsulinemic obese mice.

In obesity-related insulin resistance, pancreatic islets compensate for insulin resistance by increasing secretory capacity. Here, we report the identification of sex-determining region Y-box 6 (SOX6), a member of the high mobility group box superfamily of transcription factors, as a co-repressor for pancreatic-duodenal homeobox factor-1 (PDX1). SOX6 mRNA levels were profoundly reduced by both a long term high fat feeding protocol in normal mice and in genetically obese ob/ob mice on a normal chow diet. Interestingly, we show that SOX6 is expressed in adult pancreatic insulin-producing beta-cells and that overexpression of SOX6 decreased glucose-stimulated insulin secretion, which was accompanied by decreased ATP/ADP ratio, Ca(2+) mobilization, proinsulin content, and insulin gene expression. In a complementary fashion, depletion of SOX6 by small interfering RNAs augmented glucose-stimulated insulin secretion in insulinoma mouse MIN6 and rat INS-1E cells. These effects can be explained by our mechanistic studies that show SOX6 acts to suppress PDX1 stimulation of the insulin II promoter through a direct protein/protein interaction. Furthermore, SOX6 retroviral expression decreased acetylation of histones H3 and H4 in chromatin from the promoter for the insulin II gene, suggesting that SOX6 may decrease PDX1 stimulation through changes in chromatin structure at specific promoters. These results suggest that perturbations in transcriptional regulation that are coordinated through SOX6 and PDX1 in beta-cells may contribute to the beta-cell adaptation in obesity-related insulin resistance.

Restoration of liver insulin signaling in Insr knockout mice fails to normalize hepatic insulin action.

Partial restoration of insulin receptor Insr expression in brain, liver, and pancreatic beta cells is sufficient for rescuing Insr knockout mice from neonatal death, preventing diabetes ketoacidosis, and normalizing life span and reproductive function. However, the transgenically rescued mice (referred to as L1) have marked hyperinsulinemia, and approximately 30% develop late-onset type 2 diabetes. Analyses of protein expression indicated that L1 mice had modestly reduced Insr content but normal insulin-stimulated Akt phosphorylation in the liver. Conversely, L1 mice had a near complete ablation of Insr protein product in the arcuate and paraventricular nuclei of the hypothalamus, which was associated with a failure to undergo insulin-dependent Akt phosphorylation in the hypothalamus. To test whether reconstitution of insulin signaling in the liver is sufficient for restoring in vivo hepatic insulin action, we performed euglycemic hyperinsulinemic clamp studies in conscious L1 and WT mice. During the clamp, L1 mice required an approximately 50% lower rate of glucose infusion than did WT controls, while the rate of glucose disappearance was not significantly altered. Conversely, the rate of glucose production was increased approximately 2-fold in L1 mice. Thus, restoration of hepatic insulin signaling in Insr knockout mice fails to normalize the in vivo response to insulin.

Dietary trans-10,cis-12 conjugated linoleic acid induces hyperinsulinemia and fatty liver in the mouse.

Conjugated linoleic acids (CLA) are a class of positional, geometric, conjugated dienoic isomers of linoleic acid (LA). Dietary CLA supplementation results in a dramatic decrease in body fat mass in mice, but also causes considerable liver steatosis. However, little is known of the molecular mechanisms leading to hepatomegaly. Although c9,t11- and t10,c12-CLA isomers are found in similar proportions in commercial preparations, the respective roles of these two molecules in liver enlargement has not been studied. We show here that mice fed a diet enriched in t10,c12-CLA (0.4% w/w) for 4 weeks developed lipoatrophy, hyperinsulinemia, and fatty liver, whereas diets enriched in c9,t11-CLA and LA had no significant effect. In the liver, dietary t10,c12-CLA triggered the ectopic production of peroxisome proliferator-activated receptor gamma (PPARgamma), adipocyte lipid-binding protein and fatty acid transporter mRNAs and induced expression of the sterol responsive element-binding protein-1a and fatty acid synthase genes. In vitro transactivation assays demonstrated that t10,c12- and c9,t11-CLA were equally efficient at activating PPARalpha, beta/delta, and gamma and inhibiting liver-X-receptor. Thus, the specific effect of t10,c12-CLA is unlikely to result from direct interaction with these nuclear receptors. Instead, t10,c12-CLA-induced hyperinsulinemia may trigger liver steatosis, by inducing both fatty acid uptake and lipogenesis.

Identification of a familial hyperinsulinism-causing mutation in the sulfonylurea receptor 1 that prevents normal trafficking and function of KATP channels.

Mutations in the pancreatic ATP-sensitive potassium (K(ATP)) channel subunits sulfonylurea receptor 1 (SUR1) and the inwardly rectifying potassium channel Kir6.2 cause persistent hyperinsulinemic hypoglycemia of infancy. We have identified a SUR1 mutation, L1544P, in a patient with the disease. Channels formed by co-transfection of Kir6.2 and the mutant SUR1 in COS cells have reduced response to MgADP ( approximately 10% that of the wild-type channels) and reduced surface expression ( approximately 19% that of the wild-type channels). However, the steady-state level of the SUR1 protein is unaffected. Treating cells with lysosomal or proteasomal inhibitors did not improve surface expression of the mutant channels, suggesting that increased degradation of mutant channels by either pathway is unlikely to account for the reduced surface expression. Removal of the RKR endoplasmic reticulum retention/retrieval trafficking motif in either SUR1 or Kir6.2 increased the surface expression of the mutant channel by approximately 35 and approximately 20%, respectively. The simultaneous removal of the RKR motif in both channel subunits restored surface expression of the mutant channel to the wild-type channel levels. Thus, the L1544P mutation may interfere with normal trafficking of K(ATP) channels by causing improper shielding of the RKR endoplasmic reticulum retention/retrieval trafficking signals in the two channel subunits.

Conditional deletion of brain-derived neurotrophic factor in the postnatal brain leads to obesity and hyperactivity.

Brain-derived neurotrophic factor has been associated previously with the regulation of food intake. To help elucidate the role of this neurotrophin in weight regulation, we have generated conditional mutants in which brain-derived neurotrophic factor has been eliminated from the brain after birth through the use of the cre-loxP recombination system. Brain-derived neurotrophic factor conditional mutants were hyperactive after exposure to stressors and had higher levels of anxiety when evaluated in the light/dark exploration test. They also had mature onset obesity characterized by a dramatic 80-150% increase in body weight, increased linear growth, and elevated serum levels of leptin, insulin, glucose, and cholesterol. In addition, the mutants had an abnormal starvation response and elevated basal levels of POMC, an anorexigenic factor and the precursor for alpha-MSH. Our results demonstrate that brain derived neurotrophic factor has an essential maintenance function in the regulation of anxiety-related behavior and in food intake through central mediators in both the basal and fasted state.

A comparison of the effects of troglitazone and vitamin E on the fatty acid composition of serum phospholipids in an experimental model of insulin resistance.

The aim of this study was to investigate the effects of troglitazone (TRO)--a new insulin-sensitizing agent--on some metabolic parameters in an experimental model of hypertriglyceridemia and insulin resistance, hereditary hypertriglyceridemic rats, and to compare its effects with those of vitamin E, an antioxidant agent. Three groups of the above rats were fed diets with a high content of sucrose (70% of energy as sucrose) for four weeks. The first group was supplemented with TRO (120 mg/kg diet), the second one with vitamin E (500 mg/kg diet), and the third group served as the control. Vitamin E supplementation did not lower serum triglycerides (2.42 +/- 0.41 vs. 3.39 +/- 0.37 mmol/l, N.S.) while TRO did (1.87 +/- 0.24 vs. 3.39 +/- 0.37 mmol/l, p < 0.01). Neither TRO nor vitamin E influenced the serum levels of free fatty acids (FFA). Both drugs influenced the spectrum of fatty acids in serum phospholipids--TRO increased the levels of polyunsaturated fatty acids (PUFA) n-6 (36.04 +/- 1.61 vs. 19.65 +/- 1.56 mol %, p < 0.001), vitamin E increased the levels of PUFA n-3 (13.3 +/- 0.87 vs. 6.79 +/- 0.87 mol %, p < 0.001) and decreased the levels of saturated fatty acids (32.97 +/- 0.58 vs. 51.45 +/- 4.01 mol %, p < 0.01). In conclusion, TRO lowered the level of serum triglycerides but vitamin E did not have this effect in hypertriglyceridemic rats. Compared with TRO, vitamin E had a different effect on the spectrum of fatty acids in serum phospholipids.

Monkey leptin receptor mRNA: sequence, tissue distribution, and mRNA expression in the adipose tissue of normal, hyperinsulinemic, and type 2 diabetic rhesus monkeys.

OBJECTIVE: We have cloned the rhesus monkey leptin receptor and examined its mRNA expression levels in the adipose tissue of monkeys to investigate the regulation of gene expression of the leptin receptor. RESEARCH METHODS AND PROCEDURES: Monkey leptin receptor cDNA was cloned by reverse transcriptase-polymerase chain reaction (RT-PCR). Tissue distribution of monkey leptin receptor was examined by Northern blot analysis and RT-PCR. The mRNA levels of monkey leptin receptor in adipose tissue of normal (n=10), hyperinsulinemic obese (n=8), and type 2 diabetic monkeys (n=8) were measured by quantitative RT-PCR. RESULTS: Monkey leptin receptor cDNA had at least two alternatively spliced isoforms (long and short forms). The long form of the leptin receptor mRNA was expressed relatively highly in liver, adipose tissue, hypothalamus, and choroid plexus, whereas the total leptin receptors were expressed in every tissue examined. The mRNA levels of the long form of the leptin receptor in adipose tissue were not correlated to body weight, fasting plasma insulin, plasma glucose, or plasma leptin levels. The mRNA levels of the long form of the leptin receptor were highly correlated to that of the total leptin receptor (long and short form). DISCUSSION: The long form of leptin receptor mRNA existed in adipose tissue as well as in liver and hypothalamus, suggesting that the leptin receptor in adipose tissue may be functional in adipose tissue. The expression of the leptin receptor mRNA in adipose tissue is not affected by obesity, hyperinsulinemia, or diabetes.

Inactivation of the first nucleotide-binding fold of the sulfonylurea receptor, and familial persistent hyperinsulinemic hypoglycemia of infancy.

Familial persistent hyperinsulinemic hypoglycemia of infancy is a disorder of glucose homeostasis and is characterized by unregulated insulin secretion and profound hypoglycemia. Loss-of-function mutations in the second nucleotide-binding fold of the sulfonylurea receptor, a subunit of the pancreatic-islet beta-cell ATP-dependent potassium channel, has been demonstrated to be causative for persistent hyperinsulinemic hypoglycemia of infancy. We now describe three additional mutations in the first nucleotide-binding fold of the sulfonylurea-receptor gene. One point mutation disrupts the highly conserved Walker A motif of the first nucleotide-binding-fold region. The other two mutations occur in noncoding sequences required for RNA processing and are predicted to disrupt the normal splicing pathway of the sulfonylurea-receptor mRNA precursor. These data suggest that both nucleotide-binding-fold regions of the sulfonylurea receptor are required for normal regulation of beta-cell ATP-dependent potassium channel activity and insulin secretion.