Hypothalamic miR-1983 Targets Insulin Receptor ß and the Insulin-mediated miR-1983 Increase Is Blocked by Metformin.

MicroRNAs (miRNAs) expressed in the hypothalamus are capable of regulating energy balance and peripheral metabolism by inhibiting translation of target messenger RNAs (mRNAs). Hypothalamic insulin resistance is known to precede that in the periphery, thus a critical unanswered question is whether central insulin resistance creates a specific hypothalamic miRNA signature that can be identified and targeted. Here we show that miR-1983, a unique miRNA, is upregulated in vitro in 2 insulin-resistant immortalized hypothalamic neuronal neuropeptide Y-expressing models, and in vivo in hyperinsulinemic mice, with a concomitant decrease of insulin receptor ß subunit protein, a target of miR-1983. Importantly, we demonstrate that miR-1983 is detectable in human blood serum and that its levels significantly correlate with blood insulin and the homeostatic model assessment of insulin resistance. Levels of miR-1983 are normalized with metformin exposure in mouse hypothalamic neuronal cell culture. Our findings provide evidence for miR-1983 as a unique biomarker of cellular insulin resistance, and a potential therapeutic target for prevention of human metabolic disease.

Immunofluorescence of GFAP and TNF-α in the Mouse Hypothalamus.

Immunofluorescence is a reliable method for identifying specific proteins in neuronal and glial cell populations of the hypothalamus. Several immunofluorescence protocols are available to detect protein markers and neuropeptides in the hypothalamus; however, published methods may vary in subtle details that can potentially impact the final outcome of the procedure. Here, we provide a detailed protocol suitable for thin cryostat sections, which has been successful for specific antibodies directed against key markers of hypothalamic neurons and glial cells. We include every detail concerning brain tissue collection, processing, sectioning, and labeling with optimal dilutions of antibodies with the aim of reducing non-specific background. Our background-optimized immunostaining protocol has been routinely used in the lab and allows efficient detection of specific neuropeptides, glial cells, and markers of inflammation and endoplasmic reticulum stress in the hypothalamus.

Insulin signalling in hypothalamic neurones.

Subsequent to the discovery of insulin by Banting and Best in the Department of Physiology at the University of Toronto 100 years ago, the field of insulin signalling and action has grown at a remarkable pace. Yet, the recognition that insulin action in the brain is critical for whole body homeostasis has only recently been appreciated. The hypothalamus is a key region in the brain that responds to circulating insulin by engaging a complex signalling cascade resulting in the ultimate release of neuropeptides that control hunger and feeding. Disruption of this important feedback system can lead to a phenomenon called cellular insulin resistance, where the neurones cease to sense insulin. The factors contributing to insulin resistance, as well as the resulting detrimental effects, include the induction of neuroinflammation, endoplasmic reticulum stress and alterations in the architecture of the blood-brain barrier that allow transport of insulin into the brain. These manifestations usually change energy balance, causing weight gain, often resulting in obesity and its deadly comorbidities, including type 2 diabetes mellitus, cardiovascular disease and metabolic syndrome. Nonetheless, there is still hope because the signal transduction pathways can be targeted at a number of levels by neurone-specific therapeutics. With the advent of unique cell models for investigating the mechanisms involved in these processes, the discovery of novel targets is increasingly possible. Although we are still looking for a cure for diabetes, Banting and Best would be impressed at how far their discovery has advanced and the contemporary knowledge that has been accumulated based on insulin action.

A case report: An aortobifemoral bypass implant found during cadaver dissection promotes inquiry-based learning.

INTRODUCTION: An unusual vascular implant found during routine cadaver dissection influenced instructors and students to learn more about the underlying disease and the surgical technique used to treat this disorder triggering an inquiry-based learning. PRESENTATION OF CASE: This report describes a case of peripheral arterial disease (PAD) with an aortobifemoral arterial bypass implant that was never found before in any of the previously dissected cadavers at Gannon University. DISCUSSION: PAD develops due to impaired blood flow to the lower extremities that causes numbness, weakness, and lower leg pain. The treatments can aim to improve the long-term cardiovascular outcomes. If therapeutic medications do not improve outcomes of PAD, revascularization by endovascular repair or aortofemoral bypass grafting is considered. CONCLUSION: We would like to note that accidental discoveries of pathologies or surgical procedures during routine cadaveric dissections present a unique possibility for inquiry-based learning among future healthcare providers.

Resveratrol Inhibits Neointimal Growth after Arterial Injury in High-Fat-Fed Rodents: The Roles of SIRT1 and AMPK.

We have shown that both insulin and resveratrol (RSV) decrease neointimal hyperplasia in chow-fed rodents via mechanisms that are in part overlapping and involve the activation of endothelial nitric oxide synthase (eNOS). However, this vasculoprotective effect of insulin is abolished in high-fat-fed insulin-resistant rats. Since RSV, in addition to increasing insulin sensitivity, can activate eNOS via pathways that are independent of insulin signaling, such as the activation of sirtuin 1 (SIRT1) and AMP-activated kinase (AMPK), we speculated that unlike insulin, the vasculoprotective effect of RSV would be retained in high-fat-fed rats. We found that high-fat feeding decreased insulin sensitivity and increased neointimal area and that RSV improved insulin sensitivity (p < 0.05) and decreased neointimal area in high-fat-fed rats (p < 0.05). We investigated the role of SIRT1 in the effect of RSV using two genetic mouse models. We found that RSV decreased neointimal area in high-fat-fed wild-type mice (p < 0.05), an effect that was retained in mice with catalytically inactive SIRT1 (p < 0.05) and in heterozygous SIRT1-null mice. In contrast, the effect of RSV was abolished in AMKPα2-null mice. Thus, RSV decreased neointimal hyperplasia after arterial injury in both high-fat-fed rats and mice, an effect likely not mediated by SIRT1 but by AMPKα2.

Metabolomics in plasma of Malawian children 7 years after surviving severe acute malnutrition: "ChroSAM" a cohort study.

BACKGROUND: More children are now surviving severe acute malnutrition (SAM), but evidence suggests that early-life malnutrition is associated with increased risk of long-term cardio-metabolic disorders. To better understand potential mechanisms, we studied the metabolite profiles of children seven years after treatment for SAM. METHODS: We followed-up children (n = 352) treated for SAM in 2006-2007, at Queen Elizabeth Central Hospital, in Malawi. Using nuclear magnetic resonance spectroscopy, tandem mass spectrometry and enzyme-linked immunosorbent assay, we measured circulating metabolites in fasting blood in a subset of SAM survivors (n = 69, 9·6 ±â€¯1·6 years), siblings (n = 44, 10·5 ±â€¯2·7 years), and age and sex-matched community controls (n = 37, 9·4 ±â€¯1·8 years). Data were analysed using univariate and sparse partial least square (sPLS) methods. Differences associated with SAM survival, oedema status, and anthropometry were tested, adjusting for age, sex, HIV, and wealth index. FINDINGS: Based on 194 measured metabolites, the profiles of SAM survivors were similar to those of siblings and community controls. IGF1, creatinine, and FGF21, had loading values >0·3 and ranked stably in the top 10 distinguishing metabolites, but did not differ between SAM survivors and controls with univariate analysis. Current stunting was associated with IGF1 (ß = 15·2, SE = 3·5, partial R2 = 12%, p < 0·0001) and this relationship could be influenced by early childhood SAM (ß = 17·4, SE = 7·7, partial R2 = 2·8%, p = 0·025). No metabolites were associated with oedema status, duration of hospital stay, anthropometry measured during hospitalization, nor with changes in anthropometry since hospitalization. INTERPRETATION: In this group of survivors, SAM was not associated with longer-term global metabolic changes 7 years after treatment. However, SAM may influence the relationship between current stunting and IGF1. Further risk markers for NCDs in SAM survivors may only be revealed by direct metabolic challenge or later in life.

Long-term metabolic effects of malnutrition: Liver steatosis and insulin resistance following early-life protein restriction.

Early postnatal-life malnutrition remains prevalent globally, and about 45% of all child deaths are linked to malnutrition. It is not clear whether survivors of childhood malnutrition suffer from long-term metabolic effects, especially when they are later in life exposed to a fat and carbohydrate rich obesogenic diet. The lack of knowledge around this dietary "double burden" warrants studies to understand the long-term consequences of children previously exposed to malnutrition. We hypothesized that an early-life nutritional insult of low protein consumption in mice would lead to long-term metabolic disturbances that would exacerbate the development of diet-induced insulin resistance and non-alcoholic fatty liver disease (NAFLD). We investigated the effects of feeding a low protein diet (4% wt/wt) immediately after weaning for four weeks and subsequent feeding of a high carbohydrate high fat feeding for 16 weeks on metabolic function and development of NAFLD. Mice exposed to early-life protein restriction demonstrated a transient glucose intolerance upon recovery by regular chow diet feeding. However, protein restriction after weaning in mice did not exacerbate an obesogenic diet-induced insulin resistance or progression to NAFLD. These data suggest that transient protein restriction in early-life does not exacerbate an obesogenic diet-induced NAFLD and insulin resistance.

Divergent Regulation of ER and Kiss Genes by 17ß-Estradiol in Hypothalamic ARC Versus AVPV Models.

Kisspeptin (Kiss) and G-protein-coupled receptor (Gpr)54 have emerged as key regulators of reproduction. 17ß-estradiol (E2)-mediated regulation of these neurons is nuclei specific, where anteroventral periventricular (AVPV) Kiss neurons are positively regulated by E2, whereas arcuate nucleus (ARC) neurons are inhibited. We have generated immortalized Kiss cell lines from male and female adult-derived murine hypothalamic primary culture, as well as cell lines from microdissected AVPV and ARC from female Kiss-green fluorescent protein (GFP) mice. All exhibit endogenous Kiss-1 expression, estrogen receptors (ER)s (ERα, ERß, and Gpr30), as well as known markers of AVPV Kiss neurons in the mHypoA-50 and mHypoA-Kiss/GFP-4, vs markers of ARC Kiss neurons in the mHypoA-55 and the mHypoA-Kiss/GFP-3 lines. There was an increase in Kiss-1 mRNA expression at 24 hours in the AVPV lines and a repression of Kiss-1 mRNA at 4 hours in the ARC lines. An E2-mediated decrease in ERα mRNA expression at 24 hours in the AVPV cell lines was detected, and a significant decrease in Gpr30, ERα, and ERß mRNA levels at 4 hours in the ARC cell lines was evident. ER agonists and antagonists determined the specific ERs responsible for mediating changes in gene expression. In the AVPV, ERα is required but not ERß or GPR30, vs the ARC Kiss-expressing cell lines that require GPR30, and either ERα and/or ERß. We determined cAMP response element-binding protein 1 was necessary for the down-regulation of Kiss-1 mRNA expression using small interfering RNA knockdown in the ARC cell model. These studies elucidate some of the molecular events involved in the differential E2-mediated regulation of unique and specific Kiss neuronal models.

Carbohydrate malabsorption in acutely malnourished children and infants: a systematic review.

CONTEXT: Severe acute malnutrition (SAM) accounts for approximately 1 million child deaths per year. High mortality is linked with comorbidities, such as diarrhea and pneumonia. OBJECTIVE: The aim of this systematic review was to determine the extent to which carbohydrate malabsorption occurs in children with SAM. DATA SOURCES: The PubMed and Embase databases were searched. Reference lists of selected articles were checked. DATA EXTRACTION: All observational and controlled intervention studies involving children with SAM in which direct or indirect measures of carbohydrate absorption were analyzed were eligible for inclusion. A total of 20 articles were selected for this review. DATA SYNTHESIS: Most studies reported carbohydrate malabsorption, particularly lactose malabsorption, and suggested an increase in diarrhea and reduced weight gain in children on a lactose-containing diet. As most studies reviewed were observational, there was no conclusive scientific evidence of a causal relationship between lactose malabsorption and a worse clinical outcome among malnourished children. CONCLUSION: The combined data indicate that carbohydrate malabsorption is prevalent in children with SAM. Additional well-designed intervention studies are needed to determine whether outcomes of SAM complicated by carbohydrate malabsorption could be improved by altering the carbohydrate/lactose content of therapeutic feeds and to elucidate the precise mechanisms involved.

The effect of insulin to decrease neointimal growth after arterial injury is endothelial nitric oxide synthase-dependent.

In vitro, insulin has mitogenic effects on vascular smooth muscle cells (VSMC) but also has protective effects on endothelial cells by stimulating nitric oxide (NO) production and endothelial nitric oxide synthase (eNOS) expression. Furthermore, NOS inhibition attenuates the effect of insulin to inhibit VSMC migration in vitro. Using an in vivo model, we have previously shown that insulin decreases neointimal growth and cell migration and increases re-endothelialization after arterial injury in normal rats. Since insulin can stimulate NOS, and NO can decrease neointimal growth, we hypothesized that NOS, and more specifically eNOS was required for the effects of insulin in vivo. Rats were given subcutaneous insulin implants (3 U/day) alone or with the NOS inhibitor l-NAME (2 mg kg(-1) day(-1)) 3 days before arterial (carotid or aortic) balloon catheter injury. Insulin decreased both neointimal area (P < 0.01) and cell migration (P < 0.01), and increased re-endothelialization (P < 0.05). All of these effects were prevented by the co-administration of l-NAME. Insulin was found to decrease inducible NOS expression (P < 0.05) but increase eNOS phosphorylation (P < 0.05). These changes were also translated at the functional level where insulin improved endothelial-dependent vasorelaxation. To further study the NOS isoform involved in insulin action, s.c. insulin (0.1 U/day) was given to wild-type and eNOS knockout mice. We found that insulin was effective at decreasing neointimal formation in wild-type mice after wire injury of the femoral artery, whereas this effect of insulin was absent in eNOS knockout mice. These results show that the vasculoprotective effect of insulin after arterial injury is mediated by an eNOS-dependent mechanism.

Potentiation of the hepatic toxicity of carbon disulfide by chlordane.

OBJECTIVES: In this study, we investigated whether cytochrome P450s (CYPs) induced by a typical chlorinated hydrocarbon insecticide chlordane (CLD) potentiate hepatic toxicity of carbon disulfide (CS2). MATERIALS AND METHODS: Male Sprague-Dawley rats were treated with CLD (25 mg/kg, intraperitoneally (i.p.)) daily for 4 days, and 24 h after the final injection the rats were treated with CS2 (380 mg/kg, i.p.) in corn oil; while controls received the vehicle alone. The rats were then sacrificed at 3, 6, and 24 h following the CS2 treatment. RESULTS: It was found that at 3 h post-treatment, total hepatic glutathione (GSH) decreased modestly, but lipid peroxidation increased markedly, while all CLD-inducible CYPs (1A1, 2B1, 2E1, and 3A2) were inhibited by CS2 variably but significantly. On the other hand, samples taken at 24 h following the CS2 treatment showed a significant increase in relative liver weights, hepatic GSH and lipid peroxidation, microsomal reactive oxygen species (ROS), and serum alanine transaminase (ALT) level. Activity of the CYPs was also increased, but remained significantly depressed, especially that of CYP2B1. Livers removed at 3 and 6 h after CS2 treatment showed subtle to distinct apoptotic changes, while a severe lesion of hydropic degeneration of the centrilobular cells with apoptosis was microscopically distinguishable in samples taken at 24 h. CONCLUSIONS: These results suggest that the metabolism of CS2 by CLD-induced CYPs and the generation of lipid peroxides may have in concert contributed to the distinct hepatocellular damage.

The cytokine ciliary neurotrophic factor (CNTF) activates hypothalamic urocortin-expressing neurons both in vitro and in vivo.

Ciliary neurotrophic factor (CNTF) induces neurogenesis, reduces feeding, and induces weight loss. However, the central mechanisms by which CNTF acts are vague. We employed the mHypoE-20/2 line that endogenously expresses the CNTF receptor to examine the direct effects of CNTF on mRNA levels of urocortin-1, urocortin-2, agouti-related peptide, brain-derived neurotrophic factor, and neurotensin. We found that treatment of 10 ng/ml CNTF significantly increased only urocortin-1 mRNA by 1.84-fold at 48 h. We then performed intracerebroventricular injections of 0.5 mg/mL CNTF into mice, and examined its effects on urocortin-1 neurons post-exposure. Through double-label immunohistochemistry using specific antibodies against c-Fos and urocortin-1, we showed that central CNTF administration significantly activated urocortin-1 neurons in specific areas of the hypothalamus. Taken together, our studies point to a potential role for CNTF in regulating hypothalamic urocortin-1-expressing neurons to mediate its recognized effects on energy homeostasis, neuronal proliferaton/survival, and/or neurogenesis.

Direct regulation of the proglucagon gene by insulin, leptin, and cAMP in embryonic versus adult hypothalamic neurons.

The proglucagon gene is expressed not only in the pancreas and intestine but also in the hypothalamus. Proglucagon-derived peptides have emerged as potential regulators of energy homeostasis. Whether leptin, insulin, or cAMP activation controls proglucagon gene expression in the hypothalamus is not known. A key reason for this has been the inaccessibility of hypothalamic proglucagon-expressing neurons and the lack of suitable neuronal cell lines. Herein we describe the mechanisms involved in the direct regulation of the proglucagon gene by insulin, leptin, and cAMP in hypothalamic cell models. Insulin, through an Akt-dependent manner, significantly induced proglucagon mRNA expression by 70% in adult-derived mHypoA-2/10 neurons and significantly suppressed it by 45% in embryonic-derived mHypoE-39 neurons. Leptin, via the Janus kinase-2/ signal transducer and activator of transcription-3 pathway, caused an initial increase by 66 and 43% at 1 h followed by a decrease by 45 and 34% at 12 h in mHypoA-2/10 and mHypoE-39 cells, respectively. Furthermore, cAMP activation by forskolin up-regulated proglucagon expression by 87% in mHypoE-39 neurons and increased proglucagon mRNA, through Epac activation, in the mHypoE-20/2 neurons. Specific regions of the proglucagon promoter were regulated by cAMP signaling, as determined by transient transfections, whereas mRNA stability assays demonstrate that insulin and leptin increase proglucagon mRNA stability in the adult cells. These findings suggest that insulin, leptin, and cAMP act directly, but differentially, on specific hypothalamic neurons to regulate proglucagon gene expression. Because proglucagon-derived peptides are potential regulators of energy homeostasis, an understanding of hypothalamic proglucagon neurons is important to further expand our knowledge of alternative feeding circuits.

Glucagon-like peptide-2 directly regulates hypothalamic neurons expressing neuropeptides linked to appetite control in vivo and in vitro.

Glucagon-like peptide-2 (GLP-2), a proglucagon-derived peptide, has been postulated to affect appetite at the level of the hypothalamus. To gain better insight into this process, a degradation-resistant GLP-2 analog, human (Gly(2))GLP-2(1-33) [h(Gly(2))GLP-2] was intracerebroventricularly injected into mice to examine its action on food and water intake and also activation of hypothalamic anorexigenic α-melanocyte-stimulating hormone/proopiomelanocortin, neurotensin, and orexigenic neuropeptide Y, and ghrelin neurons. Central h(Gly(2))GLP-2 administration significantly suppressed food and water intake with acute weight loss at 2 h. Further, central h(Gly(2))GLP-2 robustly induced c-Fos activation in the hypothalamic arcuate, dorsomedial, ventromedial, paraventricular, and the lateral hypothalamic nuclei. We found differential colocalization of neuropeptides with c-Fos in specific regions of the hypothalamus. To assess whether hypothalamic neuropeptides are directly regulated by GLP-2 in vitro, we used an adult-derived clonal, immortalized hypothalamic cell line, mHypoA-2/30, that endogenously expresses functional GLP-2 receptors (GLP-2R) and two of the feeding-related neuropeptides linked to GLP-2R activation in vivo: neurotensin and ghrelin. Treatment with h(Gly(2))GLP-2 stimulated c-Fos expression and phosphorylation of cAMP response element-binding protein/activating transcription factor-1. In addition, treatment with h(Gly(2))GLP-2 significantly increased neurotensin and ghrelin mRNA transcript levels by 50 and 95%, respectively, at 24 h after treatment in protein kinase A-dependent manner. Taken together, these findings implicate the protein kinase A pathway as the means by which GLP-2 can up-regulate hypothalamic neuropeptide mRNA levels and provide evidence for a link between central GLP-2R activation and specific hypothalamic neuropeptides involved in appetite regulation.

Glucagon-like peptide-1 receptor agonist, exendin-4, regulates feeding-associated neuropeptides in hypothalamic neurons in vivo and in vitro.

Exendin-4, a long-acting glucagon-like peptide-1 receptor (GLP-1R) agonist, is a potential regulator of feeding behavior through its ability to inhibit gastric emptying, reduce food intake, and induce satiety. GLP-1R activation by exendin-4 induces anorexia; however, the specific populations of neuropeptidergic neurons activated by exendin-4 within the hypothalamus, the central regulator of energy homeostasis, remain unclear. This study determines whether exendin-4 regulates hypothalamic neuropeptide expression and explores the signaling mechanisms involved. The distribution and quantity of exendin-4-induced c-Fos immunoreactivity were evaluated to determine activation of α-melanocyte-stimulating hormone/proopiomelanocortin, neuropeptide Y, neurotensin (NT), and ghrelin neurons in hypothalamic nuclei during exendin-4-induced anorexia in mice. Additionally, exendin-4 action on NT and ghrelin transcript regulation was examined in immortalized hypothalamic neurons. With anorexia induced by intracerebroventricular exendin-4, α-melanocyte-stimulating hormone/proopiomelanocortin and neuropeptide Y neurons were activated in the arcuate nucleus, with simultaneous activation of NT-expressing neurons in the paraventricular nucleus, and ghrelin-expressing neurons in the arcuate nucleus, paraventricular nucleus, and periventricular hypothalamus, suggesting that neurons in one or more of these areas mediate the anorexic action of exendin-4. In the hypothalamic neuronal cell models, exendin-4 increased cAMP, cAMP response element-binding protein/activating transcription factor-1 and c-Fos activation, and via a protein kinase A-dependent mechanism regulated NT and ghrelin mRNA expression, indicating that these neuropeptides may serve as downstream mediators of exendin-4 action. These findings provide a previously unrecognized link between central GLP-1R activation by exendin-4 and the regulation of hypothalamic NT and ghrelin. Further understanding of this central GLP-1R activation may lead to safe and effective therapeutics for the treatment of metabolic disorders.

Immortalized neurons for the study of hypothalamic function.

The hypothalamus is a vital part of the central nervous system: it harbors control systems implicated in regulation of a wide range of homeostatic processes, including energy balance and reproduction. Structurally, the hypothalamus is a complex neuroendocrine tissue composed of a multitude of unique neuronal cell types that express a number of neuromodulators, including hormones, classical neurotransmitters, and specific neuropeptides that play a critical role in mediating hypothalamic function. However, neuropeptide and receptor gene expression, second messenger activation, and electrophysiological and secretory properties of these hypothalamic neurons are not yet fully defined, primarily because the heterogeneity and complex neuronal architecture of the neuroendocrine hypothalamus make such studies challenging to perform in vivo. To circumvent this problem, our research group recently generated embryonic- and adult-derived hypothalamic neuronal cell models by utilizing the novel molecular techniques of ciliary neurotrophic factor-induced neurogenesis and SV40 T antigen transfer to primary hypothalamic neuronal cell cultures. Significant research with these cell lines has demonstrated their value as a potential tool for use in molecular genetic analysis of hypothalamic neuronal function. Insights gained from hypothalamic immortalized cells used in conjunction with in vivo models will enhance our understanding of hypothalamic functions such as neurogenesis, neuronal plasticity, glucose sensing, energy homeostasis, circadian rhythms, and reproduction. This review discusses the generation and use of hypothalamic cell models to study mechanisms underlying the function of individual hypothalamic neurons and to gain a more complete understanding of the overall physiology of the hypothalamus.

Ciliary neurotrophic factor recruitment of glucagon-like peptide-1 mediates neurogenesis, allowing immortalization of adult murine hypothalamic neurons.

The distinct lack of cell lines derived from the adult brain is evident. Ciliary neurotrophic factor (CNTF) triggers neurogenesis in primary culture from adult mouse hypothalamus, as detected by bromodeoxyuridine and Ki67 immunostaining. Using SV-40 T-antigen, we immortalized dividing neurons and generated clonal cell lines expressing neuropeptides and receptors involved in neuroendocrine function. We hypothesized that proglucagon-derived peptides may be the mechanistic downstream effectors of CNTF due to documented neuroprotective and proliferative effects. Indeed, proglucagon gene expression was induced by CNTF, and exposure of primary cells to glucagon-like peptide-1 receptor (GLP-1) agonist, exendin-4, induced cell proliferation. Intracerebroventricular injection of CNTF into adult mice caused increased expression of proglucagon peptide in the hypothalamus. Using a specific GLP-1-receptor antagonist, we found that neurogenesis was significantly attenuated and primary culture from GLP-1-receptor-knockout mice lacked CNTF-mediated neuronal proliferation, thus linking the induction of neurogenesis in the hypothalamus to GLP-1-receptor signaling.

Regulation of brain insulin mRNA by glucose and glucagon-like peptide 1.

Whether the brain synthesizes insulin is currently debated. Two clonal, immortalized mouse hypothalamic cell lines from e17, mHypoE-39 and mHypoE-46, express insulin 2 (Ins2), but not Ins1. We analyzed regions necessary for basal gene activity and found that the mouse Ins2 region -110/+183 bp stimulates promoter activity in hypothalamic neurons. The rat Ins2 showed moderate activity, whereas the human promoter construct is repressed below basal levels. In MIN6 pancreatic beta-cells, all of the Ins1 and Ins2 promoter constructs display high levels of transcriptional activity. The cell lines also express components of glucose-sensing machinery and the endogenous glucagon-like peptide 1 receptor (Glp-1R). We observed that 16.7 mM glucose induces Ins2 mRNA, while forskolin and a Glp-1 agonist, exendin-4, induce a biphasic Ins2 mRNA response in mHypoE-39 neurons. The insulin cis-regulatory regions differ between the pancreas and the hypothalamus, and glucose and Glp-1 regulate the expression of hypothalamic insulin.

Cytochrome P450-mediated activation and toxicity of chlorpyrifos in male and female rats.

This study compared CYP-mediated activation and toxicity of chlorpyrifos (CPF) in male and female rats, since gender difference in CPF toxicity in rats has been reported. A dose of 50 mg/kg of CPF in corn oil was administered ip to 2 groups of male and female rats while the respective control groups received the vehicle alone. Measurement of cholinesterase activity in brain showed no difference in cholinesterase inhibition between male and female rats 3 h following CPF administration. In contrast, inhibition of plasma cholinesterase was significantly greater in females than males. The activities of microsomal CYP 1A1, 2B1, 2E1 and 3AV 2 determined whether CPF, a suicide substrate of cytochrome P450 enzymes, was metabolized by the liver CYP enzymes. The CYP 1A1 and 2B1 activities were significantly decreased in both male and female rats, with the CYP 1A1 decrease in females markedly greater than that in males. CPF produced a significant inhibition of only CYP 3A1/2 activity, but not CYP 2E1 activity, irrespective of gender effect. These results demonstrated that CYP 1A1, 2B1 and 3A1/2 were differentially involved in the metabolism of CPF to CPF-oxon in both genders and the extent of plasma cholinesterase inhibition was significantly greater in female than male rats.

Effect of cytochrome P450 inducers on the metabolism and toxicity of thiram in rats.

Thiram is a dithiocarbamate compound widely used as an agricultural fungicide. This study examined the effect of cytochrome P450 (CYP) inducers on the metabolism and toxicity of thiram in rats. Rats were pretreated with 3-methyl cholathrene (3-MC), phenobarbital (PB), isoniazid (INH), or pregnenolone-16a-carbonitrile (PCN) as selective inducers of CYP 1A1, 2B1, 2E1 and 3A2, respectively. Thiram was administered ip to induced rats at 0.1 or 0.5 mmol/kg, and the animals were sacrificed 3 or 24 h later to assess P450 interaction and liver damage, respectively. No significant inhibition of 3-me-induced CYP1A1 was observed with either thiram dose at 3 or 24 h after treatment; similar results were noted for rats induced with PB or PCN. By contrast, when INH was the selective inducer of CYP2E1, there was significant inhibition by thiram 3 h and 24 h after treatment, suggesting that thiram was metabolized by the induced CYP2E1; there was a significant increase in ALT activity reflective of liver damage in the rats treated with thiram. The results suggest that CYP2EI induced by INH may be significantly involved in the metabolism of thiram, and the associated liver damage.