Mechanisms underlying the efficacy of a rodent model of vertical sleeve gastrectomy - A focus on energy expenditure.

OBJECTIVE: Bariatric surgery remains the only effective and durable treatment option for morbid obesity. Vertical Sleeve Gastrectomy (VSG) is currently the most widely performed of these surgeries primarily because of its proven efficacy in generating rapid onset weight loss, improved glucose regulation and reduced mortality compared with other invasive procedures. VSG is associated with reduced appetite, however, the relative importance of energy expenditure to VSG-induced weight loss and changes in glucose regulation, particularly that in brown adipose tissue (BAT), remains unclear. The aim of this study was to investigate the role of BAT thermogenesis in the efficacy of VSG in a rodent model. METHODS: Diet-induced obese male Sprague-Dawley rats were either sham-operated, underwent VSG surgery or were pair-fed to the food consumed by the VSG group. Rats were also implanted with biotelemetry devices between the interscapular lobes of BAT to assess local changes in BAT temperature as a surrogate measure of thermogenic activity. Metabolic parameters including food intake, body weight and changes in body composition were assessed. To further elucidate the contribution of energy expenditure via BAT thermogenesis to VSG-induced weight loss, a separate cohort of chow-fed rats underwent complete excision of the interscapular BAT (iBAT lipectomy) or chemical denervation using 6-hydroxydopamine (6-OHDA). To localize glucose uptake in specific tissues, an oral glucose tolerance test was combined with an intraperitoneal injection of 14C-2-deoxy-d-glucose (14C-2DG). Transneuronal viral tracing was used to identify 1) sensory neurons directed to the stomach or small intestine (H129-RFP) or 2) chains of polysynaptically linked neurons directed to BAT (PRV-GFP) in the same animals. RESULTS: Following VSG, there was a rapid reduction in body weight that was associated with reduced food intake, elevated BAT temperature and improved glucose regulation. Rats that underwent VSG had elevated glucose uptake into BAT compared to sham operated animals as well as elevated gene markers related to increased BAT activity (Ucp1, Dio2, Cpt1b, Cox8b, Ppargc) and markers of increased browning of white fat (Ucp1, Dio2, Cited1, Tbx1, Tnfrs9). Both iBAT lipectomy and 6-OHDA treatment significantly attenuated the impact of VSG on changes in body weight and adiposity in chow-fed animals. In addition, surgical excision of iBAT following VSG significantly reversed VSG-mediated improvements in glucose tolerance, an effect that was independent of circulating insulin levels. Viral tracing studies highlighted a patent neural link between the gut and BAT that included groups of premotor BAT-directed neurons in the dorsal raphe and raphe pallidus. CONCLUSIONS: Collectively, these data support a role for BAT in mediating the metabolic sequelae following VSG surgery, particularly the improvement in glucose regulation, and highlight the need to better understand the contribution from this tissue in human patients.

Yap regulates skeletal muscle fatty acid oxidation and adiposity in metabolic disease.

Obesity is a major risk factor underlying the development of metabolic disease and a growing public health concern globally. Strategies to promote skeletal muscle metabolism can be effective to limit the progression of metabolic disease. Here, we demonstrate that the levels of the Hippo pathway transcriptional co-activator YAP are decreased in muscle biopsies from obese, insulin-resistant humans and mice. Targeted disruption of Yap in adult skeletal muscle resulted in incomplete oxidation of fatty acids and lipotoxicity. Integrated 'omics analysis from isolated adult muscle nuclei revealed that Yap regulates a transcriptional profile associated with metabolic substrate utilisation. In line with these findings, increasing Yap abundance in the striated muscle of obese (db/db) mice enhanced energy expenditure and attenuated adiposity. Our results demonstrate a vital role for Yap as a mediator of skeletal muscle metabolism. Strategies to enhance Yap activity in skeletal muscle warrant consideration as part of comprehensive approaches to treat metabolic disease.

No evidence of white adipocyte browning after endurance exercise training in obese men.

BACKGROUND/OBJECTIVES: The phenomenon of adipocyte 'beiging' involves the conversion of non-classic brown adipocytes to brown-like adipose tissue with thermogenic, fat-burning properties, and this phenomenon has been shown in rodents to slow the progression of obesity-associated metabolic diseases. Rodent studies consistently report adipocyte beiging after endurance exercise training, indicating that increased thermogenic capacity in these adipocytes may underpin the improved health benefits of exercise training. The aim of this study was to determine whether prolonged endurance exercise training induces beige adipogenesis in subcutaneous adipose tissues of obese men. SUBJECTS/METHODS: Molecular markers of beiging were examined in adipocytes obtained from abdominal subcutaneous (AbSC) and gluteofemoral (GF) subcutaneous adipose tissues before and after 6 weeks of endurance exercise training in obese men (n=6, 37.3±2.3 years, 30.1±2.3 kg m-2). RESULTS: The mRNAs encoding the brown or beige adipocyte-selective proteins were very lowly expressed in AbSC and GF adipose tissues and exercise training did not alter the mRNA expression of UCP1, CD137, CITED, TBX1, LHX8 and TCF21. Using immunohistochemistry, neither multilocular adipocytes, nor UCP1 or CD137-positive adipocytes were detected in any sample. MicroRNAs known to regulate brown and/or beige adipose development were highly expressed in white adipocytes but endurance exercise training did not impact their expression. CONCLUSIONS: The present study reaffirms emerging data in humans demonstrating no evidence of white adipose tissue beiging in response to exercise training, and supports a growing body of work demonstrating divergence of brown/beige adipose location, molecular characterization and physiological function between rodents and humans.

Serotonin in the ventral hippocampus modulates anxiety-like behavior during amphetamine withdrawal.

Withdrawal from amphetamine is associated with increased anxiety and sensitivity to stressors which are thought to contribute to relapse. Rats undergoing amphetamine withdrawal fail to exhibit stress-induced increases in serotonin (5-HT) release in the ventral hippocampus and show heightened anxiety-like behaviors. Therefore, we tested the hypothesis that reducing 5-HT levels in the ventral hippocampus is a causal mechanism in increasing anxiety-like behaviors during amphetamine withdrawal. First, we tested whether reducing 5-HT levels in the ventral hippocampus directly increases anxiety behavior. Male rats were bilaterally infused with 5,7-dihydroxytryptamine (5,7-DHT) into the ventral hippocampus, which produced a 83% decrease in ventral hippocampus 5-HT content, and were tested on the elevated plus maze (EPM) for anxiety-like behavior. Reducing ventral hippocampus 5-HT levels decreased the time spent in the open arms of the maze, suggesting that diminished ventral hippocampus 5-HT levels increases anxiety-like behavior. Next, we tested whether increasing 5-HT levels in the ventral hippocampus reverses anxiety behavior exhibited by rats undergoing amphetamine withdrawal. Rats were treated daily with either amphetamine (2.5-mg/kg, i.p.) or saline for 2weeks, and at 2weeks withdrawal, were infused with the selective serotonin reuptake inhibitor paroxetine (0.5µM) bilaterally into the ventral hippocampus and tested for anxiety-like behavior on the EPM. Rats pre-treated with amphetamine exhibited increased anxiety-like behavior on the EPM. This effect was reversed by ventral hippocampus infusion of paroxetine. Our results suggest that 5-HT levels in the ventral hippocampus are critical for regulating anxiety behavior. Increasing 5-HT levels during withdrawal may be an effective strategy for reducing anxiety-induced drug relapse.

Exercise training does not enhance hypothalamic responsiveness to leptin or ghrelin in male mice.

The detection of hormone and nutrient signals by the hypothalamus is blunted in obesity and contributes to dysregulated energy homeostasis. We investigated whether aerobic exercise training would improve long-term hypothalamic sensitivity to both leptin and ghrelin, independent of acute exercise-induced signalling. Male C57Bl/6J mice were fed either a chow or high-fat diet for 6 weeks, then remained sedentary on their respective diet, or completed 6 weeks of treadmill exercise training with a progressive increase in exercise volume and intensity. Food intake and hypothalamic signalling were assessed in mice injected with leptin or ghrelin at least 24 h after the last exercise bout. Exercise training reduced body mass, increased daily food intake and improved glucose tolerance. Intraperitoneal leptin administration reduced food intake in lean and obese mice, and this was not enhanced after exercise training. Leptin-mediated activation of phosphorylated signal transducer and activator of transcription 3 in the arcuate nucleus and ventromedial nucleus of the hypothalamus was not enhanced with exercise training. Ghrelin increased food intake and c-Fos positive neurones in the hypothalamus in lean and obese mice, and these physiological and molecular responses were not enhanced with exercise training. This suggests that the previously reported exercise effects on sensitising hypothalamic signalling and food intake responses may be limited to the period immediately after an exercise bout, and are not a result of stable structural or molecular changes that occur with exercise training.

Regulation of plasma ceramide levels with fatty acid oversupply: evidence that the liver detects and secretes de novo synthesised ceramide.

AIMS/HYPOTHESIS: Plasma ceramide concentrations correlate with insulin sensitivity, inflammation and atherosclerotic risk. We hypothesised that plasma ceramide concentrations are increased in the presence of elevated fatty acid levels and are regulated by increased liver serine C-palmitoyltransferase (SPT) activity. METHODS: Lean humans and rats underwent an acute lipid infusion and plasma ceramide levels were determined. One group of lipid-infused rats was administered myriocin to inhibit SPT activity. Liver SPT activity was determined in lipid-infused rats, and obese, insulin resistant mice. The time and palmitate dose-dependent synthesis of intracellular and secreted ceramide was determined in HepG2 liver cells. RESULTS: Plasma ceramide levels were increased during lipid infusion in humans and rats, and in obese, insulin-resistant mice. The increase in plasma ceramide was not associated with changes in liver SPT activity, and inhibiting SPT activity by ~50% did not alter plasma ceramide levels in lipid-infused rats. In HepG2 liver cells, palmitate incorporation into extracellular ceramide was both dose- and time-dependent, suggesting the liver cells rapidly secreted the newly synthesised ceramide. CONCLUSIONS/INTERPRETATION: Elevated systemic fatty acid availability increased plasma ceramide but this was not associated with changes in hepatic SPT activity, suggesting that liver ceramide synthesis is driven by substrate availability rather than increased SPT activity. This report also provides evidence that the liver is sensitive to the intracellular ceramide concentration, and an increase in liver ceramide secretion may help protect the liver from the deleterious effects of intracellular ceramide accumulation.

T cell protein tyrosine phosphatase (TCPTP) deficiency in muscle does not alter insulin signalling and glucose homeostasis in mice.

AIMS/HYPOTHESIS: Insulin activates insulin receptor protein tyrosine kinase and downstream phosphatidylinositol-3-kinase (PI3K)/Akt signalling in muscle to promote glucose uptake. The insulin receptor can serve as a substrate for the protein tyrosine phosphatase (PTP) 1B and T cell protein tyrosine phosphatase (TCPTP), which share a striking 74% sequence identity in their catalytic domains. PTP1B is a validated therapeutic target for the alleviation of insulin resistance in type 2 diabetes. PTP1B dephosphorylates the insulin receptor in liver and muscle to regulate glucose homeostasis, whereas TCPTP regulates insulin receptor signalling and gluconeogenesis in the liver. In this study we assessed for the first time the role of TCPTP in the regulation of insulin receptor signalling in muscle. METHODS: We generated muscle-specific TCPTP-deficient (Mck-Cre;Ptpn2(lox/lox)) mice (Mck, also known as Ckm) and assessed the impact on glucose homeostasis and muscle insulin receptor signalling in chow-fed versus high-fat-fed mice. RESULTS: Blood glucose and insulin levels, insulin and glucose tolerance, and insulin-induced muscle insulin receptor activation and downstream PI3K/Akt signalling remained unaltered in chow-fed Mck-Cre;Ptpn2(lox/lox) versus Ptpn2(lox/lox) mice. In addition, body weight, adiposity, energy expenditure, insulin sensitivity and glucose homeostasis were not altered in high-fat-fed Mck-Cre;Ptpn2(lox/lox) versus Ptpn2(lox/lox) mice. CONCLUSIONS/INTERPRETATION: These results indicate that TCPTP deficiency in muscle has no effect on insulin signalling and glucose homeostasis, and does not prevent high-fat diet-induced insulin resistance. Thus, despite their high degree of sequence identity, PTP1B and TCPTP contribute differentially to insulin receptor regulation in muscle. Our results are consistent with the notion that these two highly related PTPs make distinct contributions to insulin receptor regulation in different tissues.

Adolescent social defeat increases adult amphetamine conditioned place preference and alters D2 dopamine receptor expression.

Components of the brain's dopaminergic system, such as dopamine receptors, undergo final maturation in adolescence. Exposure to social stress during human adolescence contributes to substance abuse behaviors. We utilized a rat model of adolescent social stress to investigate the neural mechanisms underlying this correlation. Rats exposed to repeated social defeat in adolescence (P35-P39) exhibited increased conditioned place preference (CPP) for amphetamine (1 mg/kg) in adulthood (P70). In contrast, rats experiencing foot-shock during the same developmental period exhibited amphetamine CPP levels similar to non-stressed controls. Our previous experiments suggested adolescent defeat alters dopamine activity in the mesocorticolimbic system. Furthermore, dopamine receptors have been implicated in the expression of amphetamine CPP. Therefore, we hypothesized that alteration to dopamine receptor expression in the mesocorticolimbic system may be associated with to heightened amphetamine CPP of adult rats exposed to adolescence defeat. We measured D1 and D2 dopamine receptor protein content in the medial prefrontal cortex, nucleus accumbens (NAc), and dorsal striatum following either adolescent social defeat or foot-shock stress and then adult amphetamine CPP. In controls, amphetamine CPP training reduced D2 receptor protein content in the NAc core. However, this down-regulation of NAc core D2 receptors was blocked by exposure to social defeat but not foot-shock stress in adolescence. These results suggest social defeat stress in adolescence alters the manner in which later amphetamine exposure down-regulates D2 receptors. Furthermore, persistent alterations to adult D2 receptor expression and amphetamine responses may depend on the type of stress experienced in adolescence.

Homozygous staggerer (sg/sg) mice display improved insulin sensitivity and enhanced glucose uptake in skeletal muscle.

AIMS/HYPOTHESIS: Homozygous staggerer (sg/sg) mice, which have decreased and dysfunctional Rorα (also known as Rora) expression in all tissues, display a lean and dyslipidaemic phenotype. They are also resistant to (high fat) diet-induced obesity. We explored whether retinoic acid receptor-related orphan receptor (ROR) α action in skeletal muscle was involved in the regulation of glucose metabolism. METHODS: We used a three-armed genomic approach, including expression profiling, ingenuity analysis and quantitative PCR validation to identify the signalling pathway(s) in skeletal muscle that are perturbed in sg/sg mice. Moreover, western analysis, functional insulin and glucose tolerance tests, and ex vivo glucose uptake assays were used to phenotypically characterise the impact of aberrant v-AKT murine thymoma viral oncogene homologue (AKT) signalling. RESULTS: Homozygous and heterozygous (sg/sg and sg/+) animals exhibited decreased fasting blood glucose levels, mildly improved glucose tolerance and increased insulin sensitivity. Illumina expression profiling and bioinformatic analysis indicated the involvement of RORα in metabolic disease and phosphatidylinositol 3-kinase-AKT signalling. Quantitative PCR and western analysis validated increased AKT2 (mRNA and protein) and phosphorylation in sg/sg mice in the basal state. This was associated with increased expression of Tbc1d1 and Glut4 (also known as Slc2a4) mRNA and protein. Finally, in agreement with the phenotype, we observed increased (absolute) levels of AKT and phosphorylated AKT (in the basal and insulin stimulated states), and of (ex vivo) glucose uptake in skeletal muscle from sg/sg mice relative to wild-type littermates. CONCLUSIONS/INTERPRETATION: We propose that Rorα plays an important role in regulation of the AKT2 signalling cascade, which controls glucose uptake in skeletal muscle.

Adipose triacylglycerol lipase is a major regulator of hepatic lipid metabolism but not insulin sensitivity in mice.

AIMS/HYPOTHESIS: Hepatic steatosis is characterised by excessive triacylglycerol accumulation and is strongly associated with insulin resistance. An inability to efficiently mobilise liver triacylglycerol may be a key event mediating hepatic steatosis. Adipose triacylglycerol lipase (ATGL) is a key triacylglycerol lipase in the liver and we hypothesised that liver-specific overproduction of ATGL would reduce steatosis and enhance insulin action in obese rodents. METHODS: Studies of fatty acid metabolism were conducted in primary hepatocytes isolated from wild-type and Atgl (also known as Pnpla2)⁻(/)⁻ mice. An ATGL adenovirus was utilised to overproduce ATGL in the livers of obese insulin-resistant C57Bl/6 mice (Ad-ATGL). Blood chemistry, hepatic lipid content and insulin sensitivity were assessed in mice. RESULTS: Triacylglycerol content was increased in Atgl⁻(/)⁻ hepatocytes and was associated with increased fatty acid uptake and impaired fatty acid oxidation. ATGL adenovirus administration in obese mice increased the production of hepatic ATGL protein and reduced triacylglycerol, diacylglycerol and ceramide content in the liver. Overproduction of ATGL improved insulin signal transduction in the liver but did not affect fasting glycaemia or insulinaemia. Inflammatory signalling was not suppressed by ATGL overproduction. While ATGL overproduction increased plasma non-esterified fatty acids, neither lipid deposition nor insulin-stimulated glucose uptake were affected in skeletal muscle. CONCLUSIONS/INTERPRETATION: Liver ATGL overproduction decreases hepatic steatosis and mildly enhances liver insulin sensitivity. These effects are not sufficient to improve fasting glycaemia or insulinaemia in rodent obesity.

Interleukin-6-deficient mice develop hepatic inflammation and systemic insulin resistance.

AIMS/HYPOTHESIS: The role of IL-6 in the development of obesity and hepatic insulin resistance is unclear and still the subject of controversy. We aimed to determine whether global deletion of Il6 in mice (Il6 (-/-)) results in standard chow-induced and high-fat diet (HFD)-induced obesity, hepatosteatosis, inflammation and insulin resistance. METHODS: Male, 8-week-old Il6 (-/-) and littermate control mice were fed a standard chow or HFD for 12 weeks and phenotyped accordingly. RESULTS: Il6 (-/-) mice displayed obesity, hepatosteatosis, liver inflammation and insulin resistance when compared with control mice on a standard chow diet. When fed a HFD, the Il6 (-/-) and control mice had marked, equivalent gains in body weight, fat mass and ectopic lipid deposition in the liver relative to chow-fed animals. Despite this normalisation, the greater liver inflammation, damage and insulin resistance observed in chow-fed Il6 (-/-) mice relative to control persisted when both were fed the HFD. Microarray analysis from livers of mice fed a HFD revealed that genes associated with oxidative phosphorylation, the electron transport chain and tricarboxylic acid cycle were uniformly decreased in Il6 (-/-) relative to control mice. This coincided with reduced maximal activity of the mitochondrial enzyme ß-hydroxyacyl-CoA-dehydrogenase and decreased levels of mitochondrial respiratory chain proteins. CONCLUSIONS/INTERPRETATION: Our data suggest that IL-6 deficiency exacerbates HFD-induced hepatic insulin resistance and inflammation, a process that appears to be related to defects in mitochondrial metabolism.

Brain-derived neurotrophic factor is produced by skeletal muscle cells in response to contraction and enhances fat oxidation via activation of AMP-activated protein kinase.

AIMS/HYPOTHESIS: Brain-derived neurotrophic factor (BDNF) is produced in skeletal muscle, but its functional significance is unknown. We aimed to determine the signalling processes and metabolic actions of BDNF. METHODS: We first examined whether exercise induced BDNF expression in humans. Next, C2C12 skeletal muscle cells were electrically stimulated to mimic contraction. L6 myotubes and isolated rat extensor digitorum longus muscles were treated with BDNF and phosphorylation of the proteins AMP-activated protein kinase (AMPK) (Thr(172)) and acetyl coenzyme A carboxylase beta (ACCbeta) (Ser(79)) were analysed, as was fatty acid oxidation (FAO). Finally, we electroporated a Bdnf vector into the tibialis cranialis muscle of mice. RESULTS: BDNF mRNA and protein expression were increased in human skeletal muscle after exercise, but muscle-derived BDNF appeared not to be released into the circulation. Bdnf mRNA and protein expression was increased in muscle cells that were electrically stimulated. BDNF increased phosphorylation of AMPK and ACCbeta and enhanced FAO both in vitro and ex vivo. The effect of BDNF on FAO was AMPK-dependent, since the increase in FAO was abrogated in cells infected with an AMPK dominant negative adenovirus or treated with Compound C, an inhibitor of AMPK. Electroporation of a Bdnf expression vector into the tibialis cranialis muscle resulted in increased BDNF protein production and tropomyosin-related kinase B (TrkB(Tyr706/707)) and extracellular signal-regulated protein kinase (p44/42 Thr(202)/Tyr(204)) phosphorylation in these muscles. In addition, phosphorylation of ACCbeta was markedly elevated in the Bdnf electroporated muscles. CONCLUSIONS/INTERPRETATION: These data identify BDNF as a contraction-inducible protein in skeletal muscle that is capable of enhancing lipid oxidation in skeletal muscle via activation of AMPK.

Examination of 'lipotoxicity' in skeletal muscle of high-fat fed and ob/ob mice.

Excess lipid accumulation resulting from an elevated supply of plasma fatty acids is linked to the pathogenesis of the metabolic syndrome and heart disease. The term 'lipotoxicity' was coined to describe how lipid accumulation leads to cellular dysfunction and death in non-adipose tissues including the heart, pancreas and liver. While lipotoxicity has been shown in cultured skeletal muscle cells, the degree of lipotoxicity in vivo and the functional consequences are unresolved. We studied three models of fatty acid overload in male mice: 5 h Intralipid((R)) and heparin infusion, prolonged high fat feeding (HFF) and genetic obesity induced by leptin deficiency (ob/ob mice). Markers of apoptosis, proteolysis and autophagy were assessed as readouts of lipotoxicity. The Intralipid((R)) infusion increased caspase 3 activity in skeletal muscle, demonstrating that enhancing fatty acid flux activates pro-apoptotic pathways. HFF and genetic obesity increased tissue lipid content but did not influence apoptosis. Gene array analysis revealed that HFF reduced the expression of 31 pro-apoptotic genes. Markers of autophagy (LC3beta and beclin-1 expression) were unaffected by HFF and were associated with enhanced Bcl(2) protein expression. Proteolytic activity was similarly unaffected by HFF or in ob/ob mice. Thus, contrary to our previous findings in muscle culture in vitro and in other non-adipose tissues in vivo, lipid overload did not induce apoptosis, autophagy or proteolysis in skeletal muscle. A broad transcriptional suppression of pro-apoptotic proteins may explain this resistance to lipid-induced cell death in skeletal muscle.

Corticotropin releasing factor influences aggression and monoamines: modulation of attacks and retreats.

Salmonids establish social hierarchies as a result of aggressive social interactions. The establishment of dominant or subordinate status is strongly linked to neuroendocrine responses mediated through the stress axis. In this study, we tested the effects of introcerebroventricular (icv) corticotropin releasing factor (CRF) on the behavioral outcome, plasma cortisol and monoamine function in trout subjected to a socially aggressive encounter. Rainbow trout were treated with an icv injection of artificial cerebrospinal fluid (aCSF), 500 or 2000 ng ovine CRF, or not injected. Fish were allowed to interact with a similarly sized conspecific for 15 min. Following the behavioral interaction, plasma cortisol and central monoamine concentrations were analyzed. Trout treated with CRF were victorious in approximately 66% of the aggressive encounters against aCSF-treated opponents. Trout injected with CRF exhibited a reduction in the total number of attacks and decreased latency to attack. When trout were divided into winners and losers, only victorious CRF-treated fish exhibited a reduced latency to attack and fewer retreats. Social stress increased cortisol levels in both winners and losers of aggressive interaction. This effect was enhanced with the additional stress incurred from icv injection of aCSF. However, icv CRF in addition to social stress decreased plasma cortisol in both winners and losers. While aggression stimulated significant changes in serotonergic and dopaminergic activity, the magnitude and direction were dependent on limbic brain region, CRF dose, and outcome of social aggression. With broad effects on aggressive behavior, anxiety, stress responsiveness, and central monoaminergic activity, CRF plays an important role in modulating the behavioral components of social interaction.

Stearoyl CoA desaturase 1 is elevated in obesity but protects against fatty acid-induced skeletal muscle insulin resistance in vitro.

AIMS/HYPOTHESIS: Stearoyl CoA desaturase 1 (SCD1) is implicated in mediating obesity and insulin resistance. Paradoxically, SCD1 converts saturated fatty acids, the lipid species implicated in mediating insulin resistance, to monounsaturated fatty acids. The aim of the present study was to assess the molecular mechanisms that implicate SCD1 in the aetiology of fatty acid-induced insulin resistance. METHODS: SCD1 protein was transiently decreased or increased in rat L6 skeletal muscle myotubes using SCD1 short interfering RNA (siRNA) or liposome-mediated transfection of pcDNA3.1/Hygro-mSCD1, respectively. RESULTS: Reducing SCD1 protein resulted in marked esterification of exogenous fatty acids into diacylglycerol (DAG) and ceramide. Insulin-stimulated Akt activity and phosphorylation and 2-deoxyglucose uptake were reduced with SCD1 siRNA. Exposure of L6 myotubes to palmitate abolished insulin-stimulated glucose uptake in both control and SCD1 siRNA myotubes. Overexpression of SCD1 resulted in triacylglycerol esterification but attenuated ceramide and DAG accumulation and protected myotubes from fatty acid-induced insulin resistance. CONCLUSIONS/INTERPRETATION: SCD1 protects from cellular toxicity in L6 myotubes by preventing excessive accumulation of bioactive lipid metabolites.

Corticotropin-releasing factor in the dorsal raphe elicits temporally distinct serotonergic responses in the limbic system in relation to fear behavior.

The neurotransmitters serotonin and corticotrophin-releasing factor are thought to play an important role in fear and anxiety behaviors. This study aimed to determine the relationship between corticotrophin-releasing factor-evoked changes in serotonin levels within discrete regions of the limbic system and the expression of fear behavior in rats. The effects of corticotrophin-releasing factor administration to the serotonin cell body regions of the dorsal raphe nucleus on fear behavior, behavioral activity, and extracellular serotonin levels were assessed in freely moving rats with microdialysis probes implanted into the central nucleus of the amygdala and the medial prefrontal cortex. Infusion of corticotrophin-releasing factor (0.5 microg) into the dorsal raphe rapidly induced freezing behavior, which was positively correlated with an immediate increase in serotonin release in the central nucleus of the amygdala. In contrast, cessation of freezing behavior correlated with a delayed and prolonged increase in serotonin release within the medial prefrontal cortex. Our findings suggest that corticotrophin-releasing factor-induced freezing behavior is associated with regionally and temporally distinct serotonergic responses in the limbic system that may reflect differing roles for these regions in the expression of fear/anxiety behavior.