Abstract: Laforin is a dual specificity protein phosphatase involved in Lafora disease (LD), a fatal form of progressive myoclonus epilepsy characterized by neurodegeneration and the presence of intracellular polyglucosan inclusions (Lafora bodies) in different tissues. In this work, we describe that mice lacking laforin (epm2a-/-) have enhanced insulin response leading to altered whole-body energy balance. This enhanced insulin response overactivates the Akt pathway which increases glucose uptake in the heart, resulting in increased glycogen levels and the formation of polyglucosan inclusions. In addition, enhanced insulin response resulted in increased liver lipid biosynthesis, resulting in hepatic steatosis. On the contrary, overexpression in rat hepatoma FTO2B cells of native laforin but not of a form lacking phosphatase activity (C266S) resulted in attenuation of insulin signaling. These results define laforin as a new regulator of insulin sensitivity, which provides novel insights into LD pathogenesis and identifies this phosphatase as a potential novel component of the insulin signaling cascade.
Abstract: In diet-induced obesity, hypothalamic inflammation is triggered as an outcome of prolonged exposure to dietary fats. Toll-like receptor 4 (TLR4) activation plays a central role in this process, inducing endoplasmic reticulum stress and activating inflammatory cytokine gene transcription. Although saturated fatty acids can induce endoplasmic reticulum stress in the hypothalamus, it is unknown whether inflammatory cytokines alone can activate this mechanism. Here, rats were treated with TNF-α or lyposaccharide (LPS) and endoplasmic reticulum stress and unfolded protein response were evaluated by immunoblot and polymerase chain reaction (PCR). Activation of TLR4 by LPS was capable of inducing a complete endoplasmic reticulum stress and unfolded protein response through the PERK/eIF2α and IRE1α/XBP1 pathways. Conversely, TNF-α, injected either locally or systemically, was unable to induce a complete program of unfolded protein response, although the activation of endoplasmic reticulum stress was achieved to a certain degree. Thus, in the hypothalamus, the isolated action of TNF-α is insufficient to produce the activation of a complete program of unfolded protein response.
Abstract: Atypical antipsychotic drugs (AAPDs) induce hyperphagia and body weight gain as a deleterious side effect. However, the mechanism whereby these drugs affect the neuronal pathways regulating energy balance has yet to be fully elucidated. The present study was conducted to investigate the respective and interaction effects of olanzapine and agonism of the melanin-concentrating hormone (MCH) receptor (MCHR1) on body weight, food intake, adiposity and expressions of genes liable of being involved in the anabolic action of AAPDs and MCH agonism. MCH is a hypothalamic neuropeptide, which exerts stimulating effects on food intake and body weight gain. Male Wistar rats received olanzapine (1 mg/kg of rat/day per os) and/or an intracerebroventricular (ICV) infusion of a MCHR1 agonist (30 microg/rat/day) during 13 days. Food intake and body weight were recorded daily, whereas adipose tissue depots were weighed at day 13. At the end of the experiment, we also measured brain levels of the messengers RNAs (mRNAs) encoding for MCH, MCHR1, neuropeptides-Y (NPY) and agouti-related peptide (AgRP) using in situ hybridization. The 13-day treatments combining olanzapine and the MCHR1 agonist exerted additive effects in enhancing food intake and adiposity. Consistently, each treatment differently affected brain expression of genes influencing energy balance. While the MCHR1 agonist treatment increased NPY mRNA expression in the hypothalamic arcuate nucleus, olanzapine treatment specifically increased MCHR1 mRNA expression in the nucleus accumbens shell (NAcSh). AAPDs and MCH agonism exert additive effects on energy balance and selective effects on the brain expression of energy balance-related genes.
Abstract: The nuclear receptor chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) is an important coordinator of glucose homeostasis. We report, for the first time, a unique differential regulation of its expression by the nutritional status in the mouse hypothalamus compared to peripheral tissues.
Abstract: BACKGROUND: Several effects of leptin in the immune system rely on its capacity to modulate cytokine expression and apoptosis in the thymus. Surprisingly, some of these effects are dependent on signal transduction through the IRS1/PI3-kinase, but not on the activation of JAK2. Since all the well known effects of leptin in different cell types and tissues seem to be dependent on JAK2 activation, we hypothesized that, at least for the control of thymic function, another, unknown kinase could mediate the transduction of the leptin signal from the ObR towards the IRS1/PI3-kinase signaling cascade. METHODOLOGY/PRINCIPAL FINDINGS: Here, by employing immunoblot, real-time PCR and flow citometry we show that the tyrosine kinase, Fyn, is constitutively associated with the ObR in thymic cells. Following a leptin stimulus, Fyn undergoes an activating tyrosine phosphorylation and a transient association with IRS1. All these effects are independent of JAK2 activation and, upon Fyn inhibition, the signal transduction towards IRS1/PI3-kinase is abolished. In addition, the inhibition of Fyn significantly modifies the effects of leptin on thymic cytokine expression. CONCLUSION/SIGNIFICANCE: Therefore, in the thymus, Fyn acts as a tyrosine kinase that transduces the leptin signal independently of JAK2 activation, and mediates some of the immunomodulatory effects of leptin in this tissue.
Abstract: Infiltrating macrophages play an important role in the production of inflammatory mediators by the adipose tissue of obese subjects. To reach the adipose tissue, peripheral monocytes are recruited by locally produced chemoattractants. However, little is known about the activation of monocytes in the peripheral blood of obese subjects. The objective of this study was to determine reactive oxygen species and endoplasmic reticulum stress as early markers of monocytic commitment with an inflammatory phenotype in the peripheral blood of nondiabetic obese patients. Patients were recruited from an academic general hospital; controls were voluntary students. Seven lean controls and 6 nondiabetic obese patients were included in the study. Monocytes were prepared from peripheral blood. Immunoblot, flow cytometry, and polymerase chain reaction were used to determine reactive oxygen species and endoplasmic reticulum stress. Increased reactive oxygen species and activation of endoplasmic reticulum stress were detected in the monocytes from obese patients. Reducing endoplasmic reticulum stress with a chemical chaperone reversed monocytic activation, as determined by the reduction of reactive oxygen species production. Thus, monocytes from nondiabetic obese patients are already committed with an inflammatory phenotype in peripheral blood; and reducing endoplasmic reticulum stress negatively modulates their activation.
Abstract: In diet-induced obesity, hypothalamic and systemic inflammatory factors trigger intracellular mechanisms that lead to resistance to the main adipostatic hormones, leptin and insulin. Tumor necrosis factor-alpha (TNF-alpha) is one of the main inflammatory factors produced during this process and its mechanistic role as an inducer of leptin and insulin resistance has been widely investigated. Most of TNF-alpha inflammatory signals are delivered by TNF receptor 1 (R1); however, the role played by this receptor in the context of obesity-associated inflammation is not completely known. Here, we show that TNFR1 knock-out (TNFR1 KO) mice are protected from diet-induced obesity due to increased thermogenesis. Under standard rodent chow or a high-fat diet, TNFR1 KO gain significantly less body mass despite increased caloric intake. Visceral adiposity and mean adipocyte diameter are reduced and blood concentrations of insulin and leptin are lower. Protection from hypothalamic leptin resistance is evidenced by increased leptin-induced suppression of food intake and preserved activation of leptin signal transduction through JAK2, STAT3, and FOXO1. Under the high-fat diet, TNFR1 KO mice present a significantly increased expression of the thermogenesis-related neurotransmitter, TRH. Further evidence of increased thermogenesis includes increased O(2) consumption in respirometry measurements, increased expressions of UCP1 and UCP3 in brown adipose tissue and skeletal muscle, respectively, and increased O(2) consumption by isolated skeletal muscle fiber mitochondria. This demonstrates that TNF-alpha signaling through TNFR1 is an important mechanism involved in obesity-associated defective thermogenesis.
Abstract: In animal models of diet-induced obesity, the activation of an inflammatory response in the hypothalamus produces molecular and functional resistance to the anorexigenic hormones insulin and leptin. The primary events triggered by dietary fats that ultimately lead to hypothalamic cytokine expression and inflammatory signaling are unknown. Here, we test the hypothesis that dietary fats act through the activation of toll-like receptors 2/4 and endoplasmic reticulum stress to induce cytokine expression in the hypothalamus of rodents. According to our results, long-chain saturated fatty acids activate predominantly toll-like receptor 4 signaling, which determines not only the induction of local cytokine expression but also promotes endoplasmic reticulum stress. Rats fed on a monounsaturated fat-rich diet do not develop hypothalamic leptin resistance, whereas toll-like receptor 4 loss-of-function mutation and immunopharmacological inhibition of toll-like receptor 4 protects mice from diet-induced obesity. Thus, toll-like receptor 4 acts as a predominant molecular target for saturated fatty acids in the hypothalamus, triggering the intracellular signaling network that induces an inflammatory response, and determines the resistance to anorexigenic signals.
Abstract: Uncoupling protein 2 (UCP2) is highly expressed in the hypothalamus; however, little is known about the functions it exerts in this part of the brain. Here, we hypothesized that UCP2 protects hypothalamic cells from oxidative and pro-apoptotic damage generated by inflammatory stimuli. Intracerebroventricular injection of tumor necrosis factor alpha (TNF-alpha)-induced an increase of UCP2 expression in the hypothalamus, which was accompanied by increased expression of markers of oxidative stress and pro-apoptotic proteins. The inhibition of UCP2 expression by an antisense oligonucleotide enhanced the damaging effects of TNF-alpha. Conversely, increasing the hypothalamic expression of UCP2 by cold exposure reversed most of the effects of the cytokine. Thus, UCP2 acts as a protective factor against cellular damage induced by an inflammatory stimulus in the hypothalamus.
Abstract: Peroxisome proliferator-activated receptor-gamma (PPARgamma) activation up-regulates thermogenesis-related genes in rodent white and brown adipose tissues (WAT and BAT) without increasing whole-body energy expenditure. We tested here whether such dissociation is the result of a negative modulation of sympathetic activity to WAT and BAT and thyroid axis components by PPARgamma activation. Administration of the PPARgamma agonist rosiglitazone (15 mg/kg.d) for 7 d to male Sprague Dawley rats increased food intake (10%), feed efficiency (31%), weight gain (45%), spontaneous motor activity (60%), and BAT and WAT mass and reduced whole-body oxygen consumption. Consistent with an anabolic setting, rosiglitazone markedly reduced sympathetic activity to BAT and WAT (>50%) and thyroid status as evidenced by reduced levels of plasma thyroid hormones (T(4) and T(3)) and mRNA levels of BAT and liver T(3)-generating enzymes iodothyronine type 2 (-40%) and type 1 (-32%) deiodinases, respectively. Rosiglitazone also decreased mRNA levels of the thyroid hormone receptor (THR) isoforms alpha1 (-34%) and beta (-66%) in BAT and isoforms alpha1 (-20%) and alpha2 (-47%) in retroperitoneal WAT. These metabolic effects were associated with a reduction in mRNA levels of the pro-energy expenditure peptides CRH and CART in specific hypothalamic nuclei. A direct central action of rosiglitazone is, however, unlikely based on its low brain uptake and lack of metabolic effects of intracerebroventricular administration. In conclusion, a reduction in BAT sympathetic activity and thyroid status appears to, at least partly, explain the PPARgamma-induced reduction in energy expenditure and the fact that up-regulation of thermogenic gene expression does not translate into functional stimulation of whole-body thermogenesis in vivo.
Abstract: The effects of the cannabinoid-1 receptor (CB(1)) antagonist rimonabant on energy metabolism and fasting-induced hypothalamic-pituitary-adrenal (HPA) axis and neuronal activation were investigated. Lean and obese Zucker rats were treated orally with a daily dose of 10 mg/kg rimonabant for 14 days. A comprehensive energy balance profile based on whole-carcass analyses further demonstrated the potential of CB(1) antagonists for decreasing energy gain through reducing food intake and potentially increasing brown adipose tissue thermogenesis. Rimonabant also reduced plasma glucose, insulin, and homeostasis model assessment of insulin resistance, which further confirms the ability of CB(1) antagonists to improve insulin sensitivity. To test the hypothesis that rimonabant attenuates the effect of fasting on HPA axis activation in the obese Zucker model, rats were either ad libitum-fed or food-deprived for 8 h. Contrary to expectation, rimonabant increased basal circulating corticosterone levels and enhanced the HPA axis response to food deprivation in obese rats. Rimonabant also exacerbated the neuronal activation seen in the arcuate nucleus (ARC) after short-term deprivation. In conclusion, the present study demonstrates that CB(1) blockade does not prevent the hypersensitivity to food deprivation occurring at the level of HPA axis and ARC activation in the obese Zucker rats. This, however, does not prevent CB(1) antagonism from exerting beneficial effects on energy and glucose metabolism.
Abstract: Rats normally eat about 85% of their food at night. Lactation increases food intake 3- to 4-fold, but the diurnal pattern of food intake persists. The mechanisms responsible for the diurnal and lactation-induced changes in food intake are still unresolved, hence we have further investigated the possible roles of serum leptin and hypothalamic expression of neuropeptide Y (NPY), agouti-related peptide (AgRP) and pro-opiomelanocortin (POMC) in rats. Suppressor of cytokine signalling-3 (SOCS-3) acts as a feedback inhibitor of leptin signalling in the hypothalamus, hence changes in expression of SOCS-3 were also investigated. Changes in expression of NPY, AgRP or POMC alone could not account for the diurnal changes in intake and their alteration by lactation. However, there were increased AgRP mRNA:POMC mRNA ratios at night and also during lactation, which were very similar to estimated changes in food intake. Such changes in expression may result in dominance of the orexigenic AgRP peptide over the appetite-suppressing POMC-derived peptides, and so could contribute to the hyperphagia in these states. Diurnal and lactation-related changes in the AgRP mRNA:POMC mRNA ratio and food intake are not due to changes in leptin alone. However, hypoleptinaemia, possibly through increased expression of NPY, may contribute to the hyperphagia of lactation. In the dark, expression of SOCS-3 was decreased in non-lactating rats; lactation decreased SOCS-3 expression in both light and dark phases. However, such changes are likely to enhance the ability of leptin-responsive neurones to transmit the leptin signal, and so are unlikely to contribute to either the nocturnal increase in appetite or the hyperphagia of lactation.
Abstract: The factors regulating serum leptin concentration and its relationship to the hyperphagia of lactation have been investigated in rats. Lactation results in hypoleptinaemia and loss, or at least marked attenuation, of the nocturnal rise in serum leptin. Litter removal resulted in a fall in food intake and restoration of the nocturnal rise in serum leptin. Returning the litter to the mother after a 48-h absence increased food intake and began to reinitiate milk production, but the nocturnal serum leptin levels were still increased at 48 h after litter restoration. Adjusting litter size to four, eight, ten or fourteen pups at parturition resulted in different rates of litter growth and food intake during the subsequent lactation, but had no effect on the degree of hypoleptinaemia. Reducing litter size from ten to four pups at mid-lactation resulted in a transient increase in both serum leptin and pup growth rate, while food intake fell to a level found in rats suckling four pups throughout lactation. Reducing milk production by injection of bromocriptine increased serum leptin, but did not restore the nocturnal rise in serum leptin; food intake decreased, but remained much higher than in non-lactating rats. Feeding a varied, high-energy diet resulted in a decrease in the weight of food ingested, but no change in calorie intake, and had no effect on the hypoleptinaemia. These studies suggested that the hypoleptinaemia of lactating rats is due to negative energy balance, but the loss of the nocturnal rise in serum leptin is due to the suckling stimulus. The negative energy balance of lactation does not appear to be caused by a physical constraint on food intake. While the hypoleptinaemia should facilitate the hyperphagia of lactation, other orexigenic signals must also be involved.
Abstract: Adiponectin levels are decreased in subjects with obesity, diabetes and coronary artery disease. In the present study, we have investigated whether the decrease in the levels and mRNA expression of adiponectin is due to obesity or to the diet itself. Wistar rats were either fed standard laboratory chow throughout (controls) or given a fat-enriched, glucose-enriched diet (diet-fed) for 2 days or 16 weeks. After 2 days of diet feeding, total body weight, fat pad masses and the plasma levels of glucose, insulin and leptin were all comparable between the two groups, while plasma NEFA (non-esterified fatty acid) and triacylglycerol levels were increased in the diet-fed animals (P<0.01 for both). There was a marked (P<0.01) decrease in plasma adiponectin levels. After 16 weeks of diet feeding, diet-fed rats had significantly higher body weight, fat pad mass and plasma levels of leptin, adiponectin, NEFA and triacylglycerol (P<0.001 for all) compared with chow-fed controls, whereas plasma levels of glucose and insulin were similar in the two groups. After 2 days of diet feeding, there were no significant changes in Ob mRNA levels in epididymal fat, whereas there was a marked decrease in adiponectin mRNA levels. After 16 weeks of diet feeding, rats had significantly increased levels of Ob mRNA, but decreased adiponectin mRNA levels, in epididymal fat compared with the chow-fed group (P<0.001 for both). These findings suggest that obesity per se is not a factor in the decreased adiponectin levels observed in obese subjects. We propose that the lipid profile of the plasma and/or the constituents of the diet consumed by rats may contribute to adiponectin levels more than obesity per se.
Abstract: We investigated the effects of lactation on diurnal changes in serum leptin and hypothalamic expression of the leptin receptor isoforms, Ob-Ra, -Rb, -Rc, -Re and -Rf in rats. In non-lactating rats, serum leptin concentration was increased at night while hypothalamic mRNA levels of Ob-Rb, -Rc and -Re decreased; by contrast, expression of Ob-Ra and Ob-Rf was unchanged at night. There were significant negative correlations between serum leptin and mRNA expression of Ob-Rb (P<0.001) and Ob-Re (P<0.05), which were independent of time of day. In lactating rats, the nocturnal rise in serum leptin was attenuated. Daytime hypothalamic Ob-Rb mRNA levels were significantly lower than in non-lactating controls, and the normal nocturnal decreases in expression of Ob-Rb, -Rc and -Re were lost. The relationship between serum leptin and Ob-Re expression was not changed by lactation. Lactation had no effect on the expression of Ob-Ra mRNA in the hypothalamus. Decreased daytime Ob-Rb expression could lead to reduced hypothalamic sensitivity to leptin, and thus contribute to increased daytime appetite in lactating rats. Moreover, maintaining high levels of Ob-Re expression could, by increasing hypothalamic leptin-binding protein concentration and reducing local leptin bioavailability, further accentuate hyperphagia. Thus, selective changes in expression of specific isoforms of the leptin receptor may contribute to the hyperphagia of lactation in rats.
Abstract: Lactation markedly increases nutrient requirements in both rodents and ruminants. This is met mostly by increased food intake, but there are also adaptations to increase metabolic efficiency. Despite such changes, lactating animals usually experience periods of negative energy balance. This is not due to a physical constraint on food intake, at least in the rat. Leptin, a hormone secreted by adipocytes, plays an important role in the regulation of appetite and energy balance. During lactation, serum leptin concentration is decreased in both rodents and ruminants, and the nocturnal rise in concentration is lost in rats. Hypoleptinaemia in lactation is primarily a result of negative energy balance. There is also increased clearance of serum leptin, and the attenuation of the nocturnal rise in leptin in rats is at least partly due to the suckling stimulus. Hypoleptinaemia is not the major factor driving hyperphagia in lactating rats, but it probably facilitates the increased food intake. Leptin may play a more important role in this respect in lactating ruminants. Leptin is probably involved in other adaptations that increase metabolic efficiency during lactation. The ability of hypothalamic neuropeptides to respond to leptin does not appear to be altered by lactation in either rodents or ruminants. The reason why lactating animals do not respond to hypoleptinaemia with a further increase in appetite, thereby achieving energy balance, appears to be due to a failure to respond to changes in neuropeptides which mediate the effects of leptin.
Abstract: Adipose tissue, a reserve of energy, has played an essential role in mammalian evolution. Adipose tissue differs from other tissues in that its mass has considerable capacity to expand, which while beneficial in decreasing the risk of starvation, increases the risk of predation. Adipose tissue mass is thus under tight control in nondomestic species. Adipose tissue secretes a variety of factors, some of which (leptin, tumor necrosis factor (TNF) alpha, resistin) are thought to be involved in modulation of adipose mass. Leptin has a variety of functions, primarily targetting the hypothalamus where it acts to decrease appetite and increase energy expenditure. Leptin is also involved in the adaptations to fasting, and leptin is also required for normal reproductive and immune function. TNF alpha and resistin appear to have key paracrine roles, attenuating the anabolic effects of insulin on adipose tissue metabolism.
Abstract: Orexins are hypothalamic peptides implicated in the regulation of ingestive and other behaviours. Here we investigated prepro-orexin expression and hypothalamic orexin-A and -B levels in lactating rats, which display marked hyperphagia, with or without food restriction for 2 days or treatment with bromocriptine, which inhibits milk production and thus reduces the energy losses of lactation. Neither prepro-orexin gene expression nor hypothalamic orexin-A peptide levels were changed in any of these lactating groups compared with age-matched virgin controls. However, hypothalamic orexin-B levels were significantly higher in lactating rats that were food-restricted for 2 days (P<0.05) compared with non-lactating controls and with lactating rats that were either freely-fed or bromocriptine-treated. Thus, food restriction superimposed on lactation selectively increases hypothalamic orexin-B levels, suggesting that orexin-A and -B may be differentially released or cleared. Changes in orexin-B availability may influence physiological activities other than energy homeostasis, perhaps inducing arousal.
Abstract: The activities of some enzymes of glycerolipid synthesis and fatty acid oxidation were measured in subcellular fractions of the yolk sac membrane (YSM), an extra-embryonic tissue that mediates the transfer of lipid from the yolk to the circulation of the chick embryo. The activities of monoacylglycerol acyltransferase and carnitine palmitoyl transferase-1 in the YSM (respectively, 284.8+/-13.2 nmol/min/mg microsomal protein and 145.6+/-9.1 nmol/min/mg mitochondrial protein; mean +/- SE; n = 4) at day 12 of development appear to be the highest yet reported for any animal tissue. Also, the carnitine palmitoyl transferase-1 of the YSM was very insensitive to inhibition by malonyl CoA. The maximal activities of glycerol-3-phosphate acyltransferase and diacylglycerol acyltransferase in the YSM (respectively, 26.7+/-2.2 and 36.1+/-2.1 nmol/min/mg microsomal protein) were also high compared with the reported values for various animal tissues. The very high enzymic capacity for glycerolipid synthesis supports the hypothesis that the yolk-derived lipids are subjected to hydrolysis followed by reesterification during transit across the YSM. The monoacylglycerol pathway appears to be the main route for glycerolipid resynthesis in the YSM. The results also suggest that the YSM has the capacity to perform simultaneously beta-oxidation at a high rate in order to provide energy for the lipid transfer process.
