Talita Romanatto

High-fat diet induces apoptosis of hypothalamic neurons.

Consumption of dietary fats is amongst the most important environmental factors leading to obesity. In rodents, the consumption of fat-rich diets blunts leptin and insulin anorexigenic signaling in the hypothalamus by a mechanism dependent on the in situ activation of inflammation. Since inflammatory signal transduction can lead to the activation of apoptotic signaling pathways, we evaluated the effect of high-fat feeding on the induction of apoptosis of hypothalamic cells. Here, we show that consumption of dietary fats induce apoptosis of neurons and a reduction of synaptic inputs in the arcuate nucleus and lateral hypothalamus. This effect is dependent upon diet composition, and not on caloric intake, since pair-feeding is not sufficient to reduce the expression of apoptotic markers. The presence of an intact TLR4 receptor, protects cells from further apoptotic signals. In diet-induced inflammation of the hypothalamus, TLR4 exerts a dual function, on one side activating pro-inflammatory pathways that play a central role in the development of resistance to leptin and insulin, and on the other side restraining further damage by controlling the apoptotic activity.

Inhibition of Hypothalamic Inflammation Reverses Diet-Induced Insulin Resistance in the Liver

Defective liver gluconeogenesis is the main mechanism leading to fasting hyperglycemia in type 2 diabetes, and, in concert with steatosis, it is the hallmark of hepatic insulin resistance. Experimental obesity results, at least in part, from hypothalamic inflammation, which leads to leptin resistance and defective regulation of energy homeostasis. Pharmacological or genetic disruption of hypothalamic inflammation restores leptin sensitivity and reduces adiposity. Here, we evaluate the effect of a hypothalamic anti-inflammatory approach to regulating hepatic responsiveness to insulin. Obese rodents were treated by intracerebroventricular injections, with immunoneutralizing antibodies against Toll-like receptor (TLR)4 or tumor necrosis factor (TNF)α, and insulin signal transduction, hepatic steatosis, and gluconeogenesis were evaluated. The inhibition of either TLR4 or TNFα reduced hypothalamic inflammation, which was accompanied by the reduction of hypothalamic resistance to leptin and improved insulin signal transduction in the liver. This was accompanied by reduced liver steatosis and reduced hepatic expression of markers of steatosis. Furthermore, the inhibition of hypothalamic inflammation restored defective liver glucose production. All these beneficial effects were abrogated by vagotomy. Thus, the inhibition of hypothalamic inflammation in obesity results in improved hepatic insulin signal transduction, leading to reduced steatosis and reduced gluconeogenesis. All these effects are mediated by parasympathetic signals delivered by the vagus nerve.

Deletion of tumor necrosis factor-alpha-receptor 1 (TNFR1) protects against diet-induced obesity by means of increased thermogenesis

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-α (TNF-α) 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-α 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 O2 consumption in respirometry measurements, increased expressions of UCP1 and UCP3 in brown adipose tissue and skeletal muscle, respectively, and increased O2 consumption by isolated skeletal muscle fiber mitochondria. This demonstrates that TNF-α signaling through TNFR1 is an important mechanism involved in obesity-associated defective thermogenesis.

TNF-α acts in the hypothalamus inhibiting food intake and increasing the respiratory quotient—Effects on leptin and insulin signaling pathways

Acting in the hypothalamus, tumor necrosis factor-alpha (TNF-alpha) produces a potent anorexigenic effect. However, the molecular mechanisms involved in this phenomenon are poorly characterized. In this study, we investigate the capacity of TNF-alpha to activate signal transduction in the hypothalamus through elements of the pathways employed by the anorexigenic hormones insulin and leptin. High dose TNF-alpha promotes a reduction of 25% in 12h food intake, which is an inhibitory effect that is marginally inferior to that produced by insulin and leptin. In addition, high dose TNF-alpha increases body temperature and respiratory quotient, effects not reproduced by insulin or leptin. TNF-alpha, predominantly at the high dose, is also capable of activating canonical pro-inflammatory signal transduction in the hypothalamus, inducing JNK, p38, and NFkappaB, which results in the transcription of early responsive genes and expression of proteins of the SOCS family. Also, TNF-alpha activates signal transduction through JAK-2 and STAT-3, but does not activate signal transduction through early and intermediary elements of the insulin/leptin signaling pathways such as IRS-2, Akt, ERK and FOXO1. When co-injected with insulin or leptin, TNF-alpha, at both high and low doses, partially impairs signal transduction through IRS-2, Akt, ERK and FOXO1 but not through JAK-2 and STAT-3. This effect is accompanied by the partial inhibition of the anorexigenic effects of insulin and leptin, when the low, but not the high dose of TNF-alpha is employed. In conclusion, TNF-alpha, on a dose-dependent way, modulates insulin and leptin signaling and action in the hypothalamus.

Hypothalamic Actions of Tumor Necrosis Factor α Provide the Thermogenic Core for the Wastage Syndrome in Cachexia

TNFalpha is an important mediator of catabolism in cachexia. Most of its effects have been characterized in peripheral tissues, such as skeletal muscle and fat. However, by acting directly in the hypothalamus, TNFalpha can activate thermogenesis and modulate food intake. Here we show that high concentration TNFalpha in the hypothalamus leads to increased O(2) consumption/CO(2) production, increased body temperature, and reduced caloric intake, resulting in loss of body mass. Most of the thermogenic response is produced by beta 3-adrenergic signaling to the brown adipose tissue (BAT), leading to increased BAT relative mass, reduction in BAT lipid quantity, and increased BAT mitochondria density. The expression of proteins involved in BAT thermogenesis, such as beta 3-adrenergic receptor, peroxisomal proliferator-activated receptor-gamma coactivator-1 alpha, and uncoupling protein-1, are increased. In the hypothalamus, TNFalpha produces reductions in neuropeptide Y, agouti gene-related peptide, proopiomelanocortin, and melanin-concentrating hormone, and increases CRH and TRH. The activity of the AMP-activated protein kinase signaling pathway is also decreased in the hypothalamus of TNFalpha-treated rats. Upon intracerebroventricular infliximab treatment, tumor-bearing and septic rats present a significantly increased survival. In addition, the systemic inhibition of beta 3-adrenergic signaling results in a reduced body mass loss and increased survival in septic rats. These data suggest hypothalamic TNFalpha action to be important mediator of the wastage syndrome in cachexia.

Tumor necrosis factor-alpha activates signal transduction in hypothalamus and modulates the expression of pro-inflammatory proteins and orexigenic/anorexigenic neurotransmitters.

Tumor necrosis factor‐α (TNF‐α) is known to participate in the wastage syndrome that accompanies cancer and severe infectious diseases. More recently, a role for TNF‐α in the pathogenesis of type 2 diabetes mellitus and obesity has been shown. Much of the regulatory action exerted by TNF‐α upon the control of energy stores depends on its action on the hypothalamus. In this study, we show that TNF‐α activates canonical pro‐inflammatory signal transduction pathways in the hypothalamus of rats. These signaling events lead to the transcriptional activation of an early responsive gene and to the induction of expression of cytokines and a cytokine responsive protein such as interleukin‐1β, interleukin‐6, interleukin‐10 and suppressor of cytokine signalling‐3, respectively. In addition, TNF‐α induces the expression of neurotransmitters involved in the control of feeding and thermogenesis. Thus, TNF‐α may act directly in the hypothalamus inducing a pro‐inflammatory response and the modulation of expression of neurotransmitters involved in energy homeostasis.

Central leptin action improves skeletal muscle AKT, AMPK, and PGC1α activation by hypothalamic PI3K-dependent mechanism

Central leptin action requires PI3K activity to modulate glucose homeostasis and peripheral metabolism. However, the mechanism behind this phenomenon is not clearly understood. We hypothesize that hypothalamic PI3K activity is important for the modulation of the AMP-activated protein kinase (AMPK)/acetyl-CoA carboxylase (ACC) pathway, PGC1 alpha, and AKT in skeletal muscle (SM). To address this issue, we injected leptin into the lateral ventricle of rats. Hypothalamic JAK2 and AKT were activated by intracerebroventricular (ICV) injection of leptin in a time-dependent manner. Central leptin improved tolerance to glucose (GTT), increased PGC1 alpha expression, and AKT, AMPK, ACC and JAK2 phosphorylation in the soleus muscle. Previous ICV administration of either LY294002 or propranolol (IP) blocked these effects. We concluded that the activation of the hypothalamic PI3K pathway is important for leptin-induced AKT phosphorylation, as well as for active catabolic pathway through AMPK and PGC1 alpha in SM. Thus, a defective leptin signalling PI3K pathway in the hypothalamus may contribute to peripheral resistance to insulin associated to diet-induced obesity.

TNF-α transiently induces endoplasmic reticulum stress and an incomplete unfolded protein response in the hypothalamus

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.

Intracerebroventricular injection of citrate inhibits hypothalamic AMPK and modulates feeding behavior and peripheral insulin signaling

We hypothesized that citrate might modulate the AMP-activated protein kinase/acetyl-CoA carboxylase (AMPK)/(ACC) pathway and participate in neuronal feeding control and glucose homeostasis. To address this issue, we injected citrate into the lateral ventricle of rats. Intracerebroventricular (ICV) injection of citrate diminished the phosphorylation of hypothalamic AMPK/ACC, increased the expression of anorexigenic neuropeptide (pro-opiomelanocortin and corticotropin-releasing hormone), elevated the level of malonyl-CoA in the hypothalamus, and reduced food intake. No change was observed in the concentration of blood insulin after the injection of citrate. With a euglycemic-hyperinsulinemic clamp, the glucose infusion rate was higher in the citrate group than in the control group (28.6+/-0.8 vs 19.3+/-0.2 mU/kg body weight/min respectively), and so was glucose uptake in skeletal muscle and the epididymal fat pad. Concordantly, insulin receptor (IR), IR substrate type 1 (IRS1), IRS2, and protein kinase B (AKT) phosphorylation in adipose tissue and skeletal muscle was improved by citrate ICV treatment. Moreover, the treatment with citrate for 7 days promoted body weight loss and decreased the adipose tissue. Our results suggest that citrate and glucose may serve as signals of energy and nutrient availability to hypothalamic cells.

Citrate diminishes hypothalamic acetyl-CoA carboxylase phosphorylation and modulates satiety signals and hepatic mechanisms involved in glucose homeostasis in rats

The hypothalamic AMP-activated protein kinase (AMPK)/acetyl-CoA carboxylase (ACC) pathway is known to play an important role in the control of food intake and energy expenditure. Here, we hypothesize that citrate, an intermediate metabolite, activates hypothalamic ACC and is involved in the control of energy mobilization. Initially, we showed that ICV citrate injection decreased food intake and diminished weight gain significantly when compared to control and pair-fed group results. In addition, we showed that intracerebroventricular (ICV) injection of citrate diminished (80% of control) the phosphorylation of ACC, an important AMPK substrate. Furthermore, citrate treatment inhibited (75% of control) hypothalamic AMPK phosphorylation during fasting. In addition to its central effect, ICV citrate injection led to low blood glucose levels during glucose tolerance test (GTT) and high glucose uptake during hyperglycemic-euglycemic clamp. Accordingly, liver glycogen content was higher in animals given citrate (ICV) than in the control group (23.3+/-2.5 vs. 2.7+/-0.5 microg mL(-1) mg(-1), respectively). Interestingly, liver AMPK phosphorylation was reduced (80%) by the citrate treatment. The pharmacological blockade of beta3-adrenergic receptor (SR 59230A) blocked the effect of ICV citrate and citrate plus insulin on liver AMPK phosphorylation. Consistently with these results, rats treated with citrate (ICV) presented improved insulin signal transduction in liver, skeletal muscle, and epididymal fat pad. Similar results were obtained by hypothalamic administration of ARA-A, a competitive inhibitor of AMPK. Our results suggest that the citrate produced by mitochondria may modulate ACC phosphorylation in the hypothalamus, controlling food intake and coordinating a multiorgan network that controls glucose homeostasis and energy uptake through the adrenergic system.