Altered profile of plasma phospholipids in woman with recurrent pregnancy loss and recurrent implantation failure treated with lipid emulsion therapy.

BACKGROUND: Recurrent Pregnancy Loss (RPL) and Recurrent Implantation Failure (RIF) are highly heterogeneous condition and many of the mechanisms involved still require elucidation. The aim was to analyze the lipidomic profile in plasma of women with RPL and RIF before and after receiving the Lipid Emulsion Therapy (LET) containing 10% fish oil (SMOFlipid® 20%). METHODS: This study included twenty-six women with RPL or RIF from immunological or inflammatory causes, with elevated natural killer cell levels and divided into a Pregnancy Loss or a Live Birth group according to the outcome. The women received intravenous LET and sample collecting was done before the first, third and fifth dose of LET in the pregnant women. Ultra-performance liquid chromatography quadrupole time of flight mass spectrometry (UPLC-QTOF MS) and multivariate statistical methods were performed to evaluate the profile of phospholipids present in the women's plasma. RESULTS: An increase of phosphatidylcholines (PC) 40:8 and 36:5 levels with predominance of n6 polyunsaturated fatty acids (PUFA) was observed in plasma lipids of the Pregnancy Loss Group compared to Live Birth Group. We also observed an increase in the relative abundance of n3 PUFA-PC species (42:10 and 36:6) and LysoPC 15:0 with the long term use of LET. CONCLUSION: The greater availability of n3 PUFA in plasma of the pregnant women stemming from LET use can be considered advantageous regarding the alteration of the phospholipid profile and its postulated anti-inflammatory and immunomodulatory role.

The orphan G protein-coupled receptor, GPR139, is expressed in the hypothalamus and is involved in the regulation of body mass, blood glucose, and insulin.

GPR139 is an orphan G-protein-coupled receptor that is expressed in restricted areas of the nervous system, including the hypothalamus. In this study, we hypothesized that GPR139 could be involved in the regulation of energy balance and metabolism. In the first part of the study, we confirmed that GPR139 is expressed in the hypothalamus and particularly in proopiomelanocortin and agouti-related peptide neurons of the mediobasal hypothalamus. Using a lentivirus with a short-hairpin RNA, we inhibited the expression of GPR139 bilaterally in the mediobasal hypothalamus of mice. The intervention promoted a 40% reduction in the hypothalamic expression of GPR139, which was accompanied by an increase in body mass, a reduction in fasting blood glucose levels, and an increase in insulin levels. In the hypothalamus, inhibition of GPR139 was accompanied by a reduction in the expression of orexin. As previous studies using a pharmacological antagonist of orexin showed a beneficial impact on type 2 diabetes and glucose metabolism, we propose that the inhibition of hypothalamic GPR139 could be acting indirectly through the orexin system to control systemic glucose and insulin. In conclusion, this study advances the characterization of GPR139 in the hypothalamus, demonstrating its involvement in the regulation of body mass, blood insulin, and glycemia.

The orphan receptor GPR68 is expressed in the hypothalamus and is involved in the regulation of feeding.

Currently, up to 35% off all drugs approved for the treatment of human diseases belong to the G-protein-coupled receptor (GPCR) family. Out of the almost 800 existing GPCRs, 25% have no known endogenous ligands and are regarded as orphan receptors; many of these are currently under investigation as potential pharmacological targets. Here, we hypothesised that orphan GPCRs expressed in the hypothalamus could be targets for the treatment of obesity and other metabolic diseases. Using bioinformatic tools, we identified 78 class A orphan GPCRs that are expressed in the hypothalamus of mice. Initially, we selected two candidates and determined their responsivities to nutritional interventions: GPR162, the GPCR with highest expression in the hypothalamus, and GPR68, a GPCR with intermediate expression in the hypothalamus and that has never been explored for its potential involvement in metabolic regulation. GPR162 expression was not modified by fasting/feeding or by the consumption of a high-fat diet, and was therefore not subsequently evaluated. Conversely, GPR68 expression increased in response to the consumption of a high-fat diet and reduced under fasting conditions. Using immunofluorescence, GPR68 was identified in both proopiomelanocortin-expressing and agouti-related peptide-expressing neurons in the hypothalamic arcuate nucleus. Acute inhibition of GPR68 with an allosteric modulator promoted an increase in the expression of the orexigenic agouti-related peptide and neuropeptide Y, whereas 4- and 12-h inhibition of GPR68 resulted in increased caloric intake. Thus, GPR68 has emerged as an orphan GPCR that is expressed in the hypothalamus and is involved in the regulation of feeding.

Proopiomelanocortin Processing in the Hypothalamus Is Directly Regulated by Saturated Fat: Implications for the Development of Obesity.

In outbred mice, susceptibility or resistance to diet-induced obesity is associated with rapid changes in hypothalamic proopiomelanocortin (POMC) levels. Here, we evaluated 3 hypotheses that potentially explain the development of the different obesity phenotypes in outbred Swiss mice. First, rapid and differential changes in the gut microbiota in obesity-prone (OP) and obesity-resistant (OR) mice fed on a high-fat diet (HFD) might cause differential efficiencies in fatty acid harvesting leading to changes in systemic fatty acid concentrations that in turn affect POMC expression and processing. Second, independently of the gut microbiota, OP mice might have increased blood fatty acid levels after the introduction of a HFD, which could affect POMC expression and processing. Third, fatty acids might act directly in the hypothalamus to differentially regulate POMC expression and/or processing in OP and OR mice. We evaluated OP and OR male Swiss mice using 16S rRNA sequencing for the determination of gut microbiota; gas chromatography for blood lipid determination; and immunoblot and real-time polymerase chain reaction for protein and transcript determination and indirect calorimetry. Some experiments were performed with human pluripotent stem cells differentiated into hypothalamic neurons. We did not find evidence supporting the first 2 hypotheses. However, we found that in OP but not in OR mice, palmitate induces a rapid increase in hypothalamic POMC, which is followed by increased expression of proprotein convertase subtilisin/kexin type 1 PC1/3. Lentiviral inhibition of hypothalamic PC1/3 increased caloric intake and body mass in both OP and OR mice. In human stem cell-derived hypothalamic cells, we found that palmitate potently suppressed the production of POMC-derived peptides. Palmitate directly regulates PC1/3 in OP mice and likely has a functional impact on POMC processing.

Swimming reduces fatty acids-associated hypothalamic damage in mice.

The arcuate and the paraventricular and lateral hypothalamic nuclei, related to hunger and satiety control, are generally compromised by excess fatty acids. In this situation, fatty acids cause inflammation via TLR4 (toll like receptor 4) and the nuclei become less responsive to the hormones leptin and insulin, contributing to the development of obesity. In this work, these nuclei were analyzed in animals fed with high-fat diet and submitted to swimming without and with load for two months. For this, frontal sections of the hypothalamus were immunolabelled with GFAP (glial fibrillary acidic protein), synaptophysin, IL-6 (interleukin 6) and TLR4. Also, proteins extracted from the hypothalamus were analyzed using Western blotting (GFAP and synaptophysin), fluorometric analysis for caspases 3 and 7, and CBA (cytometric bead array) for Th1, Th2, and Th17 profiles. The high-fat diet significantly caused overweight and, in the hypothalamus, decreased synapses and increased astrocytic reactivity. The swimming with load, especially 80 % of the maximum load, reduced those consequences. The high-fat diet increased TLR4 in the arcuate nucleus and the swimming exercise with 80 % of the maximum load showed a tendency of reducing this expression. Swimming did not significantly influence the inflammatory or anti-inflammatory cytokines in the hypothalamus or in plasma. The high-fat diet in sedentary animals increased the expression of caspases 3 and 7 and swimming practice reduced this increment to levels compatible with animals fed on a normal diet. The set of results conclude that the impact of swimming on the damage caused in the hypothalamus by a high-fat diet is positive. The different aspects analyzed in here point to better cellular viability and conservation of the synapses in the hypothalamic nuclei of overweight animals that practiced swimming with a load.

Hypothalamic neuronal cellular and subcellular abnormalities in experimental obesity.

The characterization of the hypothalamic neuronal network, that controls food intake and energy expenditure, has provided great advances in the understanding of the pathophysiology of obesity. Most of the advances in this field were obtained thanks to the development of a number of genetic and nongenetic animal models that, at least in part, overtook the anatomical constraints that impair the study of the human hypothalamus. Despite the undisputed differences between human and rodent physiology, most seminal studies undertaken in rodents that have unveiled details of the neural regulation of energy homeostasis were eventually confirmed in humans; thus, placing experimental studies in the forefront of obesity research. During the last 15 years, researchers have provided extensive experimental proof that supports the existence of hypothalamic dysfunction, which leads to a progressive whole-body positive energy balance, and thus, to obesity. Here, we review the experimental work that unveiled the mechanisms behind hypothalamic dysfunction in obesity.

The partial inhibition of hypothalamic IRX3 exacerbates obesity.

BACKGROUND: The Iroquois homeobox 3 (Irx3) gene has been identified as a functional long-range target of obesity-associated variants within the fat mass and obesity-associated protein (FTO) gene. It is highly expressed in the hypothalamus, and both whole-body knockout and hypothalamic restricted abrogation of its expression results in a lean phenotype, which is mostly explained by the resulting increased energy expenditure in the brown adipose tissue. Because of its potential implication in the pathogenesis of obesity, we evaluated the hypothalamic cell distribution of Irx3 and the outcomes of inhibiting its expression in a rodent model of diet-induced obesity. METHODS: Bioinformatics tools were used to evaluate the correlations between hypothalamic Irx3 and neurotransmitters, markers of thermogenesis and obesity related phenotypes. Droplet-sequencing analysis in >20,000 hypothalamic cells was used to explore the types of hypothalamic cells expressing Irx3. Lentivirus was used to inhibit hypothalamic Irx3 and the resulting phenotype was studied. FINDINGS: IRX3 is expressed predominantly in POMC neurons. Its expression is inhibited during prolonged fasting, as well as when mice are fed a high-fat diet. The partial inhibition of hypothalamic Irx3 using a lentivirus resulted in increased diet-induced body mass gain and adiposity due to increased caloric intake and reduced energy expenditure. INTERPRETATION: Contrary to the results obtained when lean mice are submitted to complete inhibition of Irx3, partial inhibition of hypothalamic Irx3 in obese mice causes an exacerbation of the obese phenotype. These data suggest that at least some of the Irx3 functions in the hypothalamus are regulated according to a hormetic pattern, and modulation of its expression can be a novel approach to modifying the body's energy-handling regulation. FUND: Sao Paulo Research Foundation grants 2013/07607-8 (LAV) and 2017/02983-2 (JDJ); NIH grants R01DK083567 (YBK).

Hypothalamic mitochondrial abnormalities occur downstream of inflammation in diet-induced obesity.

Hypothalamic dysfunction is a common feature of experimental obesity. Studies have identified at least three mechanisms involved in the development of hypothalamic neuronal defects in diet-induced obesity: i, inflammation; ii, endoplasmic reticulum stress; and iii, mitochondrial abnormalities. However, which of these mechanisms is activated earliest in response to the consumption of large portions of dietary fats is currently unknown. Here, we used immunoblot, real-time PCR, mitochondrial respiration assays and transmission electron microscopy to evaluate markers of inflammation, endoplasmic reticulum stress and mitochondrial abnormalities in the hypothalamus of Swiss mice fed a high-fat diet for up to seven days. In the present study we show that the expression of the inflammatory chemokine fractalkine was the earliest event detected. Its hypothalamic expression increased as early as 3 h after the introduction of a high-fat diet and was followed by the increase of cytokines. GPR78, an endoplasmic reticulum chaperone, was increased 6 h after the introduction of a high-fat diet, however the actual triggering of endoplasmic reticulum stress was only detected three days later, when IRE-1α was increased. Mitofusin-2, a protein involved in mitochondrial fusion and tethering of mitochondria to the endoplasmic reticulum, underwent a transient reduction 24 h after the introduction of a high-fat diet and then increased after seven days. There were no changes in hypothalamic mitochondrial respiration during the experimental period, however there were reductions in mitochondria/endoplasmic reticulum contact sites, beginning three days after the introduction of a high-fat diet. The inhibition of TNF-α with infliximab resulted in the normalization of mitofusin-2 levels 24 h after the introduction of the diet. Thus, inflammation is the earliest mechanism activated in the hypothalamus after the introduction of a high-fat diet and may play a mechanistic role in the development of mitochondrial abnormalities in diet-induced obesity.

Polyunsaturated fatty acid receptors, GPR40 and GPR120, are expressed in the hypothalamus and control energy homeostasis and inflammation.

BACKGROUND: The consumption of large amounts of dietary fats is one of the most important environmental factors contributing to the development of obesity and metabolic disorders. GPR120 and GPR40 are polyunsaturated fatty acid receptors that exert a number of systemic effects that are beneficial for metabolic and inflammatory diseases. Here, we evaluate the expression and potential role of hypothalamic GPR120 and GPR40 as targets for the treatment of obesity. METHODS: Male Swiss (6-weeks old), were fed with a high fat diet (HFD, 60% of kcal from fat) for 4 weeks. Next, mice underwent stereotaxic surgery to place an indwelling cannula into the right lateral ventricle. intracerebroventricular (icv)-cannulated mice were treated twice a day for 6 days with 2.0 µL saline or GPR40 and GPR120 agonists: GW9508, TUG1197, or TUG905 (2.0 µL, 1.0 mM). Food intake and body mass were measured during the treatment period. At the end of the experiment, the hypothalamus was collected for real-time PCR analysis. RESULTS: We show that both receptors are expressed in the hypothalamus; GPR120 is primarily present in microglia, whereas GPR40 is expressed in neurons. Upon intracerebroventricular treatment, GW9508, a non-specific agonist for both receptors, reduced energy efficiency and the expression of inflammatory genes in the hypothalamus. Reducing GPR120 hypothalamic expression using a lentivirus-based approach resulted in the loss of the anti-inflammatory effect of GW9508 and increased energy efficiency. Intracerebroventricular treatment with the GPR120- and GPR40-specific agonists TUG1197 and TUG905, respectively, resulted in milder effects than those produced by GW9508. CONCLUSIONS: GPR120 and GPR40 act in concert in the hypothalamus to reduce energy efficiency and regulate the inflammation associated with obesity. The combined activation of both receptors in the hypothalamus results in better metabolic outcomes than the isolated activation of either receptor alone.

Metabolic Impact of Light Phase-Restricted Fructose Consumption Is Linked to Changes in Hypothalamic AMPK Phosphorylation and Melatonin Production in Rats.

Recent studies show that the metabolic effects of fructose may vary depending on the phase of its consumption along with the light/dark cycle. Here, we investigated the metabolic outcomes of fructose consumption by rats during either the light (LPF) or the dark (DPF) phases of the light/dark cycle. This experimental approach was combined with other interventions, including restriction of chow availability to the dark phase, melatonin administration or intracerebroventricular inhibition of adenosine monophosphate-activated protein kinase (AMPK) with Compound C. LPF, but not DPF rats, exhibited increased hypothalamic AMPK phosphorylation, glucose intolerance, reduced urinary 6-sulfatoxymelatonin (6-S-Mel) (a metabolite of melatonin) and increased corticosterone levels. LPF, but not DPF rats, also exhibited increased chow ingestion during the light phase. The mentioned changes were blunted by Compound C. LPF rats subjected to dark phase-restricted feeding still exhibited increased hypothalamic AMPK phosphorylation but failed to develop the endocrine and metabolic changes. Moreover, melatonin administration to LPF rats reduced corticosterone and prevented glucose intolerance. Altogether, the present data suggests that consumption of fructose during the light phase results in out-of-phase feeding due to increased hypothalamic AMPK phosphorylation. This shift in spontaneous chow ingestion is responsible for the reduction of 6-S-Mel and glucose intolerance.

Resolvin RvD2 reduces hypothalamic inflammation and rescues mice from diet-induced obesity.

BACKGROUND: Diet-induced hypothalamic inflammation is an important mechanism leading to dysfunction of neurons involved in controlling body mass. Studies have shown that polyunsaturated fats can reduce hypothalamic inflammation. Here, we evaluated the presence and function of RvD2, a resolvin produced from docosahexaenoic acid, in the hypothalamus of mice. METHODS: Male Swiss mice were fed either chow or a high-fat diet. RvD2 receptor and synthetic enzymes were evaluated by real-time PCR and immunofluorescence. RvD2 was determined by mass spectrometry. Dietary and pharmacological approaches were used to modulate the RvD2 system in the hypothalamus, and metabolic phenotype consequences were determined. RESULTS: All enzymes involved in the synthesis of RvD2 were detected in the hypothalamus and were modulated in response to the consumption of dietary saturated fats, leading to a reduction of hypothalamic RvD2. GPR18, the receptor for RvD2, which was detected in POMC and NPY neurons, was also modulated by dietary fats. The substitution of saturated by polyunsaturated fats in the diet resulted in increased hypothalamic RvD2, which was accompanied by reduced body mass and improved glucose tolerance. The intracerebroventricular treatment with docosahexaenoic acid resulted in increased expression of the RvD2 synthetic enzymes, increased expression of anti-inflammatory cytokines and improved metabolic phenotype. Finally, intracerebroventricular treatment with RvD2 resulted in reduced adiposity, improved glucose tolerance and increased hypothalamic expression of anti-inflammatory cytokines. CONCLUSIONS: Thus, RvD2 is produced in the hypothalamus, and its receptor and synthetic enzymes are modulated by dietary fats. The improved metabolic outcomes of RvD2 make this substance an attractive approach to treat obesity.

Defective regulation of POMC precedes hypothalamic inflammation in diet-induced obesity.

Obesity is the result of a long-term positive energy balance in which caloric intake overrides energy expenditure. This anabolic state results from the defective activity of hypothalamic neurons involved in the sensing and response to adiposity. However, it is currently unknown what the earliest obesity-linked hypothalamic defect is and how it orchestrates the energy imbalance present in obesity. Using an outbred model of diet-induced obesity we show that defective regulation of hypothalamic POMC is the earliest marker distinguishing obesity-prone from obesity-resistant mice. The early inhibition of hypothalamic POMC was sufficient to transform obesity-resistant in obesity-prone mice. In addition, the post-prandial change in the blood level of ß-endorphin, a POMC-derived peptide, correlates with body mass gain in rodents and humans. Taken together, these results suggest that defective regulation of POMC expression, which leads to a change of ß-endorphin levels, is the earliest hypothalamic defect leading to obesity.

Hypothalamic S1P/S1PR1 axis controls energy homeostasis in Middle-Aged Rodents: the reversal effects of physical exercise.

Recently, we demonstrated that the hypothalamic S1PR1/STAT3 axis plays a critical role in the control of food consumption and energy expenditure in rodents. Here, we found that reduction of hypothalamic S1PR1 expression occurs in an age-dependent manner, and was associated with defective thermogenic signaling and weight gain. To address the physiological relevance of these findings, we investigated the effects of chronic and acute exercise on the hypothalamic S1PR1/STAT3 axis. Chronic exercise increased S1PR1 expression and STAT3 phosphorylation in the hypothalamus, restoring the anorexigenic and thermogenic signals in middle-aged mice. Acutely, exercise increased sphingosine-1-phosphate (S1P) levels in the cerebrospinal fluid (CSF) of young rats, whereas the administration of CSF from exercised young rats into the hypothalamus of middle-aged rats at rest was sufficient to reduce the food intake. Finally, the intracerebroventricular (ICV) administration of S1PR1 activators, including the bioactive lipid molecule S1P, and pharmacological S1PR1 activator, SEW2871, induced a potent STAT3 phosphorylation and anorexigenic response in middle-aged rats. Overall, these results suggest that hypothalamic S1PR1 is important for the maintenance of energy balance and provide new insights into the mechanism by which exercise controls the anorexigenic and thermogenic signals in the central nervous system during the aging process.

n-3 Fatty Acids Induce Neurogenesis of Predominantly POMC-Expressing Cells in the Hypothalamus.

Apoptosis of hypothalamic neurons is believed to play an important role in the development and perpetuation of obesity. Similar to the hippocampus, the hypothalamus presents constitutive and stimulated neurogenesis, suggesting that obesity-associated hypothalamic dysfunction can be repaired. Here, we explored the hypothesis that n-3 polyunsaturated fatty acids (PUFAs) induce hypothalamic neurogenesis. Both in the diet and injected directly into the hypothalamus, PUFAs were capable of increasing hypothalamic neurogenesis to levels similar or superior to the effect of brain-derived neurotrophic factor (BDNF). Most of the neurogenic activity induced by PUFAs resulted in increased numbers of proopiomelanocortin but not NPY neurons and was accompanied by increased expression of BDNF and G-protein-coupled receptor 40 (GPR40). The inhibition of GPR40 was capable of reducing the neurogenic effect of a PUFA, while the inhibition of BDNF resulted in the reduction of global hypothalamic cell. Thus, PUFAs emerge as a potential dietary approach to correct obesity-associated hypothalamic neuronal loss.

Saturated fatty acids modulate autophagy's proteins in the hypothalamus.

Autophagy is an important process that regulates cellular homeostasis by degrading dysfunctional proteins, organelles and lipids. In this study, the hypothesis that obesity could lead to impairment in hypothalamic autophagy in mice was evaluated by examining the hypothalamic distribution and content of autophagic proteins in animal with obesity induced by 8 or 16 weeks high fat diet to induce obesity and in response to intracerebroventricular injections of palmitic acid. The results showed that chronic exposure to a high fat diet leads to an increased expression of inflammatory markers and downregulation of autophagic proteins. In obese mice, autophagic induction leads to the downregulation of proteins, such as JNK and Bax, which are involved in the stress pathways. In neuron cell-line, palmitate has a direct effect on autophagy even without inflammatory activity. Understanding the cellular and molecular bases of overnutrition is essential for identifying new diagnostic and therapeutic targets for obesity.

Alzheimer-associated Aß oligomers impact the central nervous system to induce peripheral metabolic deregulation.

Alzheimer's disease (AD) is associated with peripheral metabolic disorders. Clinical/epidemiological data indicate increased risk of diabetes in AD patients. Here, we show that intracerebroventricular infusion of AD-associated Aß oligomers (AßOs) in mice triggered peripheral glucose intolerance, a phenomenon further verified in two transgenic mouse models of AD. Systemically injected AßOs failed to induce glucose intolerance, suggesting AßOs target brain regions involved in peripheral metabolic control. Accordingly, we show that AßOs affected hypothalamic neurons in culture, inducing eukaryotic translation initiation factor 2α phosphorylation (eIF2α-P). AßOs further induced eIF2α-P and activated pro-inflammatory IKKß/NF-κB signaling in the hypothalamus of mice and macaques. AßOs failed to trigger peripheral glucose intolerance in tumor necrosis factor-α (TNF-α) receptor 1 knockout mice. Pharmacological inhibition of brain inflammation and endoplasmic reticulum stress prevented glucose intolerance in mice, indicating that AßOs act via a central route to affect peripheral glucose homeostasis. While the hypothalamus has been largely ignored in the AD field, our findings indicate that AßOs affect this brain region and reveal novel shared molecular mechanisms between hypothalamic dysfunction in metabolic disorders and AD.

Hypothalamic S1P/S1PR1 axis controls energy homeostasis.

Sphingosine 1-phosphate receptor 1 (S1PR1) is a G-protein-coupled receptor for sphingosine-1-phosphate (S1P) that has a role in many physiological and pathophysiological processes. Here we show that the S1P/S1PR1 signalling pathway in hypothalamic neurons regulates energy homeostasis in rodents. We demonstrate that S1PR1 protein is highly enriched in hypothalamic POMC neurons of rats. Intracerebroventricular injections of the bioactive lipid, S1P, reduce food consumption and increase rat energy expenditure through persistent activation of STAT3 and the melanocortin system. Similarly, the selective disruption of hypothalamic S1PR1 increases food intake and reduces the respiratory exchange ratio. We further show that STAT3 controls S1PR1 expression in neurons via a positive feedback mechanism. Interestingly, several models of obesity and cancer anorexia display an imbalance of hypothalamic S1P/S1PR1/STAT3 axis, whereas pharmacological intervention ameliorates these phenotypes. Taken together, our data demonstrate that the neuronal S1P/S1PR1/STAT3 signalling axis plays a critical role in the control of energy homeostasis in rats.

Defective regulation of the ubiquitin/proteasome system in the hypothalamus of obese male mice.

In both human and experimental obesity, inflammatory damage to the hypothalamus plays an important role in the loss of the coordinated control of food intake and energy expenditure. Upon prolonged maintenance of increased body mass, the brain changes the defended set point of adiposity, and returning to normal weight becomes extremely difficult. Here we show that in prolonged but not in short-term obesity, the ubiquitin/proteasome system in the hypothalamus fails to maintain an adequate rate of protein recycling, leading to the accumulation of ubiquitinated proteins. This is accompanied by an increased colocalization of ubiquitin and p62 in the arcuate nucleus and reduced expression of autophagy markers in the hypothalamus. Genetic protection from obesity is accompanied by the normal regulation of the ubiquitin/proteasome system in the hypothalamus, whereas the inhibition of proteasome or p62 results in the acceleration of body mass gain in mice exposed for a short period to a high-fat diet. Thus, the defective regulation of the ubiquitin/proteasome system in the hypothalamus may be an important mechanism involved in the progression and autoperpetuation of obesity.

Fractalkine (CX3CL1) is involved in the early activation of hypothalamic inflammation in experimental obesity.

Hypothalamic inflammation is a common feature of experimental obesity. Dietary fats are important triggers of this process, inducing the activation of toll-like receptor-4 (TLR4) signaling and endoplasmic reticulum stress. Microglia cells, which are the cellular components of the innate immune system in the brain, are expected to play a role in the early activation of diet-induced hypothalamic inflammation. Here, we use bone marrow transplants to generate mice chimeras that express a functional TLR4 in the entire body except in bone marrow-derived cells or only in bone marrow-derived cells. We show that a functional TLR4 in bone marrow-derived cells is required for the complete expression of the diet-induced obese phenotype and for the perpetuation of inflammation in the hypothalamus. In an obesity-prone mouse strain, the chemokine CX3CL1 (fractalkine) is rapidly induced in the neurons of the hypothalamus after the introduction of a high-fat diet. The inhibition of hypothalamic fractalkine reduces diet-induced hypothalamic inflammation and the recruitment of bone marrow-derived monocytic cells to the hypothalamus; in addition, this inhibition reduces obesity and protects against diet-induced glucose intolerance. Thus, fractalkine is an important player in the early induction of diet-induced hypothalamic inflammation, and its inhibition impairs the induction of the obese and glucose intolerance phenotypes.

Melatonin acts through MT1/MT2 receptors to activate hypothalamic Akt and suppress hepatic gluconeogenesis in rats.

Melatonin can contribute to glucose homeostasis either by decreasing gluconeogenesis or by counteracting insulin resistance in distinct models of obesity. However, the precise mechanism through which melatonin controls glucose homeostasis is not completely understood. Male Wistar rats were administered an intracerebroventricular (icv) injection of melatonin and one of following: an icv injection of a phosphatidylinositol 3-kinase (PI3K) inhibitor, an icv injection of a melatonin receptor (MT) antagonist, or an intraperitoneal (ip) injection of a muscarinic receptor antagonist. Anesthetized rats were subjected to pyruvate tolerance test to estimate in vivo glucose clearance after pyruvate load and in situ liver perfusion to assess hepatic gluconeogenesis. The hypothalamus was removed to determine Akt phosphorylation. Melatonin injections in the central nervous system suppressed hepatic gluconeogenesis and increased hypothalamic Akt phosphorylation. These effects of melatonin were suppressed either by icv injections of PI3K inhibitors and MT antagonists and by ip injection of a muscarinic receptor antagonist. We conclude that melatonin activates hypothalamus-liver communication that may contribute to circadian adjustments of gluconeogenesis. These data further suggest a physiopathological relationship between the circadian disruptions in metabolism and reduced levels of melatonin found in type 2 diabetes patients.