CETP Expression in Bone-Marrow-Derived Cells Reduces the Inflammatory Features of Atherosclerosis in Hypercholesterolemic Mice.

CETP activity reduces plasma HDL-cholesterol concentrations, a correlate of an increased risk of atherosclerotic events. However, our recent findings suggest that CETP expression in macrophages promotes an intracellular antioxidant state, reduces free cholesterol accumulation and phagocytosis, and attenuates pro-inflammatory gene expression. To determine whether CETP expression in macrophages affects atherosclerosis development, we transplanted bone marrow from transgenic mice expressing simian CETP or non-expressing littermates into hypercholesterolemic LDL-receptor-deficient mice. The CETP expression did not change the lipid-stained lesion areas but decreased the macrophage content (CD68), neutrophil accumulation (LY6G), and TNF-α aorta content of young male transplanted mice and decreased LY6G, TNF-α, iNOS, and nitrotyrosine (3-NT) in aged female transplanted mice. These findings suggest that CETP expression in bone-marrow-derived cells reduces the inflammatory features of atherosclerosis. These novel mechanistic observations may help to explain the failure of CETP inhibitors in reducing atherosclerotic events in humans.

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.

A20 deubiquitinase controls PGC-1α expression in the adipose tissue.

BACKGROUND: Peroxisome proliferator-activated receptor γ coactivator- 1alpha (PGC-1α) plays an important role in whole body metabolism and, particularly in glucose homeostasis. Its expression is highly regulated and, small variations in tissue levels can have a major impact in a number of physiological and pathological conditions. Recent studies have shown that the ubiquitin/proteasome system plays a role in the control of PGC-1α degradation. METHODS: Here we evaluated the interaction of PGC-1α with the protein A20, which plays a dual-role in the control of the ubiquitin/proteasome system acting as a deubiquitinase and as an E3 ligase. We employed immunoprecipitation, quantitative real-time PCR and immunofluorescence staining to evaluate PGC-1α, A20, PPARγ and ubiquitin in the adipose tissue of humans and mice. RESULTS: In distinct sites of the adipose tissue, A20 binds to PGC-1α. At least in the subcutaneous fat of humans and mice the levels of PGC-1α decrease during obesity, while its physical association with A20 increases. The inhibition of A20 leads to a reduction of PGC-1α and PPARγ expression, suggesting that A20 acts as a protective factor against PGC-1α disposal. CONCLUSION: We provide evidence that mechanisms regulating PGC-1α ubiquitination are potentially involved in the control of the function of this transcriptional co-activator.

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.

Cross Talk Between Brain Innate Immunity and Serotonin Signaling Underlies Depressive-Like Behavior Induced by Alzheimer's Amyloid-ß Oligomers in Mice.

Considerable clinical and epidemiological evidence links Alzheimer's disease (AD) and depression. However, the molecular mechanisms underlying this connection are largely unknown. We reported recently that soluble Aß oligomers (AßOs), toxins that accumulate in AD brains and are thought to instigate synapse damage and memory loss, induce depressive-like behavior in mice. Here, we report that the mechanism underlying this action involves AßO-induced microglial activation, aberrant TNF-α signaling, and decreased brain serotonin levels. Inactivation or ablation of microglia blocked the increase in brain TNF-α and abolished depressive-like behavior induced by AßOs. Significantly, we identified serotonin as a negative regulator of microglial activation. Finally, AßOs failed to induce depressive-like behavior in Toll-like receptor 4-deficient mice and in mice harboring a nonfunctional TLR4 variant in myeloid cells. Results establish that AßOs trigger depressive-like behavior via a double impact on brain serotonin levels and microglial activation, unveiling a cross talk between brain innate immunity and serotonergic signaling as a key player in mood alterations in AD. SIGNIFICANCE STATEMENT: Alzheimer's disease (AD) is a progressive neurodegenerative disorder and the main cause of dementia in the world. Brain accumulation of amyloid-ß oligomers (AßOs) is a major feature in the pathogenesis of AD. Although clinical and epidemiological data suggest a strong connection between AD and depression, the underlying mechanisms linking these two disorders remain largely unknown. Here, we report that aberrant activation of the brain innate immunity and decreased serotonergic tonus in the brain are key players in AßO-induced depressive-like behavior in mice. Our findings may open up new possibilities for the development of effective therapeutics for AD and depression aimed at modulating microglial function.

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.

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.

TLR4 expression in bone marrow-derived cells is both necessary and sufficient to produce the insulin resistance phenotype in diet-induced obesity.

The anomalous activation of toll-like receptor 4 (TLR4) by dietary fats is one of the most important mechanisms linking obesity to insulin resistance. TLR4 is expressed in most tissues of the body, but its activity in the cells of the immune system is expected to underlie its most important roles of inducing inflammation and insulin resistance. Here we explore the hypothesis that TLR4 expression in bone marrow-derived cells mediates most of the actions of this receptor as an inducer of insulin resistance. Wild type and TLR4-mutant mice were used in bone marrow transplant experiments producing chimeras that harbored the functional receptor in all cells of the body except bone marrow-derived cells or only in bone marrow-derived cells. Transplanted mice were fed chow or a high-fat diet, and glucose homeostasis was evaluated by glucose and insulin tolerance tests. Insulin signal transduction and the expression of markers of inflammation were evaluated in the liver and white adipose tissue. In addition, we performed liver histology and evaluated the expression of gluconeogenic enzymes. The expression of TLR4 in bone marrow-derived cells only, but not in non-bone marrow-derived tissues only, was a determining factor in the induction of diet-induced insulin resistance, which was accompanied by an increased expression of inflammatory markers in both white adipose tissue and liver as well as increased liver steatosis and increased expression of gluconeogenic enzymes. TLR4 expressed in bone marrow-derived cells is an important mediator of obesity-associated insulin resistance in mice.

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.

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.

Infiltration of a mixture of immune cells may be related to good prognosis in patients with differentiated thyroid carcinoma.

OBJECTIVE: Immune responses against differentiated thyroid carcinomas (DTC) have long been recognized. We aimed to investigate the role of immune cell infiltration in the progression of DTC. DESIGN: We studied 398 patients - 253 with papillary and 13 with follicular thyroid cancers, as well as 132 with nonmalignant tissues. PATIENTS AND MEASUREMENTS: Immune cell infiltration was identified using CD3, CD4, CD8, CD20, CD68 and FoxP3 immunohistochemical markers. In addition, we assessed colocalization of CD4 and IL-17 to identify Th17 lymphocytic infiltration and colocalization of CD33 and CD11b to identify infiltration of myeloid-derived suppressor cells (MDSC). RESULTS: Immune cells infiltrated malignant tissues more often than benign lesions. The presence of chronic lymphocytic thyroiditis (CLT) concurrent to DTC, CD68+, CD4+, CD8+, CD20+, FoxP3+ and Th17 lymphocytes but not MDSCs was associated with clinical and pathological features of lower tumour aggressiveness and a more favourable patient outcome. A log-rank test confirmed an association between concurrent CLT, tumour-associated macrophage infiltration, and CD8+ lymphocytes and an increased in disease-free survival, suggesting that evidence of these immune reactions is associated with a favourable prognosis. CONCLUSION: Our data suggest that the tumour or peri-tumoural microenvironment may act to modify the observed pattern of immune response. Immune cell infiltration and the presence of concurrent CLT helped characterize specific tumour histotypes associated with favourable prognostic features.

Reduction of endoplasmic reticulum stress--a novel mechanism of action of statins in the protection against atherosclerosis.

Fatty-acid-induced endoplasmic reticulum stress has been recently described as a novel mechanism involved in the genesis of atherosclerosis. Here we show that statins, a class of drug widely employed in the clinical management of hypercholesterolemia, reduces lipid-induced macrophage endoplasmic reticulum stress in an isolated cell system and in LDL receptor knockout mice. Given the importance of endoplasmic reticulum stress as an inducer of inflammation, we suspect that the novel mechanism of action herein described for statins may play a major role on its beneficial effects in the prevention of cardiovascular disease.