Expression of Clock Genes in Human Subcutaneous and Visceral Adipose Tissues

Disrupted circadian rhythms are associated with obesity and metabolic alterations, but little is known about the participation of peripheral circadian clock machinery in these processes. The aim of the present study was to analyze RNA expression of clock genes in subcutaneous (SAT) and visceral (VAT) adipose tissues of male and female subjects in AM (morning) and PM (afternoon) periods, and its interactions with body mass index (BMI). Ninety-one subjects (41 +/- 11 yrs of age) presenting a wide range of BMI (21.4 to 48.6 kg/m(2)) were included. SAT and VAT biopsies were obtained from patients undergoing abdominal surgeries. Clock genes expressions were evaluated by qRT-PCR. The only clock gene that showed higher expression (p < .0001) in SAT in comparison to VAT was PER1 of female (372%) and male (326%) subjects. Different patterns of expression between the AM and PM periods were observed, in particular REV-ERBa, which was reduced (p < .05) at the PM period in SAT and VAT of both women and men (women: similar to 53% lower; men: similar to 78% lower), whereas CLOCK expression was not altered. Relationships between clock genes were different in SAT vs. VAT. BMI was negatively correlated with SATPER1 (r = -.549; p = .001) and SATPER2 (r = -.613; p = .0001) and positively with VATCLOCK (r = .541; p = .001) and VATBMAL1 (r = .468; p = .007) only in women. These data suggest that the circadian clock machinery of adipose tissue depots differs between female and male subjects, with a sex-specific effect observed for some genes. BMI correlated with clock genes, but at this moment it is not possible to establish the cause-effect relationship. (Author correspondence: mzanquetta@gmail.com)

Dichotomous effects of VEGF-A on adipose tissue dysfunction

Obese fat pads are frequently undervascularized and hypoxic, leading to increased fibrosis, inflammation, and ultimately insulin resistance. We hypothesized that VEGF-A-induced stimulation of angiogenesis enables sustained and sufficient oxygen and nutrient exchange during fat mass expansion, thereby improving adipose tissue function. Using a doxycycline (Dox)-inducible adipocyte-specific VEGF-A overexpression model, we demonstrate that the local up-regulation of VEGF-A in adipocytes improves vascularization and causes a "browning" of white adipose tissue (AT), with massive up-regulation of UCP1 and PGC1 alpha. This is associated with an increase in energy expenditure and resistance to high fat diet-mediated metabolic insults. Similarly, inhibition of VEGF-A-induced activation of VEGFR2 during the early phase of high fat diet-induced weight gain, causes aggravated systemic insulin resistance. However, the same VEGF-A-VEGFR2 blockade in ob/ob mice leads to a reduced body-weight gain, an improvement in insulin sensitivity, a decrease in inflammatory factors, and increased incidence of adipocyte death. The consequences of modulation of angiogenic activity are therefore context dependent. Proangiogenic activity during adipose tissue expansion is beneficial, associated with potent protective effects on metabolism, whereas antiangiogenic action in the context of preexisting adipose tissue dysfunction leads to improvements in metabolism, an effect likely mediated by the ablation of dysfunctional proinflammatory adipocytes.

Omega-6 polyunsaturated fatty acids prevent atherosclerosis development in LDLr-KO mice, in spite of displaying a pro-inflammatory profile similar to trans fatty acids

The development of atherosclerosis and the inflammatory response were investigated in LDLr-KO mice on three high-fat diets (40% energy as fat) for 16 weeks: trans (TRANS), saturated (SAFA) or omega-6 polyunsaturated (PUFA) fats. The following parameters were measured: plasma lipids, aortic root total cholesterol (TC), lesion area (Oil Red-O), ABCA1 content and macrophage infiltration (immunohistochemistry), collagen content (Picrosirius-red) and co-localization of ABCA1 and macrophage (confocal microscopy) besides the plasma inflammatory markers (IL-6, TNF-alpha) and the macrophage inflammatory response to lipopolysaccharide from Escherichia coli (LPS). As expected, plasma TC and TG concentrations were lower on the PUFA diet than on TRANS or SAFA diets. Aortic intima macrophage infiltration, ABCA1 content, and lesion area on PUFA group were lower compared to TRANS and SAFA groups. Macrophages and ABCA1 markers did not co-localize in the atherosclerotic plaque, suggesting that different cell types were responsible for the ABCA1 expression in plaques. Compared to PUFA, TRANS and SAFA presented higher collagen content and necrotic cores in atherosclerotic plaques. In the artery wall, TC was lower on PUFA compared to TRANS group; free cholesterol was lower on PUFA compared to TRANS and SAFA; cholesteryl ester concentration did not vary amongst the groups. Plasma TNF-alpha concentration on PUFA and TRANS-fed mice was higher compared to SAFA. No difference was observed in IL-6 concentration amongst groups. Regarding the macrophage inflammatory response to LPS, TRANS and PUFA presented higher culture medium concentrations of IL-6 and TNF-alpha as compared to SAFA. The PUFA group showed the lowest amount of the anti-inflammatory marker IL-10 compared to TRANS and SAFA groups. In conclusion, PUFA intake prevented atherogenesis, even in a pro-inflammatory condition. (c) 2012 Elsevier Ireland Ltd. All rights reserved.

The Role of Dyslipidemia on Ocular Surface, Lacrimal and Meibomian Gland Structure and Function

Purpose: Dyslipidemia is characterized by high lipid blood levels that are risk factors for cardiovascular diseases, which are leading causes of death. However, it is unclear whether dyslipidemia is a cause of the dry eye syndrome (DES). Therefore we determined in transgenic mice models of dyslipidemia, whether there is an association with DES development. Methods: Dyslipidemic models included male and female adult mice overexpressing apolipoprotein CIII (Apo CIII), LDL receptor knockout (LDLR-KO) and ApoE knockout (ApoE-KO). They were compared with age-and gender-matched C57BL/6 mice. Ocular health was evaluated based on corneal slit lamp assessment, phenol red thread test (PRT) and impression cytology. Blood lipid profiles and histology of meibomian and lacrimal glands were also evaluated. Effects of high-fat diet and aging were observed in LDLR-KO and ApoCIII strains, respectively. Results: Body weight and lacrimal gland weight were significantly higher in male mice compared to females of the same strain (P < 0.05). Body weight was significantly lower in LDLRKO mice receiving high lipid diet compared to their controls (P = 0.0043). ApoE-KO were hypercholesterolemic and ApoCIII hypertriglyceridemic while LDLR-KO showed increases in both parameters. The PRT test was lower in male LDLR-KO mice with high-fat diet than control mice with standard diet (P = 0.0273). Aging did not affect lacrimal structural or functional parameters of ApoCIII strain. Conclusions: DES development is not solely dependent on dyslipidemia in relevant mice models promoting this condition. On the other hand, lacrimal gland structure and function are differentially impacted by lipid profile changes in male and female mice. This dissociation suggests that other factors beside dyslipidemia impact on tear film dysfunction and DES development.

Effect of Echium oil compared with marine oils on lipid profile and inhibition of hepatic steatosis in LDLr knockout mice

Abstract Background In an effort to identify new alternatives for long-chain n-3 polyunsaturated fatty acids (LC n-3 PUFA) supplementation, the effect of three sources of omega 3 fatty acids (algae, fish and Echium oils) on lipid profile and inflammation biomarkers was evaluated in LDL receptor knockout mice. Methods The animals received a high fat diet and were supplemented by gavage with an emulsion containing water (CON), docosahexaenoic acid (DHA, 42.89%) from algae oil (ALG), eicosapentaenoic acid (EPA, 19.97%) plus DHA (11.51%) from fish oil (FIS), and alpha-linolenic acid (ALA, 26.75%) plus stearidonic acid (SDA, 11.13%) from Echium oil (ECH) for 4 weeks. Results Animals supplemented with Echium oil presented lower cholesterol total and triacylglycerol concentrations than control group (CON) and lower VLDL than all of the other groups, constituting the best lipoprotein profile observed in our study. Moreover, the Echium oil attenuated the hepatic steatosis caused by the high fat diet. However, in contrast to the marine oils, Echium oil did not affect the levels of transcription factors involved in lipid metabolism, such as Peroxisome Proliferator Activated Receptor α (PPAR α) and Liver X Receptor α (LXR α), suggesting that it exerts its beneficial effects by a mechanism other than those observed to EPA and DHA. Echium oil also reduced N-6/N-3 FA ratio in hepatic tissue, which can have been responsible for the attenuation of steatosis hepatic observed in ECH group. None of the supplemented oils reduced the inflammation biomarkers. Conclusion Our results suggest that Echium oil represents an alternative as natural ingredient to be applied in functional foods to reduce cardiovascular disease risk factors.

Diet-Sensitive Sources of Reactive Oxygen Species in Liver Mitochondria: Role of Very Long Chain Acyl-CoA Dehydrogenases

High fat diets and accompanying hepatic steatosis are highly prevalent conditions. Previous work has shown that steatosis is accompanied by enhanced generation of reactive oxygen species (ROS), which may mediate further liver damage. Here we investigated mechanisms leading to enhanced ROS generation following high fat diets (HFD). We found that mitochondria from HFD livers present no differences in maximal respiratory rates and coupling, but generate more ROS specifically when fatty acids are used as substrates. Indeed, many acyl-CoA dehydrogenase isoforms were found to be more highly expressed in HFD livers, although only the very long chain acyl-CoA dehydrogenase (VLCAD) was more functionally active. Studies conducted with permeabilized mitochondria and different chain length acyl-CoA derivatives suggest that VLCAD is also a source of ROS production in mitochondria of HFD animals. This production is stimulated by the lack of NAD+. Overall, our studies uncover VLCAD as a novel, diet-sensitive, source of mitochondrial ROS.

Oral leucine supplementation is sensed by the brain but neither reduces food intake nor induces an anorectic pattern of gene expression in the hypothalamus

Leucine activates the intracellular mammalian target of the rapamycin (mTOR) pathway, and hypothalamic mTOR signaling regulates food intake. Although central infusion of leucine reduces food intake, it is still uncertain whether oral leucine supplementation is able to affect the hypothalamic circuits that control energy balance. We observed increased phosphorylation of p70s6k in the mouse hypothalamus after an acute oral gavage of leucine. We then assessed whether acute oral gavage of leucine induces the activation of neurons in several hypothalamic nuclei and in the brainstem. Leucine did not induce the expression of Fos in hypothalamic nuclei, but it increased the number of Fos-immunoreactive neurons in the area postrema. In addition, oral gavage of leucine acutely increased the 24 h food intake of mice. Nonetheless, chronic leucine supplementation in the drinking water did not change the food intake and the weight gain of ob/ob mice and of wild-type mice consuming a low- or a high-fat diet. We assessed the hypothalamic gene expression and observed that leucine supplementation increased the expression of enzymes (BCAT1, BCAT2 and BCKDK) that metabolize branched-chain amino acids. Despite these effects, leucine supplementation did not induce an anorectic pattern of gene expression in the hypothalamus. In conclusion, our data show that the brain is able to sense oral leucine intake. However, the food intake is not modified by chronic oral leucine supplementation. These results question the possible efficacy of leucine supplementation as an appetite suppressant to treat obesity

Studio degli effetti funzionali e tossici di derivati di Brassicaceae in modelli sperimentali

I vegetali appartenenti alla famiglia delle Brassicaceae, sono ricchi di molecole biologicamente attive note per le numerose proprietà salutari. L’effetto di un estratto di germogli di cavolo nero toscano (TBCSE) è stato investigato, in termini chemiopreventivi, sugli enzimi epatici del metabolismo degli xenobiotici e antiossidanti, in ratti trattati con TBCSE. I risultati hanno mostrato un complesso pattern di modulazione, con una prevalente inibizione, del sistema citocromo P450-dipendente, e induzioni significative degli enzimi di fase II (glutatione transferasi e glucuronosiltransferasi) e antiossidanti (catalasi, NAD(P)H:chinone reduttasi, glutatione reduttasi e perossidasi). Successivamente, l’effetto di TBCSE è stato studiato nei confronti delle alterazioni provocate da un’alimentazione iperlipidica nel ratto. Il trattamento si è dimostrato efficace nel contrastare gli effetti deleteri dei grassi presenti nella dieta, come l’iperlipidemia, l’aumento del peso corporeo e del fegato, l’indebolimento delle attività degli enzimi antiossidanti e del potenziale detossificante a livello epatico. Complessivamente, TBCSE emerge essere un promettente prodotto nutraceutico con potenziali effetti chemiopreventivi, e da impiegare come strategia alimentare per contrastare gli effetti correlati ad una dieta iperlipidica. Il consumo di dosi sovralimentari di molecole isolate dalle Brassicaceae, tramite per esempio integratori dietetici, come strategia alimentare preventiva, potrebbe tuttavia rappresentare un rischio per la salute. La potenziale tossicità del sulforafane, glucorafanina, indolo-3-carbinolo, e 3,3'-diindolimetano, è stata valutata in epatociti primari di ratto. La citotossicità e l’induzione di stress ossidativo, osservate a concentrazioni non lontane da quelle che potrebbero essere raggiunte in vivo, insieme ad una forte modulazione dell’espressione genica, riguardante principalmente il metabolismo degli xenobiotici, risposte ad alterazioni dello stato ossidoredutivo, eventi di riparazione del DNA e di proteine, induzione dell’apoptosi, e meccanismi (co)cancerogeni, sottolineano la potenzialità di queste molecole di determinare un rischio tossicologico, in seguito ad un’assunzione prolungata e ad alte dosi.

Effects of a whey protein supplementation on intrahepatocellular lipids in obese female patients

High protein diets have been shown to improve hepatic steatosis in rodent models and in high-fat fed humans. We therefore evaluated the effects of a protein supplementation on intrahepatocellular lipids (IHCL), and fasting plasma triglycerides in obese non diabetic women.

Elevated systemic monocyte chemoattractrant protein-1 in hepatic steatosis without significant hepatic inflammation

Non-alcoholic fatty liver disease (NAFLD) is strongly associated with obesity and the metabolic syndrome. It encompasses a clinico-pathologic spectrum of conditions ranging from simple steatosis to nonalcoholic steatohepatitis (NASH). The latter develops upon pro-inflammatory cell infiltration and is widely considered as the first relevant pathophysiological step in NAFLD-progression. The chemokine monocyte chemoattractant protein 1 (MCP-1) plays an important role in the progression of hepatic inflammation and fibrosis, and both increased hepatic expression and circulating serum levels have been described in NASH. Here, we aimed to investigate MCP-1 expression in simple hepatic steatosis. Upon feeding a high-fat diet mice developed hepatic steatosis in the absence of significant hepatic inflammation, but elevated hepatic MCP-1 expression compared to control mice fed a standard chow. Interestingly, high-fat diet fed mice had significantly higher MCP-1 serum levels, and MCP-1 mRNA expression was significantly increased in visceral adipose tissue. Furthermore, MCP-1 serum levels were also elevated in patients with ultrasound-diagnosed NAFLD and correlated with the body-mass index and fasting glucose. In conclusion, our data indicate both the liver and adipose tissue as cellular sources of elevated circulating MCP-1 levels already in the early phase of hepatic steatosis. Since MCP-1 derived from visceral adipose tissue reaches the liver via portal circulation at high concentrations it may significantly contribute to the progression of simple steatosis to NASH.

Effects of supplementation with essential amino acids on intrahepatic lipid concentrations during fructose overfeeding in humans

A high dietary protein intake has been shown to blunt the deposition of intrahepatic lipids in high-fat- and high-carbohydrate-fed rodents and humans.

Cholesteryl ester accumulation and accelerated cholesterol absorption in intestine-specific hormone sensitive lipase-null mice

Hormone sensitive lipase (HSL) regulates the hydrolysis of acylglycerols and cholesteryl esters (CE) in various cells and organs, including enterocytes of the small intestine. The physiological role of this enzyme in enterocytes, however, stayed elusive. In the present study we generated mice lacking HSL exclusively in the small intestine (HSLiKO) to investigate the impact of HSL deficiency on intestinal lipid metabolism and the consequences on whole body lipid homeostasis. Chow diet-fed HSLiKO mice showed unchanged plasma lipid concentrations. In addition, feeding with high fat/high cholesterol (HF/HC) diet led to unaltered triglyceride but increased plasma cholesterol concentrations and CE accumulation in the small intestine. The same effect was observed after an acute cholesterol load. Gavaging of radioactively labeled cholesterol resulted in increased abundance of radioactivity in plasma, liver and small intestine of HSLiKO mice 4h post-gavaging. However, cholesterol absorption determined by the fecal dual-isotope ratio method revealed no significant difference, suggesting that HSLiKO mice take up the same amount of cholesterol but in an accelerated manner. mRNA expression levels of genes involved in intestinal cholesterol transport and esterification were unchanged but we observed downregulation of HMG-CoA reductase and synthase and consequently less intestinal cholesterol biosynthesis. Taken together our study demonstrates that the lack of intestinal HSL leads to CE accumulation in the small intestine, accelerated cholesterol absorption and decreased cholesterol biosynthesis, indicating that HSL plays an important role in intestinal cholesterol homeostasis.

The effect of increased lipid intake on hormonal responses during aerobic exercise in endurance-trained men

In view of the growing health problem associated with obesity, clarification of the regulation of energy homeostasis is important. Peripheral signals, such as ghrelin and leptin, have been shown to influence energy homeostasis. Nutrients and physical exercise, in turn, influence hormone levels. Data on the hormonal response to physical exercise (standardized negative energy balance) after high-fat (HF) or low-fat (LF) diet with identical carbohydrate intake are currently not available. The aim of the study was to investigate whether a short-term dietary intervention with HF and LF affects ghrelin and leptin levels and their modulators, GH, insulin and cortisol, before and during aerobic exercise. Eleven healthy, endurance-trained male athletes (W(max) 365 +/- 29 W) were investigated twice in a randomized crossover design following two types of diet: 1. LF - 0.5 g fat/kg body weight (BW) per day for 2.5 days; 2. HF - 0.5 g fat/kg BW per day for 1 day followed by 3.5 g fat/kg BW per day for 1.5 days. After a standardized carbohydrate snack in the morning, metabolites and hormones (GH, ghrelin, leptin, insulin and cortisol) were measured before and at regular intervals throughout a 3-h aerobic exercise test on a cycloergometer at 50% of W(max). Diet did not significantly affect GH and cortisol concentrations during exercise but resulted in a significant increase in ghrelin and decrease in leptin concentrations after LF compared with HF diet (area under the curve (AUC) ghrelin LF vs HF: P < 0.03; AUC leptin LF vs HF: P < 0.02, Wilcoxon rank test). These data suggest that acute negative energy balance induced by exercise elicits a hormonal response with opposite changes of ghrelin and leptin. In addition, the hormonal response is modulated by the preceding intake of fat.

Interaction between dietary lipids and physical inactivity on insulin sensitivity and on intramyocellular lipids in healthy men

OBJECTIVE: To assess the effect of a possible interaction between dietary fat and physical inactivity on whole-body insulin sensitivity and intramyocellular lipids (IMCLs). RESEARCH DESIGN AND METHODS: Eight healthy male volunteers were studied on two occasions. After 2 days of an equilibrated diet and moderate physical activity, participants remained inactive (bed rest) for 60 h and consumed either a high-saturated fat (45% fat, of which approximately 60% was saturated fat [BR-HF]) or a high-carbohydrate (70% carbohydrate [BR-HCHO]) diet. To evaluate the effect of a high-fat diet alone, six of the eight volunteers were restudied after a 2-day equilibrated diet followed by 60 h on a high-saturated fat diet and controlled physical activity (PA-HF). Insulin sensitivity was measured by hyperinsulinemic-euglycemic clamp and IMCL concentrations by (1)H-magnetic resonance spectroscopy. RESULTS: Insulin-mediated glucose disposal was decreased by BR-HF condition (-24 +/- 6%, P < 0.05) but did not change with BR-HCHO (+19 +/- 10%, NS). BR-HF and BR-HCHO increased IMCL levels (+32 +/- 7%, P < 0.05 and +17 +/- 8%, P < 0.0011, respectively). Although the increase in IMCL levels with PA-HF (+31 +/- 19%, P = 0.12) was similar to that during BR-HF, insulin-mediated glucose disposal (-7 +/- 9%, NS) was not decreased. CONCLUSIONS: These data indicate that physical inactivity and a high-saturated fat diet may interact to reduce whole-body insulin sensitivity. IMCL content was influenced by dietary lipid and physical inactivity but was not directly associated with insulin resistance.

Effect of GH on human skeletal muscle lipid metabolism in GH deficiency

Adult-onset growth hormone (GH) deficiency (GHD) is associated with insulin resistance and decreased exercise capacity. Intramyocellular lipids (IMCL) depend on training status, diet, and insulin sensitivity. Using magnetic resonance spectroscopy, we studied IMCL content following physical activity (IMCL-depleted) and high-fat diet (IMCL-repleted) in 15 patients with GHD before and after 4 mo of GH replacement therapy (GHRT) and in 11 healthy control subjects. Measurements of insulin resistance and exercise capacity were performed and skeletal muscle biopsies were carried out to assess expression of mRNA of key enzymes involved in skeletal muscle lipid metabolism by real-time PCR and ultrastructure by electron microscopy. Compared with control subjects, patients with GHD showed significantly higher difference between IMCL-depleted and IMCL-repleted. GHRT resulted in an increase in skeletal muscle mRNA expression of IGF-I, hormone-sensitive lipase, and a tendency for an increase in fatty acid binding protein-3. Electron microscopy examination did not reveal significant differences after GHRT. In conclusion, variation of IMCL may be increased in patients with GHD compared with healthy control subjects. Qualitative changes within the skeletal muscle (i.e., an increase in free fatty acids availability from systemic and/or local sources) may contribute to the increase in insulin resistance and possibly to the improvement of exercise capacity after GHRT. The upregulation of IGF-I mRNA suggests a paracrine/autocrine role of IGF-I on skeletal muscle.