Kristina Bardova

Sex differences during the course of diet-induced obesity in mice: adipose tissue expandability and glycemic control

Objective:Adverse effects of obesity on glucose homeostasis are linked to low-grade adipose tissue inflammation and accumulation of lipids in non-adipose tissues. The goal of this study was to evaluate the role of adipose tissue plasticity in a less severe deterioration of glucose homeostasis in females compared with males during the course of high-fat (HF) feeding in mice.Design:Mice of the C57BL/6N strain were fed either a chow or obesogenic HF diet for up to 35 weeks after weaning. Metabolic markers and hormones in plasma, glucose homeostasis, adipocyte size and inflammatory status of gonadal (gWAT) and subcutaneous (scWAT) adipose depots and liver steatosis were evaluated at 15 and 35 weeks of HF feeding.Results:HF-fed males were heavier than females until week ∼20, after which the body weights stabilized at a similar level (55–58 g) in both sexes. Greater weight gain and fat accumulation in females were associated with larger adipocytes in gWAT and scWAT at week 35. Although adipose tissue macrophage infiltration was in general less frequent in scWAT, it was reduced in both fat depots of female as compared with male mice; however, the expression of inflammatory markers in gWAT was similar in both sexes at week 35. In females, later onset of the impairment of glucose homeostasis and better insulin sensitivity were associated with higher plasma levels of adiponectin (weeks 0, 15 and 35) and reduced hepatosteatosis (weeks 15 and 35).Conclusions:Compared with males, female mice demonstrate increased capacity for adipocyte enlargement in response to a long-term HF feeding, which is associated with reduced adipose tissue macrophage infiltration and lower fat deposition in the liver, and with better insulin sensitivity. Our data suggest that adipose tissue expandability linked to adiponectin secretion might have a role in the sex differences observed in obesity-associated metabolic disorders.

Role of energy charge and AMP-activated protein kinase in adipocytes in the control of body fat stores.

As indicated by in vitro studies, both lipogenesis and lipolysis in adipocytes depend on the cellular ATP levels. Ectopic expression of mitochondrial uncoupling protein 1 (UCP1) in the white adipose tissue of the aP2-Ucp1 transgenic mice reduced obesity induced by genetic or dietary manipulations. Furthermore, respiratory uncoupling lowered the cellular energy charge in adipocytes, while the synthesis of fatty acids (FA) was inhibited and their oxidation increased. Importantly, the complex metabolic changes triggered by ectopic UCP1 were associated with the activation of AMP-activated protein kinase (AMPK), a metabolic master switch, in adipocytes. Effects of several typical treatments that reduce adiposity, such as administration of leptin, β-adrenoceptor agonists, bezafibrate, dietary n-3 polyunsaturated FA or fasting, can be compared with a phenotype of the aP2-Ucp1 mice. These situations generally lead to the upregulation of mitochondrial UCPs and suppression of the cellular energy charge and FA synthesis in adipocytes. On the other hand, FA oxidation is increased. Moreover, it has been shown that AMPK in adipocytes can be activated by adipocyte-derived hormones leptin and adiponectin, and also by insulin-sensitizes thiazolidinediones. Thus, it is evident that metabolism of adipose tissue itself is important for the control of body fat content and that the cellular energy charge and AMPK are involved in the control of lipid metabolism in adipocytes. The reciprocal link between synthesis and oxidation of FA in adipocytes represents a prospective target for the new treatment strategies aimed at reducing obesity.

Preservation of Metabolic Flexibility in Skeletal Muscle by a Combined Use of n-3 PUFA and Rosiglitazone in Dietary Obese Mice

Insulin resistance, the key defect in type 2 diabetes (T2D), is associated with a low capacity to adapt fuel oxidation to fuel availability, i.e., metabolic inflexibility. This, in turn, contributes to a further damage of insulin signaling. Effectiveness of T2D treatment depends in large part on the improvement of insulin sensitivity and metabolic adaptability of the muscle, the main site of whole-body glucose utilization. We have shown previously in mice fed an obesogenic high-fat diet that a combined use of n-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA) and thiazolidinediones (TZDs), anti-diabetic drugs, preserved metabolic health and synergistically improved muscle insulin sensitivity. We investigated here whether n-3 LC-PUFA could elicit additive beneficial effects on metabolic flexibility when combined with a TZD drug rosiglitazone. Adult male C57BL/6N mice were fed an obesogenic corn oil–based high-fat diet (cHF) for 8 weeks, or randomly assigned to various interventions: cHF with n-3 LC-PUFA concentrate replacing 15% of dietary lipids (cHF+F), cHF with 10 mg rosiglitazone/kg diet (cHF+ROSI), cHF+F+ROSI, or chow-fed. Indirect calorimetry demonstrated superior preservation of metabolic flexibility to carbohydrates in response to the combined intervention. Metabolomic and gene expression analyses in the muscle suggested distinct and complementary effects of the interventions, with n-3 LC-PUFA supporting complete oxidation of fatty acids in mitochondria and the combination with n-3 LC-PUFA and rosiglitazone augmenting insulin sensitivity by the modulation of branched-chain amino acid metabolism. These beneficial metabolic effects were associated with the activation of the switch between glycolytic and oxidative muscle fibers, especially in the cHF+F+ROSI mice. Our results further support the idea that the combined use of n-3 LC-PUFA and TZDs could improve the efficacy of the therapy of obese and diabetic patients.

Involvement of AMP-activated protein kinase in fat depot-specific metabolic changes during starvation

The mechanisms controlling fat depot‐specific metabolism are poorly understood. During starvation of mice, downregulation of lipogenic genes, suppression of fatty acid synthesis, and increases in lipid oxidation were all more pronounced in epididymal than in subcutaneous fat. In epididymal fat, relatively strong upregulation of uncoupling protein 2 and phosphoenolpyruvate carboxykinase genes was found. In mice maintained both at 20 and 30 °C, AMP‐activated protein kinase was activated in epididymal but did not change in subcutaneous fat. Our results suggest that AMPK may have a role in the different response of various fat depots to starvation.

Unmasking differential effects of rosiglitazone and pioglitazone in the combination treatment with n-3 fatty acids in mice fed a high-fat diet.

Combining pharmacological treatments and life style interventions is necessary for effective therapy of major diseases associated with obesity, which are clustered in the metabolic syndrome. Acting via multiple mechanisms, combination treatments may reduce dose requirements and, therefore, lower the risk of adverse side effects, which are usually associated with long-term pharmacological interventions. Our previous study in mice fed high-fat diet indicated additivity in preservation of insulin sensitivity and in amelioration of major metabolic syndrome phenotypes by the combination treatment using n-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA) and rosiglitazone, i.e. an anti-diabetic drug of the thiazolidinedione (TZD) family. We investigated here whether pioglitazone, a TZD-drug in clinical use, could elicit the additive beneficial effects when combined with n-3 LC-PUFA. Adult male mice (C57BL/6N) were fed an obesogenic corn oil-based high-fat diet (cHF) for 8 weeks, or randomly assigned to various dietary treatments (i) cHF+F, cHF with n-3 LC-PUFA concentrate replacing 15% of dietary lipids; (ii) cHF+ROSI, cHF with 10 mg rosiglitazone/kg diet; (iii) cHF+F+ROSI; (iv) cHF+PIO, cHF with 50 mg pioglitazone/kg diet; and (v) cHF+F+PIO, or chow-fed. Plasma concentrations of 163 metabolites were evaluated using a targeted metabolomics approach. Both TZDs preserved glucose homeostasis and normal plasma lipid levels while inducing adiponectin, with pioglitazone showing better effectiveness. The beneficial effects of TZDs were further augmented by the combination treatments. cHF+F+ROSI but not cHF+F+PIO counteracted development of obesity, in correlation with inducibility of fatty acid β-oxidation, as revealed by the metabolomic analysis. By contrast, only cHF+F+PIO eliminated hepatic steatosis and this treatment also reversed insulin resistance in dietary obese mice. Our results reveal differential effects of rosiglitazone and pioglitazone, unmasked in the combination treatment with n-3 LC-PUFA, and support the notion that n-3 LC-PUFA could be used as add-on treatment to TZDs in order to improve diabetic patients therapy.

Expression of mitochondrial uncoupling protein 3 and adenine nucleotide translocase 1 genes in developing rat heart: putative involvement in control of mitochondrial membrane potential

Postnatal maturation of the heart depends on the switch from glycolytic to oxidative metabolism and it is associated with decreasing tolerance to oxygen deprivation. Therefore, changes in composition and function of cardiac mitochondria during postnatal development require detailed characterization. Left-ventricular myocardium of prenatal, and 1-, 2-, 5-, 10-, 20-, 28-, 50-, 60-, and 90-d-old male Wistar rats was studied. The expression of uncoupling proteins (UCPs), adenine nucleotide translocase (ANT), and peroxisome proliferator-activated receptor alpha (PPARalpha) genes was characterized by northern blotting (UCP2), real-time quantitative RT-PCR (UCP2, UCP3, ANT1, ANT2, and PPARalpha), and by immunoblotting (UCP3). In isolated mitochondria, cytochromes a + a(3) were quantified by a spectrophotometry, and mitochondrial membrane potential (MMP) was measured using Rhodamine 123 (by spectrofluorimetry and flow cytometry). The specific content of cytochromes in mitochondria increased two-fold between birth and day 30, similarly, as the expression of ANT1 and PPARalpha genes. Postnatal activation in the expression of UCP2, UCP3, ANT1 and PPARalpha genes resulted in the expression maxima between days 20 and 30. The content/expression declined following day 20 (UCP2, UCP3, and PPARalpha) or 30 (cytochromes and ANT1), while expression of ANT2 declined continuously during the first month of life. In 1-d-old animals a single population of mitochondria with a relatively high MMP was observed; with increasing age, a second population of mitochondria with a significantly lower MMP appeared. The results support the view that mitochondrial energy conversion in heart changes during ontogeny and suggest the involvement of UCP3 and/or ANT1 in the control mechanism.

Impaired noradrenaline-induced lipolysis in white fat of aP2-Ucp1 transgenic mice is associated with changes in G-protein levels

In vitro experiments suggest that stimulation of lipolysis by catecholamines in adipocytes depends on the energy status of these cells. We tested whether mitochondrial uncoupling proteins (UCPs) that control the efficiency of ATP production could affect lipolysis and noradrenaline signalling in white fat in vivo. The lipolytic effect of noradrenaline was lowered by ectopic UCP1 in white adipocytes of aP2-Ucp1 transgenic mice, overexpressing the UCP1 gene from the aP2 gene promoter, reflecting the magnitude of UCP1 expression, the impaired stimulation of cAMP levels by noradrenaline and the reduction of the ATP/ADP ratio in different fat depots. Thus only subcutaneous but not epididymal fat was affected. UCP1 also down-regulated the expression of hormone-sensitive lipase and lowered its activity, and altered the expression of trimeric G-proteins in adipocytes. The adipose tissue content of the stimulatory G-protein alpha subunit was increased while that of the inhibitory G-protein alpha subunits decreased in response to UCP1 expression. Our results support the idea that the energy status of cells, and the ATP/ADP ratio in particular, modulates the lipolytic effects of noradrenaline in adipose tissue in vivo. They also demonstrate changes at the G-protein level that tend to overcome the reduction of lipolysis when ATP level in adipocytes is low. Therefore, respiratory uncoupling may exert a broad effect on hormonal signalling in adipocytes.

Modulation of Lipid Metabolism by Energy Status of Adipocytes Implications for Insulin Sensitivity

Abstract: It is becoming evident that insulin resistance of white adipose tissue is a major factor underlying the cardiovascular risk of obesity. Impaired fat storage rather than altered glucose metabolism in adipocytes probably contributes to development of insulin resistance in muscle and other tissues, in particular via increased delivery of nonesterified fatty acids into circulation. Lipid metabolism of adipose tissue is affected by the energy status of fat cells. In vitro experiments indicated the dependence of both lipogenesis and lipolysis on ATP levels in adipocytes. Thus, respiratory uncoupling in adipocytes that results in stimulation of energy dissipation and depression of ATP synthesis may contribute to the control of lipid metabolism, adiposity, and insulin sensitivity. This notion is supported by the expression of UCPs in adipocytes, for example, UCP2, UCP5, as well as some protonophoric anion transporters, and by induction of UCP1 and UCP3 in white fat by pharmacological treatments that reduce adiposity. A negative correlation between expression of UCPs in adipocytes and accumulation of white fat was also found. Expression of UCP1 from the adipose‐specific promoter in the aP2‐Ucp1 transgenic mice mitigated obesity induced by genetic or dietary factors. The obesity resistance, accompanied by respiratory uncoupling in adipocytes and increased energy expenditure, resulted from ectopic expression of UCP1 in white, but not brown fat. Probably due to depression of the ATP/ADP ratio, both fatty acid synthesis and lipolytic action of norepinephrine in adipocytes of transgenic mice were relatively low. Expression of regulatory G‐proteins, which are essential for both catecholamine and insulin signaling in adipocytes, was also altered by ectopic UCP1. These results support the role of protonophoric proteins in adipocytes in the control of adiposity and insulin sensitivity. Antidiabetic effects of thiazolidinediones, fibrates, β3‐adrenoreceptor agonists, dietary n‐3 PUFAs, and leptin may be explained at least partially by their effects on the energy and hence also the lipid metabolism of fat cells.

Induction of lipogenesis in white fat during cold exposure in mice: link to lean phenotype

Background/objective:Futile substrate cycling based on lipolytic release of fatty acids (FA) from intracellular triacylglycerols (TAG) and their re-esterification (TAG/FA cycling), as well as de novo FA synthesis (de novo lipogenesis (DNL)), represent the core energy-consuming biochemical activities of white adipose tissue (WAT). We aimed to characterize their roles in cold-induced thermogenesis and energy homeostasis.Methods:Male obesity-resistant A/J and obesity-prone C57BL/6J mice maintained at 30 °C were exposed to 6 °C for 2 or 7 days. In epididymal WAT (eWAT), TAG synthesis and DNL were determined using in vivo 2H incorporation from 2H2O into tissue TAG and nuclear magnetic resonance spectroscopy. Quantitative real-time-PCR and/or immunohistochemistry and western blotting were used to determine the expression of selected genes and proteins in WAT and liver.Results:The mass of WAT depots declined during cold exposure (CE). Plasma levels of TAG and non-esterified FA were decreased by day 2 but tended to normalize by day 7 of CE. TAG synthesis (reflecting TAG/FA cycle activity) gradually increased during CE. DNL decreased by day 2 of CE but increased several fold over the control values by day 7. Expression of genes involved in lipolysis, glyceroneogenesis, FA re-esterification, FA oxidation and mitochondrial biogenesis in eWAT was induced during CE. All these changes were more pronounced in obesity-resistant A/J than in B6 mice and occurred in the absence of uncoupling protein 1 in eWAT. Expression of markers of glyceroneogenesis in eWAT correlated negatively with hepatic FA synthesis by day 7 in both strains. Leptin and fibroblast growth factor 21 plasma levels were differentially affected by CE in the two mouse strains.Conclusions:Our results indicate integrated involvement of (i) TAG/FA cycling and DNL in WAT, and (ii) hepatic very-low-density lipoprotein-TAG synthesis in the control of blood lipid levels and provision of FA fuels for thermogenesis in cold. They suggest that lipogenesis in WAT contributes to a lean phenotype.

Levels of palmitic acid ester of hydroxystearic acid (PAHSA) are reduced in the breast milk of obese mothers

To achieve optimal development of a newborn, breastfeeding is extensively recommended, but little is known about the role of non-nutritive bioactive milk components. We aimed to characterize the fatty acid esters of hydroxy fatty acids (FAHFAs), namely palmitic acid hydroxystearic acids (PAHSAs)-endogenous lipids with anti-inflammatory and anti-diabetic properties, in human breast milk. Breast milk samples from 30 lean (BMI=19-23) and 23 obese (BMI>30) women were collected 72h postpartum. Adipose tissue and milk samples were harvested from C57BL/6J mice. FAHFA lipid profiles were measured using reverse phase and chiral liquid chromatography-mass spectrometry method. PAHSA regioisomers as well as other FAHFAs were present in both human and murine milk. Unexpectedly, the levels of 5-PAHSA were higher relative to other regioisomers. The separation of both regioisomers and enantiomers of PAHSAs revealed that both R- and S-enantiomers were present in the biological samples, and that the majority of the 5-PAHSA signal is of R configuration. Total PAHSA levels were positively associated with weight gain during pregnancy, and 5-PAHSA as well as total PAHSA levels were significantly lower in the milk of the obese compared to the lean mothers. Our results document for the first time the presence of lipid mediators from the FAHFA family in breast milk, while giving an insight into the stereochemistry of PAHSAs. They also indicate the negative effect of obesity on 5-PAHSA levels. Future studies will be needed to explore the role and mechanism of action of FAHFAs in breast milk.