Jozef Ukropec

Effects of obesity, diabetes and exercise on Fndc5 gene expression and irisin release in human skeletal muscle and adipose tissue: in vivo and in vitro studies

Considerable controversy exists regarding the role of irisin, a putative exercise‐induced myokine, in human metabolism. We therefore studied irisin and its precursor Fndc5 in obesity, type 2 diabetes and exercise. Complex clinical studies combined with cell culture work revealed that Fndc5/irisin was decreased in type 2 diabetes in vivo, but not in muscle cells in vitro, indicating that diabetes‐related factor(s) regulate Fndc5/irisin in vivo. Several attributes of type 2 diabetes, such as hyperglycaemia, triglyceridaemia, visceral adiposity and extramyocellular lipid deposition were negatively associated with adipose tissue Fndc5 mRNA and circulating irisin. Moreover, mimicking diabetic status in vitro by treating muscle cells with palmitate and glucose lowered Fndc5 mRNA. Neither exercise training nor an acute exercise bout modulated circulating irisin or muscle Fndc5 expression. However, the associations between intensity of habitual physical activity, muscle volume, strength, contractility and circulating irisin provide a link between irisin and positive outcomes of increased physical activity.

UCP1-independent Thermogenesis in White Adipose Tissue of Cold-acclimated Ucp1-/- Mice

Apart from UCP1-based nonshivering thermogenesis in brown adipocytes, the identity of thermogenic mechanisms that can be activated to reduce a positive energy balance is largely unknown. To identify potentially useful mechanisms, we have analyzed physiological and molecular mechanisms that enable mice, genetically deficient in UCP1 and sensitive to acute exposure to the cold at 4 °C, to adapt to long term exposure at 4 °C. UCP1-deficient mice that can adapt to the cold have increased oxygen consumption and show increased oxidation of both fat and glucose as indicated from serum metabolite levels and liver glycogen content. Enhanced energy metabolism in inguinal fat was also indicated by increased oxygen consumption and fat oxidation in tissue suspensions and increased AMP kinase activity in dissected tissues. Analysis of gene expression in skeletal muscle showed surprisingly little change between cold-adapted Ucp1+/+ and Ucp1-/- mice, whereas in inguinal fat a robust induction occurred for type 2 deiodinase, sarcoendoplasmic reticulum Ca2+-ATPase, mitochondrial glycerol 3-phosphate dehydrogenase, PGC1α, CoxII, and mitochondrial DNA content. Western blot analysis showed an induction of total phospholamban and its phosphorylated form in inguinal fat and other white fat depots, but no induction was apparent in muscle. We conclude that alternative thermogenic mechanisms, based in part upon the enhanced capacity for ion and substrate cycling associated with brown adipocytes in white fat depots, are induced in UCP1-deficient mice by gradual cold adaptation.

The hypotriglyceridemic effect of dietary n−3 FA is associated with increased β-oxidation and reduced leptin expression

To study the mechanisms responsible for the hypotriglyceridemic effect of marine oils, we monitored the effects of high dietary intake of n−3 PUFA on hepatic and muscular β-oxidation, plasma leptin concentration, leptin receptor gene expression, and in vivo insulin action. Two groups of male Wistar rats were fed either a high-fat diet [28% (w/w) of saturated fat] or a high-fat diet containing 10% n−3 PUFA and 18% saturated fat for 3 wk. The hypotriglyceridemic effect of n−3 PUFA was accompanied by increased hepatic oxidation of palmitoyl-CoA (125%, P<0.005) and palmitoyl-l-carnitine (480%, P<0.005). These findings were corroborated by raised carnitine palmitoyltransferase-2 activity (154%, P<0.001) and mRNA levels (91%, P<0.01) as well as by simultaneous elevation of hepatic peroxisomal acyl-CoA oxidase activity (144%, P<0.01) and mRNA content (82%, P<0.05). In contrast, hepatic carnitine palmitoyltransferase-1 activity remained unchanged despite a twofold increased mRNA level after n−3 PUFA feeding. Skeletal muscle FA oxidation was less affected by dietary n−3 PUFA, and the stimulatory effect was found only in peroxisomes. Dietary intake of n−3 PUFA was followed by increased acyl-CoA oxidase activity (48%, P<0.05) and mRNA level (83%, P<0.05) in skeletal muscle. The increased FA oxidation after n−3 PUFA supplementation of the high-fat diet was accompanied by lower plasma leptin concentration (−38%, P<0.05) and leptin mRNA expression (−66%, P<0.05) in retroperitoneal adipose tissue, and elevated hepatic mRNA level for the leptin receptor Ob-Ra (140%, P<0.05). Supplementation of the high-fat diet with n−3 PUFA enhanced in vivo insulin sensitivity, as shown by normalization of the glucose infusion rate during euglycemic hyperinsulinemic clamp.Our results indicate that the hypotriglyceridemic effect of dietary n−3 PUFA is associated with stimulation of FA oxidation in the liver and to a smaller extent in skeletal muscle. This may ameliorate dyslipidemia, tissue lipid accumulation, and insulin action, in spite of decreased plasma leptin level and leptin mRNA in adipose tissue.

High prevalence of prediabetes and diabetes in a population exposed to high levels of an organochlorine cocktail

Aims/hypothesisA heavily polluted area of Eastern Slovakia was targeted by the PCBRISK cross-sectional survey to search for possible links between environmental pollution and both prediabetes and diabetes.MethodsAssociations of serum levels of five persistent organic pollutants (POPs), namely polychlorinated biphenyls (PCBs), 2,2′-bis(4-chlorophenyl)-1,1-dichloroethylene (p,p′-DDE), 2,2′-bis(4-chlorophenyl)-1,1,1-trichloro-ethane (p,p′-DDT), hexachlorobenzene (HCB) and β-hexachlorocyclohexane (β-HCH), with prediabetes and diabetes were investigated in 2,047 adults. Diabetes and prediabetes were diagnosed by fasting plasma glucose in all participants and by OGTT in 1,220 compliant participants.ResultsOur population was stratified in terms of individual POPs quintiles and associations between environmental pollution, prediabetes and diabetes were investigated. Prevalence of prediabetes and diabetes increased in a dose-dependent manner, with individuals in upper quintiles of individual POPs showing striking increases in prevalence of prediabetes as shown by OR and 95% CI for PCBs (2.74; 1.92–3.90), DDE (1.86; 1.17–2.95), DDT (2.48; 1.77–3.48), HCB (1.86; 1.7–2.95) and β-HCH (1.97; 1.28–3.04). Interestingly, unlike PCBs, DDT and DDE, increased levels of HCB and β-HCH seemed not to be associated with increased prevalence of diabetes. Nevertheless, individuals in the 5th quintile of the variable expressing the cumulative effect of all five POPs (sum of orders) had a more than tripled prevalence of prediabetes and more than six times higher prevalence of diabetes when compared with the 1st referent quintile.Conclusions/interpretationIncreasing serum concentrations of individual POPs considerably increased prevalence of prediabetes and diabetes in a dose-dependent manner. Interaction of industrial and agricultural pollutants in increasing prevalence of prediabetes or diabetes is likely.

Adipogenesis and insulin sensitivity in obesity are regulated by retinoid-related orphan receptor gamma.

Obesity is a well‐known risk factor for the development of secondary complications such as type 2 diabetes. However, only a part of the obese population develops secondary metabolic disorders. Here, we identify the transcription factor retinoid‐related orphan receptor gamma (RORγ) as a negative regulator of adipocyte differentiation through expression of its newly identified target gene matrix metalloproteinase 3. In vivo differentiation of adipocyte progenitor cells from Rorγ‐deficient mice is enhanced and obese Rorγ−/− mice show decreased adipocyte sizes. These small adipocytes are highly insulin sensitive, leading to an improved control of circulating free fatty acids. Ultimately, Rorγ−/− mice are protected from hyperglycemia and insulin resistance in the state of obesity. In adipose stromal‐vascular fraction from obese human subjects, Rorγ expression is correlated with adipocyte size and negatively correlated with adipogenesis and insulin sensitivity. Taken together, our findings identify RORγ as a factor, which controls adipogenesis as well as adipocyte size and modulates insulin sensitivity in obesity. RORγ might therefore serve as a novel pharmaceutical target to treat obesity‐associated insulin resistance.

Inactivation of UCP1 and the Glycerol Phosphate Cycle Synergistically Increases Energy Expenditure to Resist Diet-induced Obesity

Our current paradigm for obesity assumes that reduced thermogenic capacity increases susceptibility to obesity, whereas enhanced thermogenic capacity protects against obesity. Here we report that elimination of two major thermogenic pathways encoded by the mitochondrial uncoupling protein (Ucp1) and mitochondrial glycerol-3-phosphate dehydrogenase (Gdm) result in mice with increased resistance to diet-induced obesity when housed at 28 °C, provided prior adaptation occurred at 20 °C. Obesity resistant Gdm-/-·Ucp1-/- mice maintained at 28 °C have increased energy expenditure, in part through conversion of white to brown adipocytes in inguinal fat. Increased oxygen consumption in inguinal fat cell suspensions and the up-regulation of genes of mitochondrial function and fat metabolism indicated increased thermogenic activity, despite the absence of UCP1, whereas liver and skeletal muscle showed no changes in gene expression. Additionally, comparisons of energy expenditure in UCP1-deficient and wild type mice fed an obesogenic diet indicates that UCP1-based brown fat-based thermogenesis plays no role in so-called diet-induced thermogenesis. Accordingly, a new paradigm for obesity emerges in which the inactivation of major thermogenic pathways force the induction of alternative pathways that increase metabolic inefficiency.

Inactivation of the Mitochondrial Carrier SLC25A25 (ATP-Mg2+/Pi Transporter) Reduces Physical Endurance and Metabolic Efficiency in Mice

An ATP-Mg2+/Pi inner mitochondrial membrane solute transporter (SLC25A25), which is induced during adaptation to cold stress in the skeletal muscle of mice with defective UCP1/brown adipose tissue thermogenesis, has been evaluated for its role in metabolic efficiency. SLC25A25 is thought to control ATP homeostasis by functioning as a Ca2+-regulated shuttle of ATP-Mg2+ and Pi across the inner mitochondrial membrane. Mice with an inactivated Slc25a25 gene have reduced metabolic efficiency as evidenced by enhanced resistance to diet-induced obesity and impaired exercise performance on a treadmill. Mouse embryo fibroblasts from Slc25a25−/− mice have reduced Ca2+ flux across the endoplasmic reticulum, basal mitochondrial respiration, and ATP content. Although Slc25a25−/− mice are metabolically inefficient, the source of the inefficiency is not from a primary function in thermogenesis, because Slc25a25−/− mice maintain body temperature upon acute exposure to the cold (4 °C). Rather, the role of SLC25A25 in metabolic efficiency is most likely linked to muscle function as evidenced from the physical endurance test of mutant mice on a treadmill. Consequently, in the absence of SLC25A25 the efficiency of ATP production required for skeletal muscle function is diminished with secondary effects on adiposity. However, in the absence of UCP1-based thermogenesis, induction of Slc25a25 in mice with an intact gene may contribute to an alternative thermogenic pathway for the maintenance of body temperature during cold stress.

Adipocytes as a new source of catecholamine production.

Catecholamines are an important regulator of lipolysis in adipose tissue. Here we show that rat adipocytes, isolated from mesenteric adipose tissue, express genes of catecholamine biosynthetic enzymes and produce catecholamines de novo. Administration of tyrosine hydroxylase inhibitor, alpha‐methyl‐p‐tyrosine, in vitro significantly reduced concentration of catecholamines in isolated adipocytes. We hypothesize that the sympathetic innervation of adipose tissues is not the only source of catecholamines, since adipocytes also have the capacity to produce both norepinephrine and epinephrine.

Protein array reveals differentially expressed proteins in subcutaneous adipose tissue in obesity.

Objective: Many adipokines, inflammatory cytokines, and other proteins produced by adipose tissue have been shown to be involved in the development of obesity‐related insulin resistance. Nevertheless, new factors that play an important role in these processes are still emerging. Therefore, we screened the level of 120 different proteins in biopsies of subcutaneous adipose tissue (ScAT) of lean and obese subjects.

Adipokine Protein Expression Pattern in Growth Hormone Deficiency Predisposes to the Increased Fat Cell Size and the Whole Body Metabolic Derangements

CONTEXT GH deficiency (GHD) in adults is associated with central adiposity, dyslipidemia, and insulin resistance. OBJECTIVE The objective of the study was to test the hypothesis that GHD might change the spectrum of adipokines and thus influence the adipose tissue and the whole-body metabolic and inflammatory status leading to development of insulin resistance. DESIGN This was a single-center observational study with a cross-sectional design. PARTICIPANTS AND METHODS Protein arrays were used to characterize adipokines expressed in the sc adipose tissue obtained from young GHD adults and compared with age-, gender-, and body mass index (BMI)-matched group of healthy individuals. All subjects underwent an oral glucose tolerance test, euglycemic hyperinsulinemic clamp, and magnetic resonance imaging examination. RESULTS Presence of abdominal obesity, enlarged adipocytes, increased circulating high-sensitivity C-reactive protein, impaired glucose tolerance, and decreased insulin action were found in GHD. Changes in adipokine protein expression due to GHD were highly dependent on the obesity phenotype. Lean GHD individuals (BMI approximately 23 kg/m(2)) had decreased protein levels for stem cell factor and epithelial growth factor, indicating a possible defect in adipocyte differentiation and proliferation. Decrease of vascular endothelial growth factor, stromal cell-derived factor, angiopoietin-2, and brain-derived neurotrophic factor advocated for attenuated angiogenesis and neurogenesis. Presence of obesity (BMI approximately 31 kg/m(2)) eliminated these inhibitory effects. However, adipose tissue expansion in GHD individuals was paralleled by an elevation of adipose tissue proinflammatory cytokines (IL-1beta, interferon-gamma) and chemoattractants (interferon-inducible T cell alpha-chemoattractant, monocyte chemotactic protein-2, monocyte chemotactic protein-3, eotaxin). CONCLUSION Our data demonstrate that GHD modulates adipokine and cytokine protein expression pattern, which might influence the adipose tissue growth and differentiation and predispose to tissue hypoxia, inflammation, and a defect in the whole-body insulin action.