Identification of nesfatin-1 in human and murine adipose tissue:a novel depot-specific adipokine with increased levels in obesity

Nesfatin-1 is a recently identified anorexigenic peptide derived from its precursor protein, nonesterified fatty acid/nucleobindin 2 (NUCB2). Although the hypothalamus is pivotal for the maintenance of energy homeostasis, adipose tissue plays an important role in the integration of metabolic activity and energy balance by communicating with peripheral organs and the brain via adipokines. Currently no data exist on nesfatin-1 expression, regulation, and secretion in adipose tissue. We therefore investigated NUCB2/nesfatin-1 gene and protein expression in human and murine adipose tissue depots. Additionally, the effects of insulin, dexamethasone, and inflammatory cytokines and the impact of food deprivation and obesity on nesfatin-1 expression were studied by quantitative RT-PCR and Western blotting. We present data showing NUCB2 mRNA (P < 0.001), nesfatin-1 intracellular protein (P < 0.001), and secretion (P < 0.01) were significantly higher in sc adipose tissue compared with other depots. Also, nesfatin-1 protein expression was significantly increased in high-fat-fed mice (P < 0.01) and reduced under food deprivation (P < 0.01) compared with controls. Stimulation of sc adipose tissue explants with inflammatory cytokines (TNFa and IL-6), insulin, and dexamethasone resulted in a marked increase in intracellular nesfatin-1 levels. Furthermore, we present evidence that the secretion of nesfatin-1 into the culture media was dramatically increased during the differentiation of 3T3-L1 preadipocytes into adipocytes (P < 0.001) and after treatments with TNF-a, IL-6, insulin, and dexamethasone (P < 0.01). In addition, circulating nesfatin-1 levels were higher in high-fat-fed mice (P < 0.05) and showed positive correlation with body mass index in human. We report that nesfatin-1 is a novel depot specific adipokine preferentially produced by sc tissue, with obesity- and food deprivation-regulated expression.

Interventions against obesity through increased lipolysis in adipose tissue

Obesity is a disease of excess adiposity affecting> 17% of men and >20% of women in Britain. Clinically, it is defined by a Body Mass Index (BMI, kg/m2) of 2:30. Obesity is a confounding factor that promotes insulin resistance, hyperinsulinaemia and type 2 diabetes. Type 2 diabetes accounts for >90% of all cases of diabetes, with a prevalence of 2-6% of adults in most western societies, a majority of which are overweight or obese. Weight loss in obese patients reduces the risk of developing diabetes by >50%. This thesis has investigated the first part of a two-stage therapeutic intervention against obesity in which adipose tissue lipolysis will be combined with increased energy expenditure: the approach is also designed to consider agents that will benefit glycaemic control in coexistent obesity and diabetes by improving insulin sensitivity. Rodent and human in vitro models of adipocyte biology and skeletal muscle have been developed, characterised and evaluated. They include isolated epididymal and parametrial adipocytes of lean and obese diabetic ob/ob mice, cultured 3T3-Ll preadipocytes, isolated human omental and subcutaneous adipocytes and rat L6 cultured muscle cells. Compounds investigated for anti-obesity and anti-diabetic properties include M2 (sibutramine metabolite), 3-guanidinopropionic acid and mazindol. In vivo studies were undertaken to investigate these compounds further in lean and ob/ob mice. In vivo studies indicated that M2 and 3-guanidinopropionic acid reduced body weight gain in ob/ob mice. The three compounds increased lipolysis in adipocytes isolated from lean and ob/ob mice and human adipose depots. The direct action of these compounds was mediated via a pathway involving the f3 adrenoceptors and components of the lipolytic signalling pathway, including protein kinase A and p38 MAP kinase. In addition, M2 and mazindol were capable of increasing glucose uptake into insulin sensitive tissues. M2 and mazindol can act directly on adipose tissue and skeletal muscle to increase glucose uptake via a pathway involving new protein synthesis and activation of the glucose transporters. The M2-stimulated pathway is activated by the conversion of phosphatidylinositol bisphosphate to phosphatidylinositol trisphosphate by phosphatidylinositol 3-kinase. Thus, M2, mazindol and 3-GPA showed pharmacodynamic properties which suggested they might be potential therapeutic treatments for obesity and diabetes.

Expression profiling of type 2 diabetes susceptibility genes in the pancreatic islets, adipose tissue and liver of obese mice

Type 2 diabetes (T2D) is characterized by impaired beta cell function and insulin resistance. T2D susceptibility genes identified by Genome-wide association studies (GWAS) are likely to have roles in both impaired insulin secretion from the beta cell as well as insulin resistance. The aim of this study was to use gene expression profiling to assess the effect of the diabetic milieu on the expression of genes involved in both insulin secretion and insulin resistance. We measured the expression of 43 T2D susceptibility genes in the islets, adipose and liver of leptin-deficient Ob/Ob mice compared with Ob/+ littermates. The same panel of genes were also profiled in cultured rodent adipocytes, hepatocytes and beta cells in response to high glucose conditions, to distinguish expression effects due to elevated glycemia from those on the causal pathway to diabetes or induced by other factors in the diabetic microenviroment. We found widespread deregulation of these genes in tissues from Ob/Ob mice, with differential regulation of 23 genes in adipose, 18 genes in liver and one gene (Tcf7l2) in islets of diabetic animals (Ob/Ob) compared to control (Ob/+) animals. However, these expression changes were in most cases not noted in glucose-treated adipocyte, hepatocyte or beta cell lines, indicating that they may not be an effect of hyperglycemia alone. This study indicates that expression changes are apparent with diabetes in both the insulin producing beta cells, but also in peripheral tissues involved in insulin resistance. This suggests that incidence or progression of diabetic phenotypes in a mouse model of diabetes is driven by both secretory and peripheral defects. © J. A. Barth Verlag in Georg Thieme Verlag KG Stuttgart New York.

In vivo and ex vivo regulation of visfatin production by leptin in human and murine adipose tissue: role of mitogen-activated protein kinase and phosphatidylinositol 3-kinase signaling pathways

Visfatin is an adipogenic adipokine with increased levels in obesity, properties common to leptin. Thus, leptin may modulate visfatin production in adipose tissue (AT). Therefore, we investigated the effects of leptin on visfatin levels in 3T3-L1 adipocytes and human/murine AT, with or without a leptin antagonist. The potential signaling pathways and mechanisms regulating visfatin production in AT was also studied. Real-time RT-PCR and Western blotting were used to assess the relative mRNA and protein expression of visfatin. ELISA was performed to measure visfatin levels in conditioned media of AT explants, and small interfering RNA technology was used to reduce leptin receptor expression. Leptin significantly (P<0.01) increased visfatin levels in human and murine AT with a maximal response at leptin 10(-9) M, returning to baseline at leptin 10(-7) M. Importantly, ip leptin administration to C57BL/6 ob/ob mice further supported leptin-induced visfatin protein production in omental AT (P<0.05). Additionally, soluble leptin receptor levels rose with concentration dependency to a maximal response at leptin 10(-7) M (P<0.01). The use of a leptin antagonist negated the induction of visfatin and soluble leptin receptor by leptin. Furthermore, leptin-induced visfatin production was significantly decreased in the presence of MAPK and phosphatidylinositol 3-kinase inhibitors. Also, when the leptin eceptor gene was knocked down using small interfering RNA, eptin-induced visfatin expression was significantly decreased. Thus, leptin increases visfatin production in AT in vivo and ex vivo via pathways involving MAPK and phosphatidylinositol 3-kinase signaling. The pleiotropic effects of leptin may be partially mediated by visfatin.

Ageing, adipose tissue, fatty acids and inflammation

A common feature of ageing is the alteration in tissue distribution and composition, with a shift in fat away from lower body and subcutaneous depots to visceral and ectopic sites. Redistribution of adipose tissue towards an ectopic site can have dramatic effects on metabolic function. In skeletal muscle, increased ectopic adiposity is linked to insulin resistance through lipid mediators such as ceramide or DAG, inhibiting the insulin receptor signalling pathway. Additionally, the risk of developing cardiovascular disease is increased with elevated visceral adipose distribution. In ageing, adipose tissue becomes dysfunctional, with the pathway of differentiation of preadipocytes to mature adipocytes becoming impaired; this results in dysfunctional adipocytes less able to store fat and subsequent fat redistribution to ectopic sites. Low grade systemic inflammation is commonly observed in ageing, and may drive the adipose tissue dysfunction, as proinflammatory cytokines are capable of inhibiting adipocyte differentiation. Beyond increased ectopic adiposity, the effect of impaired adipose tissue function is an elevation in systemic free fatty acids (FFA), a common feature of many metabolic disorders. Saturated fatty acids can be regarded as the most detrimental of FFA, being capable of inducing insulin resistance and inflammation through lipid mediators such as ceramide, which can increase risk of developing atherosclerosis. Elevated FFA, in particular saturated fatty acids, maybe a driving factor for both the increased insulin resistance, cardiovascular disease risk and inflammation in older adults.

An in vitro comparison of the incorporation, growth, and chondrogenic potential of human bone marrow versus adipose tissue mesenchymal stem cells in clinically relevant cell scaffolds used for cartilage repair

Aim. To compare the incorporation, growth, and chondrogenic potential of bone marrow (BM) and adipose tissue (AT) mesenchymal stem cells (MSCs) in scaffolds used for cartilage repair. Methods. Human BM and AT MSCs were isolated, culture expanded, and characterised using standard protocols, then seeded into 2 different scaffolds, Chondro-Gide or Alpha Chondro Shield. Cell adhesion, incorporation, and viable cell growth were assessed microscopically and following calcein AM/ethidium homodimer (Live/Dead) staining. Cell-seeded scaffolds were treated with chondrogenic inducers for 28 days. Extracellular matrix deposition and soluble glycosaminoglycan (GAG) release into the culture medium was measured at day 28 by histology/immunohistochemistry and dimethylmethylene blue assay, respectively. Results. A greater number of viable MSCs from either source adhered and incorporated into Chondro-Gide than into Alpha Chondro Shield. In both cell scaffolds, this incorporation represented less than 2% of the cells that were seeded. There was a marked proliferation of BM MSCs, but not AT MSCs, in Chondro-Gide. MSCs from both sources underwent chondrogenic differentiation following induction. However, cartilaginous extracellular matrix deposition was most marked in Chondro- Gide seeded with BM MSCs. Soluble GAG secretion increased in chondrogenic versus control conditions. There was no marked difference in GAG secretion by MSCs from either cell source. Conclusion. Chondro-Gide and Alpha Chondro Shield were permissive to the incorporation and chondrogenic differentiation of human BM and AT MSCs. Chondro-Gide seeded with BM MSCs demonstrated the greatest increase in MSC number and deposition of a cartilaginous tissue.

Concentrations of brominated and chlorinated contaminants in adipose tissue of polar bears (Ursus maritimus) between 1991-2007

Adipose tissue was sampled from the western Hudson Bay (WHB) subpopulation of polar bears at intervals from 1991 to 2007 to examine temporal trends of PCB and OCP levels both on an individual and sum-contaminant basis. We also determined levels and temporal trends of emerging polybrominated diphenyl ethers (PBDEs), hexabromocyclododecane (HBCD), polybrominated biphenyls (PBBs) and other current-use brominated flame retardants. Over the 17-year period, sum DDT (and p,p'-DDE, p,p'-DDD, p,p'-DDT) decreased (-8.4%/year); alpha-hexachlorocyclohexane (alpha-HCH) decreased (-11%/year); beta-HCH increased ( + 8.3%/year); and sum PCB and sum chlordane (CHL), both contaminants at highest concentrations in all years (>1 ppm), showed no distinct trends even when compared to previous data for this subpopulation dating back to 1968. Some of the less persistent PCB congeners decreased significantly (-1.6%/year to -6.3%/year), whereas CB153 levels tended to increase (+ 3.3%/year). Parent CHLs (c-nonachlor, t-nonachlor) declined, whereas non-monotonic trends were detected for metabolites (heptachlor epoxide, oxychlordane). sum chlorobenzene, octachlorostyrene, sum mirex, sum MeSO2-PCB and dieldrin did not significantly change. Increasing sum PBDE levels (+13%/year) matched increases in the four consistently detected congeners, BDE47, BDE99, BDE100 and BDE153. Although no trend was observed, total-(alpha)-HBCD was only detected post-2000. Levels of the highest concentration brominated contaminant, BB153, showed no temporal change. As long-term ecosystem changes affecting contaminant levels may also affect contaminant patterns, we examined the influence of year (i.e., aging or "weathering" of the contaminant pattern), dietary tracers (carbon stable isotope ratios, fatty acid patterns) and biological (age/sex) group on congener/metabolite profiles. Patterns of PCBs, CHLs and PBDEs were correlated with dietary tracers and biological group, but only PCB and CHL patterns were correlated with year. DDT patterns were not associated with any explanatory variables, possibly related to local DDT sources. Contaminant pattern trends may be useful in distinguishing the possible role of ecological/diet changes on contaminant burdens from expected dynamics due to atmospheric sources and weathering.

Lipid Metabolism of Monosodium Glutamate Obese Rats after Partial Removal of Adipose Tissue

We analyzed the effects of partial fat pad removal on retroperitoneal and epididymal fat depots and carcass metabolism of control (C) and MSG-obese (M) rats. Three-month-old C and M male Wistar rats were submitted to either partial surgical excision of epididymal and retroperitoneal fat tissue (lipectomy, L) or sham surgery (S) and studied after 7 or 30 days. Retroperitoneal and epididymal tissue re-growth after lipectomy was not observed, as indicated by the low pads weight of the L groups. The lipolysis rate was stimulated in LC7 and LM7, probably due to surgical stress and low insulin levels. In LM7, but not in LC7, in vivo lipogenesis rate increased in retroperitoneal and epididymal fat tissue, as did the diet-derived lipid accumulation in epididymal fat tissue. Although these local increases were no longer present in LM30, this group showed a large increase in the percentage of small area adipocytes in both pads as well as increased carcass lipogenesis rate. The present data showed that the partial removal of fat depots affected the metabolism of control and MSG-obese rats differently. In the obese animals only, it stimulated both local and carcass lipogenesis rate as well as adipocyte differentiation, i.e. responses likely to favor excised tissue re-growth and/or compensatory growth of non-excised depots.

Adenylate cyclase activity in ob/ob mice: Discriminating alterations in adipose tissue and liver.

info:eu-repo/semantics/published

Fatty acid synthesis from U-14C glucose, U-14C fructose, L-U-14C alanine and 3H2O in liver slices and adipose tissue from Bar-Harbor obese-hyperglycemic mice.

info:eu-repo/semantics/published

Antilipolytic activity of suloctidil in rat adipose tissue

info:eu-repo/semantics/published

Effect of maternal cold exposure and nutrient restriction on insulin-like growth factor sensitivity in adipose tissue of newborn sheep

Adipose tissue mass in the newborn is determined in part by insulin-like growth factor (IGF)s, which are dependent on the maternal nutritional and metabolic environment during late gestation. The present study was designed to determine whether maternal cold exposure (CE) commencing in mid gestation could modulate some of the adaptive effects of nutrient restriction in late gestation on adipose tissue endocrine sensitivity in the resulting offspring. Twenty eight pregnant sheep were entered into the study and were either shorn, i.e. cold exposed, from 70 days gestation (term = 147 days), or remained unshorn, and were fed either their total calculated metabolisable energy (ME) requirements for body weight and pregnancy from 110 days gestation or 50% of this amount (n=7 per group). Adipose tissue was sampled from the offspring at one day of age and the mRNA abundance for IGF-I, II their receptors (R) and GH secretagogue receptor-1a (GHSR-1a) were determined. CE mothers produced larger offspring with more perirenal adipose tissue, an adaptation prevented by maternal nutrient restriction. Nutrient restriction in unshorn mothers increased IGF-I and IIR mRNA abundance. The mRNA abundances for IGF-I, II and IIR in adipose tissue were reduced by CE, adaptations independent of maternal food intake, whereas CE plus nutrient restriction increased GHSR-1a mRNA. In conclusion, maternal nutrient restriction with or without CE has very different effects on IGF sensitivity of adipose tissue and may act to ensure adequate fat stores are present in the newborn in the face of very different maternal endocrine and metabolic environments.

Establishment of a protocol for the cryopreservation of cell sheets of adipose tissue stem cells

Dissertação de Mestrado, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2016