Adipose mRNA Levels Research Articles

SUN-126 Distinct Molecular Phenotypes of Non-Diseased Breast Adipose Tissue of Pre-Menopausal Obese and Non-Obese Women May Underlie Differing Breast Cancer Risks

Obesity is a major risk factor for many chronic diseases including postmenopausal breast cancer. Paradoxically, breast cancer susceptibility is inversely linked to obesity in pre-menopausal women. Adipose tissues are active endocrine organs that play major roles in tumor development and progression; however, fat depots at different anatomical sites are biologically and functionally distinct and their singular influence on breast epithelial biology remains unclear. To study the early events by which breast adiposity may provide a microenvironment predisposing normal breast epithelial cells to tumorigenesis, we collected breast tissue from pre-menopausal (n=10/group) non-obese (NO, BMI=27.6±0.8) and obese (O, BMI=44.5±2.8) women of comparable ages (NO: 36.1± 3.3; O: 40.0±2.0) with no breast cancer and undergoing elective breast reduction surgery. Breast adipose tissue and corresponding glandular cells were analyzed histologically and evaluated for expression of genes (adipokines, cytokines, steroid hormone signaling) by QPCR and proteins (proliferation, apoptosis, inflammation) by IHC. Adipocyte size distributions from NO and O breasts did not differ (P=0.9). However, adipose mRNA levels for pro-inflammatory cytokines (IL-6, IL-8, CSF-1, MCP-1) and adipokines (LEP, CFD) were higher for O than NO (P<0.05). AdipoQ, ER-α, and ER-β transcript levels were lower for O than NO (P<0.05), while those for CYP19 and PTGS2 showed reverse trends (O>NO, P<0.05). In the corresponding glandular cells, NO had higher mRNA levels for IL-6, IL-8, ER-α, and ER-β than O (P<0.05). Immunostaining with anti-Ki67 antibodies indicated that O glandular cells were 3-fold less proliferative than those for NO, consistent with their lower Cyclin D1 mRNA levels (P<0.05). Galectin-1, a pro-fibrotic protein, showed predominant myo- vs. luminal epithelial localization, with staining intensities for O tending to be higher (P=0.07) than for NO. Perilipin immunostaining was specific for adipocytes and did not differ for O and NO. A non-targeted approach using a Human Cytokine Array (R&D Systems) was employed to further evaluate the inflammation status of O vs. NO adipose. The analyses confirmed the higher expression of IL-8, Leptin and CFD (by QPCR) in O vs. NO and identified C-reactive protein, EMMPRIN, Trefoil Factor-3, Cystatin-3 and Macrophage Migration Inhibitory Factor-1 as greater in O than NO (~2-fold). Our findings demonstrate marked differences in gene and protein expression patterns of O and NO breast adipose tissue, which were accompanied by a suppression of proliferation of O relative to NO breast epithelium. We speculate that early exposure of the breast epithelium to a highly inflammatory environment fueled by breast adiposity may promote a senescent state that confers protection from pre-menopausal breast cancer.

Lean maternal hyperglycemia alters offspring lipid metabolism and susceptibility to diet-induced obesity in mice†.

We previously developed a model of gestational diabetes mellitus (GDM) in which dams exhibit glucose intolerance, insulin resistance, and reduced insulin response to glucose challenge only during pregnancy, without accompanying obesity. Here, we aimed to determine how lean gestational glucose intolerance affects offspring risk of metabolic dysfunction. One cohort of offspring was sacrificed at 19 weeks, and one at 31 weeks, with half of the second cohort placed on a high-fat, high-sucrose diet (HFHS) at 23 weeks. Exposure to maternal glucose intolerance increased weights of HFHS-fed offspring. Chow-fed offspring of GDM dams exhibited higher body fat percentages at 4, 12, and 20 weeks of age. At 28 weeks, offspring of GDM dams fed the HFHS but not the chow diet (CD) also had higher body fat percentages than offspring of controls (CON). Exposure to GDM increased the respiratory quotient (Vol CO2/Vol O2) in offspring. Maternal GDM increased adipose mRNA levels of peroxisome proliferator-activated receptor gamma (Pparg) and adiponectin (Adipoq) in 31-week-old CD-fed male offspring, and increased mRNA levels of insulin receptor (Insr) and lipoprotein lipase (Lpl) in 31-week-old male offspring on both diets. In liver at 31 weeks, mRNA levels of peroxisome proliferator-activated receptor alpha (Ppara) were elevated in CD-fed male offspring of GDM dams, and male offspring of GDM dams exhibited higher mRNA levels of Insr on both diets. Neither fasting insulin nor glucose tolerance was affected by exposure to GDM. Our findings show that GDM comprising glucose intolerance only during pregnancy programs increased adiposity in offspring, and suggests increased insulin sensitivity of subcutaneous adipose tissue.

Acute physiological effects of glucocorticoids on fuel metabolism in humans are permissive but not direct

Background and aimsThe effects of glucocorticoids on fuel metabolism are complex. Acute glucocorticoid excess promotes lipolysis but chronic glucocorticoid excess causes visceral fat accumulation. We hypothesized that interactions between cortisol and insulin and adrenaline account for these conflicting results. We tested the effect of cortisol on lipolysis and glucose production with and without insulin and adrenaline in humans both in vivo and in vitro.Materials and methodsA total of 20 healthy men were randomized to low and high insulin groups (both n = 10). Subjects attended on 3 occasions and received low (c. 150 nM), medium (c. 400 nM) or high (c. 1400 nM) cortisol infusion in a randomized crossover design. Deuterated glucose and glycerol were infused intravenously along with a pancreatic clamp (somatostatin with replacement of glucagon, insulin and growth hormone) and adrenaline. Subcutaneous adipose tissue was obtained for analysis. In parallel, the effect of cortisol on lipolysis was tested in paired primary cultures of human subcutaneous and visceral adipocytes.Results In vivo, high cortisol increased lipolysis only in the presence of high insulin and/or adrenaline but did not alter glucose kinetics. High cortisol increased adipose mRNA levels of ATGL, HSL and CGI‐58 and suppressed G0S2. In vitro, high cortisol increased lipolysis in the presence of insulin in subcutaneous, but not visceral, adipocytes.ConclusionsThe acute lipolytic effects of cortisol require supraphysiological concentrations, are dependent on insulin and adrenaline and are observed only in subcutaneous adipose tissue. The resistance of visceral adipose tissue to cortisol's lipolytic effects may contribute to the central fat accumulation observed with chronic glucocorticoid excess.

Pharmacological Evaluation of Adipose Dysfunction via 11β-Hydroxysteroid Dehydrogenase Type 1 in the Development of Diabetes in Diet-Induced Obese Mice with Cortisone Pellet Implantation

Signals from intracellular glucocorticoids (GCs) via 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) in adipose tissues have been reported to serve as amplifiers leading to deterioration of glucose metabolism associated with obesity. To elucidate adipose dysfunction via 11β-HSD1 activation in the development of obesity-related diabetes, we established novel diabetic mice by implanting a cortisone pellet (CP) in diet-induced obesity (DIO) mice. Cortisone pellet-implanted DIO mice (DIO/CP mice) showed hyperglycemia, insulin resistance, hyperlipidemia, and ectopic fat accumulation, whereas cortisone pellet implantation in lean mice did not induce hyperglycemia. In DIO/CP mice, indexes of lipolysis such as plasma glycerol and nonesterified fatty acids (NEFAs) increased before hyperglycemia appeared. Furthermore, the adipose mRNA level of 11β-HSD1 was up-regulated in DIO/CP mice compared with sham-operated DIO mice. RU486 (mifepristone, 11β-[p-(dimethylamino)phenyl]-17β-hydroxy-17-(1-propynyl)estra-4,9-dien-3-one), a glucocorticoid receptor antagonist, decreased adipose mRNA levels of 11β-HSD1 as well as adipose triglyceride lipase. RU486 also improved plasma NEFA, glycerol, and glucose levels in DIO/CP mice. These results demonstrate that lipolysis in adipose tissues caused by GC activation via 11β-HSD1 serves as a trigger for diabetes with ectopic fat accumulation. Our findings also indicate the possibility of a vicious circle of GC signals via 11β-HSD1 up-regulation in adipose tissues, contributing to deterioration of glucose metabolism to result in diabetes. Our DIO/CP mouse could be a suitable model of type 2 diabetes to evaluate adipose dysfunction via 11β-HSD1.

Alcohol exposure increases fibroblast growth factor 21 expression

Long‐term excess alcohol exposure leads to alcohol liver disease (ALD). Alcohol‐induced hepatic fat accumulation sensitizes the liver to various “second hits” to develop more advanced liver injury. Recombinant FGF‐21 has beneficial effects in promoting adipose tissue glucose uptake, lowering circulating glucose levels, and reducing hepatic lipid content in diabetic and obese rodent and monkey models. Serum FGF‐21 is elevated in non‐alcoholic fatty liver disease, coronary heart disease and type 2 diabetes. Serum levels of FGF‐21 were significantly higher in alcoholic patients with and without liver disease. Alcohol ingestion caused a significant increase in hepatic FGF‐21 expression and elevation of serum FGF‐21 concentration in mice exposed to chronic, acute or chronic‐binge alcohol. Incubation of hepatocytes with alcohol also increased FGF‐21 mRNA expression in a dose‐dependent manner. Alcohol significantly decreases hepatic and adipose mRNA levels of FGFR4 and β‐klotho, which is a specific co‐receptor for FGF‐21. Alcohol impaired recombinant FGF‐21‐induced ERK‐1/2 phosphorylation in adipose tissue and liver in mice treated with Lieber deCarli diet containing 5% alcohol for 4 weeks. We demonstrated that alcohol exposure increases hepatic expression and serum concentration of FGF‐21, which are associated with hepatic and adipose tissue FGF‐21 resistant in ALD.

Role of perivascular adipose tissue in endothelial dysfunction of adipose triglyceride lipase-deficient mice

Perivascular adipose tissue (PVAT) is an important factor in vascular biology due to its ability to produce a variety of vasoactive substances. In addition, it is regarded as a source of proinflammatory mediators and reactive oxygen species (ROS). We have recently demonstrated that mice lacking adipose triglyceride lipase (ATGL) suffer from severe endothelial dysfunction. Since blood vessels of ATGL knockout ( ATGL ( − / − ) ) mice are covered by large amounts of PVAT, we investigated its potential contribution to the observed endothelial defect. Therefore, PVAT encompassing thoracic aortas of wild-type and ATGL ( − / − ) mice was isolated, and analyzed for expression of different adipokines, inflammation markers and sources of oxidative stress. Knockout of ATGL caused a ∼7-fold increase in PVAT wet weight. While adiponectin protein expression was not affected by ATGL deficiency, leptin levels were increased ∼3-fold. Adipose mRNA levels of the inflammation markers tumor necrosis factor a (TNFα and monocyte chemoattractant protein 1 (MCP-1) were ∼5-fold higher in ATGL-deficient PVAT. In addition, NADPH oxidase isoform NOX2 was significantly upregulated at both mRNA and protein level. To distinguish between PVAT-mediated effects and those originating from the cardiac dysfunctional phenotype of the animals, we additionally analyzed PVAT isolated from ATGL ( − / − ) mice with cardiomyocyte-specific overexpression of ATGL (rescued cardiac phenotype). Interestingly, the effect of ATGL deficiency on TNFα and leptin expression was reversible, while increased adipose NOX2 and MCP-1 expression persisted. These data suggest that PVAT-derived inflammatory and oxidative stress might contribute to endothelial dysfunction in ATGL deficiency. The functional consequences of these observations are currently being investigated.

Regulation of Small Ubiquitin-Like Modifier-1, Nuclear Receptor Coreceptor, Histone Deacetylase 3, and Peroxisome Proliferator-Activated Receptor-γ in Human Adipose Tissue

This study investigated the regulation of peroxisome proliferator-activated receptor-γ (PPARγ), the histone deacetylase 3 (HDAC3)-nuclear receptor coreceptor (NCoR) complex (a corepressor of transcription used by PPARγ), and small ubiquitin-like modifier-1 (SUMO-1) (a posttranslational modifier of PPARγ) in human adipose tissue and both adipocyte and macrophage cell lines. The objective was to determine whether there were alterations in the human adipose tissue gene expression levels of PPARγ, HDAC3, NCoR, and SUMO-1 associated either with obesity or with treatment of impaired glucose tolerance (IGT) subjects with insulin-sensitizing medications. We obtained subcutaneous adipose tissue biopsies from 86 subjects with a wide range of body mass index (BMI) and insulin sensitivity (S(I)). Additionally, adipose tissue biopsies were obtained from a randomized subgroup of IGT subjects before and after 10 weeks of treatment with either pioglitazone or metformin. The adipose mRNA levels of PPARγ, NCoR, HDAC3, and SUMO-1 correlated strongly with each other (P<0.0001); however, SUMO-1, NCoR, and HDAC3 gene expression were not significantly associated with BMI or S(I). Pioglitazone increased SUMO-1 expression by 23% (P<0.002) in adipose tissue and an adipocyte cell line (P<0.05), but not in macrophages. Small interfering RNA (siRNA)-mediated knockdown of SUMO-1 decreased PPARγ, HDAC3, and NCoR in THP-1 cells and increased tumor necrosis factor-α (TNF-α) induction in response to lipopolysaccharide (LPS). These results suggest that the coordinate regulation of SUMO-1, PPARγ1/2, HDAC3, and NCoR may be more tightly controlled in macrophages than in adipocytes in human adipose and that these modulators of PPARγ activity may be particularly important in the negative regulation of macrophage-mediated adipose inflammation by pioglitazone.

Azilsartan treatment improves insulin sensitivity in obese spontaneously hypertensive Koletsky rats

Hypertension often coexists with insulin resistance. However, most metabolic effects of the antihypertensive agents have been investigated in nomotensive animals, in which different conclusions may arise. We investigated the metabolic effects of the new angiotensin II type 1 receptor blocker azilsartan using the obese Koletsky rats superimposed on the background of the spontaneously hypertensive rats. Male Koletsky rats were treated with azilsartan (2 mg/kg/day) over 3 weeks. Blood pressure was measured by tail-cuff. Blood biochemical and hormonal parameters were determined by enzymatic or ELISA methods. Gene expression was assessed by RT-PCR. In Koletsky rats, azilsartan treatment lowered blood pressure, basal plasma insulin concentration and the homeostasis model assessment of insulin resistance index, and inhibited over-increase of plasma glucose and insulin concentrations during oral glucose tolerance test. These effects were accompanied by decreases in both food intake and body weight (BW) increase. Although two treatments showed the same effect on BW gain, insulin sensitivity was higher after azilsartan treatment than pair-feeding. Azilsartan neither affected plasma concentrations of triglyceride and free fatty acids, nor increased adipose mRNA levels of peroxisome proliferator-activated receptor (PPAR)γ and its target genes such as adiponectin, aP2. In addition, azilsartan downregulated 11β-hydroxysteroid dehydrogenase type 1 expression. These results show the insulin-sensitizing effect of azilsartan in obese Koletsky rats. This effect is independent of decreases in food intake and BW increase or of the activation of adipose PPARγ. Our findings indicate the possible usefulness of azilsartan in the treatment of metabolic syndrome.

PPARγ activation induces acute PAI-1 gene expression in the liver but not in adipose tissues of diabetic model mice

Insulin resistance in patients with type II diabetes has recently been treated with thiazolidinediones, a class of peroxisome proliferator-activated receptor γ (PPARγ) agonists. However, these compounds are possibly associated with a significant increase in the risk of cardiovascular events. We examined the effect of the PPARγ agonist rosiglitazone on the expression of plasminogen activator inhibitor-1 (PAI-1) that is the primary inhibitor of fibrinolysis in the liver of diabetic mice and cultured mouse and human hepatocytes. Concentrations of plasma PAI-1 and levels of its mRNA expression in the liver were significantly elevated in accordance with hepatic PPARγ1 and PPARγ2 mRNA accumulation in genetically diabetic db/db mice. An intraperitoneal injection of rosiglitazone significantly increased plasma PAI-1 concentrations in parallel with hepatic, but not with adipose mRNA levels in db/db mice, and did not affect these parameters in wild-type mice. Rosiglitazone as well as the PPARα agonist bezafibrate significantly induced PAI-1 mRNA expression in cultured mouse hepatocytes. Furthermore, both rosiglitazone and pioglitazone significantly induced, whereas bezafibrate did not affect PAI-1 mRNA expression in the human liver carcinoma cell line HepG2. The transient induction of PAI-1 gene expression mediated by PPARγ in the fatty liver might be involved in the increased risk of cardiovascular events associated with thiazolidinediones in diabetic patients through decreasing fibrinolytic activity.

Short-Chain Fructooligosaccharides Influence Insulin Sensitivity and Gene Expression of Fat Tissue in Obese Dogs 2

Dietary fibers may modulate insulin resistance and glucose homeostasis in dogs. Their efficacy is, however, dependent on their origin, physical properties, and fermentability in the large bowel. Eight healthy Beagle dogs were fed a commercial diet at twice their maintenance requirements until they became obese. They were then maintained in the obese state and used in a cross-over design study to evaluate the effects of short-chain fructooligosaccharide (scFOS) supplementation (1% wt:wt dry matter in the diet). The euglycemic hyperinsulinemic clamp technique was performed before and after fattening and at the end of each 6-wk cross-over period. Fat tissue biopsies were taken in food-deprived and postprandial phases to measure mRNA abundance of genes involved with fatty acid, glucose metabolism, or inflammation. Insulin resistance appeared progressively with fattening and the rate of glucose infusion during euglycemic clamp was lower (P < 0.05) at the end of the fattening period (7.39 mg·kg−1·min−1) than at baseline (21.21 mg·kg−1·min−1). In stable obese dogs, scFOS increased (P < 0.05) the rate of glucose infusion compared with control (7.77 vs. 4.72 mg·kg−1·min−1). Plasma insulin and triglyceride concentrations were greater in obese than in lean dogs but were not altered by scFOS. Whereas mRNA was not affected in food-deprived dogs, scFOS increased uncoupling protein 2 (P = 0.05) and tended to increase carnitine palmitoyl transferase 1 adipose mRNA levels during the postprandial period (P = 0.09). Adding 1% scFOS to the diet of obese dogs decreases insulin resistance and appears to modulate the transcription of genes involved in fatty acid or glucose metabolism.

Antidiabetic effects of sub-chronic administration of the cannabinoid receptor (CB 1) antagonist, AM251, in obese diabetic ( ob/ob) mice

Recent research suggests that cannabinoid CB 1 receptor antagonism reduces appetite and body weight gain. The present study was designed to assess the sub-chronic effects of the selective cannabinoid CB 1 receptor antagonist, AM251 ( N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1 H-pyrazole-3-carboxamide), in young ob/ob mice. Pair-fed animals were used as additional controls. Daily injection of AM251 (6 mg/kg body weight) for 18 days significantly ( P < 0.05) decreased daily and 18-day cumulative food intake. The corresponding body weight change did not achieve significance and values were not different from pair-fed mice. Non-fasting plasma glucose was decreased ( P < 0.05) from day 10 onwards by AM251 treatment. The glycaemic response to intraperitoneal glucose was correspondingly improved ( P < 0.05) in AM251 treated mice. In keeping with this, insulin sensitivity was enhanced ( P < 0.05) compared to controls. Furthermore, adipose mRNA levels of acetyl-CoA carboxylase 1 were significantly ( P < 0.05) reduced by 18 days AM251 treatment. There were no differences in either non-fasting or glucose-stimulated insulin release. Pair-feeding had broadly similar metabolic effects to AM251 treatment apart from increased ( P < 0.01) locomotor activity which was only observed in AM251 treated ob/ob mice. These data indicate that sub-chronic antagonism of the cannabinoid CB 1 receptor by daily treatment with AM251 counters aspects of the hyperphagia-related impairment of ob/ob mouse metabolism. Such effects seem predominantly mediated by restriction of energy intake.

A role of lipin in human obesity and insulin resistance: relation to adipocyte glucose transport and GLUT4 expression

The mouse lipin gene, Lpin1, is important for adipose tissue development and is a candidate gene for insulin resistance. Here, we investigate the adipose tissue expression levels of the human LPIN1 gene in relation to various clinical variables as well as adipocyte function. LPIN1 gene expression was induced at an early step in human preadipocyte differentiation in parallel with peroxisome proliferator-activated receptor gamma. Lipin mRNA levels were higher in fat cells than in adipose tissue segments but showed no difference between subcutaneous and omental depots. Moreover, LPIN1 expression levels were reduced in obesity, improved following weight reduction in obese subjects, and were downregulated in women with the metabolic syndrome. With respect to adipocyte function, adipose LPIN1 gene expression was strongly associated with both basal and insulin-mediated subcutaneous adipocyte glucose transport as well as mRNA levels of glucose transporter 4 (GLUT4). We show that body fat accumulation is a major regulator of human adipose LPIN1 expression and suggest a role of LPIN1 in human preadipocyte as well as mature adipocyte function.

Eotaxin and Obesity

Asthma and obesity incidence is increasing worldwide, and asthma is often more severe in the obese. Eotaxin, a CC chemokine, is important in extrinsic asthma, an inflammatory disorder. Our objective was to examine the relation between eotaxin and obesity. We conducted a comparison study of eotaxin in mice fed high-fat vs. standard chow diet for 26 wk, in obese vs. lean humans, in obese humans before and after 4-6 wk of weight loss, and in sc vs. visceral adipose tissue from patients undergoing bariatric surgery. Our clinical study occurred in an outpatient weight loss program. Patients were obese adults with metabolic syndrome (n = 40) and nine morbidly obese bariatric surgery patients. Intervention was a very-low-calorie diet. We assessed circulating eotaxin and eotaxin mRNA levels in adipose tissue. Serum eotaxin levels were significantly higher in obese mice, and adipose mRNA levels correlated positively with serum eotaxin levels. Adipose tissue explants from obese mice showed increased secretion of eotaxin compared with explants from lean mice. In obese patients, plasma eotaxin levels were significantly higher than in lean controls and significantly reduced after weight loss, and eotaxin mRNA levels were 4.7-fold higher in visceral than sc adipose tissue. Circulating eotaxin and eotaxin mRNA levels in visceral adipose tissue were increased in obesity in mice and humans. Adipose tissue explants secrete eotaxin, and the stromal/vascular component of adipose tissue seems to be the predominant source of eotaxin. Diet-induced weight loss in humans led to reduction in plasma eotaxin levels, demonstrating that clinical interventions that target obesity can modulate systemic eotaxin levels.