Peroxisome proliferator-activated receptor gamma activation promotes infiltration of alternatively activated macrophages into adipose tissue

Obesity is associated with infiltration of macrophages into adipose tissue. Adipose macrophages may contribute to an elevated inflammatory status by secreting a variety of proinflammatory mediators, including tumor necrosis factor alpha and interleukin-6 (IL-6). Recent data suggest that during diet-induced obesity the phenotype of adipose-resident macrophages changes from alternatively activated macrophages toward a more classical and pro-inflammatory phenotype. Here, we explore the effect of peroxisome proliferator-activated receptor gamma activation on obesity-induced inflammation in 129SV mice fed a high fat diet for 20 weeks. High fat feeding increased bodyweight gain, adipose tissue mass, and liver triglycerides. Rosiglitazone treatment further increased adipose mass, reduced liver triglycerides, and changed adipose tissue morphology toward smaller adipocytes. Surprisingly, rosiglitazone markedly increased the number of macrophages in adipose tissue, as shown by immunohistochemical analysis and quantification of macrophage marker genes CD68 and F4/80+. In adipose tissue, markers for classically activated macrophages including IL-18 were down-regulated, whereas markers characteristic for alternatively activated macrophages (arginase 1, IL-10) were up-regulated by rosiglitazone. Importantly, conditioned media from rosiglitazone-treated alternatively activated macrophages neutralized the inhibitory effect of macrophages on 3T3-L1 adipocyte differentiation, suggesting that alternatively activated macrophages may be involved in mediating the effects of rosiglitazone on adipose tissue morphology and mass. Our results suggest that short term rosiglitazone treatment increases infiltration of alternatively activated macrophages in adipose tissue. The alternatively activated macrophages might play a role in peroxisome proliferator-activated receptor gamma-dependent expansion and remodeling of adipose tissue.     

RANKL from bone marrow adipose lineage cells promotes osteoclast formation and bone loss

Receptor activator of NF‐κB ligand (RANKL) is essential for osteoclast formation and bone remodeling. Nevertheless, the cellular source of RANKL for osteoclastogenesis has not been fully uncovered. Different from peripheral adipose tissue, bone marrow (BM) adipose lineage cells originate from bone marrow mesenchymal stromal cells (BMSCs). Here, we demonstrate that adiponectin promoter‐driven Cre expression (Adipoq^Cre ) can target bone marrow adipose lineage cells. We cross the Adipoq^Cre mice with rankl^fl/fl mice to conditionally delete RANKL from BM adipose lineage cells. Conditional deletion of RANKL increases cancellous bone mass of long bones in mice by reducing the formation of trabecular osteoclasts and inhibiting bone resorption but does not affect cortical bone thickness or resorption of calcified cartilage. Adipoq^Cre; rankl^fl/fl mice exhibit resistance to estrogen deficiency and rosiglitazone (ROS)‐induced trabecular bone loss but show bone loss induced by unloading. BM adipose lineage cells therefore represent an essential source of RANKL for the formation of trabecula osteoclasts and resorption of cancellous bone during remodeling under physiological and pathological conditions. Targeting bone marrow adiposity is a promising way of preventing pathological bone loss.     

The regulation of acyl-CoA dehydrogenases in adipose tissue by rosiglitazone

The acyl-CoA dehydrogenases (ACADs), which catalyze the rate-limiting step in the mitochondrial beta-oxidation spiral, were investigated in white adipose tissue (WAT) of C57Bl/6 mice treated with 10 mg/kg/day rosiglitazone. Rosiglitazone was also administered to PPAR-alpha knockout mice. ACAD abundance and activity were determined using western blotting and an ACAD enzyme activity assay. Rosiglitazone increased ACAD activity in both epididymal and inguinal WAT but not in brown adipose tissue, liver, or muscle. Given the known function of PPAR-alpha in regulating the expression of ACAD genes in liver, it was hypothesized that PPAR-alpha may be involved in upregulating the ACADs during rosiglitazone-mediated adipose tissue remodeling. However, the effect of rosiglitazone on adipose tissue ACAD activity was the same in wild-type and PPAR-alpha knockout mice. In conclusion, rosiglitazone increases expression and activity of ACAD enzymes in WAT independently of PPAR-alpha.     

Liver peroxisome proliferator-activated receptor gamma contributes to hepatic steatosis,triglyceride clearance,and regulation of body fat mass

Peroxisome proliferator-activated receptor gamma (PPAR gamma) is a nuclear receptor that mediates the antidiabetic effects of thiazolidinediones. PPAR gamma is present in adipose tissue and becomes elevated in fatty livers, but the roles of specific tissues in thiazolidinedione actions are unclear. We studied the function of liver PPAR gamma in both lipoatrophic A-ZIP/F-1 (AZIP) and wild type mice. In AZIP mice, ablation of liver PPAR gamma reduced the hepatic steatosis but worsened the hyperlipidemia, triglyceride clearance, and muscle insulin resistance. Inactivation of AZIP liver PPAR gamma also abolished the hypoglycemic and hypolipidemic effects of rosiglitazone, demonstrating that, in the absence of adipose tissue, the liver is a primary and major site of thiazolidinedione action. In contrast, rosiglitazone remained effective in non-lipoatrophic mice lacking liver PPAR gamma, suggesting that adipose tissue is the major site of thiazolidinedione action in typical mice with adipose tissue. Interestingly, mice without liver PPAR gamma, but with adipose tissue, developed relative fat intolerance, increased adiposity, hyperlipidemia, and insulin resistance. Thus, liver PPAR gamma regulates triglyceride homeostasis, contributing to hepatic steatosis, but protecting other tissues from triglyceride accumulation and insulin resistance.     

Activation of peroxisome proliferator-activated receptor-γ by rosiglitazone improves lipid homeostasis at the adipose tissue-liver axis in ethanol-fed mice

The development of alcohol-induced fatty liver is associated with a reduction of white adipose tissue (WAT). Peroxisome proliferator-activated receptor (PPAR)-γ prominently distributes in the WAT and plays a crucial role in maintaining adiposity. The present study investigated the effects of PPAR-γ activation by rosiglitazone on lipid homeostasis at the adipose tissue-liver axis. Adult C57BL/6 male mice were pair fed liquid diet containing ethanol or isocaloric maltose dextrin for 8 wk with or without rosiglitazone supplementation to ethanol-fed mice for the last 3 wk. Ethanol exposure downregulated adipose PPAR-γ gene and reduced the WAT mass in association with induction of inflammation, which was attenuated by rosiglitazone. Ethanol exposure stimulated lipolysis but reduced fatty acid uptake capacity in association with dysregulation of lipid metabolism genes. Rosiglitazone normalized adipose gene expression and corrected ethanol-induced lipid dyshomeostasis. Ethanol exposure induced steatosis and upregulated inflammatory genes in the liver, which were attenuated by rosiglitazone. Hepatic peroxisomal fatty acid β-oxidation was suppressed by ethanol in associated with inhibition of acyl-coenzyme A oxidase 1. Rosiglitazone elevated plasma adiponectin level and normalized peroxisomal fatty acid β-oxidation rate. However, rosiglitazone did not affect ethanol-reduced very low-density lipoprotein secretion from the liver. These results demonstrated that activation of PPAR-γ by rosiglitazone reverses ethanol-induced adipose dysfunction and lipid dyshomeostasis at the WAT-liver axis, thereby abrogating alcoholic fatty liver.     

Dysregulation of adipose glutathione peroxidase 3 in obesity contributes to local and systemic oxidative stress

Glutathione peroxidase 3 (GPx3) accounts for the major antioxidant activity in the plasma. Here, we demonstrate that down-regulation of GPx3 in the plasma of obese subjects is associated with adipose GPx3 dysregulation, resulting from the increase of inflammatory signals and oxidative stress. Although GPx3 was abundantly expressed in kidney, lung, and adipose tissue, we observed that GPx3 expression was reduced selectively in the adipose tissue of several obese animal models as decreasing plasma GPx3 level. Adipose GPx3 expression was greatly suppressed by prooxidative conditions such as high levels of TNFalpha and hypoxia. In contrast, the antioxidant N-acetyl cysteine and the antidiabetic drug rosiglitazone increased adipose GPx3 expression in obese and diabetic db/db mice. Moreover, GPx3 overexpression in adipocytes improved high glucose-induced insulin resistance and attenuated inflammatory gene expression whereas GPx3 neutralization in adipocytes promoted expression of proinflammatory genes. Taken together, these data suggest that suppression of GPx3 expression in the adipose tissue of obese subjects might constitute a vicious cycle to expand local reactive oxygen species accumulation in adipose tissue potentially into systemic oxidative stress and obesity-related metabolic complications.     

Hormone-sensitive lipase modulates adipose metabolism through PPARγ

Hormone-sensitive lipase (HSL) is rate limiting for diacylglycerol and cholesteryl ester hydrolysis in adipose tissue and essential for complete hormone-stimulated lipolysis. Gene expression profiling in HSL-/- mice suggests that HSL is important for modulating adipogenesis and adipose metabolism. To test whether HSL is required for the supply of intrinsic ligands for PPARγ for normal adipose differentiation, HSL-/- and wild-type (WT) littermates were fed normal chow (NC) and high-fat (HF) diets supplemented with or without rosiglitazone (200 mg/kg) for 16 weeks. Results show that supplementing rosiglitazone to an NC diet completely normalized the decreased body weight and adipose depots in HSL-/- mice. Additionally, rosiglitazone resulted in similar serum glucose, total cholesterol, FFA, and adiponectin values in WT and HSL-/- mice. Furthermore, rosiglitazone normalized the expression of genes involved in adipocyte differentiation, markers of adipocyte differentiation, and enzymes involved in triacylglycerol synthesis and metabolism, and cholesteryl ester homeostasis, in HSL-/- mice. Supplementing rosiglitazone to an HF diet resulted in improved glucose tolerance in both WT and HSL-/- animals and also partial normalization in HSL-/- mice of abnormal WAT gene expression, serum chemistries, organ and body weight changes. In vitro studies showed that adipocytes from WT animals can provide ligands for activation of PPARγ and that activation is further boosted following lipolytic stimulation, whereas adipocytes from HSL-/- mice displayed attenuated activation of PPARγ, with no change following lipolytic stimulation. These results suggest that one of the mechanisms by which HSL modulates adipose metabolism is by providing intrinsic ligands or pro-ligands for PPARγ.     

Subcutaneous Abdominal Adipose Tissue Subcompartments: Potential Role in Rosiglitazone Effects

Abdominal visceral tissue (VAT) and subcutaneous adipose tissue (SAT), comprised of superficial‐SAT (sSAT) and deep‐SAT (dSAT), are metabolically distinct. The antidiabetic agents thiazolidinediones (TZDs), in addition to their insulin‐sensitizing effects, redistribute SAT suggesting that TZD action involves adipose tissue depot‐specific regulation. We investigated the expression of proteins key to adipocyte metabolism on differentiated first passage (P1) preadipocytes treated with rosiglitazone, to establish a role for the diverse depots of abdominal adipose tissue in the insulin‐sensitizing effects of TZDs. Adipocytes and preadipocytes were isolated from sSAT, dSAT, and VAT samples obtained from eight normal subjects. Preadipocytes (P1) left untreated (U) or treated with a classic differentiation cocktail (DI) including rosiglitazone (DIR) for 9 days were evaluated for strata‐specific differences in differentiation including peroxisome proliferator‐activated receptor‐γ (PPAR‐γ) and lipoprotein lipase (LPL) expression, insulin sensitivity via adiponectin and glucose transport‐4 (GLUT4), glucocorticoid metabolism with 11β‐hydroxysteroid dehydrogenase type‐1 (11βHSD1), and alterations in the adipokine leptin. While depot‐specific differences were absent with the classic differentiation cocktail, with rosiglitazone sSAT had the most potent response followed by dSAT, whereas VAT was resistant to differentiation. With rosiglitazone, universal strata effects were observed for PPAR‐γ, LPL, and leptin, with VAT in all cases expressing significantly lower basal expression levels. Clear dSAT‐specific changes were observed with decreased intracellular GLUT4. Specific sSAT alterations included decreased 11βHSD1 whereas secreted adiponectin was potently upregulated in sSAT with respect to dSAT and VAT. Overall, the subcompartments of SAT, sSAT, and dSAT, appear to participate in the metabolic changes that arise with rosiglitazone administration.     

Hypoxia and the endocrine and signalling role of white adipose tissue

White adipose tissue is a major endocrine and signalling organ. It secretes multiple protein hormones and factors, termed adipokines (such as adiponectin, leptin, IL-6, MCP-1, TNFalpha) which engage in extensive cross-talk within adipose tissue and with other tissues. Many adipokines are linked to inflammation and immunity and these include cytokines, chemokines and acute phase proteins. In obesity, adipose tissue exhibits a major inflammatory response with increased production of inflammation-related adipokines. It has been proposed that hypoxia may underlie the inflammatory response in adipose tissue and evidence that the tissue is hypoxic in obesity has been obtained in animal models. Cell culture studies have demonstrated that the expression and secretion of key adipokines, including leptin, IL-6 and VEGF, are stimulated by hypoxia, while adiponectin (with an anti-inflammatory action) production falls. Hypoxia also stimulates glucose transport by adipocytes and may have a pervasive effect on cell function within adipose tissue.     

Enhanced angiogenesis in obesity and in response to PPARgamma activators through adipocyte VEGF and ANGPTL4 production

PPARgamma activators such as rosiglitazone (RSG) stimulate adipocyte differentiation and increase subcutaneous adipose tissue mass. However, in addition to preadipocyte differentiation, adipose tissue expansion requires neovascularization to support increased adipocyte numbers. Paradoxically, endothelial cell growth and differentiation is potently inhibited by RSG in vitro, raising the question of how this drug can induce an increase in adipose tissue mass while inhibiting angiogenesis. We find that adipose tissue from mice treated with RSG have increased capillary density. To determine whether adipose tissue angiogenesis was stimulated by RSG, we developed a novel assay to study angiogenic sprout formation ex vivo. Angiogenic sprout formation from equally sized adipose tissue fragments, but not from aorta rings, was greatly increased by obesity and by TZD treatment in vivo. To define the mechanism involved in RSG-stimulated angiogenesis in adipose tissue, the expression of proangiogenic factors by adipocytes was examined. Expression of VEGFA and VEGFB, as well as of the angiopoietin-like factor-4 (ANGPTL4), was stimulated by in vivo treatment with RSG. To define the potential role of these factors, we analyzed their effects on endothelial cell growth and differentiation in vitro. We found that ANGPTL4 stimulates endothelial cell growth and tubule formation, albeit more weakly than VEGF. However, ANGPTL4 mitigates the growth inhibitory actions of RSG on endothelial cells in the presence or absence of VEGF. Thus, the interplay between VEGF and ANGPTL4 could lead to a net expansion of the adipose tissue capillary network, required for adipose tissue growth, in response to PPARgamma activators.     

Mitochondrial biogenesis and remodeling during adipogenesis and in response to the insulin sensitizer rosiglitazone

White adipose tissue is an important endocrine organ involved in the control of whole-body metabolism, insulin sensitivity, and food intake. To better understand these functions, 3T3-L1 cell differentiation was studied by using combined proteomic and genomic strategies. The proteomics approach developed here exploits velocity gradient centrifugation as an alternative to isoelectric focusing for protein separation in the first dimension. A 20- to 30-fold increase in the concentration of numerous mitochondrial proteins was observed during adipogenesis, as determined by mass spectrometry and database correlation analysis. Light and electron microscopy confirmed a large increase in the number of mitochondrion profiles with differentiation. Furthermore, mRNA profiles obtained by using Affymetrix GeneChips revealed statistically significant increases in the expression of many nucleus-encoded mitochondrial genes during adipogenesis. Qualitative changes in mitochondrial composition also occur during adipose differentiation, as exemplified by increases in expression of proteins involved in fatty acid metabolism and of mitochondrial chaperones. Furthermore, the insulin sensitizer rosiglitazone caused striking changes in mitochondrial shape and expression of selective mitochondrial proteins. Thus, although mitochondrial biogenesis has classically been associated with brown adipocyte differentiation and thermogenesis, our results reveal that mitochondrial biogenesis and remodeling are inherent to adipose differentiation per se and are influenced by the actions of insulin sensitizers.     

Effects of rosiglitazone on gene expression in subcutaneous adipose tissue in highly active antiretroviral therapy-associated lipodystrophy

Highly active antiretroviral therapy (HAART) has improved the prognosis of human immunodeficiency virus (HIV)-infected patients but is associated with severe adverse events, such as lipodystrophy and insulin resistance. Rosiglitazone did not increase subcutaneous fat in patients with HAART-associated lipodystrophy (HAL) in a randomized, double-blind, placebo-controlled trial, although it attenuated insulin resistance and decreased liver fat content. The aim of this study was to examine effects of rosiglitazone on gene expression in subcutaneous adipose tissue in 30 patients with HAL. The mRNA concentrations in subcutaneous adipose tissue were measured using real-time PCR. Twenty-four-week treatment with rosiglitazone (8 mg/day) compared with placebo significantly increased the expression of adiponectin, peroxisome proliferator-activated receptor-gamma (PPARgamma), and PPARgamma coactivator 1 and decreased IL-6 expression. Expression of other genes involved in lipogenesis, fatty acid metabolism, or glucose transport, such as acyl-CoA synthase, adipocyte lipid-binding protein, CD45, fatty acid transport protein-1 and -4, GLUT1, GLUT4, keratinocyte lipid-binding protein, lipoprotein lipase, PPARdelta, and sterol regulatory element-binding protein-1c, remained unchanged. Rosiglitazone also significantly increased serum adiponectin concentration. The change in serum adiponectin concentration was inversely correlated with the change in fasting serum insulin concentration and liver fat content. In conclusion, rosiglitazone induced significant changes in gene expression in subcutaneous adipose tissue and ameliorated insulin resistance in patients with HAL. Increased expression of adiponectin might have mediated most of the favorable insulin-sensitizing effects of rosiglitazone in these patients.     

Major involvement of mTOR in the PPARγ-induced stimulation of adipose tissue lipid uptake and fat accretion

Evidence points to a role of the mammalian target of rapamycin (mTOR) signaling pathway as a regulator of adiposity, yet its involvement as a mediator of the positive actions of peroxisome proliferator-activated receptor (PPAR)γ agonism on lipemia, fat accretion, lipid uptake, and its major determinant lipoprotein lipase (LPL) remains to be elucidated. Herein we evaluated the plasma lipid profile, triacylglycerol (TAG) secretion rates, and adipose tissue LPL-dependent lipid uptake, LPL expression/activity, and expression profile of other lipid metabolism genes in rats treated with the PPARγ agonist rosiglitazone (15 mg/kg/day) in combination or not with the mTOR inhibitor rapamycin (2 mg/kg/day) for 15 days. Rosiglitazone stimulated adipose tissue mTOR complex 1 and AMPK and induced TAG-derived lipid uptake (136%), LPL mRNA/activity (2- to 6-fold), and fat accretion in subcutaneous (but not visceral) white adipose tissue (WAT; 50%) and in brown adipose tissue (BAT; 266%). Chronic mTOR inhibition attenuated the upregulation of lipid uptake, LPL expression/activity, and fat accretion induced by PPARγ activation in both subcutaneous WAT and BAT, which resulted in hyperlipidemia. In contrast, rapamycin did not affect most of the other WAT lipogenic genes upregulated by rosiglitazone. Together these findings demonstrate that mTOR is a major regulator of adipose tissue LPL-mediated lipid uptake and a critical mediator of the hypolipidemic and lipogenic actions of PPARγ activation.     

Adipose tissue: a subtle and complex cell system

White adipose tissue (WAT) represents a large amount of all adult tissues. For a long time, it was considered as a poorly active, overgrown and undesirable tissue. It was mainly studied for its involvement in energy metabolism and disorders, as well as for its endocrine functions. WAT is composed of two main populations, matures adipocytes and stroma vascular fraction (SVF) that can be separated easily. The SVF contains two compartments, stromal and hematopoietic that have been recently characterized. The stromal population (or ADAS for Adipose Derived Stromal Cells) presents functional features of, as well as lineage relationship with, macrophages. These stromal cells, that are able to differentiate into adipocytes, also display endothelial potential, and could be considered as vascular progenitors. Differentiation of various adipose-derived cell subsets towards functional cardiomyocytes, osteoblasts, chondrocytes, muscle, hematopoietic and neural cells was also obtained in vitro or in vivo. Adipose tissue thus appears as a complex tissue composed of different cell subsets that could vary according to the nature and the location of fat pads, or to the physiological or pathological status. WAT appears as a very plastic and heterogeneous tissue that is very easy to sample. This represents a great advantage when considering adipose tissue as a potential and suitable source of stem cell for cell therapy. Further investigations in this way have to lead to the emergence of new insights fundamental to progress in our knowledge of adipose tissue biology.     

Intramuscular adipose is derived from a non-Pax3 lineage and required for efficient regeneration of skeletal muscles

Ectopic accumulation of adipose in the skeletal muscle is associated with muscle wasting, insulin resistance and diabetes. However, the developmental origin of postnatal intramuscular adipose and its interaction with muscle tissue are unclear. We report here that compared to the fast EDL muscles, slow SOL muscles are more enriched with adipogenic progenitors and have higher propensity to form adipose. Using Cre/LoxP mediated lineage tracing in mice, we show that intramuscular adipose in both EDL and SOL muscles is exclusively derived from a Pax3(-) non-myogenic lineage. In contrast, inter-scapular brown adipose is derived from the Pax3(+) lineage. To dissect the interaction between adipose and skeletal muscle tissues, we used Myf5-Cre and aP2-Cre mice in combination with ROSA26-iDTR mice to genetically ablate myogenic and adipogenic cell lineages, respectively. Whereas ablation of the myogenic cell lineage facilitated adipogenic differentiation, ablation of the adipogenic cell lineage surprisingly impaired the regeneration of acutely injured skeletal muscles. These results reveal striking heterogeneity of tissue-specific adipose and a previously unappreciated role of intramuscular adipose in skeletal muscle regeneration.     

Differentiation of mesenchymal stem cells derived from human bone marrow and subcutaneous adipose tissue into pancreatic islet-like clusters in vitro

Although stem cells are present in various adult tissues and body fluids, bone marrow has been the most popular source of stem cells for treatment of a wide range of diseases. Recent results for stem cells from adipose tissue have put it in a position to compete for being the leading therapeutic source. The major advantage of these stem cells over their counterparts is their amazing proliferative and differentiation potency. However, their pancreatic lineage transdifferentiation competence was not compared to that for bone marrow-derived stem cells. This study aims to identify an efficient source for transdifferentiation into pancreatic islet-like clusters, which would increase potential application in curative diabetic therapy. The results reveal that mesenchymal stem cells (MSC) derived from bone marrow and subcutaneous adipose tissue can differentiate into pancreatic islet-like clusters, as evidenced by their islet-like morphology, positive dithizone staining and expression of genes such as Nestin, PDX1, Isl 1, Ngn 3, Pax 4 and Insulin. The pancreatic lineage differentiation was further corroborated by positive results in the glucose challenge assay. However, the results indicate that bone marrow-derived MSCs are superior to those from subcutaneous adipose tissue in terms of differentiation into pancreatic islet-like clusters. In conclusion, bone marrow-derived MSC might serve as a better alternative in the treatment of diabetes mellitus than those from adipose tissue.     

c-Jun N-terminal kinase is involved in the suppression of adiponectin expression by TNF-alpha in 3T3-L1 adipocytes

Adiponectin, one of adipokines that is secreted from adipocytes, plays an important role in the regulation of glucose and lipid metabolism. Paradoxically, serum concentrations of adiponectin are decreased in obese and type 2 diabetic patients, although it is produced in adipose tissue. On the other hand, plasma TNF-alpha levels are increased in such subjects. In the present study, the mechanism by which adiponectin is regulated by TNF-alpha was investigated. The decreased adiponectin mRNA levels by TNF-alpha were partially recovered by treatment with a c-Jun N-terminal kinase (JNK) inhibitor or the PPAR-gamma agonist rosiglitazone in 3T3-L1 adipocytes. Interestingly, however, cotreatment with the JNK inhibitor and rosiglitazone led to a recovery of TNF-alpha-mediated adiponectin suppression to the control level. The JNK inhibitor regulated the expression of adiponectin by the increase of PPAR-gamma DNA binding activity and the recovery of its mRNA expression while rosiglitazone acted via a PPAR-gamma independent pathway which remains to be elucidated. These findings suggest that the JNK signaling pathway, activated by TNF-alpha, is involved in the regulation of adiponectin expression.