Effects of obesity on the relationship of leptin mRNA expression and adipocyte size in anatomically distinct fat depots in mice

In support of leptin's physiological role as humoral signal of fat mass, we have shown that adipocyte volume is a predominant determinant of leptin mRNA levels in anatomically distinct fat depots in lean young mice in the postabsorptive state. In this report, we investigated how obesity may affect the relationship between leptin mRNA levels and adipocyte volume in anatomically distinct fat depots in mice with genetic (Lep(ob)/Lep(ob) and A(y)/+), diet-induced, and aging-related obesity. In all of the obese mice examined, tissue leptin mRNA levels relative to the average adipocyte volume were lower in the perigonadal and/or retroperitoneal than in the inguinal fat depots and were lower than those of the lean young mice in the perigonadal fat depot. A close, positive correlation between leptin mRNA level and adipocyte volume was present from small to hypertrophic adipocytes within each perigonadal and inguinal fat pad in the obese mice, but the slopes of the regression lines relating leptin mRNA level to adipocyte volume were significantly lower in the perigonadal than in the inguinal fat pads of the same mice. These results suggest that obesity per se is associated with a decreased leptin gene expression per unit of fat mass in mice and that the positive correlation between leptin mRNA level and adipocyte volume is an intrinsic property of adipocytes that is not disrupted by adipocyte hypertrophy in obese mice.     

Fargesin improves lipid and glucose metabolism in 3T3-L1 adipocytes and high-fat diet-induced obese mice

This study examined the effects of fargesin, a neolignan isolated from Magnolia plants, on obesity and insulin resistance and the possible mechanisms involved in these effects in 3T3-L1 adipocytes and high-fat diet (HFD)-induced obese mice. Fargesin promoted the glucose uptake in 3T3-L1 adipocytes. In HFD-induced obese mice, fargesin decreased the body weight gain, white adipose tissue (WAT), and plasma triglyceride, non-esterified fatty acid and glucose levels, and improved the glucose tolerance. Fargesin increased glucose transporter 4 (GLUT4) protein expression and phosphorylation of Akt, AMP-activated protein kinase (AMPK), and acetyl-CoA carboxylase (ACC) in both 3T3-L1 adipocytes and WAT of HFD-induced obese mice. Fargesin also decreased the mRNA expression levels of fatty acid oxidation-related genes, such as peroxisome proliferator-activated receptor α (PPARα), carnitine palmitoyltransferase-1 (CPT-1), uncoupling protein-2 (UCP-2) and leptin in WAT. Taken together, the present findings suggest that fargesin improves dyslipidemia and hyperglycemia by activating Akt and AMPK in WAT. ? 2012 International Union of Biochemistry and Molecular Biology, Inc.     

Selective up-regulation of fatty acid uptake by adipocytes characterizes both genetic and diet-induced obesity in rodents.

Long chain fatty acid transport is selectively up-regulated in adipocytes of Zucker fatty rats, diverting fatty acids from sites of oxidation toward storage in adipose tissue. To determine whether this is a general feature of obesity, we studied [(3)H]oleate uptake by adipocytes and hepatocytes from 1) homozygous male obese (ob), diabetic (db), fat (fat), and tubby (tub) mice and from 2) male Harlan Sprague-Dawley rats fed for 7 weeks a diet containing 55% of calories from fat. V(max) and K(m) were compared with controls of the appropriate background strain (C57BL/6J or C57BLKS) or diet (13% of calories from fat). V(max) for adipocyte fatty acid uptake was increased 5-6-fold in ob, db, fat, and tub mice versus controls (p < 0.001), whereas no differences were seen in the corresponding hepatocytes. Similar changes occurred in fat-fed rats. Of three membrane fatty acid transporters expressed in adipocytes, plasma membrane fatty acid-binding protein mRNA was increased 9-11-fold in ob and db, which lack a competent leptin/leptin receptor system, but was not increased in fat and tub, i.e. in strains with normal leptin signaling capability; fatty acid translocase mRNA was increased 2.2-6.5-fold in tub, ob, and fat adipocytes, but not in db adipocytes; and only marginal changes in fatty acid transport protein 1 mRNA were found in any of the mutant strains. Adipocyte fatty acid uptake is generally increased in murine obesity models, but up-regulation of individual transporters depends on the specific pathophysiology. Leptin may normally down-regulate expression of plasma membrane fatty acid binding protein.     

Exposure to an organometal compound stimulates adipokine and cytokine expression in white adipose tissue

ObjectiveWhite adipose tissue (WAT) is now considered a defined tissue capable of interactions with other organ systems. WAT role in elevating the level of systemic chronic inflammation suggests that alterations in this tissue as the result of disease or environmental factors may influence the development and progression of various obesity-related pathologies. This study investigated WAT cell-specific responses to an organometal compound, trimethyltin (TMT), to determine possible contribution to induced inflammation.MethodsHuman primary mature adipocytes and macrophage differentiated THP-1 cells were cultured in TMT presence and relative toxicities and different adipokine levels were determined. The inflammatory response was examined in TMT presence for primary cells from obese ob/ob mice WAT, and after TMT injection in ob/ob mice.ResultsBoth adipocytes and macrophages were resistant to cell death induced by TMT. However, adipocytes cultured in TMT presence showed increased expression of TNFα and IL-6, and modified leptin levels. In macrophage cultures, TMT also increased TNFα and IL-6, while MCP-1 and MIP-1α were decreased. In vivo, a single injection of TMT in ob/ob mice, elevated TNFα, MIP-1α and adiponectin in WAT.ConclusionsElevation of the inflammatory related products can be induced by chemical exposure in adipocytes and macrophages, as well as murine WAT. These data suggest that numerous factors, including a systemic chemical exposure, can induce an inflammatory response from the WAT. Furthermore, when characterizing both chemical-induced toxicity and the progression of the chronic inflammation associated with elevated WAT content, such responses in this target tissue should be taken into consideration.     

The roles of transforming growth factor-β and Smad3 signaling in adipocyte differentiation and obesity

We aimed at elucidating the roles of transforming growth factor (TGF)-β and Smad3 signaling in adipocyte differentiation (adipogenesis) and in the pathogenesis of obesity. TGF-β/Smad3 signaling in white adipose tissue (WAT) was determined in genetically obese (ob/ob) mice. The effect of TGF-β on adipogenesis was evaluated in mouse embryonic fibroblasts (MEF) isolated both from WT controls and Smad3 KO mice by Oil red-O staining and gene expression analysis. Phenotypic analyses of high-fat diet (HFD)-induced obesity in Smad3 KO mice compared to WT controls were performed. TGF-β/Smad3 signaling was elevated in WAT from ob/ob mice compared to the controls. TGF-β significantly inhibited adipogenesis in MEF, but the inhibitory effects of TGF-β on adipogenesis were partially abolished in MEF from Smad3 KO mice. TGF-β inhibited adipogenesis independent from the Wnt and β-catenin pathway. Smad3 KO mice were protected against HFD-induced insulin resistance. The size of adipocytes from Smad3 KO mice on the HFD was significantly smaller compared to the controls. In conclusion, the TGF-β/Smad3 signaling pathway plays key roles not only in adipogenesis but also in development of insulin resistance.     

Up-regulation of mitochondrial activity and acquirement of brown adipose tissue-like property in the white adipose tissue of fsp27 deficient mice

Fsp27, a member of the Cide family proteins, was shown to localize to lipid droplet and promote lipid storage in adipocytes. We aimed to understand the biological role of Fsp27 in regulating adipose tissue differentiation, insulin sensitivity and energy balance. Fsp27(-/-) mice and Fsp27/lep double deficient mice were generated and we examined the adiposity, whole body metabolism, BAT and WAT morphology, insulin sensitivity, mitochondrial activity, and gene expression changes in these mouse strains. Furthermore, we isolated mouse embryonic fibroblasts (MEFs) from wildtype and Fsp27(-/-) mice, followed by their differentiation into adipocytes in vitro. We found that Fsp27 is expressed in both brown adipose tissue (BAT) and white adipose tissue (WAT) and its levels were significantly elevated in the WAT and liver of leptin-deficient ob/ob mice. Fsp27(-/-) mice had increased energy expenditure, lower levels of plasma triglycerides and free fatty acids. Furthermore, Fsp27(-/-)and Fsp27/lep double-deficient mice are resistant to diet-induced obesity and display increased insulin sensitivity. Moreover, white adipocytes in Fsp27(-/-) mice have reduced triglycerides accumulation and smaller lipid droplets, while levels of mitochondrial proteins, mitochondrial size and activity are dramatically increased. We further demonstrated that BAT-specific genes and key metabolic controlling factors such as FoxC2, PPAR and PGC1alpha were all markedly upregulated. In contrast, factors inhibiting BAT differentiation such as Rb, p107 and RIP140 were down-regulated in the WAT of Fsp27(-/-) mice. Remarkably, Fsp27(-/-) MEFs differentiated in vitro show many brown adipocyte characteristics in the presence of the thyroid hormone triiodothyronine (T3). Our data thus suggest that Fsp27 acts as a novel regulator in vivo to control WAT identity, mitochondrial activity and insulin sensitivity.     

Obese adipocytes show ultrastructural features of stressed cells and die of pyroptosis

We previously suggested that, in obese animals and humans, white adipose tissue inflammation results from the death of hypertrophic adipocytes; these are then cleared by macrophages, giving rise to distinctive structures we denominated crown-like structures. Here we present evidence that subcutaneous and visceral hypertrophic adipocytes of leptin-deficient (ob/ob and db/db) obese mice exhibit ultrastructural abnormalities (including calcium accumulation and cholesterol crystals), many of which are more common in hyperglycemic db/db versus normoglycemic ob/ob mice and in visceral versus subcutaneous depots. Degenerating adipocytes whose intracellular content disperses in the extracellular space were also noted in obese mice; in addition, increased anti-reactive oxygen species enzyme expression in obese fat pads, documented by RT-PCR and immunohistochemistry, suggests that ultrastructural changes are accompanied by oxidative stress. RT-PCR showed NLRP3 inflammasome activation in the fat pads of both leptin-deficient and high-fat diet obese mice, in which formation of active caspase-1 was documented by immunohistochemistry in the cytoplasm of several hypertrophic adipocytes. Notably, caspase-1 was not detected in FAT-ATTAC transgenic mice, where adipocytes die of apoptosis. Thus, white adipocyte overexpansion induces a stress state that ultimately leads to death. NLRP3-dependent caspase-1 activation in hypertrophic adipocytes likely induces obese adipocyte death by pyroptosis, a proinflammatory programmed cell death.     

Leptin attenuates gene expression for renal 25-hydroxyvitamin D3-1alpha-hydroxylase in mice via the long form of the leptin receptor

Leptin, the ob gene product secreted by adipocytes, controls overall energy balance. We previously showed that leptin administration to leptin-deficient obese (ob/ob) mice suppressed mRNA expression and activity of renal 25-hydroxyvitamin D(3)-1alpha-hydroxylase (CYP27B1). In leptin receptor-deficient (db/db) mice, we presently examined whether leptin affects 1alpha-hydroxylase expression in renal tubules through the active form of the leptin receptor (ObRb). Elevated serum concentrations of calcium and 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] in untreated ob/ob mice showed sharp reduction with leptin administration (4 mg/kg, i.p. every 12h for 2 days); no such reduction of elevation occurred in db/db mice. ObRb mRNA was expressed in kidney, brain, fat, lung, and bone in wild-type and ob/ob mice, but not db/db mice. The ob/ob and db/db mice showed large increases in renal 1alpha-hydroxylase mRNA expression and activity. Leptin administration (4 mg/kg) completely abrogated these increases in ob/ob but not db/db mice. Renal 25-hydroxyvitamin D(3)-24-hydroxylase (CYP24) mRNA synthesis also was greatly elevated in ob/ob and db/db mice; excesses decreased significantly with leptin administration in ob/ob mice, but increased in db/db mice. Renal tubular cells in primary culture expressed mRNAs including proximal tubules markers (1alpha-hydroxylase and megalin), parathyroid hormone receptor, and vitamin D receptor. Calcitonin receptor mRNA, synthesized mainly in distal tubules, was scant, indicating that most cultured cells were from proximal tubules. Cells did not express ObRb mRNA. Forskolin exposure at 10(-6)M for 3 or 6h significantly increased 1alpha-hydroxylase mRNA. Leptin at 10(-6)M did not change mRNA expression in either presence or absence of forskolin. Accordingly, leptin attenuates renal 1alpha-hydroxylase gene expression through ObRb. Furthermore, leptin appears to act indirectly on renal proximal tubules to regulate 1alpha-hydroxylase gene expression.     

Central leptin regulates the UCP1 and ob genes in brown and white adipose tissue via different beta-adrenoceptor subtypes

The three known subtypes of beta-adrenoreceptors (beta(1)-AR, beta(2)-AR, and beta(3)-AR) are differentially expressed in brown and white adipose tissue and mediate peripheral responses to central modulation of sympathetic outflow by leptin. To assess the relative roles of the beta-AR subtypes in mediating leptin's effects on adipocyte gene expression, mice with a targeted disruption of the beta(3)-adrenoreceptor gene (beta(3)-AR KO) were treated with vehicle or the beta(1)/beta(2)-AR selective antagonist, propranolol (20 microgram/g body weight/day) prior to intracerebroventricular (ICV) injections of leptin (0.1 microgram/g body weight/day). Leptin produced a 3-fold increase in UCP1 mRNA in brown adipose tissue of wild type (FVB/NJ) and beta(3)-AR KO mice. The response was unaltered by propranolol in wild type mice, but was completely blocked by this antagonist in beta(3)-AR KO mice. In contrast, ICV leptin had no effect on leptin mRNA in either epididymal or retroperitoneal white adipose tissue (WAT) from beta(3)-AR KOs. Moreover, propranolol did not block the ability of exogenous leptin to reduce leptin mRNA in either WAT depot site of wild type mice. These results demonstrate that the beta(3)-AR is required for leptin-mediated regulation of ob mRNA expression in WAT, but is interchangeable with the beta(1)/beta(2)-ARs in mediating leptin's effect on UCP1 mRNA expression in brown adipose tissue.     

Upregulation of uncoupling protein 2 mRNA in genetic obesity: lack of an essential role for leptin, hyperphagia, increased tissue lipid content, and TNF-alpha

Uncoupling protein 2 (UCP2) has been proposed to play a prominent role in the regulation of energy balance. UCP2 mRNA expression is upregulated in white adipose tissue (WAT) and liver, but is not altered in skeletal muscle in genetically obese ob/ob mice. The mechanisms involved in the upregulation of UCP2 in obesity have not been investigated. We have now examined the potential role of leptin, hyperphagia, increased tissue lipid content, and overexpression of tumor necrosis factor (TNF)-alpha in the upregulation of UCP2 mRNA expression in the liver and WAT in ob/ob mice. Treatment of ob/ob mice with leptin for 3 days significantly reduced their food intake but had no effect on the upregulation of UCP2 mRNA levels in the liver or WAT. To investigate the effect of feeding and higher tissue lipid content on the upregulation of UCP2 in liver and WAT, we compared UCP2 mRNA levels in ad-libitum fed and 72-h fasted control and ob/ob mice. In controls, fasting had no effect on UCP2 mRNA levels in liver, but increased UCP2 mRNA in WAT suggesting that the effects of fasting on UCP2 mRNA levels are tissue-specific. In ob/ob mice, fasting did not lower UCP2 mRNA levels in liver or WAT suggesting that the upregulation of UCP2 in ob/ob mice is not merely a direct consequence of increased food intake. 72-h fasting lowered hepatic total lipid content by 34% and 36% in control and ob/ob mice, respectively, without any corresponding decrease in hepatic UCP2 mRNA levels, suggesting that the enhanced UCP2 expression in the liver of ob/ob mice is not secondary to lipid accumulation in their livers. Although TNF-alpha has been shown to acutely increase UCP2 mRNA levels in liver and WAT, and is overexpressed in adipose tissue in obesity, deletion of the genes for both TNF receptors in ob/ob mice produces a further increase in UCP2 mRNA expression in liver and adipose tissue indicating a paradoxical inhibitory role. Taken together, these results suggest that the upregulation of UCP2 mRNA levels in the liver and WAT of ob/ob mice is not due to the lack of leptin, hyperphagia, increased tissue lipid content, or over-expression of TNF-alpha.     

IRS2 signaling in LepR-b neurons suppresses FoxO1 to control energy balance independently of leptin action

Irs2-mediated insulin/IGF1 signaling in the CNS modulates energy balance and glucose homeostasis; however, the site for Irs2 function is unknown. The hormone leptin mediates energy balance by acting on leptin receptor (LepR-b)-expressing neurons. To determine whether LepR-b neurons mediate the metabolic actions of Irs2 in the brain, we utilized Lepr(cre) together with Irs2(L/L) to ablate Irs2 expression in LepR-b neurons (Lepr(ΔIrs2)). Lepr(ΔIrs2) mice developed obesity, glucose intolerance, and insulin resistance. Leptin action was not altered in young Lepr(ΔIrs2) mice, although insulin-stimulated FoxO1 nuclear exclusion was reduced in Lepr(ΔIrs2) mice. Indeed, deletion of Foxo1 from LepR-b neurons in Lepr(ΔIrs2) mice normalized energy balance, glucose homeostasis, and arcuate nucleus gene expression. Thus, Irs2 signaling in LepR-b neurons plays a crucial role in metabolic sensing and regulation. While not required for leptin action, Irs2 suppresses FoxO1 signaling in LepR-b neurons to promote energy balance and metabolism.     

Obesity modulates the expression of haptoglobin in the white adipose tissue via TNFalpha.

Increase in adipose mass results in obesity and modulation of several factors in white adipose tissue (WAT). Two important examples are tumor necrosis factor alpha (TNFalpha) and leptin, both of which are upregulated in adipose tissue in obesity. In order to isolate genes differentially expressed in the WAT of genetically obese db/db mice compared to their lean littermates, we performed RNA fingerprinting and identified haptoglobin (Hp), which is significantly upregulated in the obese animals. Hp is a glycoprotein induced by a number of cytokines, LPS (Lipopolysaccharide), and more generally by inflammation. A significant upregulation of WAT Hp expression was also evident in several experimental obese models including the yellow agouti (/) A(y), ob/ob and goldthioglucose-treated mice (10-, 8-, and 7-fold, respectively). To identify the potential signals for an increase in Hp expression in obesity, we examined leptin and TNFalpha in vivo. Wild type animals treated with recombinant leptin did not show any alteration in WAT Hp expression compared to controls that were food restricted to the level of intake of the treated animals. On the other hand, Hp expression was induced in mice transgenically expressing TNFalpha in adipose tissue. Finally, a significant downregulation of WAT Hp mRNA was observed in ob/ob mice deficient in TNFalpha function, when compared to the ob/ob controls. These results demonstrate that haptoglobin expression in WAT is increased in obesity in rodents and TNFalpha is an important signal for this regulation.     

Absence of hormone-sensitive lipase inhibits obesity and adipogenesis in Lep ob/ob mice

Hormone-sensitive lipase (HSL) plays a crucial role in the hydrolysis of triacylglycerol and cholesteryl ester in various tissues including adipose tissues. To explore the role of HSL in the metabolism of fat and carbohydrate, we have generated mice lacking both leptin and HSL (Lep(ob/ob)/HSL(-/-)) by cross-breeding HSL(-/-) mice with genetically obese Lep(ob/ob) mice. Unexpectedly, Lep(ob/ob)/HSL(-/-) mice ate less food, gained less weight, and had lower adiposity than Lep(ob/ob)/HSL(+/+) mice. Lep(ob/ob)/HSL(-/-) mice had massive accumulation of preadipocytes in white adipose tissues with increased expression of preadipocyte-specific genes (CAAT/enhancer-binding protein beta and adipose differentiation-related protein) and decreased expression of genes characteristic of mature adipocytes (CCAAT/enhancer-binding protein alpha, peroxisome proliferator activator receptor gamma, and adipocyte determination and differentiation factor 1/sterol regulatory element-binding protein-1). Consistent with the reduced food intake, hypothalamic expression of neuropeptide Y and agouti-related peptide was decreased. Since HSL is expressed in hypothalamus, we speculate that defective generation of free fatty acids in the hypothalamus due to the absence of HSL mediates the altered expression of these orexigenic neuropeptides. Thus, deficiency of both leptin and HSL has unmasked novel roles of HSL in adipogenesis as well as in feeding behavior.     

Acylation-stimulating protein (ASP) deficiency induces obesity resistance and increased energy expenditure in ob/ob mice

Acylation-stimulating protein (ASP) acts as a paracrine signal to increase triglyceride synthesis in adipocytes. ASP administration results in more rapid postprandial lipid clearance. In mice, C3 (the precursor to ASP) knockout results in ASP deficiency and leads to reduced body fat and leptin levels. The protective potential of ASP deficiency against obesity and involvement of the leptin pathway were examined in ob/ob C3(-/-) double knockout mice (2KO). Compared with age-matched ob/ob mice, 2KO mice had delayed postprandial triglyceride and fatty acid clearance; associated with decreased body weight (4-17 weeks age: male: -13.7%, female: -20.6%, p < 0.0001) and HOMA (homeostasis model assessment) index (-37.7%), suggesting increased insulin sensitivity. By contrast, food intake in 2KO mice was +9.1% higher over ob/ob mice (p < 0.001, 2KO 5.1 +/- 0.2 g/day, ob/ob 4.5 +/- 0.2 g/day, wild type 2.6 +/- 0.1 g/day). The hyperphagia/leanness was balanced by a 28.5% increase in energy expenditure (oxygen consumption: 2KO, 131 +/- 8.9 ml/h; ob/ob, 102 +/- 4.5 ml/h; p < 0.01; wild type, 144 +/- 8.9 ml/h). These results suggest that the ASP regulation of energy storage may influence energy expenditure and dynamic metabolic balance.     

Nutritional regulation of adipose tissue apolipoprotein E expression

Apolipoprotein E (apoE) is a multifunctional protein that is highly expressed in human and murine adipose tissue. Endogenous adipocyte apoE expression influences adipocyte triglyceride turnover and modulates the expression of genes involved in lipid synthesis and oxidation. We now demonstrate the regulation of adipose tissue apoE expression by nutritional status in lean and obese mice. Obesity induced by high-fat diet, or by hyperphagia in ob/ob mice, produces significant reduction of adipose tissue apoE expression at the protein and messenger RNA level. Fasting in C57BL/6J mice for 24 h significantly increased apoE protein and messenger RNA levels. In ob/ob mice, transplantation of adipose tissue from lean littermate controls to restore circulating leptin levels produced significant weight loss over 12 wk and also produced an increase in adipose tissue apoE expression. The increase in adipose tissue apoE expression in this model, however, did not require leptin. Adipose tissue apoE was also significantly increased in ob/ob mice after a 48-h fast or after 7 days of caloric restriction. In summary, obesity suppresses adipose tissue apoE expression, whereas fasting or weight loss increases it. From our previous observations, these changes in adipose tissue apoE expression will have significant impact on adipose tissue lipid flux and lipoprotein metabolism. Furthermore, these results suggest adipose tissue apoE participates in defending adipose tissue and organismal energy homeostasis in response to nutritional perturbation.