Increased insulin mediated glucose metabolism in fat cells from I versus C57BL mice

The purpose of this study was to determine whether adipocytes from I strain mice, which are characterized by a greater in vivo glucose tolerance than most other strains, had a higher capacity to utilize glucose in response to physiological concentrations of insulin. Using C57BL mice as a control strain, we examined the effect of insulin on glucose metabolism in epididymal and inguinal adipocytes from 2-month-old male mice. Body weight was only slightly less (7%) for the I mice than for the C57BL mice, but fat pad sizes were 60 and 20% less for epididymal and inguinal depots, respectively, in the I mice. Fat cell size was also smaller in epididymal adipocytes from the I mice than from the C57BL mice. Fat cell size of inguinal adipocytes was similar in the two strains. Without insulin the rates of [U-14C]glucose incorporation into CO2 or lipids were twofold higher in cells from the I mice than in those from the C57BL mice. Maximal insulin concentration (2.5 nM) increased glucose metabolism by 140 and 500% in epididymal and inguinal adipose cells, respectively, in the I mice versus 30 and 50% in the C57BL mice. The maximal effect of insulin was reached at a much higher insulin concentration in the I mice than in the C57BL mice. The activity of fatty acid synthetase was four- to sixfold higher in fat cells from I than in those from C57BL mice. These results demonstrate an increased insulin responsiveness of glucose metabolism in fat cells from the I mice related to an increased lipogenic capacity. Furthermore, they show that adipose tissue in mice exhibits significant regional differences in terms of insulin responsiveness of glucose metabolism.     

Regulation of lipolysis in isolated adipocytes of rainbow trout (Oncorhynchus mykiss): the role of insulin and glucagon

In the present study, we have examined the effects of insulin and glucagon on the lipolysis of rainbow trout (Oncorhynchus mykiss). To this end, adipocytes were isolated from mesenteric fat and incubated in the absence (basal lipolysis) or presence of different concentrations of insulin and glucagon. In addition, to further elucidate the effects of these hormones in vivo on adipocyte lipolysis, both fasting and intraperitoneal glucagon injection experiments were performed. Basal lipolysis, measured as the glycerol released in the adipocyte medium, increased proportionally with cell concentration and incubation time. Cell viability was verified by measuring the release of lactate dehydrogenase (LDH) activity in the medium. Insulin (at doses of 35 and 350 nM) decreased lipolysis in isolated adipocytes of rainbow trout in vitro, while glucagon was clearly lipolytic at concentrations of 10 and 100 nM. Furthermore, hypoinsulinemia induced by fasting, as well as glucagon injection, significantly increased lipolysis in isolated adipocytes approximately 1.5- and 1.4-fold, respectively, when compared with adipocytes from control fish. Our data demonstrate that lipolysis, as measured in isolated adipocytes of rainbow trout, can be regulated by both insulin and glucagon. These results not only indicate that insulin is an important hormone in lipid deposition via its anti-lipolytic effects on rainbow trout adipocytes, but also reveal glucagon as a lipolytic hormone, as shown by both in vitro and in vivo experiments.     

Retinol-binding protein 4 inhibits insulin signaling in adipocytes by inducing proinflammatory cytokines in macrophages through a c-Jun N-terminal kinase- and toll-like receptor 4-dependent and retinol-independent mechanism

Retinol-binding protein 4 (RBP4), the sole retinol transporter in blood, is secreted from adipocytes and liver. Serum RBP4 levels correlate highly with insulin resistance, other metabolic syndrome factors, and cardiovascular disease. Elevated serum RBP4 causes insulin resistance, but the molecular mechanisms are unknown. Here we show that RBP4 induces expression of proinflammatory cytokines in mouse and human macrophages and thereby indirectly inhibits insulin signaling in cocultured adipocytes. This occurs through activation of c-Jun N-terminal protein kinase (JNK) and Toll-like receptor 4 (TLR4) pathways independent of the RBP4 receptor, STRA6. RBP4 effects are markedly attenuated in JNK1-/- JNK2-/- macrophages and TLR4-/- macrophages. Because RBP4 is a retinol-binding protein, we investigated whether these effects are retinol dependent. Unexpectedly, retinol-free RBP4 (apo-RBP4) is as potent as retinol-bound RBP4 (holo-RBP4) in inducing proinflammatory cytokines in macrophages. Apo-RBP4 is likely to be physiologically significant since RBP4/retinol ratios are increased in serum of lean and obese insulin-resistant humans compared to ratios in insulin-sensitive humans, indicating that higher apo-RBP4 is associated with insulin resistance independent of obesity. Thus, RBP4 may cause insulin resistance by contributing to the development of an inflammatory state in adipose tissue through activation of proinflammatory cytokines in macrophages. This process reveals a novel JNK- and TLR4-dependent and retinol- and STRA6-independent mechanism of action for RBP4.     

Augmentation of RBP4/STRA6 signaling leads to insulin resistance and inflammation and the plausible therapeutic role of vildagliptin and metformin

A role of Retinol Binding Protein-4 (RBP4) in insulin resistance is widely studied. However, there is paucity of information on its receptor viz., Stimulated by Retinoic Acid-6 (STRA6) with insulin resistance. To address this, we investigated the regulation of RBP4/STRA6 expression in 3T3-L1 adipocytes exposed to glucolipotoxicity (GLT) and in visceral adipose tissue (VAT) from high fat diet (HFD) fed insulin-resistant rats. 3T3-L1 adipocytes were subjected to GLT and other experimental maneuvers with and without vildagliptin or metformin. Real-time PCR and western-blot experiments were performed to analyze RBP4, STRA6, PPARγ gene and protein expression. Adipored staining and glucose uptake assay were performed to evaluate lipid and glucose metabolism. Oral glucose tolerance test (OGTT) and Insulin Tolerance Test (ITT) were performed to determine the extent of insulin resistance in HFD fed male Wistar rats. Total serum RBP4 was measured by quantitative sandwich enzyme-linked immunosorbent assay kit. Adipocytes under GLT exhibited significantly increased RBP4/STRA6 expressions and decreased insulin sensitivity/glucose uptake. Vildagliptin and metformin not only restored the above but also decreased the expression of IL-6, NFκB, SOCS-3 along with lipid accumulation. Furthermore, HFD fed rats exhibited significantly increased serum levels of RBP4 along with VAT expression of RBP4, STRA6, PPARγ, IL-6. These molecules were significantly altered by the vildagliptin/ metformin treatment. We conclude that RBP4/STRA6 pathway is primarily involved in mediating inflammation and insulin resistance in adipocytes and visceral adipose tissues under glucolipotoxicity and in insulin resistant rats.

Graphic abstract

     

Resistin mRNA levels are downregulated by estrogen in vivo and in vitro

Resistin, a hormone secreted by adipocytes, is suggested to be an important link between obesity and diabetes. The aim of this study was to evaluate the regulatory effect of estrogen on adipocyte resistin gene expression in ovariectomized (OVX) rats and in isolated rat adipocytes in vitro. Subcutaneous injection of estradiol benzoate reduced resistin mRNA levels in adipocytes isolated from the inguinal, parametrial, perirenal, retroperitoneal, or periovarian fat deposits of OVX rats, while an in vitro study showed that estradiol treatment decreased resistin mRNA levels in cultured rat periovarian fat adipocytes. Results of Western blotting analysis also showed that estrogen decreased adipose resistin contents in vivo and in vitro. These data suggest that estrogen is a pivotal negative regulator of resistin gene expression.     

Serum retinol-binding protein is more highly expressed in visceral than in subcutaneous adipose tissue and is a marker of intra-abdominal fat mass

Intra-abdominal fat is associated with insulin resistance and cardiovascular risk. Levels of serum retinol-binding protein (RBP4), secreted by fat and liver cells, are increased in obesity and type 2 diabetes (T2D). Here we report that, in 196 subjects, RBP4 is preferentially expressed in visceral (Vis) versus subcutaneous (SC) fat. Vis fat RBP4 mRNA was increased approximately 60-fold and 12-fold in Vis and SC obese subjects respectively versus lean subjects, and approximately 2-fold with impaired glucose tolerance/T2D subjects versus normoglycemic subjects. In obese subjects, serum RBP4 was increased 2- to 3-fold, and serum transthyretin, which stabilizes RBP4 in the circulation, was increased 35%. Serum RBP4 correlated positively with adipose RBP4 mRNA and intra-abdominal fat mass and inversely with insulin sensitivity, independently of age, gender, and body mass index. RBP4 mRNA correlated inversely with GLUT4 mRNA in Vis fat and positively with adipocyte size in both depots. RBP4 levels are therefore linked to Vis adiposity, and Vis fat may be a major source of RBP4 in insulin-resistant states.     

Suppression of retinol-binding protein 4 with RNA oligonucleotide prevents high-fat diet-induced metabolic syndrome and non-alcoholic fatty liver disease in mice

Conflicting data have been reported regarding the role of retinol-binding protein (RBP4) in insulin resistance, obesity, type 2 diabetes and non-alcoholic fatty liver disease (NAFLD). In this study, we used pharmacological methods to investigate the role of RBP4. RNA oligonucleotide against RBP4 (anti-RBP4 oligo) was transfected into 3T3-L1 adipocytes. RT-PCR analysis showed that RBP4 mRNA expression decreased by 55% (p<0.01) compared with control cells. Validated RNA oligo was used in an in vivo study with high fat diet (HFD) fed - mice. 14 weeks of HFD feeding increased RBP4 expression (associated with elevated serum levels measured with immunoblotting and ELISA) by 56% in adipose tissue (p<0.05) and 68% in the liver (p<0.01). Adipose RBP4 levels were significantly reduced after 4 weeks treatment with anti-RBP4 oligo (25mg/kg, p<0.01) and rosiglitazone (RSG, 10mg/kg, p<0.05) compared with scrambled RNA oligo (25mg/kg) treated mice. Only anti-RBP4 oligo significantly inhibited RBP4 protein (p<0.01) and mRNA expression (p<0.01) in the liver and reduced serum RBP4 levels. Anti-RBP4 oligo and RSG showed comparable effects on impaired glucose tolerance, hyperinsulinaemia and hyperglycaemia. Anti-RBP4 oligo significantly enhanced adipose-GLUT4 expression (p<0.01) but did not increase muscle-GLUT4. Both RSG and anti-RBP4 oligo significantly reduced hepatic phosphoenolpyruvate carboxykinase expression (both p<0.05). Histological analysis revealed that anti-RBP4 oligo ameliorated hepatic steatosis and reduced lipid droplets associated with normalized liver function. Histological and pharmacological results of this study indicate that RBP4 is not only an adipocytokine, but also a hepatic cytokine leading to metabolic syndrome, NAFLD and type 2 diabetes.     

Downregulation of STRA6 in Adipocytes and Adipose Stromovascular Fraction in Obesity and Effects of Adipocyte-Specific STRA6 Knockdown In Vivo

To investigate the mechanisms by which elevated retinol-binding protein 4 (RBP4) causes insulin resistance, we studied the role of the high-affinity receptor for RBP4, STRA6 (stimulated by retinoic acid), in insulin resistance and obesity. In high-fat-diet-fed and ob/ob mice, STRA6 expression was decreased 70 to 95% in perigonadal adipocytes and both perigonadal and subcutaneous adipose stromovascular cells. To determine whether downregulation of STRA6 in adipocytes contributes to insulin resistance, we generated adipose-Stra6^−/− mice. Adipose-Stra6^−/− mice fed chow had decreased body weight, fat mass, leptin levels, insulin levels, and adipocyte number and increased expression of brown fat-selective markers in white adipose tissue. When fed a high-fat diet, these mice had a mild improvement in insulin sensitivity at an age when adiposity was unchanged. STRA6 has been implicated in retinol uptake, but retinol uptake and the expression of retinoid homeostatic genes (encoding retinoic acid receptor β [RARβ], CYP26A1, and lecithin retinol acyltransferase) were not altered in adipocytes from adipose-Stra6^−/− mice, indicating that retinoid homeostasis was maintained with STRA6 knockdown. Thus, STRA6 reduction in adipocytes in adipose-Stra6^−/− mice fed chow resulted in leanness, which may contribute to their increased insulin sensitivity. However, in wild-type mice with high-fat-diet-induced obesity and in ob/ob mice, the marked downregulation of STRA6 in adipocytes and adipose stromovascular cells does not compensate for obesity-associated insulin resistance.     

Visceral adiposity is associated with serum retinol binding protein-4 levels in healthy women

Objective: Retinol binding protein‐4 (RBP4) has been reported to impair insulin sensitivity throughout the body. We investigated the relationship between serum RBP4 levels and adiposity indices as well as metabolic risk variables. Research Methods and Procedure: We recruited a total of 102 healthy women 21 to 67 years old. We assessed body composition by computed tomography and divided the study population into four groups based on body weight and visceral fat area (non‐obese without visceral adiposity, non‐obese with visceral adiposity, obese without visceral adiposity, and obese with visceral adiposity). Serum RBP4 levels were measured by radioimmunoassay. Results: Despite similar levels of total body fat, non‐obese women had lower systolic blood pressure, total cholesterol, triglyceride (TG), low‐density lipoprotein (LDL)‐cholesterol levels, insulin resistance indices, and RBP4 levels than non‐obese women with visceral adiposity and had higher high‐density lipoprotein‐cholesterol levels. Similarly, obese women without visceral adiposity had lower blood pressure, total cholesterol, TG levels, insulin resistance indices, and RBP4 levels than obese women with visceral adiposity. In addition, despite having increased body fat, obese women without visceral adiposity had lower TGs, insulin resistance indices, and serum RBP4 levels than non‐obese women with visceral adiposity. By step‐wise multiple regression analysis, visceral fat areas and LDL‐cholesterol levels independently affected RBP4 levels. Discussion: We determined that serum RBP4 levels are independently associated with visceral fat and LDL‐cholesterol levels. These results suggest that, irrespective of body weight, visceral obesity is an independent predictor of serum RBP4 levels, and RBP4 may represent a link between visceral obesity and cardiovascular disease.     

Growth Hormone Treatment of Hypophysectomized Rats Increases Catecholamine-induced Lipolysis and the Number of β-Adrenergic Receptors in Adipocytes: No Differences in the Effects of Growth Hormone on Different Fat Depots

Growth hormone (GH) has a lipolytic effect in adipose tissue but this effect may differ in adipose tissue from various fat depots. This latter possibility was investigated in the present study, in which the effects of GH in vivo on catecholamine-induced lipolysis and the number of β-adrenergic receptors in isolated adipocytes from different fat depots of hypophysectomized rats were investigated. Female and male Sprague-Dawley rats were hypophysectomized or sham-operated at 45 days of age. One week after the operation, hormonal replacement therapy with L-thyroxine and hydrocortisone acetate was given. In addition, groups of rats were treated with GH (1.33 mg/kg per day, given as two daily subcutaneous injections). After 1 week of hormonal treatment, adipocytes were isolated from the parametrial, epididymal and inguinal fat pads, and glycerol release after catecholamine-stimulation and ¹²⁵I-cyanopindolol binding were measured. Hypophysectomy resulted in a marked decrease in the lipolytic response to catecholamines. GH treatment significantly increased catecholamine-induced lipolysis with similar effects in adipocytes from parametrial or epididymal and inguinal fat depots in both female and male rats. There were no differences between norepinephrine compared with isoproterenol-induced responses. ¹²⁵I-cyanopindolol binding was reduced after hypophysectomy and normalized by GH treatment, without differences between parametrial and inguinal adipose tissue regions. We conclude that the lipolytic effects of GH in the rat may partly be mediated by a stimulatory effect on β-adrenergic receptors in adipocytes. In addition, GH exerted similar effect on catecholamine-induced lipolysis and β-adrenergic receptors in adipocytes from parametrial, epididymal and inguinal fat depots.     

Decreased clearance of serum retinol-binding protein and elevated levels of transthyretin in insulin-resistant ob/ob mice

Serum retinol-binding protein (RBP4) is secreted by liver and adipocytes and is implicated in systemic insulin resistance in rodents and humans. RBP4 normally binds to the larger transthyretin (TTR) homotetramer, forming a protein complex that reduces renal clearance of RBP4. To determine whether alterations in RBP4-TTR binding contribute to elevated plasma RBP4 levels in insulin-resistant states, we investigated RBP4-TTR interactions in leptin-deficient ob/ob mice and high-fat-fed obese mice (HFD). Gel filtration chromatography of plasma showed that 88-94% of RBP4 is contained within the RBP4-TTR complex in ob/ob and lean mice. Coimmunoprecipitation with an RBP4 antibody brought down stoichiometrically equal amounts of TTR and RBP4, indicating that TTR was not more saturated with RBP4 in ob/ob mice than in controls. However, plasma TTR levels were elevated approximately fourfold in ob/ob mice vs. controls. RBP4 injected intravenously in lean mice cleared rapidly, whereas the t(1/2) for disappearance was approximately twofold longer in ob/ob plasma. Urinary fractional excretion of RBP4 was reduced in ob/ob mice, consistent with increased retention. In HFD mice, plasma TTR levels and clearance of injected RBP4 were similar to chow-fed controls. Hepatic TTR mRNA levels were elevated approximately twofold in ob/ob but not in HFD mice. Since elevated circulating RBP4 causes insulin resistance and glucose intolerance in mice, these findings suggest that increased TTR or alterations in RBP4-TTR binding may contribute to insulin resistance by stabilizing RBP4 at higher steady-state concentrations in circulation. Lowering TTR levels or interfering with RBP4-TTR binding may enhance insulin sensitivity in obesity and type 2 diabetes.     

Impaired glucose transport in inguinal adipocytes after short-term high-sucrose feeding in mice

Diets enriched in sucrose severely impair metabolic regulation and are associated with obesity, insulin resistance and glucose intolerance. In the current study, we investigated the effect of 4 weeks high-sucrose diet (HSD) feeding in C57BL6/J mice, with specific focus on adipocyte function. Mice fed HSD had slightly increased adipose tissue mass but displayed similar hepatic triglycerides, glucose and insulin levels, and glucose clearance capacity as chow-fed mice. Interestingly, we found adipose depot-specific differences, where both the non- and insulin-stimulated glucose transports were markedly impaired in primary adipocytes isolated from the inguinal fat depot from HSD-fed mice. This was accompanied by decreased protein levels of both GLUT4 and AS160. A similar but much less pronounced trend was observed in the retroperitoneal depot. In contrast, both GLUT4 expression and insulin-stimulated glucose uptake were preserved in adipocytes isolated from epididymal adipose tissue with HSD. Further, we found a slight shift in cell size distribution towards larger cells with HSD and a significant decrease of ACC and PGC-1α expression in the inguinal adipose tissue depot. Moreover, fructose alone was sufficient to decrease GLUT4 expression in cultured, mature adipocytes.Altogether, we demonstrate that short-term HSD feeding has deleterious impact on insulin response and glucose transport in the inguinal adipose tissue depot, specifically. These changes occur before the onset of systemic glucose dysmetabolism and therefore could provide a mechanistic link to overall impaired energy metabolism reported after prolonged HSD feeding, alone or in combination with HFD.     

Interleukin-1β Downregulates RBP4 Secretion in Human Adipocytes

Aims/hypothesis

The excessive accumulation of adipose tissue in the obese state is linked to an altered secretion profile of adipocytes, chronic low-grade inflammation and metabolic complications. RBP4 has been implicated in these alterations, especially insulin resistance. The aim of the present study was to determine if a local inflammatory micro-environment in adipose tissue regulates RBP4 expression and secretion.

Methods

Human SGBS and primary adipocytes cultured with conditioned media from human THP-1 macrophages were used as an in vitro model for adipose inflammation. Adipocytes were exposed to recombinant TNF-α, IL-1β, IL-6 or IL-8. In addition, coexpression of IL-1β and RBP4 was measured in adipose tissue samples from 18 healthy females. RBP4 expression was studied by quantitative PCR and ELISA.

Results

RBP4 mRNA expression and secretion was significantly reduced upon incubation with macrophage-conditioned media in SGBS adipocytes and human primary adipocytes. Out of several factors studied we identified IL-1β as a new factor regulating RBP4. IL-1β significantly downregulated RBP4 mRNA and secretion in a time- and dose-dependent manner. IL-1β mediated its inhibitory effects on RBP4 expression via IL-1 receptor and NF-κB, as incubation with the IL-1 receptor blocking antibody and the NF-κB inhibitors CAPE and SC-514 reversed its effect. Most interestingly, RBP4 mRNA was negatively correlated with IL-1β mRNA in subcutaneous adipose tissue.

Conclusions

Adipose tissue inflammation as found in the obese state might lead to a downregulation in local RBP4 levels. IL-1β was identified as a major factor contributing to the decrease in RBP4. The increase in circulating RBP4 that often precedes the development of systemic insulin resistance is most likely unrelated to inflammatory processes in adipose tissue.     

In Vivo Dedifferentiation of Adult Adipose Cells

Introduction

Adipocytes can dedifferentiate into fibroblast-like cells in vitro and thereby acquire proliferation and multipotent capacities to participate in the repair of various organs and tissues. Whether dedifferentiation occurs under physiological or pathological conditions in vivo is unknown.

Methods

A tissue expander was placed under the inguinal fat pads of rats and gradually expanded by injection of water. Samples were collected at various time points, and morphological, histological, cytological, ultrastructural, and gene expression analyses were conducted. In a separate experiment, purified green fluorescent protein+ adipocytes were transplanted into C57 mice and collected at various time points. The transplanted adipocytes were assessed by bioluminescence imaging and whole-mount staining.

Results

The expanded fat pad was obviously thinner than the untreated fat pad on the opposite side. It was also tougher in texture and with more blood vessels attached. Hematoxylin and eosin staining and transmission electron microscopy indicated there were fewer monolocular adipocytes in the expanded fat pad and the morphology of these cells was altered, most notably their lipid content was discarded. Immunohistochemistry showed that the expanded fat pad contained an increased number of proliferative cells, which may have been derived from adipocytes. Following removal of the tissue expander, many small adipocytes were observed. Bioluminescence imaging suggested that some adipocytes survived when transplanted into an ischemic-hypoxic environment. Whole-mount staining revealed that surviving adipocytes underwent a process similar to adipocyte dedifferentiation in vitro. Monolocular adipocytes became multilocular adipocytes and then fibroblast-like cells.

Conclusions

Mature adipocytes may be able to dedifferentiate in vivo, and this may be an adipose tissue self-repair mechanism. The capacity of adipocytes to dedifferentiate into stem cell-like cells may also have a more general role in the regeneration of many tissues, notably in fat grafting.     

Resveratrol enhances fatty acid oxidation capacity and reduces resistin and Retinol-Binding Protein 4 expression in white adipocytes

Resveratrol is a naturally occurring polyphenol known to affect energy metabolism and insulin sensitivity in mice and lipogenic gene expression in adipocytes. Here, we sought to get further insight into the impact of resveratrol on adipocyte biology by studying its effects on oxidative metabolism and the expression of the insulin resistance-related adipokines resistin and Retinol-Binding Protein 4 (RBP4) in mature adipocytes. Effects were assessed in 3T3-L1 adipocytes and in adipocytes derived from primary mouse embryonic fibroblasts (MEF). Besides reducing triacylglycerol content and the mRNA levels of lipogenic genes, resveratrol treatment resulted in both models in increased mRNA levels of carnitine palmitoyltransferase 1 (a rate-limiting enzyme in mitochondrial fatty acid oxidation), reduced mRNA levels of receptor interacting protein 140 (a suppressor of oxidative metabolism), and signs of enhanced flux through the fatty acid beta-oxidation pathway. In primary MEF-derived adipocytes, the treatment also increased mitochondrial DNA content and the mRNA levels of subunit II of cytochrome oxidase (a component of the mitochondrial respiratory chain) and of uncoupling protein 1. Expression of resistin and RBP4 was reduced in both adipocyte models following resveratrol treatment. The results indicate that resveratrol directly acts in mature white adipocytes to favor a remodeling toward increased oxidative capacity and reduced lipogenesis, while down-regulating two putative insulin resistance factors. These results constitute novel insights into resveratrol action in adipocytes that add to the potential of this food phytochemical and its synthetic analogues for the control of obesity and related metabolic disorders.     

Retinoids and retinoid-binding protein expression in rat adipocytes.

Adipose tissue has been reported to contain relatively high levels of the specific mRNA for retinol-binding protein (RBP) (Makover A., Soprano, D.R., Wyatt, M. L., and Goodman, D.S. (1989) J. Lipid Res. 30, 171-180). Studies were conducted to explore retinoid and retinoid-binding protein storage and metabolism in adipose tissue. In these studies, we measured RBP and cellular retinol-binding protein (CRBP) mRNA levels and retinoid levels in 6 adipose depots in male rats. Total RNA was isolated from inguinal, dorsal, mesenteric, epididymal, perinephric, and brown adipose tissue, and average RBP and CRBP mRNA levels were determined by Northern blot analysis. The relative levels of RBP mRNA in these 6 anatomically different adipose depots averaged, respectively, 6.3, 6.7, 16, 34, 37, and 21% of the level in a rat liver RNA standard. Retinoid levels in the 6 depots were similar and averaged approximately 6-7 micrograms of retinol eq/g of adipose tissue. Since adipose tissue contains several cell types, the cellular localizations of RBP and CRBP expression and retinoid storage were examined. RNA was prepared from isolated rat adipocytes and stromal-vascular cells. Cellular levels of the mRNAs for RBP, CRBP, apolipoprotein E (apoE), lipoprotein lipase, adipocyte P2, and adipsin were measured by Northern blot analysis. RBP was expressed almost exclusively in the adipocytes and only weakly in the stromal-vascular cells. Both CRBP and apoE mRNA levels were relatively high in the stromal-vascular cell preparations and only very low mRNA levels were found in the adipocytes. Lipoprotein lipase, adipsin, and adipocyte P2 mRNAs were found in substantial levels in both the adipocytes and stromal-vascular cells, but with higher levels present in the adipocytes. Cultured adipocytes synthesized RBP protein and secreted it into the medium. Only adipocytes (not stromal-vascular cells) contained retinol, at levels between 0.65-0.8 micrograms of retinol eq/10(6) cells. These studies demonstrate that adipocytes store retinoid and synthesize and secrete RBP, and suggest that rat adipocytes may be dynamically involved in retinoid storage and metabolism.     

p66Shc-generated oxidative signal promotes fat accumulation

Reactive oxygen species (ROS) and insulin signaling in the adipose tissue are critical determinants of aging and age-associated diseases. It is not clear, however, if they represent independent factors or they are mechanistically linked. We investigated the effects of ROS on insulin signaling using as model system the p66(Shc)-null mice. p66(Shc) is a redox enzyme that generates mitochondrial ROS and promotes aging in mammals. We report that insulin activates the redox enzyme activity of p66(Shc) specifically in adipocytes and that p66(Shc)-generated ROS regulate insulin signaling through multiple mechanisms, including AKT phosphorylation, Foxo localization, and regulation of selected insulin target genes. Deletion of p66(Shc) resulted in increased mitochondrial uncoupling and reduced triglyceride accumulation in adipocytes and in vivo increased metabolic rate and decreased fat mass and resistance to diet-induced obesity. In addition, p66(Shc-/-) mice showed impaired thermo-insulation. These findings demonstrate that p66(Shc)-generated ROS regulate the effect of insulin on the energetic metabolism in mice and suggest that intracellular oxidative stress might accelerate aging by favoring fat deposition and fat-related disorders.     

Targeted overexpression of inducible 6-phosphofructo-2-kinase in adipose tissue increases fat deposition but protects against diet-induced insulin resistance and inflammatory responses

Increasing evidence demonstrates the dissociation of fat deposition, the inflammatory response, and insulin resistance in the development of obesity-related metabolic diseases. As a regulatory enzyme of glycolysis, inducible 6-phosphofructo-2-kinase (iPFK2, encoded by PFKFB3) protects against diet-induced adipose tissue inflammatory response and systemic insulin resistance independently of adiposity. Using aP2-PFKFB3 transgenic (Tg) mice, we explored the ability of targeted adipocyte PFKFB3/iPFK2 overexpression to modulate diet-induced inflammatory responses and insulin resistance arising from fat deposition in both adipose and liver tissues. Compared with wild-type littermates (controls) on a high fat diet (HFD), Tg mice exhibited increased adiposity, decreased adipose inflammatory response, and improved insulin sensitivity. In a parallel pattern, HFD-fed Tg mice showed increased hepatic steatosis, decreased liver inflammatory response, and improved liver insulin sensitivity compared with controls. In both adipose and liver tissues, increased fat deposition was associated with lipid profile alterations characterized by an increase in palmitoleate. Additionally, plasma lipid profiles also displayed an increase in palmitoleate in HFD-Tg mice compared with controls. In cultured 3T3-L1 adipocytes, overexpression of PFKFB3/iPFK2 recapitulated metabolic and inflammatory changes observed in adipose tissue of Tg mice. Upon treatment with conditioned medium from iPFK2-overexpressing adipocytes, mouse primary hepatocytes displayed metabolic and inflammatory responses that were similar to those observed in livers of Tg mice. Together, these data demonstrate a unique role for PFKFB3/iPFK2 in adipocytes with regard to diet-induced inflammatory responses in both adipose and liver tissues.