Crosstalk between corepressor NRIP1 and cAMP signaling on adipocyte thermogenic programming.

OBJECTIVES: Nuclear receptor interacting protein 1 (NRIP1) suppresses energy expenditure via repression of nuclear receptors, and its depletion markedly elevates uncoupled respiration in mouse and human adipocytes. We tested whether NRIP1 deficient adipocytes implanted into obese mice would enhance whole body metabolism. Since ß-adrenergic signaling through cAMP strongly promotes adipocyte thermogenesis, we tested whether the effects of NRIP1 knock-out (NRIP1KO) require the cAMP pathway. METHODS: NRIP1KO adipocytes were implanted in recipient high-fat diet (HFD) fed mice and metabolic cage studies conducted. The Nrip1 gene was disrupted by CRISPR in primary preadipocytes isolated from control vs adipose selective GsαKO (cAdGsαKO) mice prior to differentiation to adipocytes. Protein kinase A inhibitor was also used. RESULTS: Implanting NRIP1KO adipocytes into HFD fed mice enhanced whole-body glucose tolerance by increasing insulin sensitivity, reducing adiposity, and enhancing energy expenditure in the recipients. NRIP1 depletion in both control and GsαKO adipocytes was equally effective in upregulating uncoupling protein 1 (UCP1) and adipocyte beiging, while ß-adrenergic signaling by CL 316,243 was abolished in GsαKO adipocytes. Combining NRIP1KO with CL 316,243 treatment synergistically increased Ucp1 gene expression and increased the adipocyte subpopulation responsive to beiging. Estrogen-related receptor α (ERRα) was dispensable for UCP1 upregulation by NRIPKO. CONCLUSIONS: The thermogenic effect of NRIP1 depletion in adipocytes causes systemic enhancement of energy expenditure when such adipocytes are implanted into obese mice. Furthermore, NRIP1KO acts independently but cooperatively with the cAMP pathway in mediating its effect on adipocyte beiging.

Acetyl-CoA carboxylase 1 is a suppressor of the adipocyte thermogenic program.

Disruption of adipocyte de novo lipogenesis (DNL) by deletion of fatty acid synthase (FASN) in mice induces browning in inguinal white adipose tissue (iWAT). However, adipocyte FASN knockout (KO) increases acetyl-coenzyme A (CoA) and malonyl-CoA in addition to depletion of palmitate. We explore which of these metabolite changes triggers adipose browning by generating eight adipose-selective KO mouse models with loss of ATP-citrate lyase (ACLY), acetyl-CoA carboxylase 1 (ACC1), ACC2, malonyl-CoA decarboxylase (MCD) or FASN, or dual KOs ACLY/FASN, ACC1/FASN, and ACC2/FASN. Preventing elevation of acetyl-CoA and malonyl-CoA by depletion of adipocyte ACLY or ACC1 in combination with FASN KO does not block the browning of iWAT. Conversely, elevating malonyl-CoA levels in MCD KO mice does not induce browning. Strikingly, adipose ACC1 KO induces a strong iWAT thermogenic response similar to FASN KO while also blocking malonyl-CoA and palmitate synthesis. Thus, ACC1 and FASN are strong suppressors of adipocyte thermogenesis through promoting lipid synthesis rather than modulating the DNL intermediates acetyl-CoA or malonyl-CoA.

De novo lipogenesis fuels adipocyte autophagosome and lysosome membrane dynamics.

Adipocytes robustly synthesize fatty acids (FA) from carbohydrate through the de novo lipogenesis (DNL) pathway, yet surprisingly DNL contributes little to their abundant triglyceride stored in lipid droplets. This conundrum raises the hypothesis that adipocyte DNL instead enables membrane expansions to occur in processes like autophagy, which requires an abundant supply of phospholipids. We report here that adipocyte Fasn deficiency in vitro and in vivo markedly impairs autophagy, evident by autophagosome accumulation and severely compromised degradation of the autophagic substrate p62. Our data indicate the impairment occurs at the level of autophagosome-lysosome fusion, and indeed, loss of Fasn decreases certain membrane phosphoinositides necessary for autophagosome and lysosome maturation and fusion. Autophagy dependence on FA produced by Fasn is not fully alleviated by exogenous FA in cultured adipocytes, and interestingly, imaging studies reveal that Fasn colocalizes with nascent autophagosomes. Together, our studies identify DNL as a critical source of FAs to fuel autophagosome and lysosome maturation and fusion in adipocytes.

The adipocyte supersystem of insulin and cAMP signaling.

Adipose tissue signals to brain, liver, and muscles to control whole body metabolism through secreted lipid and protein factors as well as neurotransmission, but the mechanisms involved are incompletely understood. Adipocytes sequester triglyceride (TG) in fed conditions stimulated by insulin, while in fasting catecholamines trigger TG hydrolysis, releasing glycerol and fatty acids (FAs). These antagonistic hormone actions result in part from insulin's ability to inhibit cAMP levels generated through such G-protein-coupled receptors as catecholamine-activated ß-adrenergic receptors. Consistent with these antagonistic signaling modes, acute actions of catecholamines cause insulin resistance. Yet, paradoxically, chronically activating adipocytes by catecholamines cause increased glucose tolerance, as does insulin. Recent results have helped to unravel this conundrum by revealing enhanced complexities of these hormones' signaling networks, including identification of unexpected common signaling nodes between these canonically antagonistic hormones.

CRISPR-enhanced human adipocyte browning as cell therapy for metabolic disease.

Obesity and type 2 diabetes are associated with disturbances in insulin-regulated glucose and lipid fluxes and severe comorbidities including cardiovascular disease and steatohepatitis. Whole body metabolism is regulated by lipid-storing white adipocytes as well as "brown" and "brite/beige" adipocytes that express thermogenic uncoupling protein 1 (UCP1) and secrete factors favorable to metabolic health. Implantation of brown fat into obese mice improves glucose tolerance, but translation to humans has been stymied by low abundance of primary human beige adipocytes. Here we apply methods to greatly expand human adipocyte progenitors from small samples of human subcutaneous adipose tissue and then disrupt the thermogenic suppressor gene NRIP1 by CRISPR. Ribonucleoprotein consisting of Cas9 and sgRNA delivered ex vivo are fully degraded by the human cells following high efficiency NRIP1 depletion without detectable off-target editing. Implantation of such CRISPR-enhanced human or mouse brown-like adipocytes into high fat diet fed mice decreases adiposity and liver triglycerides while enhancing glucose tolerance compared to implantation with unmodified adipocytes. These findings advance a therapeutic strategy to improve metabolic homeostasis through CRISPR-based genetic enhancement of human adipocytes without exposing the recipient to immunogenic Cas9 or delivery vectors.

Single-Cell RNA Profiling Reveals Adipocyte to Macrophage Signaling Sufficient to Enhance Thermogenesis.

Adipocytes deficient in fatty acid synthase (iAdFASNKO) emit signals that mimic cold exposure to enhance the appearance of thermogenic beige adipocytes in mouse inguinal white adipose tissues (iWATs). Both cold exposure and iAdFASNKO upregulate the sympathetic nerve fiber (SNF) modulator Neuregulin 4 (Nrg4), activate SNFs, and require adipocyte cyclic AMP/protein kinase A (cAMP/PKA) signaling for beige adipocyte appearance, as it is blocked by adipocyte Gsα deficiency. Surprisingly, however, in contrast to cold-exposed mice, neither iWAT denervation nor Nrg4 loss attenuated adipocyte browning in iAdFASNKO mice. Single-cell transcriptomic analysis of iWAT stromal cells revealed increased macrophages displaying gene expression signatures of the alternately activated type in iAdFASNKO mice, and their depletion abrogated iWAT beiging. Altogether, these findings reveal that divergent cellular pathways are sufficient to cause adipocyte browning. Importantly, adipocyte signaling to enhance alternatively activated macrophages in iAdFASNKO mice is associated with enhanced adipose thermogenesis independent of the sympathetic neuron involvement this process requires in the cold.

Control of Adipocyte Thermogenesis and Lipogenesis through ß3-Adrenergic and Thyroid Hormone Signal Integration.

Here, we show that ß adrenergic signaling coordinately upregulates de novo lipogenesis (DNL) and thermogenesis in subcutaneous white adipose tissue (sWAT), and both effects are blocked in mice lacking the cAMP-generating G protein-coupled receptor Gs (Adipo-GsαKO) in adipocytes. However, UCP1 expression but not DNL activation requires rapamycin-sensitive mTORC1. Furthermore, ß3-adrenergic agonist CL316243 readily upregulates thermogenic but not lipogenic genes in cultured adipocytes, indicating that additional regulators must operate on DNL in sWAT in vivo. We identify one such factor as thyroid hormone T3, which is elevated locally by adrenergic signaling. T3 administration to wild-type mice enhances both thermogenesis and DNL in sWAT. Mechanistically, T3 action on UCP1 expression in sWAT depends upon cAMP and is blocked in Adipo-GsαKO mice even as elevated DNL persists. Thus, T3 enhances sWAT thermogenesis by amplifying cAMP signaling, while its control of adipocyte DNL can be mediated independently of both cAMP and rapamycin-sensitive mTORC1.

Molecular pathways linking adipose innervation to insulin action in obesity and diabetes mellitus.

Adipose tissue comprises adipocytes and many other cell types that engage in dynamic crosstalk in a highly innervated and vascularized tissue matrix. Although adipose tissue has been studied for decades, it has been appreciated only in the past 5 years that extensive arborization of nerve fibres has a dominant role in regulating the function of adipose tissue. This Review summarizes the latest literature, which suggests that adipocytes signal to local sensory nerve fibres in response to perturbations in lipolysis and lipogenesis. Such adipocyte signalling to the central nervous system causes sympathetic output to distant adipose depots and potentially other metabolic tissues to regulate systemic glucose homeostasis. Paracrine factors identified in the past few years that mediate such adipocyte-neuron crosstalk are also reviewed. Similarly, immune cells and endothelial cells within adipose tissue communicate with local nerve fibres to modulate neurotransmitter tone, blood flow, adipocyte differentiation and energy expenditure, including adipose browning to produce heat. This understudied field of neurometabolism related to adipose tissue biology has great potential to reveal new mechanistic insights and potential therapeutic strategies for obesity and type 2 diabetes mellitus.

Toll-Like Receptor-4 Disruption Suppresses Adipose Tissue Remodeling and Increases Survival in Cancer Cachexia Syndrome.

Cancer-induced cachexia, characterized by systemic inflammation, body weight loss, adipose tissue (AT) remodeling and muscle wasting, is a malignant metabolic syndrome with undefined etiology. Here, we show that both genetic ablation and pharmacological inhibition of TLR4 were able to attenuate the main clinical markers of cachexia in mice bearing Lewis lung carcinoma (LLC). AT remodelling was not found in LLC tumor-bearing (TB) TLR4-/- mice due to reduced macrophage infiltration and adipocyte atrophy. TLR4-/- mice were also resistant to cold-induced browning of subcutaneous AT (scAT). Importantly, pharmacological inhibition of TLR4 (Atorvastatin) reproduced the main protective effect against AT remodeling found in TLR4-/- TB mice. Moreover, the treatment was effective in prolonging survival and attenuating tumor mass growth when compared to non-treated-TB animals. Furthermore, tumor-induced elevation of circulating pro-inflammatory cytokines was similarly abolished in both genetic ablation and pharmacological inhibition of TLR4. These data suggest that TLR4 is a critical mediator and a promising target for novel anti-cachexia therapies.

Neuronal modulation of brown adipose activity through perturbation of white adipocyte lipogenesis.

OBJECTIVE: Crosstalk between adipocytes and local neurons may be an important regulatory mechanism to control energy homeostasis. We previously reported that perturbation of adipocyte de novo lipogenesis (DNL) by deletion of fatty acid synthase (FASN) expands sympathetic neurons within white adipose tissue (WAT) and stimulates the appearance of "beige" adipocytes. Here we tested whether WAT DNL activity can also influence neuronal regulation and thermogenesis in brown adipose tissue (BAT). METHODS AND RESULTS: Induced deletion of FASN in all adipocytes in mature mice (iAdFASNKO) enhanced sympathetic innervation and neuronal activity as well as UCP1 expression in both WAT and BAT. This increased sympathetic innervation could be observed at both 22 °C and 30 °C, indicating it is not a response to heat loss but rather adipocyte signaling. In contrast, selective ablation of FASN in brown adipocytes of mice (iUCP1FASNKO) failed to modulate sympathetic innervation and the thermogenic program in BAT. Surprisingly, DNL in brown adipocytes was also dispensable in maintaining euthermia when UCP1FASNKO mice were cold-exposed. CONCLUSION: These results indicate that DNL in white adipocytes influences long distance signaling to BAT, which can modify BAT sympathetic innervation and expression of genes involved in thermogenesis.

Adipocyte lipid synthesis coupled to neuronal control of thermogenic programming.

BACKGROUND: The de novo biosynthesis of fatty acids (DNL) through fatty acid synthase (FASN) in adipocytes is exquisitely regulated by nutrients, hormones, fasting, and obesity in mice and humans. However, the functions of DNL in adipocyte biology and in the regulation of systemic glucose homeostasis are not fully understood. METHODS & RESULTS: Here we show adipocyte DNL controls crosstalk to localized sympathetic neurons that mediate expansion of beige/brite adipocytes within inguinal white adipose tissue (iWAT). Induced deletion of FASN in white and brown adipocytes of mature mice (iAdFASNKO mice) enhanced glucose tolerance, UCP1 expression, and cAMP signaling in iWAT. Consistent with induction of adipose sympathetic nerve activity, iAdFASNKO mice displayed markedly increased neuronal tyrosine hydroxylase (TH) and neuropeptide Y (NPY) content in iWAT. In contrast, brown adipose tissue (BAT) of iAdFASNKO mice showed no increase in TH or NPY, nor did FASN deletion selectively in brown adipocytes (UCP1-FASNKO mice) cause these effects in iWAT. CONCLUSIONS: These results demonstrate that downregulation of fatty acid synthesis via FASN depletion in white adipocytes of mature mice can stimulate neuronal signaling to control thermogenic programming in iWAT.

Early suppression of adipocyte lipid turnover induces immunometabolic modulation in cancer cachexia syndrome.

Cancer cachexia is a multifactorial syndrome characterized by body weight loss, atrophy of adipose tissue (AT) and systemic inflammation. However, there is limited information regarding the mechanisms of immunometabolic response in AT from cancer cachexia. Male Wistar rats were inoculated with 2 × 107 of Walker 256 tumor cells [tumor bearing (TB) rats]. The mesenteric AT (MeAT) was collected on d 0, 4, 7 (early stage), and 14 (cachexia stage) after tumor cell injection. Surgical biopsies for MeAT were obtained from patients who had gastrointestinal cancer with cachexia. Lipolysis showed an early decrease in glycerol release in TB d 4 (TB4) rats in relation to the control, followed by a 6-fold increase in TB14 rats, whereas de novo lipogenesis was markedly lower in the incorporation of glucose into fatty acids in TB14 rats during the development of cachexia. CD11b and CD68 were positive in TB7 and TB14 rats, respectively. In addition, we found cachexia stage results similar to those of animals in MeAT from patients: an increased presence of CD68+, iNOS2+, TNFα+, and HSL+ cells. In summary, translational analysis of MeAT from patients and an animal model of cancer cachexia enabled us to identify early disruption in Adl turnover and subsequent inflammatory response during the development of cancer cachexia.-Henriques, F. S., Sertié, R. A. L., Franco, F. O., Knobl, P., Neves, R. X., Andreotti, S., Lima, F. B., Guilherme, A., Seelaender, M., Batista, M. L., Jr. Early suppression of adipocyte lipid turnover induces immunometabolic modulation in cancer cachexia syndrome.

Activation of mTORC1 is essential for ß-adrenergic stimulation of adipose browning.

A classic metabolic concept posits that insulin promotes energy storage and adipose expansion, while catecholamines stimulate release of adipose energy stores by hydrolysis of triglycerides through ß-adrenergic receptor (ßARs) and protein kinase A (PKA) signaling. Here, we have shown that a key hub in the insulin signaling pathway, activation of p70 ribosomal S6 kinase (S6K1) through mTORC1, is also triggered by PKA activation in both mouse and human adipocytes. Mice with mTORC1 impairment, either through adipocyte-specific deletion of Raptor or pharmacologic rapamycin treatment, were refractory to the well-known ßAR-dependent increase of uncoupling protein UCP1 expression and expansion of beige/brite adipocytes (so-called browning) in white adipose tissue (WAT). Mechanistically, PKA directly phosphorylated mTOR and RAPTOR on unique serine residues, an effect that was independent of insulin/AKT signaling. Abrogation of the PKA site within RAPTOR disrupted ßAR/mTORC1 activation of S6K1 without affecting mTORC1 activation by insulin. Conversely, a phosphomimetic RAPTOR augmented S6K1 activity. Together, these studies reveal a signaling pathway from ßARs and PKA through mTORC1 that is required for adipose browning by catecholamines and provides potential therapeutic strategies to enhance energy expenditure and combat metabolic disease.

A major role of insulin in promoting obesity-associated adipose tissue inflammation.

OBJECTIVE: Adipose tissue (AT) inflammation is associated with systemic insulin resistance and hyperinsulinemia in obese rodents and humans. A longstanding concept is that hyperinsulinemia may promote systemic insulin resistance through downregulation of its receptor on target tissues. Here we tested the novel hypothesis that insulin also impairs systemic insulin sensitivity by specifically enhancing adipose inflammation. METHODS: Circulating insulin levels were reduced by about 50% in diet-induced and genetically obese mice by treatments with diazoxide or streptozotocin, respectively. We then examined AT crown-like structures, macrophage markers and pro-inflammatory cytokine expression in AT. AT lipogenesis and systemic insulin sensitivity was also monitored. Conversely, insulin was infused into lean mice to determine its affects on the above parameters. RESULTS: Lowering circulating insulin levels in obese mice by streptozotocin treatment decreased macrophage content in AT, enhancing insulin stimulated Akt phosphorylation and de novo lipogenesis (DNL). Moreover, responsiveness of blood glucose levels to injected insulin was improved by streptozotocin and diazoxide treatments of obese mice without changes in body weight. Remarkably, even in lean mice, infusion of insulin under constant euglycemic conditions stimulated expression of cytokines in AT. Consistent with these findings, insulin treatment of 3T3-L1 adipocytes caused a 10-fold increase in CCL2 mRNA levels within 6 h, which was blocked by the ERK inhibitor PD98059. CONCLUSION: Taken together, these results indicate that obesity-associated hyperinsulinemia unexpectedly drives AT inflammation in obese mice, which in turn contributes to factors that suppress insulin-stimulated adipocyte DNL and systemic insulin sensitivity.

The Lipid Droplet Protein Hypoxia-inducible Gene 2 Promotes Hepatic Triglyceride Deposition by Inhibiting Lipolysis.

The liver is a major site of glucose, fatty acid, and triglyceride (TG) synthesis and serves as a major regulator of whole body nutrient homeostasis. Chronic exposure of humans or rodents to high-calorie diets promotes non-alcoholic fatty liver disease, characterized by neutral lipid accumulation in lipid droplets (LD) of hepatocytes. Here we show that the LD protein hypoxia-inducible gene 2 (Hig2/Hilpda) functions to enhance lipid accumulation in hepatocytes by attenuating TG hydrolysis. Hig2 expression increased in livers of mice on a high-fat diet and during fasting, two states associated with enhanced hepatic TG content. Hig2 expressed in primary mouse hepatocytes localized to LDs and promoted LD TG deposition in the presence of oleate. Conversely, tamoxifen-inducible Hig2 deletion reduced both TG content and LD size in primary hepatocytes from mice harboring floxed alleles of Hig2 and a cre/ERT2 transgene controlled by the ubiquitin C promoter. Hepatic TG was also decreased by liver-specific deletion of Hig2 in mice with floxed Hig2 expressing cre controlled by the albumin promoter. Importantly, we demonstrate that Hig2-deficient hepatocytes exhibit increased TG lipolysis, TG turnover, and fatty acid oxidation as compared with controls. Interestingly, mice with liver-specific Hig2 deletion also display improved glucose tolerance. Taken together, these data indicate that Hig2 plays a major role in promoting lipid sequestration within LDs in mouse hepatocytes through a mechanism that impairs TG degradation.

Map4k4 suppresses Srebp-1 and adipocyte lipogenesis independent of JNK signaling.

Adipose tissue lipogenesis is paradoxically impaired in human obesity, promoting ectopic triglyceride (TG) deposition, lipotoxicity, and insulin resistance. We previously identified mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4), a sterile 20 protein kinase reported to be upstream of c-Jun NH2-terminal kinase (JNK) signaling, as a novel negative regulator of insulin-stimulated glucose transport in adipocytes. Using full-genome microarray analysis we uncovered a novel role for Map4k4 as a suppressor of lipid synthesis. We further report here the surprising finding that Map4k4 suppresses adipocyte lipogenesis independently of JNK. Thus, while Map4k4 silencing in adipocytes enhances the expression of lipogenic enzymes, concomitant with increased conversion of (14)C-glucose and (14)C-acetate into TGs and fatty acids, JNK1 and JNK2 depletion causes the opposite effects. Furthermore, high expression of Map4k4 fails to activate endogenous JNK, while Map4k4 depletion does not attenuate JNK activation by tumor necrosis factor α. Map4k4 silencing in cultured adipocytes elevates both the total protein expression and cleavage of sterol-regulated element binding protein-1 (Srebp-1) in a rapamycin-sensitive manner, consistent with Map4k4 signaling via mechanistic target of rapamycin complex 1 (mTORC1). We show Map4k4 depletion requires Srebp-1 upregulation to increase lipogenesis and further show that Map4k4 promotes AMP-protein kinase (AMPK) signaling and the phosphorylation of mTORC1 binding partner raptor (Ser792) to inhibit mTORC1. Our results indicate that Map4k4 inhibits adipose lipogenesis by suppression of Srebp-1 in an AMPK- and mTOR-dependent but JNK-independent mechanism.

Association of common genetic variants in the MAP4K4 locus with prediabetic traits in humans.

Mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) is expressed in all diabetes-relevant tissues and mediates cytokine-induced insulin resistance. We investigated whether common single nucleotide polymorphisms (SNPs) in the MAP4K4 locus associate with glucose intolerance, insulin resistance, impaired insulin release, or elevated plasma cytokines. The best hit was tested for association with type 2 diabetes. Subjects (N = 1,769) were recruited from the Tübingen Family (TÜF) study for type 2 diabetes and genotyped for tagging SNPs. In a subgroup, cytokines were measured. Association with type 2 diabetes was tested in a prospective case-cohort study (N = 2,971) derived from the EPIC-Potsdam study. Three SNPs (rs6543087, rs17801985, rs1003376) revealed nominal and two SNPs (rs11674694, rs11678405) significant associations with 2-hour glucose levels. SNPs rs6543087 and rs11674694 were also nominally associated with decreased insulin sensitivity. Another two SNPs (rs2236936, rs2236935) showed associations with reduced insulin release, driven by effects in lean subjects only. Three SNPs (rs11674694, rs13003883, rs2236936) revealed nominal associations with IL-6 levels. SNP rs11674694 was significantly associated with type 2 diabetes. In conclusion, common variation in MAP4K4 is associated with insulin resistance and ß-cell dysfunction, possibly via this gene's role in inflammatory signalling. This variation's impact on insulin sensitivity may be more important since its effect on insulin release vanishes with increasing BMI.

Map4k4 negatively regulates peroxisome proliferator-activated receptor (PPAR) gamma protein translation by suppressing the mammalian target of rapamycin (mTOR) signaling pathway in cultured adipocytes.

The receptor peroxisome proliferator-activated receptor gamma (PPARgamma) is considered a master regulator of adipocyte differentiation and promotes glucose and lipid metabolism in mature adipocytes. We recently identified the yeast Sterile 20 (Ste20) protein kinase ortholog, Map4k4, in an RNA interference-based screen as an inhibitor of PPARgamma expression in cultured adipocytes. Here, we show that RNA interference-mediated silencing of Map4k4 elevates the levels of both PPARgamma1 and PPARgamma2 proteins in 3T3-L1 adipocytes without affecting PPARgamma mRNA levels, suggesting that Map4k4 regulates PPARgamma at a post-transcriptional step. PPARgamma degradation rates are remarkably rapid as measured in the presence of cycloheximide (t(1/2) = 2 h), but silencing Map4k4 had no effect on PPARgamma degradation. However, depletion of Map4k4 significantly enhances [(35)S]methionine/cysteine incorporation into proteins, suggesting that Map4k4 signaling decreases protein translation. We show a function of Map4k4 is to inhibit rapamycin-sensitive mammalian target of rapamycin (mTOR) activity, decreasing 4E-BP1 phosphorylation. In addition, our results show mTOR and 4E-BP1 are required for the increased PPARgamma protein expression upon Map4k4 knockdown. Consistent with this concept, adenovirus-mediated expression of Map4k4 decreased PPARgamma protein levels and mTOR phosphorylation. These data show that Map4k4 negatively regulates PPARgamma post-transcriptionally, by attenuating mTOR signaling and a 4E-BP1-dependent mechanism.

Tumor necrosis factor-alpha induces caspase-mediated cleavage of peroxisome proliferator-activated receptor gamma in adipocytes.

The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand-dependent transcription factor that acts as a primary regulator of adipogenesis and controls adipocyte metabolism and insulin action. Increased expression of tumor necrosis factor (TNFalpha) in adipose tissue of obese subjects potently suppresses the expression of PPARgamma and attenuates adipocyte functions. Here we show that PPARgamma is a substrate of caspase-3 and caspase-6 during TNFalpha receptor signaling in adipocytes, and the consequent PPARgamma cleavage disrupts its nuclear localization. TNFalpha treatment of 3T3-L1 adipocytes decreases full-length PPARgamma while increasing the level of a 45-kDa immunoreactive PPARgamma fragment. Specific inhibitors of caspase-3 and caspase-6 attenuate the cleavage of PPARgamma protein in response to TNFalpha in cultured adipocytes. Incubation of nuclear fractions with recombinant caspase-3 and caspase-6 also generates a 45-kDa PPARgamma cleavage product. Dispersion of nuclear PPARgamma to the cytoplasm in response to TNFalpha treatment occurs in parallel with detection of activated caspase-3. We suggest that activation of the caspase cascade by TNFalpha down-regulates PPARgamma protein and PPARgamma-mediated metabolic processes in adipose cells.

Adipocyte dysfunctions linking obesity to insulin resistance and type 2 diabetes.

Acquired resistance to the action of insulin to stimulate glucose transport in skeletal muscle is associated with obesity and promotes the development of type 2 diabetes. In skeletal muscle, insulin resistance can result from high levels of circulating fatty acids that disrupt insulin signalling pathways. However, the severity of insulin resistance varies greatly among obese people. Here we postulate that this variability might reflect differences in levels of lipid-droplet proteins that promote the sequestration of fatty acids within adipocytes in the form of triglycerides, thereby lowering exposure of skeletal muscle to the inhibitory effects of fatty acids.