Adipocyte subpopulations mediate lipolysis and obesity-induced insulin resistance.

Studies in humans and mice have determined that distinct subpopulations of adipocytes reside even within individual adipose tissue depots. Previously, our lab defined three white adipocyte subpopulations with stable and unique gene expression profiles, which were termed type 1, 2, and 3 adipocytes, respectively. Our previous studies demonstrated that type 2 adipocytes were highly responsive to the inflammatory cytokine, tumor necrosis factor alpha (TNFα). This study extends these findings to investigate the role of type 2 adipocytes in obesity. We found that treatment with TNFα increased lipolysis specifically in type 2 adipocytes, at least in part, through the reduction of fat-specific protein 27 (FSP27) expression. To assess the physiological role of lipolysis from this adipocyte subpopulation, a type2Ad-hFSP27tg mouse model was generated by overexpressing human FSP27 specifically in type 2 adipocytes. Glucose and insulin tolerance test analysis showed that male type2Ad-hFSP27tg mice on 60% high-fat diet exhibited improved glucose tolerance and insulin sensitivity, with no change in body weight compared to controls. These metabolic changes may, at least in part, be explained by the reduced lipolysis rate in the visceral fat of type2Ad-hFSP27tg mice. Although FSP27 overexpression in primary type 2 adipocytes was sufficient to acutely reduce TNFα-induced apoptosis in vitro, it failed to reduce macrophage infiltration in obesity in vivo. Taken together, these results strongly suggest that type 2 adipocytes contribute to the regulation of lipolysis and could serve as a potential therapeutic target for obesity-associated insulin resistance.

Endothelial-Specific Expression of CIDEC Improves High-Fat Diet-Induced Vascular and Metabolic Dysfunction.

Cell death-inducing DNA fragmentation factor-α-like effector C (CIDEC), originally identified to be a lipid droplet-associated protein in adipocytes, positively associates with insulin sensitivity. Recently, we discovered that it is expressed abundantly in human endothelial cells and regulates vascular function. The current study was designed to characterize the physiological effects and molecular actions of endothelial CIDEC in the control of vascular phenotype and whole-body glucose homeostasis. To achieve this, we generated a humanized mouse model expressing endothelial-specific human CIDEC (E-CIDECtg). E-CIDECtg mice exhibited protection against high-fat diet-induced glucose intolerance, insulin resistance, and dyslipidemia. Moreover, these mice displayed improved insulin signaling and endothelial nitric oxide synthase activation, enhanced endothelium-dependent vascular relaxation, and improved vascularization of adipose tissue, skeletal muscle, and heart. Mechanistically, we identified a novel interplay of CIDEC-vascular endothelial growth factor A (VEGFA)-vascular endothelial growth factor receptor 2 (VEGFR2) that reduced VEGFA and VEGFR2 degradation, thereby increasing VEGFR2 activation. Overall, our results demonstrate a protective role of endothelial CIDEC against obesity-induced metabolic and vascular dysfunction, in part, by modulation of VEGF signaling. These data suggest that CIDEC may be investigated as a potential future therapeutic target for mitigating obesity-related cardiometabolic disease.

Human CIDEC transgene improves lipid metabolism and protects against high-fat diet-induced glucose intolerance in mice.

Cell death-inducing DNA fragmentation factor-like effector C (CIDEC) expression in adipose tissue positively correlates with insulin sensitivity in obese humans. Further, E186X, a single-nucleotide CIDEC variant is associated with lipodystrophy, hypertriglyceridemia, and insulin resistance. To establish the unknown mechanistic link between CIDEC and maintenance of systemic glucose homeostasis, we generated transgenic mouse models expressing CIDEC (Ad-CIDECtg) and CIDEC E186X variant (Ad-CIDECmut) transgene specifically in the adipose tissue. We found that Ad-CIDECtg but not Ad-CIDECmut mice were protected against high-fat diet-induced glucose intolerance. Furthermore, we revealed the role of CIDEC in lipid metabolism using transcriptomics and lipidomics. Serum triglycerides, cholesterol, and low-density lipoproteins were lower in high-fat diet-fed Ad-CIDECtg mice compared to their littermate controls. Mechanistically, we demonstrated that CIDEC regulates the enzymatic activity of adipose triglyceride lipase via interacting with its activator, CGI-58, to reduce free fatty acid release and lipotoxicity. In addition, we confirmed that CIDEC is indeed a vital regulator of lipolysis in adipose tissue of obese humans, and treatment with recombinant CIDEC decreased triglyceride breakdown in visceral human adipose tissue. Our study unravels a central pathway whereby adipocyte-specific CIDEC plays a pivotal role in regulating adipose lipid metabolism and whole-body glucose homeostasis. In summary, our findings identify human CIDEC as a potential 'drug' or a 'druggable' target to reverse obesity-induced lipotoxicity and glucose intolerance.

Lipid Composition of the Cell Membrane Outer Leaflet Regulates Endocytosis of Nanomaterials through Alterations in Scavenger Receptor Activity.

Understanding the principles that guide the uptake of engineered nanomaterials (ENMs) by cells is of interest in biomedical and occupational health research. While evidence has started to accumulate on the role of membrane proteins in ENM uptake, the role of membrane lipid chemistry in regulating ENM endocytosis has remained largely unexplored. Here, we have addressed this issue by altering the plasma membrane lipid composition directly in live cells using a methyl-α-cyclodextrin (MαCD)-catalyzed lipid exchange method. Our observations, in an alveolar epithelial cell line and using silica nanoparticles, reveal that the lipid composition of the plasma membrane outer leaflet plays a significant role in ENM endocytosis and the intracellular fate of ENMs, by affecting nonspecific ENM diffusion into the cell, changing membrane fluidity, and altering the activity of scavenger receptors (SRs) involved in active endocytosis. These results have implications for understanding ENM uptake in different subsets of cells, depending on cell membrane lipid composition.

Fsp27 plays a crucial role in muscle performance.

Fsp27 was previously identified as a lipid droplet-associated protein in adipocytes. Various studies have shown that it plays a role in the regulation of lipid homeostasis in adipose tissue and liver. However, its function in muscle, which also accumulate and metabolize fat, remains completely unknown. Our present study identifies a novel role of Fsp27 in muscle performance. Here, we demonstrate that Fsp27-/- and Fsp27+/- mice, both males and females, had severely impaired muscle endurance and exercise capacity compared with wild-type controls. Liver and muscle glycogen stores were similar among all groups fed or fasted, and before or after exercise. Reduced muscle performance in Fsp27-/- and Fsp27+/- mice was associated with severely decreased fat content in the muscle. Furthermore, results in heterozygous Fsp27+/- mice indicate that Fsp27 haploinsufficiency undermines muscle performance in both males and females. In summary, our physiological findings reveal that Fsp27 plays a critical role in muscular fat storage, muscle endurance, and muscle strength.NEW & NOTEWORTHY This is the first study identifying Fsp27 as a novel protein associated with muscle metabolism. The Fsp27-knockout model shows that Fsp27 plays a role in muscular-fat storage, muscle endurance, and muscle strength, which ultimately impacts limb movement. In addition, our study suggests a potential metabolic paradox in which FSP27-knockout mice presumed to be metabolically healthy based on glucose utilization and oxidative metabolism are unhealthy in terms of exercise capacity and muscular performance.

Effect of growth hormone on insulin signaling.

Growth hormone (GH) is a pleiotropic hormone that coordinates an array of physiological processes, including effects on bone, muscle, and fat, ultimately resulting in growth. Metabolically, GH promotes anabolic action in most tissues except adipose, where its catabolic action causes the breakdown of stored triglycerides into free fatty acids (FFA). GH antagonizes insulin action via various molecular pathways. Chronic GH secretion suppresses the anti-lipolytic action of insulin and increases FFA flux into the systemic circulation; thus, promoting lipotoxicity, which causes pathophysiological problems, including insulin resistance. In this review, we will provide an update on GH-stimulated adipose lipolysis and its consequences on insulin signaling in liver, skeletal muscle, and adipose tissue. Furthermore, we will discuss the mechanisms that contribute to the diabetogenic action of GH.

Emerging Mechanisms of GH-Induced Lipolysis and Insulin Resistance.

Growth hormone (GH) is a pleiotropic hormone that coordinates an array of physiological processes including growth and metabolism. GH promotes anabolic action in all tissues except adipose, where it catabolizes stored fat to release energy for the promotion of growth in other tissues. However, chronic stimulation of lipolysis by GH results in an increased flux of free fatty acids (FFAs) into systemic circulation. Hence, a sustained release of high levels of GH contributes significantly to the development of insulin resistance by antagonizing the anti-lipolytic action of insulin. The molecular pathways associated with the lipolytic effect of GH in adipose tissue however, remain elusive. Recent studies have provided molecular insights into GH-induced lipolysis and impairment of insulin signaling. This review discusses the physiological and metabolic actions of GH on adipose tissue as well as GH-mediated deregulation of the FSP27-PPARγ axis which alters adipose tissue homeostasis and contributes to the development of insulin resistance and Type 2 diabetes.

Fat-Specific Protein 27 Regulation of Vascular Function in Human Obesity.

Background Pathophysiological mechanisms that connect obesity to cardiovascular disease are incompletely understood. FSP27 (Fat-specific protein 27) is a lipid droplet-associated protein that regulates lipolysis and insulin sensitivity in adipocytes. We unexpectedly discovered extensive FSP27 expression in human endothelial cells that is downregulated in association with visceral obesity. We sought to examine the functional role of FSP27 in the control of vascular phenotype. Methods and Results We biopsied paired subcutaneous and visceral fat depots from 61 obese individuals (body mass index 44±8 kg/m2, age 48±4 years) during planned bariatric surgery. We characterized depot-specific FSP27 expression in relation to adipose tissue microvascular insulin resistance, endothelial function and angiogenesis, and examined differential effects of FSP27 modification on vascular function. We observed markedly reduced vasodilator and angiogenic capacity of microvessels isolated from the visceral compared with subcutaneous adipose depots. Recombinant FSP27 and/or adenoviral FSP27 overexpression in human tissue increased endothelial nitric oxide synthase phosphorylation and nitric oxide production, and rescued vasomotor and angiogenic dysfunction (P<0.05), while siRNA-mediated FSP27 knockdown had opposite effects. Mechanistically, we observed that FSP27 interacts with vascular endothelial growth factor-A and exerts robust regulatory control over its expression. Lastly, in a subset of subjects followed longitudinally for 12±3 months after their bariatric surgery, 30% weight loss improved metabolic parameters and increased angiogenic capacity that correlated positively with increased FSP27 expression (r=0.79, P<0.05). Conclusions Our data strongly support a key role and functional significance of FSP27 as a critical endogenous modulator of human microvascular function that has not been previously described. FSP27 may serve as a previously unrecognized regulator of arteriolar vasomotor capacity and angiogenesis which are pivotal in the pathogenesis of cardiometabolic diseases linked to obesity.

Growth hormone acts along the PPARγ-FSP27 axis to stimulate lipolysis in human adipocytes.

The lipolytic effects of growth hormone (GH) have been known for half a century and play an important physiological role for substrate metabolism during fasting. In addition, sustained GH-induced lipolysis is causally linked to insulin resistance. However, the underlying molecular mechanisms remain elusive. In the present study, we obtained experimental data in human subjects and used human adipose-derived stromal vascular cells (hADSCs) as a model system to elucidate GH-triggered molecular signaling that stimulates adipose tissue lipolysis and insulin resistance in human adipocytes. We discovered that GH downregulates the expression of fat-specific protein (FSP27), a negative regulator of lipolysis, by impairing the transcriptional ability of the master transcriptional regulator, peroxisome proliferator-activated receptor-γ (PPARγ) via MEK/ERK activation. Ultimately, GH treatment promotes phosphorylation of PPARγ at Ser273 and causes its translocation from nucleus to the cytosol. Surprisingly, FSP27 overexpression inhibited PPARγ Ser273 phosphorylation and promoted its nuclear retention. GH antagonist treatment had similar effects. Our study identifies a novel signaling mechanism by which GH transcriptionally induces lipolysis via the MEK/ERK pathway that acts along PPARγ-FSP27 in human adipose tissue.

Growth hormone controls lipolysis by regulation of FSP27 expression.

Growth hormone (GH) has long been known to stimulate lipolysis and insulin resistance; however, the molecular mechanisms underlying these effects are unknown. In the present study, we demonstrate that GH acutely induces lipolysis in cultured adipocytes. This effect is secondary to the reduced expression of a negative regulator of lipolysis, fat-specific protein 27 (FSP27; aka Cidec) at both the mRNA and protein levels. These effects are mimicked in vivo as transgenic overexpression of GH leads to a reduction of FSP27 expression. Mechanistically, we show GH modulation of FSP27 expression is mediated through activation of both MEK/ERK- and STAT5-dependent intracellular signaling. These two molecular pathways interact to differentially manipulate peroxisome proliferator-activated receptor gamma activity (PPARγ) on the FSP27 promoter. Furthermore, overexpression of FSP27 is sufficient to fully suppress GH-induced lipolysis and insulin resistance in cultured adipocytes. Taken together, these data decipher a molecular mechanism by which GH acutely regulates lipolysis and insulin resistance in adipocytes.

Effects of a High Fat Diet and Voluntary Wheel Running Exercise on Cidea and Cidec Expression in Liver and Adipose Tissue of Mice.

Cidea and Cidec play an important role in regulating triglyceride storage in liver and adipose tissue. It is not known if the Cidea and Cidec genes respond to a high fat diet (HFD) or exercise training, two interventions that alter lipid storage. The purpose of the present study was to determine the effect of a HFD and voluntary wheel running (WR) on Cidea and Cidec mRNA and protein expression in adipose tissue and liver of mice. A HFD promoted a significant increase in Cidea and Cidec mRNA levels in adipose tissue and liver. The increase in Cidea and Cidec mRNAs in adipose tissue and liver in response to a HFD was prevented by WR. Similar to the changes in Cidea mRNA, Cidea protein levels in adipose tissue significantly increased in response to a HFD, a process that was, again, prevented by WR. However, in adipose tissue the changes in Cidec mRNA did not correspond to the changes in Cidec protein levels, as a HFD decreased Cidec protein abundance. Interestingly, in adipose tissue Cidea protein expression was significantly related to body weight (R=.725), epididymal adipose tissue (EWAT) mass (R=.475) and insulin resistance (R=.706), whereas Cidec protein expression was inversely related to body weight (R=-.787), EWAT mass (R=-.706), and insulin resistance (R=-.679). Similar to adipose tissue, Cidea protein expression in liver was significantly related to body weight (R=.660), EWAT mass (R=.468), and insulin resistance (R=.599); however, unlike adipose tissue, Cidec protein levels in liver were not related to body weight or EWAT mass and only moderately associated with insulin resistance (R=-.422, P=0.051). Overall, our findings indicate that Cidea is highly associated with adiposity and insulin resistance, whereas Cidec is related to insulin sensitivity. The present study suggests that Cide proteins might play an important functional role in the development of obesity, hepatic steatosis, as well as the pathogenesis of type 2 diabetes.

Fat-specific protein 27 inhibits lipolysis by facilitating the inhibitory effect of transcription factor Egr1 on transcription of adipose triglyceride lipase.

Lipolysis in fat tissue represents a major source of circulating fatty acids. Previously, we have found that lipolysis in adipocytes is controlled by early growth response transcription factor Egr1 that directly inhibits transcription of adipose triglyceride lipase, ATGL (Chakrabarti, P., Kim, J. Y., Singh, M., Shin, Y. K., Kim, J., Kumbrink, J., Wu, Y., Lee, M. J., Kirsch, K. H., Fried, S. K., and Kandror, K. V. (2013) Mol. Cell. Biol. 33, 3659-3666). Here we demonstrate that knockdown of the lipid droplet protein FSP27 (a.k.a. CIDEC) in human adipocytes increases expression of ATGL at the level of transcription, whereas overexpression of FSP27 has the opposite effect. FSP27 suppresses the activity of the ATGL promoter in vitro, and the proximal Egr1 binding site is responsible for this effect. FSP27 co-immunoprecipitates with Egr1 and increases its association with and inhibition of the ATGL promoter. Knockdown of Egr1 attenuates the inhibitory effect of FSP27. These results provide a new model of transcriptional regulation of ATGL.

Fat-specific protein 27 (FSP27) interacts with adipose triglyceride lipase (ATGL) to regulate lipolysis and insulin sensitivity in human adipocytes.

In adipocytes, lipolysis is a highly regulated process involving hormonal signals, lipid droplet-associated proteins, and lipases. The discovery of new lipid droplet-associated proteins added complexity to the current model of lipolysis. In this study, we used cultured human adipocytes to demonstrate that fat-specific protein 27 (FSP27), an abundantly expressed protein in adipocytes, regulates both basal and stimulated lipolysis by interacting with adipose triglyceride lipase (ATGL, also called desnutrin or PNPLA2). We identified a core domain of FSP27, amino acids 120-220, that interacts with ATGL to inhibit its lipolytic function and promote triglyceride storage. We also defined the role of FSP27 in free fatty acid-induced insulin resistance in adipocytes. FSP27 depletion in human adipocytes increased lipolysis and inhibited insulin signaling by decreasing AKT phosphorylation. However, reducing lipolysis by either depletion of ATGL or expression of exogenous full-length FSP27 or amino acids 120-220 protected human adipocytes against the adverse effects of free fatty acids on insulin signaling. In embryonic fibroblasts derived from ATGL KO mice, exogenous free fatty acids did not affect insulin sensitivity. Our results demonstrate a crucial role for FSP27-ATGL interactions in regulating lipolysis, triglyceride accumulation, and insulin signaling in human adipocytes.

ERbeta impedes prostate cancer EMT by destabilizing HIF-1alpha and inhibiting VEGF-mediated snail nuclear localization: implications for Gleason grading.

High Gleason grade prostate carcinomas are aggressive, poorly differentiated tumors that exhibit diminished estrogen receptor beta (ERbeta) expression. We report that a key function of ERbeta and its specific ligand 5alpha-androstane-3beta,17beta-diol (3beta-adiol) is to maintain an epithelial phenotype and repress mesenchymal characteristics in prostate carcinoma. Stimuli (TGF-beta and hypoxia) that induce an epithelial-mesenchymal transition (EMT) diminish ERbeta expression, and loss of ERbeta is sufficient to promote an EMT. The mechanism involves ERbeta-mediated destabilization of HIF-1alpha and transcriptional repression of VEGF-A. The VEGF-A receptor neuropilin-1 drives the EMT by promoting Snail1 nuclear localization. Importantly, this mechanism is manifested in high Gleason grade cancers, which exhibit significantly more HIF-1alpha and VEGF expression, and Snail1 nuclear localization compared to low Gleason grade cancers.

Targeting the Notch1 and mTOR pathways in a mouse T-ALL model.

Mutations in NOTCH1 are frequently detected in patients with T-cell acute lymphoblastic leukemia (T-ALL) and in mouse T-ALL models. Treatment of mouse or human T-ALL cell lines in vitro with gamma-secretase inhibitors (GSIs) results in growth arrest and/or apoptosis. These studies suggest GSIs as potential therapeutic agents in the treatment of T-ALL. To determine whether GSIs have antileukemic activity in vivo, we treated near-end-stage Tal1/Ink4a/Arf+/- leukemic mice with vehicle or with a GSI developed by Merck (MRK-003). We found that GSI treatment significantly extended the survival of leukemic mice compared with vehicle-treated mice. Notch1 target gene expression was repressed and increased numbers of apoptotic cells were observed in the GSI-treated mice, demonstrating that Notch1 inhibition in vivo induces apoptosis. T-ALL cell lines also exhibit PI3K/mTOR pathway activation, indicating that rapamycin may also have therapeutic benefit. When GSIs are administered in combination with rapamycin, mTOR kinase activity is ablated and apoptosis induced. Moreover, GSI and rapamycin treatment inhibits human T-ALL growth and extends survival in a mouse xenograft model. This work supports the idea of targeting NOTCH1 in T-ALL and suggests that inhibition of the mTOR and NOTCH1 pathways may have added efficacy.

The Notch1/c-Myc pathway in T cell leukemia.

The Notch receptor family and its ligands (Delta-like and Jagged) have been found deregulated in several human cancers. We and the Aster/Pear group recently identified c-myc as a direct transcriptional target gene of the Notch1 pathway in T cell acute lymphoblastic leukemia (T-ALL). Although the oncogenic roles of c-Myc and Notch1 are established, a direct link between Notch1 and c-Myc had not been demonstrated. Importantly, our work in mouse tal1 tumor cell lines revealed that leukemic growth/survival remains dependent on the Notch1-c-Myc pathway. Studies by the Efstratiadis group provide genetic evidence that the Notch1-c-Myc pathway also contributes to mouse mammary tumorigenesis. Taken together, these studies demonstrate that Notch1 mediates T cell and epithelial cell transformation at least in part by sustaining c-Myc lev.

Histone deacetylases RPD3 and HOS2 regulate the transcriptional activation of DNA damage-inducible genes.

DNA microarray and genetic studies of Saccharomyces cerevisiae have demonstrated that histone deacetylases (HDACs) are required for transcriptional activation and repression, but the mechanism by which they activate transcription remains poorly understood. We show that two HDACs, RPD3 and HOS2, are required for the activation of DNA damage-inducible genes RNR3 and HUG1. Using mutants specific for the Rpd3L complex, we show that the complex is responsible for regulating RNR3. Furthermore, unlike what was described for the GAL genes, Rpd3L regulates the activation of RNR3 by deacetylating nucleosomes at the promoter, not at the open reading frame. Rpd3 is recruited to the upstream repression sequence of RNR3, which surprisingly does not require Tup1 or Crt1. Chromatin remodeling and TFIID recruitment are largely unaffected in the Deltarpd3/Deltahos2 mutant, but the recruitment of RNA polymerase II is strongly reduced, arguing that Rpd3 and Hos2 regulate later stages in the assembly of the preinitiation complex or facilitate multiple rounds of polymerase recruitment. Furthermore, the histone H4 acetyltransferase Esa1 is required for the activation of RNR3 and HUG1. Thus, reduced or unregulated constitutive histone H4 acetylation is detrimental to promoter activity, suggesting that HDAC-dependent mechanisms are in place to reset promoters to allow high levels of transcription.

Notch1 contributes to mouse T-cell leukemia by directly inducing the expression of c-myc.

Recent work with mouse models and human leukemic samples has shown that gain-of-function mutation(s) in Notch1 is a common genetic event in T-cell acute lymphoblastic leukemia (T-ALL). The Notch1 receptor signals through a gamma-secretase-dependent process that releases intracellular Notch1 from the membrane to the nucleus, where it forms part of a transcriptional activator complex. To identify Notch1 target genes in leukemia, we developed mouse T-cell leukemic lines that express intracellular Notch1 in a doxycycline-dependent manner. Using gene expression profiling and chromatin immunoprecipitation, we identified c-myc as a novel, direct, and critical Notch1 target gene in T-cell leukemia. c-myc mRNA levels are increased in primary mouse T-cell tumors that harbor Notch1 mutations, and Notch1 inhibition decreases c-myc mRNA levels and inhibits leukemic cell growth. Retroviral expression of c-myc, like intracellular Notch1, rescues the growth arrest and apoptosis associated with gamma-secretase inhibitor treatment or Notch1 inhibition. Consistent with these findings, retroviral insertional mutagenesis screening of our T-cell leukemia mouse model revealed common insertions in either notch1 or c-myc genes. These studies define the Notch1 molecular signature in mouse T-ALL and importantly provide mechanistic insight as to how Notch1 contributes to human T-ALL.

Molecular structure of D-hydantoinase from Bacillus sp. AR9: evidence for mercury inhibition.

Stereospecific conversion of hydantoins into their carbamoyl acid derivatives could be achieved by using the enzyme hydantoinase. Specific hydantoinases convert either the D-form or the L-form of the hydantoin and the amino acids responsible for stereospecificity have not been identified. Structural studies on hydantoinases from a few bacterial species were published recently. The structure of a thermostable D-hydantoinase from Bacillus sp. AR9 (bar9HYD) was solved to 2.3 angstroms resolution. The usual modification of carboxylation of the active-site residue Lys150 did not happen in bar9HYD. Two manganese ions were modelled in the active site. Through biochemical studies, it was shown that mercury inhibits the activity of the enzyme. The mercury derivative provided some information about the binding site of the mercuric inhibitors and a possible reason for inhibition is presented.