The Next Frontier: Translational Development of Ubiquitination, SUMOylation, and NEDDylation in Cancer.

Post-translational modifications of proteins ensure optimized cellular processes, including proteostasis, regulated signaling, cell survival, and stress adaptation to maintain a balanced homeostatic state. Abnormal post-translational modifications are associated with cellular dysfunction and the occurrence of life-threatening diseases, such as cancer and neurodegenerative diseases. Therefore, some of the frequently seen protein modifications have been used as disease markers, while others are targeted for developing specific therapies. The ubiquitin and ubiquitin-like post-translational modifiers, namely, small ubiquitin-like modifier (SUMO) and neuronal precursor cell-expressed developmentally down-regulated protein 8 (NEDD8), share several features, such as protein structures, enzymatic cascades mediating the conjugation process, and targeted amino acid residues. Alterations in the regulatory mechanisms lead to aberrations in biological processes during tumorigenesis, including the regulation of tumor metabolism, immunological modulation of the tumor microenvironment, and cancer stem cell stemness, besides many more. Novel insights into ubiquitin and ubiquitin-like pathways involved in cancer biology reveal a potential interplay between ubiquitination, SUMOylation, and NEDDylation. This review outlines the current understandings of the regulatory mechanisms and assay capabilities of ubiquitination, SUMOylation, and NEDDylation. It will further highlight the role of ubiquitination, SUMOylation, and NEDDylation in tumorigenesis.

A next generation sequencing based approach to identify extracellular vesicle mediated mRNA transfers between cells.

BACKGROUND: Exosomes and other extracellular vesicles (EVs) have emerged as an important mechanism of cell-to-cell communication. However, previous studies either did not fully resolve what genetic materials were shuttled by exosomes or only focused on a specific set of miRNAs and mRNAs. A more systematic method is required to identify the genetic materials that are potentially transferred during cell-to-cell communication through EVs in an unbiased manner. RESULTS: In this work, we present a novel next generation of sequencing (NGS) based approach to identify EV mediated mRNA exchanges between co-cultured adipocyte and macrophage cells. We performed molecular and genomic profiling and jointly considered data from RNA sequencing (RNA-seq) and genotyping to track the "sequence varying mRNAs" transferred between cells. We identified 8 mRNAs being transferred from macrophages to adipocytes and 21 mRNAs being transferred in the opposite direction. These mRNAs represented biological functions including extracellular matrix, cell adhesion, glycoprotein, and signal peptides. CONCLUSIONS: Our study sheds new light on EV mediated RNA communications between adipocyte and macrophage cells, which may play a significant role in developing insulin resistance in diabetic patients. This work establishes a new method that is applicable to examining genetic material exchanges in many cellular systems and has the potential to be extended to in vivo studies as well.

Intrinsic Properties of Brown and White Adipocytes Have Differential Effects on Macrophage Inflammatory Responses.

Obesity is marked by chronic, low-grade inflammation. Here, we examined whether intrinsic differences between white and brown adipocytes influence the inflammatory status of macrophages. White and brown adipocytes were characterized by transcriptional regulation of UCP-1, PGC1α, PGC1ß, and CIDEA and their level of IL-6 secretion. The inflammatory profile of PMA-differentiated U937 and THP-1 macrophages, in resting state and after stimulation with LPS/IFN-gamma and IL-4, was assessed by measuring IL-6 secretion and transcriptional regulation of a panel of inflammatory genes after mono- or indirect coculture with white and brown adipocytes. White adipocyte monocultures show increased IL-6 secretion compared to brown adipocytes. White adipocytes cocultured with U937 and THP-1 macrophages induced a greater increase in IL-6 secretion compared to brown adipocytes cocultured with both macrophages. White adipocytes cocultured with macrophages increased inflammatory gene expression in both types. In contrast, macrophages cocultured with brown adipocytes induced downregulation or no alterations in inflammatory gene expression. The effects of adipocytes on macrophages appear to be independent of stimulation state. Brown adipocytes exhibit an intrinsic ability to dampen inflammatory profile of macrophages, while white adipocytes enhance it. These data suggest that brown adipocytes may be less prone to adipose tissue inflammation that is associated with obesity.

Shox2 is a molecular determinant of depot-specific adipocyte function.

Visceral and s.c. fat exhibit different intrinsic properties, including rates of lipolysis, and are associated with differential risk for the development of type 2 diabetes. These effects are in part related to cell autonomous differences in gene expression. In the present study, we show that expression of Shox2 (Short stature homeobox 2) is higher in s.c. than visceral fat in both rodents and humans and that levels are further increased in humans with visceral obesity. Fat-specific disruption of Shox2 in male mice results in protection from high fat diet-induced obesity, with a preferential loss of s.c. fat. The reduced adipocyte size is secondary to a twofold increase in the expression of ß3 adrenergic receptor (Adrb3) at both the mRNA and protein level and a parallel increase in lipolytic rate. These effects are mimicked by knockdown of Shox2 in C3H10T1/2 cells. Conversely, overexpression of Shox2 leads to a repression of Adrb3 expression and decrease lipolytic rate. Shox2 does not affect differentiation but directly interacts with CCAAT/enhancer binding protein alpha and attenuates its transcriptional activity of the Adrb3 promoter. Thus, Shox2 can regulate the expression of Adrb3 and control the rate of lipolysis and, in this way, exerts control of the phenotypic differences between visceral and s.c. adipocytes.

Mesodermal developmental gene Tbx15 impairs adipocyte differentiation and mitochondrial respiration.

Increased intraabdominal (visceral) fat is associated with a high risk of diabetes and metabolic syndrome. We have previously shown that the mesodermal developmental transcription factor Tbx15 is highly differentially expressed between visceral and subcutaneous (s.c.) fat in both humans and rodents, and in humans visceral fat Tbx15 expression is decreased in obesity. Here we show that, in mice, Tbx15 is 260-fold more highly expressed in s.c. preadipocytes than in epididymal preadipocytes. Overexpression of Tbx15 in 3T3-L1 preadipocytes impairs adipocyte differentiation and decreases triglyceride content. This defect in differentiation can be corrected by stimulating cells with the PPARγ agonist rosiglitazone (Rosi). However, triglyceride accumulation remains decreased by ∼50%, due to a decrease in basal lipogenic rate and increase in basal lipolytic rate. 3T3-L1 preadipocytes overexpressing Tbx15 also have a 15% reduction in mitochondrial mass and a 28% reduction in basal mitochondrial respiration (P = 0.004) and ATP turnover (P = 0.02), and a 45% (P = 0.003) reduction in mitochondrial respiratory capacity. Thus, differential expression of Tbx15 between fat depots plays an important role in the interdepot differences in adipocyte differentiation, triglyceride accumulation, and mitochondrial function that may contribute to the risk of diabetes and metabolic disease.

SIRT2 regulates adipocyte differentiation through FoxO1 acetylation/deacetylation.

The family of mammalian Sirtuin proteins comprises seven members homologous to yeast Sir2. Here we show that SIRT2, a cytoplasmic sirtuin, is the most abundant sirtuin in adipocytes. Sirt2 expression is downregulated during preadipocyte differentiation in 3T3-L1 cells. Overexpression of SIRT2 inhibits differentiation, whereas reducing SIRT2 expression promotes adipogenesis. Both effects are accompanied by corresponding changes in the expression of PPARgamma, C/EBPalpha, and genes marking terminal adipocyte differentiation, including Glut4, aP2, and fatty acid synthase. The mechanism underlying the effects of reduced SIRT2 in 3T3-L1 adipocytes includes increased acetylation of FOXO1, with direct interaction between SIRT2 and FOXO1. This interaction enhances insulin-stimulated phosphorylation of FOXO1, which in turn regulates FOXO1 nuclear and cytosolic localization. Thus, Sirt2 acts as an important regulator of adipocyte differentiation through modulation of FOXO1 acetylation/phosphorylation and activity and may play a role in controlling adipose tissue mass and function.

Ubc1 turnover contributes to the spindle assembly checkpoint in Saccharomyces cerevisiae.

The spindle assembly checkpoint protects the integrity of the genome by ensuring that chromosomes are properly attached to the mitotic spindle before they are segregated during anaphase. Activation of the spindle checkpoint results in inhibition of the Anaphase-Promoting Complex (APC), an E3 ubiquitin ligase that triggers the metaphase-anaphase transition. Here, we show that levels of Ubc1, an E2 enzyme that functions in complex with the APC, modulate the response to spindle checkpoint activation in Saccharomyces cerevisiae. Overexpression of Ubc1 increased resistance to microtubule poisons, whereas Ubc1 shut-off sensitized cells. We also found that Ubc1 levels are regulated by the spindle checkpoint. Checkpoint activation or direct APC inhibition led to a decrease in Ubc1 levels, charging, and half-life. Additionally, stabilization of Ubc1 prevented its down-regulation by the spindle checkpoint and increased resistance to checkpoint-activating drugs. These results suggest that down-regulation of Ubc1 in response to spindle checkpoint signaling is necessary for a robust cell cycle arrest.

Elevated levels of mitochondrial CoQ10 induce ROS-mediated apoptosis in pancreatic cancer.

Reactive oxygen species (ROS) are implicated in triggering cell signalling events and pathways to promote and maintain tumorigenicity. Chemotherapy and radiation can induce ROS to elicit cell death allows for targeting ROS pathways for effective anti-cancer therapeutics. Coenzyme Q10 is a critical cofactor in the electron transport chain with complex biological functions that extend beyond mitochondrial respiration. This study demonstrates that delivery of oxidized Coenzyme Q10 (ubidecarenone) to increase mitochondrial Q-pool is associated with an increase in ROS generation, effectuating anti-cancer effects in a pancreatic cancer model. Consequent activation of cell death was observed in vitro in pancreatic cancer cells, and both human patient-derived organoids and tumour xenografts. The study is a first to demonstrate the effectiveness of oxidized ubidecarenone in targeting mitochondrial function resulting in an anti-cancer effect. Furthermore, these findings support the clinical development of proprietary formulation, BPM31510, for treatment of cancers with high ROS burden with potential sensitivity to ubidecarenone.

TRB3 blocks adipocyte differentiation through the inhibition of C/EBPbeta transcriptional activity.

TRB3 has been implicated in the regulation of several biological processes in mammalian cells through its ability to influence Akt and other signaling pathways. In this study, we investigated the role of TRB3 in regulating adipogenesis and the activity of adipogenic transcription factors. We find that TRB3 is expressed in 3T3-L1 preadipocytes, and this expression is transiently suppressed during the initial days of differentiation concomitant with induction of C/EBPbeta. This event appears to be a prerequisite for adipogenesis. Overexpression of TRB3 blocks differentiation of 3T3-L1 cells at a step downstream of C/EBPbeta. Ectopic expression of TRB3 in mouse fibroblasts also inhibits the C/EBPbeta-dependent induction of PPARgamma2 and blocks their differentiation into adipocytes. This inhibition of preadipocyte differentiation by TRB3 appears to be the result of two complementary effects. First, TRB3 inhibits extracellular signal-regulated kinase activity, which prevents the phosphorylation of regulatory sites on C/EBPbeta. Second, TRB3 directly interacts with the DR1 domain of C/EBPbeta in the nucleus, further inhibiting both its ability to bind its response element and its ability to transactivate the C/EBPalpha and a-FABP promoters. Thus, TRB3 is an important negative regulator of adipogenesis that acts at an early step in the differentiation cascade to block the C/EBPbeta proadipogenic function.

High levels of ubidecarenone (oxidized CoQ10) delivered using a drug-lipid conjugate nanodispersion (BPM31510) differentially affect redox status and growth in malignant glioma versus non-tumor cells.

Metabolic reprogramming in cancer cells, vs. non-cancer cells, elevates levels of reactive oxygen species (ROS) leading to higher oxidative stress. The elevated ROS levels suggest a vulnerability to excess prooxidant loads leading to selective cell death, a therapeutically exploitable difference. Co-enzyme Q10 (CoQ10) an endogenous mitochondrial resident molecule, plays an important role in mitochondrial redox homeostasis, membrane integrity, and energy production. BPM31510 is a lipid-drug conjugate nanodispersion specifically formulated for delivery of supraphysiological concentrations of ubidecarenone (oxidized CoQ10) to the cell and mitochondria, in both in vitro and in vivo model systems. In this study, we sought to investigate the therapeutic potential of ubidecarenone in the highly treatment-refractory glioblastoma. Rodent (C6) and human (U251) glioma cell lines, and non-tumor human astrocytes (HA) and rodent NIH3T3 fibroblast cell lines were utilized for experiments. Tumor cell lines exhibited a marked increase in sensitivity to ubidecarenone vs. non-tumor cell lines. Further, elevated mitochondrial superoxide production was noted in tumor cells vs. non-tumor cells hours before any changes in proliferation or the cell cycle could be detected. In vitro co-culture experiments show ubidecarenone differentially affecting tumor cells vs. non-tumor cells, resulting in an equilibrated culture. In vivo activity in a highly aggressive orthotopic C6 glioma model demonstrated a greater than 25% long-term survival rate. Based on these findings we conclude that high levels of ubidecarenone delivered using BPM31510 provide an effective therapeutic modality targeting cancer-specific modulation of redox mechanisms for anti-cancer effects.

Diablo ubiquitination analysis by sandwich immunoassay.

Ubiquitin plays an essential role in modulating protein function, and deregulation of the ubiquitin system leads to the development of a variety of human diseases. E3 Ubiquitin ligases that mediate ubiquitination and degradation of caspases prevent apoptosis, and as such belong to the family of inhibitors of apoptosis proteins (IAPs). Diablo is a substrate of IAPs but also a negative regulator of IAPs in apoptotic pathway as it blocks the interaction between IAPs and caspases. In efforts to identify IAP inhibitors, we developed sandwich immunoassays in conjunction with an electrochemical luminescence (ECL) platform for quantitation of total Diablo, ubiquitinated Diablo, and ubiquitinated Diablo with K48-specific linkage. The assay panel detects Diablo ubiquitination level changes in the presence of IAP inhibitor or proteasome inhibitor, demonstrating its potential as a cost-efficient high-throughput method for drug discovery involving IAP ubiquitination cascade. The ECL based sandwich assay panel performance was subsequently evaluated for precision, linearity, and limit of quantification.

Hsp90ß knockdown in DIO mice reverses insulin resistance and improves glucose tolerance.

BACKGROUND: Inhibition of Hsp90 has been shown to improve glucose tolerance and insulin sensitivity in mouse models of diabetes. In the present report, the specific isoform Hsp90ab1, was identified as playing a major role in regulating insulin signaling and glucose metabolism. METHODS: In a diet-induced obese (DIO) mouse model of diabetes, expression of various Hsp90 isoforms in skeletal tissue was examined. Subsequent experiments characterized the role of Hsp90ab1 isoform in glucose metabolism and insulin signaling in primary human skeletal muscle myoblasts (HSMM) and a DIO mouse model. RESULTS: In DIO mice Hsp90ab1 mRNA was upregulated in skeletal muscle compared to lean mice and knockdown using anti-sense oligonucleotide (ASO) resulted in reduced expression in skeletal muscle that was associated with improved glucose tolerance, reduced fed glucose and fed insulin levels compared to DIO mice that were treated with a negative control oligonucleotide. In addition, knockdown of HSP90ab1 in DIO mice was associated with reduced pyruvate dehydrogenase kinase-4 mRNA and phosphorylation of the muscle pyruvate dehydrogenase complex (at serine 232, 293 and 300), but increased phosphofructokinase 1, glycogen synthase 1 and long-chain specific acyl-CoA dehydrogenase mRNA. In HSMM, siRNA knockdown of Hsp90ab1 induced an increase in substrate metabolism, mitochondrial respiration capacity, and insulin sensitivity, providing further evidence for the role of Hsp90ab1 in metabolism. CONCLUSIONS: The data support a novel role for Hsp90ab1 in arbitrating skeletal muscle plasticity via modulation of substrate utilization including glucose and fatty acids in normal and disease conditions. Hsp90ab1 represents a novel target for potential treatment of metabolic disease including diabetes.

Dysregulation of the calcium handling protein, CCDC47, is associated with diabetic cardiomyopathy.

BACKGROUND: Diabetes mellitus is associated with an increased risk in diabetic cardiomyopathy (DCM) that is distinctly not attributed to co-morbidities with other vasculature diseases. To date, while dysregulation of calcium handling is a key hallmark in cardiomyopathy, studies have been inconsistent in the types of alterations involved. In this study human cardiomyocytes were exposed to an environmental nutritional perturbation of high glucose, fatty acids, and l-carnitine to model DCM and iTRAQ-coupled LC-MS/MS proteomic analysis was used to capture proteins affected by the perturbation. The proteins captured were then compared to proteins currently annotated in the cardiovascular disease (CVD) gene ontology (GO) database to identify proteins not previously described as being related to CVD. Subsequently, GO analysis for calcium regulating proteins and endoplasmic/sarcoplasmic reticulum (ER/SR) associated proteins was carried out. RESULTS: Here, we identified CCDC47 (calumin) as a unique calcium regulating protein altered in our in vitro nutritional perturbation model. The cellular and functional role of CCDC47 was then assessed in rat cardiomyocytes. In rat H9C2 myocytes, overexpression of CCDC47 resulted in increase in ionomycin-induced calcium release and reuptake. Of interest, in a diet-induced obese (DIO) rat model of DCM, CCDC47 mRNA expression was increased in the atrium and ventricle of the heart, but CCDC47 protein expression was significantly increased only in the atrium of DIO rats compared to lean control rats. Notably, no changes in ANP, BNP, or ß-MHC were observed between DIO rats and lean control rats. CONCLUSIONS: Together, our in vitro and in vivo studies demonstrate that CCDC47 is a unique calcium regulating protein that is associated with early onset hypertrophic cardiomyopathy.

TNFalpha up-regulates apelin expression in human and mouse adipose tissue.

We have recently identified apelin as a novel adipokine up-regulated by insulin and obesity. Since obesity and insulin resistance are associated with chronically elevated levels of both insulin and TNFalpha, the present study was performed to investigate a putative regulation of apelin expression in adipocytes by TNFalpha. Herein, we report a tight correlation between apelin and TNFalpha expression in adipose tissue of lean and obese humans. Apelin regulation by TNFalpha was further studied in cultured explants of human adipose tissue. The endogenous expression of TNFalpha in adipocytes isolated from the explants was accompanied by a 6-9 h subsequent increase of apelin expression in adipocytes. This increase was reversed by inhibiting TNFalpha expression with 100 microM isobutylmethylxanthine. In different mouse models of obesity, expression of both TNFalpha and apelin was also significantly increased in adipocytes of obese mice. Furthermore, short-term exposure to an i.p. injection of TNFalpha in C57Bl6/J mice induced an increase of apelin expression in adipose tissue as well as apelin plasma levels. Finally, a direct positive effect of TNFalpha has been shown in differentiated 3T3F442A adipocytes on apelin expression and secretion. The signaling pathways of TNFalpha for the induction of apelin were dependent of PI3-kinase, c-Jun NH2-terminal kinase (JNK), and MAPK but not PKC activation. All together, these findings suggest that apelin might be a candidate to better understand potential links between obesity and associated disorders such as inflammation and insulin resistance.

Identification of Filamin-A and -B as potential biomarkers for prostate cancer.

AIM: A novel strategy for prostate cancer (PrCa) biomarker discovery is described. MATERIALS & METHODS: In vitro perturbation biology, proteomics and Bayesian causal analysis identified biomarkers that were validated in in vitro models and clinical specimens. RESULTS: Filamin-B (FLNB) and Keratin-19 were identified as biomarkers. Filamin-A (FLNA) was found to be causally linked to FLNB. Characterization of the biomarkers in a panel of cells revealed differential mRNA expression and regulation. Moreover, FLNA and FLNB were detected in the conditioned media of cells. Last, in patients without PrCa, FLNA and FLNB blood levels were positively correlated, while in patients with adenocarcinoma the relationship is dysregulated. CONCLUSION: These data support the strategy and the potential use of the biomarkers for PrCa.

Reduced expression of collagen VI alpha 3 (COL6A3) confers resistance to inflammation-induced MCP1 expression in adipocytes.

OBJECTIVE: Collagen VI alpha 3 (COL6A3) is associated with insulin resistance and adipose tissue inflammation. In this study, the role of COL6A3 in human adipocyte function was characterized. METHODS: Immortalized human preadipocyte cell lines stably expressing control or COL6A3 shRNA were used to study adipocyte function and inflammation. RESULTS: COL6A3 knockdown increased triglyceride content, lipolysis, insulin-induced Akt phosphorylation, and mRNA expression of key adipogenic genes (peroxisome proliferator-activated receptor-γ, glucose transporter, adiponectin, and fatty acid binding protein), indicating increased adipocyte function and insulin sensitivity. However, COL6A3 knockdown decreased basal adipocyte chemokine (C-C motif) ligand 2 [CCL2, monocyte chemoattractant protein (MCP1)] mRNA expression, reduced secreted protein levels, and abrogated tumor necrosis factor-α- and lipopolysaccharide-induced MCP1 mRNA expression. In addition, while control adipocytes co-cultured with THP1 macrophages showed a threefold increase in adipocyte MCP1 mRNA expression, in COL6A3 knockdown adipocytes MCP1 mRNA expression was unaltered by co-culturing. Lastly, in normal differentiated adipocytes, matrix metalloproteinase-11 treatment reduced expression of COL6A3 protein, MCP1 mRNA, MCP1 secretion, and abrogated tumor necrosis factor-α- and lipopolysaccharide-induced MCP1 mRNA expression and protein secretion. CONCLUSIONS: COL6A3 knockdown in adipocytes leads to the development of a unique state of inflammatory resistance via suppression of MCP1 induction.

Apelin, a newly identified adipokine up-regulated by insulin and obesity.

The results presented herein demonstrate that apelin is expressed and secreted by both human and mouse adipocytes. Apelin mRNA levels in isolated adipocytes are close to other cell types present in white adipose tissue or other organs known to express apelin such as kidney, heart, and to a lesser extent brown adipose tissue. Apelin expression is increased during adipocyte differentiation stage. A comparison of four different models of obesity in mice showed a large increase in both apelin expression in fat cells and apelin plasma levels in all the hyperinsulinemia-associated obesities and clearly demonstrated that obesity or high-fat feeding are not the main determinants of the rise of apelin expression. The lack of insulin in streptozotocin-treated mice is associated with a decreased expression of apelin in adipocytes. Furthermore, apelin expression in fat cells is strongly inhibited by fasting and recovered after refeeding, in a similar way to insulin. A direct regulation of apelin expression by insulin is observed in both human and mouse adipocytes and clearly associated with the stimulation of phosphatidylinositol 3-kinase, protein kinase C, and MAPK. These data provide evidence that insulin exerts a direct control on apelin gene expression in adipocytes. In obese patients, both plasma apelin and insulin levels were significantly higher, suggesting that the regulation of apelin by insulin could influence blood concentrations of apelin. The present work identifies apelin as a novel adipocyte endocrine secretion and focuses on its potential link with obesity-associated variations of insulin sensitivity status.

MDR1-deficient genotype in Collie dogs hypersensitive to the P-glycoprotein substrate ivermectin.

Multidrug resistance (MDR) phenotypes in cancer cells are associated with overexpression of the drug carrier P-glycoprotein. The antiparasitic drug ivermectin, one of its substrates, abnormally accumulates in the brain of transgenic mice lacking the P-glycoprotein, resulting in neurotoxicity. Similarly, an enhanced sensitivity to ivermectin has been reported in certain dogs of the Collie breed. To explore the basis of this phenotype, we analyzed the canine P-glycoprotein-encoding MDR1 gene, and we report the first characterization of the cDNA for wild-type (Beagle) P-glycoprotein. The corresponding transcripts from ivermectin-sensitive Collies revealed a homozygous 4-bp exonic deletion. We established, by genetic testings, that the MDR1 frame shift is predictable. Accordingly, no P-glycoprotein was detected in the homozygote-deficient dogs. In conclusion, we characterized a unique case of naturally occurring gene invalidation. This provides a putative novel model that remains to be exploited in the field of human therapeutics and that might significantly affect tissue distribution and drug bioavailability studies.

Intrinsic differences in adipocyte precursor cells from different white fat depots.

Obesity and body fat distribution are important risk factors for the development of type 2 diabetes and metabolic syndrome. Evidence has accumulated that this risk is related to intrinsic differences in behavior of adipocytes in different fat depots. In the current study, we demonstrate that adipocyte precursor cells (APCs) isolated from visceral and subcutaneous white adipose depots of mice have distinct patterns of gene expression, differentiation potential, and response to environmental and genetic influences. APCs derived from subcutaneous fat differentiate well in the presence of classical induction cocktail, whereas those from visceral fat differentiate poorly but can be induced to differentiate by addition of bone morphogenetic protein (BMP)-2 or BMP-4. This difference correlates with major differences in gene expression signature between subcutaneous and visceral APCs. The number of APCs is higher in obesity-prone C57BL/6 mice than obesity-resistant 129 mice, and the number in both depots is increased by up to 270% by exposure of mice to high-fat diet. Thus, APCs from visceral and subcutaneous depots are dynamic populations, which have intrinsic differences in gene expression, differentiation properties, and responses to environmental/genetic factors. Regulation of these populations may provide a new target for the treatment and prevention of obesity and its metabolic complications.

The differential role of Hif1ß/Arnt and the hypoxic response in adipose function, fibrosis, and inflammation.

In obesity, adipocytes distant from vasculature become hypoxic and dysfunctional. This hypoxic response is mediated by hypoxia-inducible factors (Hif1α, Hif2α, and Hif3α) and their obligate partner, Hif1ß (Arnt). We show that mice lacking Hif1ß in fat (FH1ßKO) are lean, exhibit reduced adipocyte size, and are protected from age- and diet-induced glucose intolerance. There is also reduced Vegf and vascular permeability in FH1ßKO fat, but diet-induced inflammation and fibrosis is unchanged. Adipocytes from FH1ßKO mice have reduced glucose uptake due to decreased Glut1 and Glut4, which is mirrored in 3T3-L1 adipocytes with Hif1ß knockdown. Hif1ß knockdown cells also fail to respond appropriately to hypoxia with reduced cellular respiration and reduced mitochondrial gene expression. Some, but not all, of these effects are reproduced by Hif1α knockdown. Thus, Hif1ß/Arnt regulates glucose uptake, mitochondrial gene expression, and vascular permeability to control adipose mass and function, providing a target for obesity therapy.