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Supplementary Figure 2 from Cdc42-Interacting Protein 4 Promotes Breast Cancer Cell Invasion and Formation of Invadopodia through Activation of N-WASp

In the earliest stages of metastasis, breast cancer cells must reorganize the cytoskeleton to affect cell shape change and promote cell invasion and motility. These events require the cytoskeletal regulators Cdc42 and Rho, their effectors such as N-WASp/WAVE, and direct inducers of actin polymerization such as Arp2/3. Little consideration has been given to molecules that shape the cell membrane. The F-BAR proteins CIP4, TOCA-1, and FBP17 generate membrane curvature and act as scaffolding proteins for activated Cdc42 and N-WASp. We found that expression of CIP4, but not TOCA-1 or FBP17, was increased in invasive breast cancer cell lines in comparison with weakly or noninvasive breast cancer cell lines. Endogenous CIP4 localized to the leading edge of migrating cells and to invadopodia in cells invading gelatin. Because CIP4 serves as a scaffolding protein for Cdc42, Src, and N-WASp, we tested whether loss of CIP4 could result in decreased N-WASp function. Interaction between CIP4 and N-WASp was epidermal growth factor responsive, and CIP4 silencing by small interfering RNA caused decreased tyrosine phosphorylation of N-WASp at a Src-dependent activation site (Y256). CIP4 silencing also impaired the migration and invasion of MDA-MB-231 cells and was associated with decreased formation of invadopodia and gelatin degradation. This study presents a new role for CIP4 in the promotion of migration and invasion of MDA-MB-231 breast cancer cells and establishes the contribution of F-BAR proteins to cancer cell motility and invasion. Cancer Res; 70(21); 8347–56. ©2010 AACR.

Supplementary Figure 3 from Cdc42-Interacting Protein 4 Promotes Breast Cancer Cell Invasion and Formation of Invadopodia through Activation of N-WASp

Supplementary Figure 4 from Cdc42-Interacting Protein 4 Promotes Breast Cancer Cell Invasion and Formation of Invadopodia through Activation of N-WASp

Supplementary Figure 1 from Cdc42-Interacting Protein 4 Promotes Breast Cancer Cell Invasion and Formation of Invadopodia through Activation of N-WASp

In mammals, meiotically competent oocytes develop cyclically during ovarian folliculogenesis. During folliculogenesis, prophase I arrested oocytes are transcriptionally active, producing and storing transcripts required for their growth and for early stages of embryogenesis prior to the maternal to zygotic transition. Defective oocyte development during folliculogenesis leads to meiotic defects, aneuploidy, follicular atresia, or non-viable embryos. Here we generated a novel oocyte-specific knockout of the SUMO E2 ligase,Ube2i, usingZp3-creto test its function during folliculogenesis.Ube2i Zp3-cre+ female mice are sterile with oocytes that arrest in meiosis I with defective spindles and chromosome alignment. Fully grown mutant oocytes abnormally maintain transcription but downregulate maternal effect genes and prematurely activate the zygotic transcriptional program. Thus, this work uncovers UBE2i as a novel orchestrator of chromatin and transcriptional regulation in mouse oocytes.TeaserOocyte-specific deletion ofUbe2icauses loss of transcriptional repression and premature activation of the zygotic genome.

Funder: National Institute for Health Research (NIHR), Serotonin reuptake inhibitors and receptor agonists are used to treat obesity, anxiety and depression. Here we studied the role of the serotonin 2C receptor (5-HT2CR) in weight regulation and behavior. Using exome sequencing of 2,548 people with severe obesity and 1,117 control individuals without obesity, we identified 13 rare variants in the gene encoding 5-HT2CR (HTR2C) in 19 unrelated people (3 males and 16 females). Eleven variants caused a loss of function in HEK293 cells. All people who carried variants had hyperphagia and some degree of maladaptive behavior. Knock-in male mice harboring a human loss-of-function HTR2C variant developed obesity and reduced social exploratory behavior; female mice heterozygous for the same variant showed similar deficits with reduced severity. Using the 5-HT2CR agonist lorcaserin, we found that depolarization of appetite-suppressing proopiomelanocortin neurons was impaired in knock-in mice. In conclusion, we demonstrate that 5-HT2CR is involved in the regulation of human appetite, weight and behavior. Our findings suggest that melanocortin receptor agonists might be effective in treating severe obesity in individuals carrying HTR2C variants. We suggest that HTR2C should be included in diagnostic gene panels for severe childhood-onset obesity.

Flavin adenine dinucleotide (FAD) interacts with flavoproteins to mediate oxidation-reduction reactions required for cellular energy demands. Not surprisingly, mutations that alter FAD binding to flavoproteins cause rare inborn errors of metabolism (IEMs) that disrupt liver function and render fasting intolerance, hepatic steatosis, and lipodystrophy. In our study, depleting FAD pools in mice with a vitamin B2 deficient diet (B2D) caused phenotypes associated with organic acidemias and other IEMs, including reduced body weight, hypoglycemia, and fatty liver disease. Integrated discovery approaches revealed B2D tempered fasting activation of target genes for the nuclear receptor PPARα, including those required for gluconeogenesis. Treatment with the PPARα agonist fenofibrate activated the integrated stress response and refilled amino acid substrates to rescue fasting glucose availability and overcome B2D phenotypes. Overall, these findings reveal PPARα governs metabolic responses to FAD availability and nominate its pharmacologic activation as strategies for organic acidemias.

OBJECTIVE: Mitophagy removes damaged mitochondria to maintain cellular homeostasis. Aryl hydrocarbon receptor (AhR) expression in the liver plays a crucial role in supporting normal liver functions, but its impact on mitochondrial function is unclear. Here, we identified a new role of AhR in the regulation of mitophagy to control hepatic energy homeostasis. METHODS: In this study, we utilized primary hepatocytes from AhR knockout (KO) mice and AhR knockdown AML12 hepatocytes. An endogenous AhR ligand, kynurenine (Kyn), was used to activate AhR in AML12 hepatocytes. Mitochondrial function and mitophagy process were comprehensively assessed by MitoSOX and mt-Keima fluorescence imaging, Seahorse XF-based oxygen consumption rate measurement, and Mitoplate S-1 mitochondrial substrate utilization analysis. RESULTS: Transcriptomic analysis indicated that mitochondria-related gene sets were dysregulated in AhR KO liver. In both primary mouse hepatocytes and AML12 hepatocyte cell lines, AhR inhibition strongly suppressed mitochondrial respiration rate and substrate utilization. AhR inhibition also blunted the fasting response of several essential autophagy genes and the mitophagy process. We further identified BCL2 interacting protein 3 (BNIP3), a mitophagy receptor that senses nutrient stress, as an AhR target gene. AhR is directly recruited to the Bnip3 genomic locus, and Bnip3 transcription was enhanced by AhR endogenous ligand treatment in wild-type liver and abolished entirely in AhR KO liver. Mechanistically, overexpression of Bnip3 in AhR knockdown cells mitigated the production of mitochondrial reactive oxygen species (ROS) and restored functional mitophagy. CONCLUSIONS: AhR regulation of the mitophagy receptor BNIP3 coordinates hepatic mitochondrial function. Loss of AhR induces mitochondrial ROS production and impairs mitochondrial respiration. These findings provide new insight into how endogenous AhR governs hepatic mitochondrial homeostasis.

Accumulating evidence suggests that chronic inflammation of metabolic tissues plays a causal role in obesity-induced insulin resistance. Yet, how specific endothelial factors impact metabolic tissues remains undefined. Bone morphogenetic protein (BMP)–binding endothelial regulator (BMPER) adapts endothelial cells to inflammatory stress in diverse organ microenvironments. Here, we demonstrate that BMPER is a driver of insulin sensitivity. Both global and endothelial cell-specific inducible knockout of BMPER cause hyperinsulinemia, glucose intolerance and insulin resistance without increasing inflammation in metabolic tissues in mice. BMPER can directly activate insulin signaling, which requires its internalization and interaction with Niemann-Pick C1 (NPC1), an integral membrane protein that transports intracellular cholesterol. These results suggest that the endocrine function of the vascular endothelium maintains glucose homeostasis. Of potential translational significance, the delivery of BMPER recombinant protein or its overexpression alleviates insulin resistance and hyperglycemia in high-fat diet-fed mice and Leprdb/db (db/db) diabetic mice. We conclude that BMPER exhibits therapeutic potential for the treatment of diabetes., Type 2 diabetes is associated with chronic inflammation and is characterized by insulin resistance. Here, the authors identify a crucial role for endothelial BMPER function in glucose homeostasis, and BMPER overexpression was shown to alleviate insulin resistance and hyperglycemia in diabetic mice.

CD11c+ macrophages/dendritic cells (MDCs) are increased and display the classically activated M1-like phenotype in obese adipose tissue (AT) and may contribute to AT inflammation and insulin resistance. Stat1 is a key transcription factor for MDC polarization into the M1-like phenotype. Here, we examined the role of Stat1 in obesity-induced AT MDC polarization and inflammation and insulin resistance using mice with specific knockout of Stat1 in MDCs (cKO). Stat1 was upregulated and phosphorylated, indicating activation, early and persistently in AT and AT MDCs of wild-type mice fed a high-fat diet (HFD). Compared with littermate controls, cKO mice fed an HFD (16 weeks) had reductions in MDC (mainly CD11c+ macrophage) M1-like polarization and interferon-γ–expressing T-helper type 1 (Th1) cells but increases in interleukin 5–expressing Th2 cells and eosinophils in perigonadal and inguinal AT, and enhanced inguinal AT browning, with increased energy expenditure. cKO mice compared with controls also had significant reductions in triglyceride content in the liver and skeletal muscle and exhibited improved insulin sensitivity and glucose tolerance. Taken together, our results demonstrate that Stat1 in MDCs plays an important role in obesity-induced MDC M1-like polarization and AT inflammation and contributes to insulin resistance and metabolic dysfunctions in obese mice.

MicroRNA-30a (miR-30a) impacts adipocyte function, and its expression in white adipose tissue (WAT) correlates with insulin sensitivity in obesity. Bioinformatic analysis demonstrates that miR-30a expression contributes to 2% of all miRNA expression in human tissues. However, molecular mechanisms of miR-30a function in fat cells remain unclear. Here, we expanded our understanding of how miR-30a expression contributes to antidiabetic peroxisome proliferator-activated receptor-γ (PPARγ) agonist activity and metabolic functions in adipocytes. We found that WAT isolated from diabetic patients shows reduced miR-30a levels and diminished expression of the canonical PPARγ target genes ADIPOQ and FABP4 relative to lean counterparts. In human adipocytes, miR-30a required PPARγ for maximal expression, and the PPARγ agonist rosiglitazone robustly induced miR-30a but not other miR-30 family members. Transcriptional activity studies in human adipocytes also revealed that ectopic expression of miR-30a enhanced the activity of rosiglitazone coupled with higher expression of fatty acid and glucose metabolism markers. Diabetic mice that overexpress ectopic miR-30a in subcutaneous WAT display durable reductions in serum glucose and insulin levels for more than 30 days. In agreement with our in vitro findings, RNA-seq coupled with Gene Set Enrichment Analysis (GSEA) suggested that miR-30a enabled activation of the beige fat program in vivo, as evidenced by enhanced mitochondrial biogenesis and induction of UCP1 expression. Metabolomic and gene expression profiling established that the long-term effects of ectopic miR-30a expression enable accelerated glucose metabolism coupled with subcutaneous WAT hyperplasia. Together, we establish a putative role of miR-30a in mediating PPARγ activity and advancing metabolic programs of white to beige fat conversion.

Our early-life environment has a profound influence on developing organs that impacts metabolic function and determines disease susceptibility across the life-course. Using a rat model for exposure to an endocrine disrupting chemical (EDC), we show that early-life chemical exposure causes metabolic dysfunction in adulthood and reprograms histone marks in the developing liver to accelerate acquisition of an adult epigenomic signature. This epigenomic reprogramming persists long after the initial exposure, but many reprogrammed genes remain transcriptionally silent with their impact on metabolism not revealed until a later life exposure to a Western-style diet. Diet-dependent metabolic disruption was largely driven by reprogramming of the Early Growth Response 1 (EGR1) transcriptome and production of metabolites in pathways linked to cholesterol, lipid and one-carbon metabolism. These findings demonstrate the importance of epigenome:environment interactions, which early in life accelerate epigenomic aging, and later in adulthood unlock metabolically restricted epigenetic reprogramming to drive metabolic dysfunction., Early life exposure to environmental stressors, including endocrine disrupting chemicals (EDCs), can impact health later in life. Here, the authors show that neonatal EDC exposure in rats causes epigenetic reprogramming in the liver, which is transcriptionally silent until animals are placed on a Western-style diet.

Dynamin-Related-Protein 1 (DRP1) critically regulates mitochondrial and peroxisomal fission in multicellular organisms. However, the impact of DRP1 on other organelles, especially its direct influence on ER functions remains largely unclear. Here, we report that DRP1 translocates to endoplasmic reticulum (ER) in response to β-adrenergic stimulation. To further investigate the function of DRP1 on ER-lipid droplet (LD) dynamics and the metabolic subsequences, we generated an adipose tissue-specific DRP1 knockout model (Adipo-Drp1(flx/flx)). We found that the LDs in adipose tissues of Adipo-Drp1(flx/flx) mice exhibited more unilocular morphology with larger sizes, and formed less multilocular structures upon cold exposure. Mechanistically, we discovered that abnormal LD morphology occurs because newly generated micro-LDs fail to dissociate from the ER due to DRP1 ablation. Conversely, the ER retention of LDs can be rescued by the overexpressed DRP1 in the adipocytes. The alteration of LD dynamics, combined with abnormal mitochondrial and autophagy functions in adipose tissue, ultimately lead to abnormalities in lipid metabolism in Adipo-Drp1(flx/flx) mice.

Type 4 sphingosine-1-phosphate receptor, which mediates the activation of the Nod-like receptor (NLR) family pyrin domain containing 3 inflammasome, emerges as a new therapeutic target for nonalcoholic steatohepatitis. We developed a selective functional antagonist for type 4 sphingosine-1-phosphate receptor (SLB736), which protected mice against nonalcoholic steatohepatitis and fibrosis, Supported by the National Research Foundation of Korea, funded by the Ministry of Education, Science, and Technology, Korea, grants 2017R1E1A1A01073206 (K.-U.L.), 2020R1A2B5B02098524 (E.H.K.), 2019R1C1C1003567 (J.-Y.Y.), and 2021R1A2C1005520 (J.W.C.), the Korea Drug Development Fund funded by the Ministry of Science and ICT, Ministry of Trade, Industry, and Energy, and Ministry of Health and Welfare grant KDDF-201406-03 (K.-U.L.), and Asan Institute for Life Sciences, Korea, grant 2020IP0016-1 (E.H.K.). This work also was supported by the Mid-Career Researcher Programs grant NRF-2019R1A2C2009905 (S.K.) of the National Research Foundation of Korea, funded by the Government of Korea (MSIP); and by grants PID2019-111669RB-I00 and PID2020-115055RB-I00 from Plan Nacional de Investigación y Desarrollo, Spain, by the Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas, Instituto de Salud Carlos III, Spain; by grant P50AA011999 from the Southern California Research Center for Alcoholic Liver and Pancreatic Diseases and Cirrhosis funded by National Institute of Alcohol Abuse and Alcoholism; by Agency for Administration of University and Research Grants of the Generalitat de Cataluña SGR-2017-1112 and the “ER stress-mitochondrial cholesterol axis in obesity-associated insulin resistance and comorbidities” Ayudas Fundación Banco Bilbao Vizcaya Argentaria a Equipos de Investigación Científica 2017, the Red Nacional 2018-102799-T de Enfermedades Metabólicas y Cáncer, and by project 201916/31 Contribution of mitochondrial oxysterol and bile acid metabolism to liver carcinogenesis 2019 by Fundació Marato Televisión 3. None of the funders had a role in the study design, collection, analysis, or interpretation of data.

An extensive literature base combined with advances in sequencing technologies demonstrate microRNA levels correlate with various metabolic diseases. Mechanistic studies also establish microRNAs regulate central metabolic pathways and thus play vital roles in maintaining organismal energy balance and metabolic homeostasis. This review highlights research progress on the roles and regulation of microRNAs in the peripheral tissues that confer insulin sensitivity. We discuss sequencing technologies used to comprehensively define the target spectrum of microRNAs in metabolic disease that complement studies reporting physiologic roles for microRNAs in the regulation of glucose and lipid metabolism in animal models. We also discuss the emerging roles of exosomal microRNAs as endocrine signals to regulate lipid and carbohydrate metabolism.

Low-grade, sustained inflammation in white adipose tissue (WAT) characterizes obesity and coincides with type 2 diabetes mellitus (T2DM). However, pharmacological targeting of inflammation lacks durable therapeutic effects in insulin-resistant conditions. Through a computational screen, we discovered that the FDA-approved rheumatoid arthritis drug auranofin improved insulin sensitivity and normalized obesity-associated abnormalities, including hepatic steatosis and hyperinsulinemia in mouse models of T2DM. We also discovered that auranofin accumulation in WAT depleted inflammatory responses to a high-fat diet without altering body composition in obese wild-type mice. Surprisingly, elevated leptin levels and blunted beta-adrenergic receptor activity achieved by leptin receptor deletion abolished the antidiabetic effects of auranofin. These experiments also revealed that the metabolic benefits of leptin reduction were superior to immune impacts of auranofin in WAT. Our studies uncover important metabolic properties of anti-inflammatory treatments and contribute to the notion that leptin reduction in the periphery can be accomplished to treat obesity and T2DM.

DNA Methyltransferase 3A (DNMT3A) is an important facilitator of differentiation of both embryonic and hematopoietic stem cells. Heterozygous germline mutations in DNMT3A lead to Tatton-Brown-Rahman Syndrome (TBRS), characterized by obesity and excessive height. While DNMT3A is known to impact feeding behavior via the hypothalamus, here we investigated a role in adipocyte progenitors utilizing heterozygous knockout mice that recapitulate cardinal TBRS phenotypes. These mice become morbidly obese due to adipocyte enlargement and tissue expansion. Adipose tissue in these mice exhibited defects in preadipocyte maturation and precocious activation of inflammatory gene networks, including interleukin-6 signaling. Adipocyte progenitor cell lines lacking DNMT3A exhibited aberrant differentiation. Furthermore, mice in which Dnmt3a was specifically ablated in adipocyte progenitors showed enlarged fat depots and increased progenitor numbers, partly recapitulating the TBRS obesity phenotypes. Loss of DNMT3A led to constitutive DNA hypomethylation, such that the DNA methylation landscape of young adipocyte progenitors resemble that of older wild-type mice. Together, our results demonstrate that DNMT3A coordinates both the central and local control of energy storage required to maintain normal weight and prevent inflammatory obesity.

SUMMARYLow-grade, sustained inflammation in white adipose tissue (WAT) characterizes obesity and frequently coincides with insulin resistance and type 2 diabetes (T2D). However, pharmacological targeting of WAT inflammation lacks durable therapeutic effects. Through a computational screen, we identified the FDA-approved rheumatoid arthritis drug auranofin is a putative small molecule for obesity treatment. We discovered that allometrically scaled safe auranofin doses homed to WAT and improved insulin sensitivity in obese wild-type mice. Auranofin treatment also normalized other obesity-associated abnormalities, including hepatic steatosis and hyperinsulinemia. Surprisingly, the anti-diabetic effects of auranofin required leptin lowering and beta-adrenergic receptors in WAT. These metabolic benefits of leptin reduction were superior to any immune impacts of auranofin in WAT. Our studies reveal important metabolic properties of anti-inflammatory treatments and contribute to the notion that leptin reduction in the periphery can be accomplished to treat obesity and T2D.

Steroid hormone receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Nuclear Hormone Receptors [74, 215, 3]) are nuclear hormone receptors of the NR3 class, with endogenous agonists that may be divided into 3-hydroxysteroids (estrone and 17β-estradiol) and 3-ketosteroids (dihydrotestosterone [DHT], aldosterone, cortisol, corticosterone, progesterone and testosterone). For rodent GR and MR, the physiological ligand is corticosterone rather than cortisol.

The pancreatic islet is a dense cellular network comprised of several cell types with endocrine function vital in the control of glucose homeostasis, metabolism, and feeding behavior. Within the islet, endocrine hormones also form an intricate paracrine network with supportive cells (endothelial, neuronal, immune) and secondary signaling molecules regulating cellular function and survival. Modulation of these signals has potential consequences for diabetes development, progression, and therapeutic intervention. Beta cell loss, reduced endogenous insulin secretion, and dysregulated glucagon secretion are hallmark features of both type 1 and 2 diabetes that not only impact systemic regulation of glucose, but also contribute to the function and survival of cells within the islet. Advancing research and technology have revealed new islet biology (cellular identity and transcriptomes) and identified previously unrecognized paracrine signals and mechanisms (somatostatin and ghrelin paracrine actions), while shifting prior views of intraislet communication. This review will summarize the paracrine signals regulating islet endocrine function and survival, the disruption and dysfunction that occur in diabetes, and potential therapeutic targets to preserve beta cell mass and function.

The immune system plays a critical role in white adipose tissue (WAT) energy homeostasis and, by extension, whole-body metabolism. Substantial evidence from mouse and human studies firmly establishes that insulin sensitivity deteriorates as a result of subclinical inflammation in the adipose tissue of individuals with diabetes. However, the relationship between adipose tissue expandability and immune cell infiltration remains a complex problem important for understanding the pathogenesis of obesity. Notably, a large body of work challenges the idea that all immune responses are deleterious to WAT function. This review highlights recent advances that describe how immune cells and adipocytes coordinately enable WAT expansion and regulation of energy homeostasis.

Persons living with HIV (PLWH) manifest chronic disorders of brown and white adipose tissues that lead to diabetes and metabolic syndrome. The mechanisms that link viral factors to defective adipose tissue function and abnormal energy balance in PLWH remain incompletely understood. Here, we explored how the HIV accessory protein viral protein R (Vpr) contributes to adaptive thermogenesis in two mouse models and human adipose tissues. Uncoupling protein 1 (UCP1) gene expression was strongly increased in subcutaneous white adipose tissue (WAT) biopsies from PLWH and in subcutaneous WAT of the Vpr mice, with near-equivalent mRNA copy number. Histology and functional studies confirmed beige transformation in subcutaneous but not visceral WAT in the Vpr mice. Measurements of energy balance indicated Vpr mice displayed metabolic inflexibility and could not shift efficiently from carbohydrate to fat metabolism during day-night cycles. Furthermore, Vpr mice showed a marked inability to defend body temperature when exposed to 4oC. Importantly, Vpr couples higher tissue catecholamine levels with UCP1 expression independent of β-adrenergic receptors. Our data reveal surprising deficits of adaptive thermogenesis that drive metabolic inefficiency in HIV-1 Vpr mouse models, providing an expanded role for viral factors in the pathogenesis of metabolic disorders in PLWH.

Although macroautophagy/autophagy deficiency causes degenerative diseases, the deletion of essential autophagy genes in adipocytes paradoxically reduces body weight. Brown adipose tissue (BAT) plays an important role in body weight regulation and metabolic control. However, the key cellular mechanisms that maintain BAT function remain poorly understood. in this study, we showed that global or brown adipocyte-specific deletion of pink1, a Parkinson disease-related gene involved in selective mitochondrial autophagy (mitophagy), induced BAT dysfunction, and obesity-prone type in mice. Defective mitochondrial function is among the upstream signals that activate the NLRP3 inflammasome. NLRP3 was induced in brown adipocyte precursors (BAPs) from pink1 knockout (KO) mice. Unexpectedly, NLRP3 induction did not induce canonical inflammasome activity. Instead, NLRP3 induction led to the differentiation of pink1 KO BAPs into white-like adipocytes by increasing the expression of white adipocyte-specific genes and repressing the expression of brown adipocyte-specific genes. nlrp3 deletion in pink1 knockout mice reversed BAT dysfunction. Conversely, adipose tissue-specific atg7 KO mice showed significantly lower expression of Nlrp3 in their BAT. Overall, our data suggest that the role of mitophagy is different from general autophagy in regulating adipose tissue and whole-body energy metabolism. Our results uncovered a new mitochondria-NLRP3 pathway that induces BAT dysfunction. The ability of the nlrp3 knockouts to rescue BAT dysfunction suggests the transcriptional function of NLRP3 as an unexpected, but a quite specific therapeutic target for obesity-related metabolic diseases. Abbreviations: ACTB: actin, beta; BAPs: brown adipocyte precursors; BAT: brown adipose tissue; BMDMs: bone marrow-derived macrophages; CASP1: caspase 1; CEBPA: CCAAT/enhancer binding protein (C/EBP), alpha; ChIP: chromatin immunoprecipitation; EE: energy expenditure; HFD: high-fat diet; IL1B: interleukin 1 beta; ITT: insulin tolerance test; KO: knockout; LPS: lipopolysaccharide; NLRP3: NLR family, pyrin domain containing 3; PINK1: PTEN induced putative kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; RD: regular diet; ROS: reactive oxygen species; RT: room temperature; UCP1: uncoupling protein 1 (mitochondrial, proton carrier); WT: wild-type.

White adipose tissue (WAT) depends on coordinated regulation of transcriptional and metabolic pathways to respond to whole-body energy demands. We highlight metabolites that contribute to biosynthetic reactions for WAT expansion. Recent studies have precisely defined how byproducts of carbohydrate and lipid metabolism affect physiological and endocrine functions in adipocytes. We emphasize the critical emerging roles of short-chain fatty acids (SCFAs) and tricarboxylic acid (TCA) cycle metabolites that connect lipogenesis to WAT energy balance and endocrine functions. These insights address how adipocytes use small molecules generated from central carbon metabolism to measure responses to nutritional stress.

Bile acids have recently emerged as key metabolic hormones with beneficial impacts in multiple metabolic diseases. We previously discovered that hepatic bile acid overload distally modulates glucose and fatty acid metabolism in adipose tissues to exert anti-obesity effects. However, the detailed mechanisms that explain the salutary effects of serum bile acid elevation remain unclear. Here, proteomic profiling identified a new hepatokine, Orosomucoid (ORM) that governs liver-adipose tissue crosstalk. Hepatic ORMs were highly induced by both genetic and dietary bile acid overload. To address the direct metabolic effects of ORM, purified ORM proteins were administered during adipogenic differentiation of 3T3-L1 cells and mouse stromal vascular fibroblasts. ORM suppressed adipocyte differentiation and strongly inhibited gene expression of adipogenic transcription factors such as C/EBPβ, KLF5, C/EBPα, and PPARγ. Taken together, our data clearly suggest that bile acid-induced ORM secretion from the liver blocks adipocyte differentiation, potentially linked to anti-obesity effect of bile acids.

CD11c+ macrophages/dendritic cells (MDCs) are increased and display classically activated M1-like phenotype in obese adipose tissue (AT) and may contribute to AT inflammation and insulin resistance. Stat1 is a key transcription factor for MDC polarization into M1-like phenotype. Here, we examined the role of Stat1 in obesity-induced AT MDC polarization and inflammation and insulin resistance using mice with specific knockout of Stat1 in MDCs (cKO). Stat1 was upregulated and phosphorylated, indicating activation, early and persistently in AT and AT MDCs of wild-type mice fed high-fat diet (HFD). Compared to littermate controls, cKO mice fed HFD (16 weeks) had reductions in MDC (mainly CD11c+ macrophage) M1-like polarization and interferon-g–expressing T helper type 1 (Th1) cells, but increases in interleukin-5–expressing Th2 cells and eosinophils in perigonadal and inguinal AT, and enhanced inguinal AT browning, with increased energy expenditure. cKO mice compared with controls also had significant reductions in triglyceride content in the liver and skeletal muscle and exhibited improved insulin sensitivity and glucose tolerance. Taken together, our results demonstrated that Stat1 in MDCs plays an important role in obesity-induced MDC M1-like polarization and AT inflammation and contributes to insulin resistance and metabolic dysfunctions in obese mice.

Sphingosine 1-phosphate (S1P) receptors (S1PRs) are a group of G protein-coupled receptors that confer a broad range of functional effects in chronic inflammatory diseases and metabolic diseases. S1PRs may also be involved in the development of non-alcoholic steatohepatitis (NASH), but the specific subtypes involved and the mechanism of action are unclear. Here we show that the livers of various mouse models of NASH as well as Kupffer cells had a particularly strong expression of S1pr4. Accordingly, genetic depletion of S1pr4 protected the mice against hepatic inflammation and fibrosis. Moreover, SLB736, a novel selective functional antagonist of SIPR4 prevented diet-induced NASH in mice without lymphopenia. S1P increased the expression of S1pr4 in Kupffer cells and activated the NLRP3 inflammasome through PLC/IP3/IP3R-dependent [Ca++] signaling. SLB736 treatment or S1pr4 depletion in Kupffer cells inhibited LPS-mediated Ca++ release and deactivated the NLRP3 inflammasome. S1PR4 antagonism may be a novel therapeutic strategy for NASH.

Obesity fosters low-grade inflammation in white adipose tissue (WAT) that may contribute to the insulin resistance that characterizes type 2 diabetes mellitus (T2DM). However, the causal relationship of these events remains unclear. The established dominance of signal transducer and activator of transcription 1 (STAT1) function in the immune response suggests an obligate link between inflammation and the co-morbidities of obesity. To this end, we sought to determine how STAT1 activity in white adipocytes affects insulin sensitivity. STAT1 expression in WAT inversely correlated with fasting plasma glucose in both obese mice and humans. Metabolomic and gene expression profiling established STAT1 deletion in adipocytes (STAT1a-KO­­­) enhanced mitochondrial function and accelerated TCA cycle flux coupled with reduced fat cell size in subcutaneous WAT depots. STAT1a-KO reduced WAT inflammation, but insulin resistance persisted in obese mice. Rather, elimination of type I cytokine interferon gamma (IFNg) activity enhanced insulin sensitivity in diet-induced obesity. Our findings reveal a permissive mechanism that bridges WAT inflammation to whole-body insulin sensitivity.

ObjectiveWhite adipose tissue (WAT) expansion regulates energy balance and overall metabolic homeostasis. WAT absence or loss occurring through lipodystrophy and lipoatrophy contributes to the development of dyslipidemia, hepatic steatosis, and insulin resistance. We previously demonstrated the sole small ubiquitin-like modifier (SUMO) E2-conjuguating enzyme Ubc9 represses human adipocyte differentiation. Germline and other tissue-specific deletions of Ubc9 frequently cause lethality in mice. As a result, the role of Ubc9 during WAT development remains unknown.MethodsTo determine how Ubc9 impacts body composition and energy balance, we generated adipocyte-specific Ubc9 knockout mice (Ubc9a-KO). CRISPR/Cas9 gene editing inserted loxP sites flanking exons 3 and 4 at the Ubc9 locus. Subsequent genetic crosses to AdipoQ-Cre transgenic mice allowed deletion of Ubc9 in white and brown adipocytes. We measured multiple metabolic endpoints that describe energy balance and carbohydrate metabolism in Ubc9a-KO and littermate controls during postnatal growth.ResultsTo our surprise, Ubc9a-KO mice developed hyperinsulinemia and hepatic steatosis. Global energy balance defects emerged from dysfunctional WAT marked by pronounced local inflammation, loss of serum adipokines, hepatomegaly, and near absence of major adipose tissue depots. We observed progressive lipoatrophy that commences in the early adolescent period.ConclusionsOur results demonstrate that Ubc9 expression in mature adipocytes is essential for maintaining WAT expansion. Deletion of Ubc9 in fat cells compromised and diminished adipocyte function that provoked WAT inflammation and ectopic lipid accumulation in the liver. Our findings reveal an indispensable role for Ubc9 during white adipocyte expansion and endocrine control of energy balance.

CD11c(+) macrophages/dendritic cells (MDCs) are increased and display the classically activated M1-like phenotype in obese adipose tissue (AT) and may contribute to AT inflammation and insulin resistance. Stat1 is a key transcription factor for MDC polarization into the M1-like phenotype. Here, we examined the role of Stat1 in obesity-induced AT MDC polarization and inflammation and insulin resistance using mice with specific knockout of Stat1 in MDCs (cKO). Stat1 was upregulated and phosphorylated, indicating activation, early and persistently in AT and AT MDCs of wild-type mice fed a high-fat diet (HFD). Compared with littermate controls, cKO mice fed an HFD (16 weeks) had reductions in MDC (mainly CD11c(+) macrophage) M1-like polarization and interferon-γ–expressing T-helper type 1 (Th1) cells but increases in interleukin 5–expressing Th2 cells and eosinophils in perigonadal and inguinal AT, and enhanced inguinal AT browning, with increased energy expenditure. cKO mice compared with controls also had significant reductions in triglyceride content in the liver and skeletal muscle and exhibited improved insulin sensitivity and glucose tolerance. Taken together, our results demonstrate that Stat1 in MDCs plays an important role in obesity-induced MDC M1-like polarization and AT inflammation and contributes to insulin resistance and metabolic dysfunctions in obese mice.

Obesity is associated with chronic low-grade inflammation, reflecting significant alterations in the composition of immune cell populations that reside in white adipose tissue (WAT). The ensuing pro-inflammatory environment likely impinges on the metabolic functions of adipocytes and may partially explain the insulin resistance that characterizes type 2 diabetes mellitus (T2DM). We recently demonstrated ectopic expression of the microRNA miR-30a in the subcutaneous fat pad of diabetic mice coupled improved insulin sensitivity and increased energy expenditure with decreased ectopic fat deposition in the liver and reduced WAT inflammation. We subsequently pursued the notion that pharmacological inhibitors of inflammation that exhibit a gene expression profile similar to ectopic miR-30a expression in WAT should represent new drugs for insulin resistance and T2DM. To this end, we used the Broad Connectivity Map to analyze a library of small molecules that induce mRNA profiles similar to that of exogenous miR-30a expression in WAT. Through this process, we nominated the rheumatoid arthritis compound auranofin. Treatment of obese mice with auranofin improved insulin sensitivity and glucose tolerance. Auranofin treatment also normalized other obesity-associated abnormalities, including hepatic steatosis, serum insulin and leptin. Mechanistically, a combination of proteomics and immunophenotyping established auranofin likely exerts antidiabetic activities by opposing low-grade inflammation in WAT and ectopic liver fat accumulation. These studies reveal important metabolic properties of anti-inflammatory treatments that may be re-purposed as therapies for insulin resistance and T2DM. Disclosure A. Cox: None. P.M. Masschelin: None. N. Chernis: None. P. Saha: None. Z. Lian: None. J.B. Felix: None. K. Kim: None. H. Wu: None. S.M. Hartig: None. Funding American Diabetes Association (1-18-IBS-105 to S.M.H.); National Institutes of Health (R01DK114356)

The HIV accessory protein Vpr contributes to cardinal metabolic defects noted in persons living with HIV (PLWH), including accelerated lipolysis, impaired fatty acid oxidation, slowed fatty acid export, and steatohepatitis (Agarwal et al, Sci Trans Med 2017). PLWH also manifest “beiging” and markers of thermogenesis within some white adipose tissue (WAT) depots, but paradoxically these changes correlate with decreased insulin sensitivity and hyperlipidemia. We sought to determine whether Vpr activity affects thermogenesis and the dynamics of beiging in two Vpr mouse models. UCP1 expression was strongly increased in subcutaneous fat of Vpr mice and showed equivalent copy number in WAT biopsies from PLWH. Histology confirmed browning of subcutaneous but not visceral WAT in the Vpr mouse models. Adipocytes derived from these depots showed increased oxygen consumption rate in subcutaneous but not visceral fat cells, also strongly indicative of beiging. Measurements of energy balance indicated Vpr mice display metabolic inflexibility and cannot shift efficiently from carbohydrates to fat during day-night cycles. Furthermore, Vpr mice mice showed a marked inability to defend body temperature when exposed to 4oC for 6 hours. Thus HIV-1 Vpr disrupts metabolic efficiency coupled with maladaptive subcutaneous WAT beiging. These data suggest an expanded role for Vpr in the pathogenesis of HIV-associated metabolic disorders. Disclosure N. Agarwal: None. P. Saha: None. S.M. Hartig: None. A. Balasubramanyam: None. Funding National Institutes of Health (R21/R33AI116208)

White adipose tissue (WAT) is an endocrine organ that dynamically expands and contracts to meet the metabolic demands of the organism. However, prolonged excessive caloric intake overwhelms WAT depots, resulting in peripheral fat deposition, systemic glucose dysregulation, tissue fibrosis, insulin resistance, and type 2 diabetes mellitus. The chronic triggers that degrade WAT function remain undefined, hindering the development of effective strategies to treat obesity and prevent its co-morbidities. We have identified a microRNA, miR-30a, that potently stimulates subcutaneous adipose tissue metabolic flexibility and enhances systemic insulin sensitivity. In follow-up studies, we created a genetically engineered mouse model to conclusively show miR-30a expression in white adipocytes is antidiabetic. Obese mice modified to express miR-30a at elevated levels in adipose tissues maintain insulin sensitivity coupled with reduced hepatic steatosis. Proteomic profiling indicated that miR-30a targets Plasminogen Activator Inhibitor-1 (PAI-1) to limit pro-fibrotic programs that would otherwise restrict subcutaneous WAT expansion and decrease insulin sensitivity. While visceral WAT depots remain a site of significant fibrosis in mice, subcutaneous WAT fibrosis is epidemiologically linked to metabolic disease risk in adult humans. In obese humans, miR-30a expression negatively correlates with PAI-1 levels in subcutaneous WAT, supporting an anti-fibrotic role for miR-30a that ultimately defends insulin sensitivity. Collectively, our observations linking miR-30a expression to insulin sensitivity suggest a new method that restores WAT homeostasis. Disclosure N. Chernis: None. P. Saha: None. J.B. Felix: None. P.M. Masschelin: None. L. Lian: None. A. Cox: None. K. Kim: None. H. Wu: None. S.M. Hartig: None. Funding American Diabetes Association (1-18-IBS-105 to S.M.H.); National Institutes of Health (R01DK114356)

Obesity fosters low-grade inflammation in white adipose tissue (WAT) that may contribute to the insulin resistance that characterizes type 2 diabetes mellitus (T2DM). However, the causal relationship of these events remains unclear. The established dominance of signal transducer and activator of transcription 1 (STAT1) function in the immune response suggests an obligate link between inflammation and the co-morbidities of obesity. To this end, we sought to determine how STAT1 activity in white adipocytes affects insulin sensitivity. STAT1 expression in WAT inversely correlated with fasting plasma glucose in both obese mice and humans. Metabolomic and gene expression profiling established STAT1 deletion in adipocytes (STAT1a-KO) enhanced mitochondrial function and accelerated TCA cycle flux coupled with reduced fat cell size in subcutaneous WAT depots. STAT1a-KO reduced WAT inflammation, but insulin resistance persisted in obese mice. Rather, elimination of type I cytokine interferon gamma (IFNγ) activity enhanced insulin sensitivity in diet-induced obesity. Our findings reveal a permissive mechanism that bridges WAT inflammation to whole-body insulin sensitivity.

Bile acid (BA) homeostasis is tightly regulated by multiple transcription factors, including farnesoid X receptor (FXR) and small heterodimer partner (SHP). We previously reported that loss of the FXR/SHP axis causes severe intrahepatic cholestasis, similar to human progressive familial intrahepatic cholestasis type 5 (PFIC5). In this study, we found that constitutive androstane receptor (CAR) is endogenously activated in Fxr:Shp double knockout (DKO) mice. To test the hypothesis that CAR activation protects DKO mice from further liver damage, we generated Fxr;Shp;Car triple knockout (TKO) mice. In TKO mice, residual adenosine triphosphate (ATP) binding cassette, subfamily B member 11 (ABCB11; alias bile salt export pump [BSEP]) function and fecal BA excretion are completely impaired, resulting in severe hepatic and biliary damage due to excess BA overload. In addition, we discovered that pharmacologic CAR activation has different effects on intrahepatic cholestasis of different etiologies. In DKO mice, CAR agonist 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP; here on TC) treatment attenuated cholestatic liver injury, as expected. However, in the PFIC2 model Bsep knockout (BKO) mice, TC treatment exhibited opposite effects that reflect increased BA accumulation and liver injury. These contrasting results may be linked to differential regulation of systemic cholesterol homeostasis in DKO and BKO livers. TC treatment selectively up-regulated hepatic cholesterol levels in BKO mice, supporting de novo BA synthesis. Conclusion: CAR activation in DKO mice is generally protective against cholestatic liver injury in these mice, which model PFIC5, but not in the PFIC2 model BKO mice. Our results emphasize the importance of the genetic and physiologic background when implementing targeted therapies to treat intrahepatic cholestasis.

Ductal carcinoma in situ (DCIS) is a non-obligate precursor to most types of invasive breast cancer (IBC). Although it is estimated only one third of untreated patients with DCIS will progress to IBC, standard of care for treatment is surgery and radiation. This therapeutic approach combined with a lack of reliable biomarker panels to predict DCIS progression is a major clinical problem. DCIS shares the same molecular subtypes as IBC including estrogen receptor (ER) and progesterone receptor (PR) positive luminal subtypes, which encompass the majority (60–70%) of DCIS. Compared to the established roles of ER and PR in luminal IBC, much less is known about the roles and mechanism of action of estrogen (E2) and progesterone (P4) and their cognate receptors in the development and progression of DCIS. This is an underexplored area of research due in part to a paucity of suitable experimental models of ER+/PR + DCIS. This review summarizes information from clinical and observational studies on steroid hormones as breast cancer risk factors and ER and PR as biomarkers in DCIS. Lastly, we discuss emerging experimental models of ER+/PR+ DCIS.

Endocrine-disrupting chemicals interact with transcription factors essential for adipocyte differentiation. Exposure to endocrine-disrupting chemicals corresponds with elevated risks of obesity, but the effects of these compounds on human cells remain largely undefined. Widespread use of bisphenol AF (BPAF) as a bisphenol A (BPA) alternative in the plastics industry presents unknown health risks. To this end, we discovered that BPAF interferes with the metabolic function of mature human adipocytes. Although 4-day exposures to BPAF accelerated adipocyte differentiation, we observed no effect on mature fat cell marker genes. Additional gene and protein expression analysis showed that BPAF treatment during human adipocyte differentiation failed to suppress the proinflammatory transcription factor STAT1. Microscopy and respirometry experiments demonstrated that BPAF impaired mitochondrial function and structure. To test the hypothesis that BPAF fosters vulnerabilities to STAT1 activation, we treated mature adipocytes previously exposed to BPAF with interferon-γ (IFNγ). BPAF increased IFNγ activation of STAT1 and exposed mitochondrial vulnerabilities that disrupt adipocyte lipid and carbohydrate metabolism. Collectively, our data establish that BPAF activates inflammatory signaling pathways that degrade metabolic activity in human adipocytes. These findings suggest how the BPA alternative BPAF contributes to metabolic changes that correspond with obesity.

Hormones produced by the anterior pituitary gland regulate an array of important physiological functions, but the causes of pituitary hormone disorders are not fully understood. Herein we report that genetically engineered mice with deletion of the hedgehog signaling receptor PATCHED1 (Ptch1) by S100a4 promoter–driven Cre recombinase (S100a4-Cre;Ptch1(fl/fl) mutants) exhibit adult-onset hypogonadotropic hypogonadism and multiple pituitary hormone disorders. During the transition from puberty to adulthood, S100a4-Cre;Ptch1(fl/fl) mice of both sexes develop hypogonadism coupled with reduced gonadotropin levels. Their pituitary glands also display severe structural and functional abnormalities, as revealed by transmission electron microscopy and expression of key genes regulating pituitary endocrine functions. S100a4-Cre activity in the anterior pituitary gland is restricted to CD45(+) cells of hematopoietic origin, including folliculo-stellate cells and other immune cell types, causing sex-specific changes in the expression of genes regulating the local microenvironment of the anterior pituitary. These findings provide in vivo evidence for the importance of pituitary hematopoietic cells in regulating fertility and endocrine function, in particular during sexual maturation and likely through sexually dimorphic mechanisms. These findings support a previously unrecognized role of hematopoietic cells in causing hypogonadotropic hypogonadism and provide inroads into the molecular and cellular basis for pituitary hormone disorders in humans.

Specific metabolic underpinnings of androgen receptor (AR)-driven growth in prostate adenocarcinoma (PCa) are largely undefined, hindering the development of strategies to leverage the metabolic dependencies of this disease when hormonal manipulations fail. Here we show that the mitochondrial pyruvate carrier (MPC), a critical metabolic conduit linking cytosolic and mitochondrial metabolism, is transcriptionally regulated by AR. Experimental MPC inhibition restricts proliferation and metabolic outputs of the citric acid cycle (TCA) including lipogenesis and oxidative phosphorylation in AR-driven PCa models. Mechanistically, metabolic disruption resulting from MPC inhibition activates the eIF2α/ATF4 integrated stress response (ISR). ISR signaling prevents cell cycle progression while coordinating salvage efforts, chiefly enhanced glutamine assimilation into the TCA, to regain metabolic homeostasis. We confirm that MPC function is operant in PCa tumors in-vivo using isotopomeric metabolic flux analysis. In turn, we apply a clinically viable small molecule targeting the MPC, MSDC0160, to pre-clinical PCa models and find that MPC inhibition suppresses tumor growth in hormone-responsive and castrate-resistant conditions. Collectively, our findings characterize the MPC as a tractable therapeutic target in AR-driven prostate tumors.

Obesity associates with chronic low-grade inflammation that likely impinges on the metabolic functions of adipocytes and may partially explain the insulin resistance that characterizes type 2 diabetes mellitus (T2DM). We recently demonstrated ectopic expression of the microRNA miR-30a in the subcutaneous fat pad of diabetic mice coupled improved insulin sensitivity and increased energy expenditure with decreased ectopic fat deposition in the liver and reduced WAT inflammation. Further, miR-30a directly inhibited STAT1, a major mediator of inflammatory signals such as interferon gamma (IFNγ). Despite the established dominance of STAT1 in the immune response and the importance of inflammation in the development of T2DM, the role of STAT1 in white adipocytes remains unknown. To this end, we sought to determine how STAT1 activity in white adipocytes affects insulin sensitivity. IFNγ treatment of human adipocytes diminished oxidative phosphorylation capacity, along with expression of mitochondrial proteins. STAT1 deletion in white adipocytes (STAT1 fKO) did not change body weight, but improved whole-body insulin sensitivity of diabetic mice. Transcriptome profiling by RNA-Seq suggested STAT1 fKO adipocytes engage GABPa and NFIA target genes that reflect oxidative phosphorylation specifically in the inguinal white adipose tissue (iWAT). Additional metabolomics profiling indicated the metabolic effects of STAT1 deletion in iWAT are associated with accelerated TCA cycle flux and aspartic acid generation, which provides intermediates to support lipid synthesis and histone modifications. These studies reveal important roles of inflammatory signals to suppress metabolism and epigenetic regulation in adipocytes that may underlie insulin resistance and T2DM. Disclosure A. Cox: None. P. Masschelin: None. N. Chernis: None. S.M. Hartig: None. Funding American Diabetes Association (1-18-IBS-105 to S.M.H.); National Institutes of Health (R01DK114356); Baylor College of Medicine (P30DK079638)