Olfactomedin-2 (OLFM2) is a pleiotropic glycoprotein emerging as a regulator of energy homeostasis. We here show the expression of OLFM2 to be adipocyte-specific and inversely associated with obesity. OLFM2 levels increase during adipogenesis and are suppressed in inflamed adipocytes. Functionally, OLFM2 deficiency impairs adipocyte differentiation, while its over-production enhances the adipogenic transformation of fat cell progenitors. Loss and gain of function experiments revealed that OLFM2 modulates key metabolic and structural pathways, including PPAR signaling, citrate cycle, fatty acid degradation, axon guidance and focal adhesion in 3T3 cell lines and primary human adipocytes. On the molecular level, OLFM2 deficiency in differentiated adipocytes predominantly downregulates genes involved in cell cycle. Extending these findings in vivo, both whole-body Olfm2 knockout and adipose-specific Olfm2 depletion in mice resulted in impaired adipose cell cycle gene expression, with the latter also displaying fat mass accretion and metabolic dysfunction. Collectively, our results underscore a critical role for OLFM2 in adipocyte biology, and support a causative link between reduced adipose OLFM2 and the pathophysiology of obesity.

CBL is rapidly phosphorylated upon insulin receptor activation. Mice whole body CBL depletion improved insulin sensitivity and glucose clearance; however, the precise mechanisms remain unknown. We depleted either CBL or its associated protein SORBS1/CAP independently in myocytes and assessed mitochondrial function and metabolism compared to control cells. CBL- and CAP-depleted cells showed increased mitochondrial mass with greater proton leak. Mitochondrial respiratory complex I activity and assembly into respirasomes were reduced. Proteome profiling revealed alterations in proteins involved in glycolysis and fatty acid degradation. Our findings demonstrate CBL/CAP pathway couples insulin signaling to efficient mitochondrial respiratory function and metabolism in muscle.

The composition of extracellular vesicles (EVs) is altered in many pathological conditions, and their molecular content provides essential information on features of parent cells and mechanisms of crosstalk between cells and organs. Metabolic Syndrome (MetS) is a cluster of clinical manifestations including obesity, insulin resistance, dyslipidemia and hypertension that increases the risk of cardiovascular disease and type 2 diabetes mellitus. Here, we investigated the crosstalk between liver and adipocytes by characterizing EVs secreted by primary hepatocytes isolated from Zucker rat model, and studied the effect they have on 3T3-L1 adipocytes. We found that steatotic hepatocytes secrete EVs with significantly reduced exosomal markers in comparison with their lean counterpart. Moreover, proteomic analysis revealed that those EVs reflect the metabolic state of the parent cell in that the majority of proteins upregulated relate to fat metabolism, fatty acid synthesis, glycolysis, and pentose phosphate pathway. In addition, hepatocytes-secreted EVs influenced lipolysis and insulin sensitivity in recipient 3T3-L1 adipocytes. Untargeted metabolomic analysis detected alterations in different adipocyte metabolic pathways in cells treated with hepatic EVs. In summary, our work showed that steatosis has a significant impact in the amount and composition of EVs secreted by hepatocytes. Moreover, our data point to the involvement of hepatic-EVs in the development of pathologies associated with MetS.

Extracellular vesicles (EVs) are membrane enclosures released by eukaryotic cells that carry bioactive molecules and serve to modulate biological responses in recipient cells. Both increased EV release and altered EV composition are associated with the development and progression of many pathologies including cancer. Hypoxia, a feature of rapidly growing solid tumours, increases the release of EVs. However, the molecular mechanisms remain unknown. The hypoxia inducible factors (HIFs) are transcription factors that act as major regulators of the cellular adaptations to hypoxia. Here, we investigated the requirement of HIF pathway activation for EV release in Human Embryonic Kidney Cells (HEK293). Time course experiments showed that EV release increased concomitantly with sustained HIF1α and HIF2α activation following the onset of hypoxia. shRNA mediated knock-down of HIF1α but not HIF2α abrogated the effect of hypoxia on EV release, suggesting HIF1α is involved in this process. However, stabilization of HIF proteins in normoxic conditions through: (i) heterologous expression of oxygen insensitive HIF1α or HIF2α mutants in normoxic cells or (ii) chemical inhibition of the prolyl hydroxylase 2 (PHD2) repressor protein, did not increase EV release, suggesting HIF activation alone is not sufficient for this process. Our findings suggest HIF1α plays an important role in the regulation of EV release during hypoxia in HEK293 cells, however other hypoxia triggered mechanisms likely contribute as stabilization of HIF1α alone in normoxia is not sufficient for EV release.

Resulting from impaired collagen turnover, fibrosis is a hallmark of adipose tissue (AT) dysfunction and obesity-associated insulin resistance (IR). Prolidase, also known as peptidase D (PEPD), plays a vital role in collagen turnover by degrading proline-containing dipeptides but its specific functional relevance in AT is unknown. Here we show that in human and mouse obesity, PEPD expression and activity decrease in AT, and PEPD is released into the systemic circulation, which promotes fibrosis and AT IR. Loss of the enzymatic function of PEPD by genetic ablation or pharmacological inhibition causes AT fibrosis in mice. In addition to its intracellular enzymatic role, secreted extracellular PEPD protein enhances macrophage and adipocyte fibro-inflammatory responses via EGFR signalling, thereby promoting AT fibrosis and IR. We further show that decreased prolidase activity is coupled with increased systemic levels of PEPD that act as a pathogenic trigger of AT fibrosis and IR. Thus, PEPD produced by macrophages might serve as a biomarker of AT fibro-inflammation and could represent a therapeutic target for AT fibrosis and obesity-associated IR and type 2 diabetes. Obesity-associated AT fibro-inflammation and metabolic disturbances are linked to PEPD activity and PEPD extracellular levels.

Aging, obesity, and insulin resistance are associated with low levels of PGC1α and PGC1β coactivators and defective mitochondrial function. We studied mice deficient for PGC1α and PGC1β [double heterozygous (DH)] to investigate their combined pathogenic contribution. Contrary to our hypothesis, DH mice were leaner, had increased energy dissipation, a pro‐thermogenic profile in BAT and WAT, and improved carbohydrate metabolism compared to wild types. WAT showed upregulation of mitochondriogenesis/oxphos machinery upon allelic compensation of PGC1α4 from the remaining allele. However, DH mice had decreased mitochondrial OXPHOS and biogenesis transcriptomes in mitochondria‐rich organs. Despite being metabolically healthy, mitochondrial defects in DH mice impaired muscle fiber remodeling and caused qualitative changes in the hepatic lipidome. Our data evidence first the existence of organ‐specific compensatory allostatic mechanisms are robust enough to drive an unexpected phenotype. Second, optimization of adipose tissue bioenergetics is sufficient to maintain a healthy metabolic phenotype despite a broad severe mitochondrial dysfunction in other relevant metabolic organs. Third, the decrease in PGC1s in adipose tissue of obese and diabetic patients is in contrast with the robustness of the compensatory upregulation in the adipose of the DH mice.

The adipokine Neuregulin 4 (Nrg4) protects against obesity-induced insulin resistance. Here, we analyze how the downregulation of Nrg4 influences insulin action and the underlying mechanisms in adipocytes. Validated shRNA lentiviral vectors were used to generate scramble (Scr) and Nrg4 knockdown (KD) 3T3-L1 adipocytes. Adipogenesis was unaffected in Nrg4 KD adipocytes, but there was a complete impairment of the insulin-induced 2-deoxyglucose uptake, which was likely the result of reduced insulin receptor and Glut4 protein. Downregulation of Nrg4 enhanced the expression of proinflammatory cytokines. Anti-inflammatory agents recovered the insulin receptor, but not Glut4, content. Proteins enriched in Glut4 storage vesicles such as the insulin-responsive aminopeptidase (IRAP) and Syntaxin-6 as well as TBC1D4, a protein involved in the intracellular retention of Glut4 vesicles, also decreased by Nrg4 KD. Insulin failed to reduce autophagy in Nrg4 KD adipocytes, observed by a minor effect on mTOR phosphorylation, at the time that proteins involved in autophagy such as LC3-II, Rab11, and Clathrin were markedly upregulated. The lysosomal activity inhibitor bafilomycin A1 restored Glut4, IRAP, Syntaxin-6, and TBC1D4 content to those found in control adipocytes. Our study reveals that Nrg4 preserves the insulin responsiveness by preventing inflammation and, in turn, benefits the insulin regulation of autophagy.

BackgroundExtracellular vesicles (EVs) are released by many cells and provide a mechanism for intercellular communication in autocrine and paracrine fashion. EVs of various sizes, namely microvesicles (ranging from 100–1000 nm) or exosomes (with sizes between 30–150 nm) are released by most eukaryotic cells. EVs are known to contain bioactive molecules including proteins lipids and nucleic acids including miRNAs and LNC RNAs that regulate gene expression and cell functions in recipient cells. For example, tumour released exosomes remodel the microenvironment allowing cancer cells to survive and thrive. Low oxygen (hypoxia) is known to increase the release of exosomes in cancer cells. Hypoxia leads to the activation of hypoxia inducible factor (HIF) transcription factors, considered master regulators of the hypoxic response. The objective of this study was to investigate whether hypoxia induced EV (exosome) release is dependent on hypoxia inducible factor (HIF) proteins.MethodsWe isolated exosomes and microvesicles by differential centrifugation of conditioned media of cells exposed to normoxia or hypoxia (1% Oxygen) for different lengths of time ranging from 1–24hrs. We determined, size distribution and concentration of vesicles by nanoparticle tracking analysis (NTA). Expression of HIF1 and HIF2 was monitored by western blot of cellular lysates. We expressed oxygen‐stable mutated HIF1 and HIF2 isoforms either alone or in combination and determined EV release by NTA. We used chemical inhibitors of dioxygenase (PHD) and von Hippel–Lindau (VHL) proteins, the two proteins involved in HIF degradation under oxygen conditions, to stabilize HIF expression in normoxic cells and analysed EV release and HIF expression. We also analysed EV release in a cell line lacking VHL and after a VHL knock‐in. Western blotting of cellular lysates was used to monitor HIFs, VHL, and VEGF proteins. NTA was used to determine EV concentration and size. ANOVA statistical analysis was performed to analyse the data.ResultsHypoxia increased the amount extracellular vesicles in the conditioned media of all cells tested. The release of small vesicles (exosomes) was most increased following the exposure to hypoxia. HIF protein expression increased steadily during the time course of hypoxia, reaching a plateau after 2hrs following exposure. Expression of mutated (oxygen stable) HIF1 and HIF2 proteins in normoxic cells, either alone or in combination, did not increase exosome release. Chemical inhibition of PHD or VHL did not affect EV release. Exosome release was not different in VHL KD or KI cells.Conclusionthese data suggest that hypoxia driven exosome release is HIF independent.Support or Funding InformationSponsored by Fundacion Ramon Areces

Background: Membrane-derived extracellular vesicles (EVs) are released to the circulation by cells found in adipose tissue, transferring microRNAs (miRNAs) that may mediate the adaptive response of recipient cells. This study investigated plasma EVs from obese vs. nonobese women and their functional impact in adipocytes. Methods: Plasma EVs were isolated by differential centrifugation. Concentration and size were examined by nanoparticle tracking analysis (NanoSight). RNA was purified from plasma and plasma EVs of 45 women (47 +/- 12 years, 58% of obesity) and profiles of mature miRNAs were assessed. Functional analyses were performed in human adipocytes. Findings: Smaller plasma EVs were found in obese when compared to nonobese women. Positive associations were identified between circulating EVs numbers and parameters of impaired glucose tolerance. Almost 40% of plasma cell-free miRNAs were also found in isolated plasma EVs, defined as Ct values < 37 in >= 75% of samples. BMI together with parameters of insulin resistance were major contributors to EVs-contained miRNA patterns. Treatments of cultured human adipocytes with EVs from obese women led to a significant reduction of genes involved in lipid biosynthesis, while increasing the expression of IRS] (12.3%, p = 0.002). Interpretation: Size, concentration and the miRNA cargo of plasma EVs are associated with obesity and parameters of insulin resistance. Plasma EVs may mediate intercellular communication relevant to metabolism in adipocytes. (C) 2019 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

Obesity leads to adipose tissue dysfunction, insulin resistance and diabetes. Adipose tissue produces adipokines that contribute to regulate insulin sensitivity. In turn, insulin stimulates the production and release of some adipokines. Casitas-b-lymphoma proteins (c-Cbl, Cbl-b and Cbl3) are intracellular adaptor signalling proteins that are rapidly phosphorylated by activation of tyrosine kinase receptors. c-Cbl is rapidly phosphorylated by insulin in adipocytes. Here, we tested the hypothesis that Cbl signalling regulates adipokine expression in adipose tissue. We determined the adipokine profile of WAT of Cbl−/− and Cbl+/+ mice in the C57BL6 background. Female Cbl−/− mice exhibited altered expression of adiponectin, leptin and RBP4 in visceral adipose tissue, while no significant changes were seen in male mice. TNFα and IL6 levels were unaffected by Cbl depletion. RBP4 expression was unchanged in liver. Adipose tissue of Cbl−/− animals showed increased basal activation of extracellular regulated kinases (ERK1/2) compared to Cbl+/+. c-Cbl knockdown in 3T3L1 adipocytes also increased basal ERK phosphorylation and RBP4 expression. Inhibition of ERK1/2 phosphorylation in Cbl-depleted 3T3L1 adipocytes or in adipose tissue explants of Cbl−/− mice reduced RBP4 mRNA. 17β-Estradiol increased RBP4 mRNA in adipocytes. Cbl depletion did not change ER expression but increased phosphorylation of ERα at S118, a target site for ERK1/2. ERK1/2 inhibition reduced phosphoER and RBP4 levels. These findings suggest that Cbl contributes to regulate RBP4 expression in adipose of female mice through ERK1/2-mediated activation of ERα. Since Cbl signalling is compromised in diabetes, these data highlight a novel mechanism that upregulates RBP4 locally.

Extracellular vesicles (EVs) are released by many cells and provide a mechanism for intercellular communication in an autocrine and paracrine fashion. EVs are also released during pathological states. Adipocytes release EVs, however, the impact of these vesicles in glucose metabolism and insulin action has not been thoroughly investigated. We sought to investigate whether EVs released by metabolically challenged adipocytes exposed to hypoxia or to macrophage conditioned media affect insulin responsiveness and glucose transport. EVs were isolated from the culture supernatant of adipocytes cultured under normoxia, hypoxia (1% oxygen) or exposed to macrophage conditioned media (15%v/v). EVs were isolated from plasma of lean and obese individuals. EVs were used to treat cells and glucose transport and activation of insulin signalling cascades was measured. EVs released from hypoxic adipocytes but not those released by adipocytes exposed to macrophage conditioned media impaired insulin‐stimulated 2‐deoxyglucose uptake and reduced insulin‐stimulated Ser473 phosphorylation of AKT. ERK signalling was intact. Insulin stimulated glucose transport in muscle cells was also inhibited. EVs from obese individuals decreased insulin stimulated 2‐deoxyglucose uptake in adipocytes (p=0.0159). EVs from obese subjects contained distinct miRNA signatures. These findings indicate that EVs released by stressed adipocytes impair insulin action.Support or Funding InformationUniversity of Livepool, MINECO and FEDERThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Scope We investigate the effects of extracellular vesicles (EVs) obtained from in vitro adipocyte cell models and from obese subjects on glucose transport and insulin responsiveness. Methods and results EVs are isolated from the culture supernatant of adipocytes cultured under normoxia, hypoxia (1% oxygen), or exposed to macrophage conditioned media (15% v/v). EVs are isolated from the plasma of lean individuals and subjects with obesity. Cultured adipocytes are incubated with EVs and activation of insulin signalling cascades and insulin‐stimulated glucose transport are measured. EVs released from hypoxic adipocytes impair insulin‐stimulated 2‐deoxyglucose uptake and reduce insulin mediated phosphorylation of AKT. Insulin‐mediated phosphorylation of extracellular regulated kinases (ERK1/2) is not affected. EVs from individuals with obesity decrease insulin stimulated 2‐deoxyglucose uptake in adipocytes (p = 0.0159). Conclusion EVs released by stressed adipocytes impair insulin action in neighboring adipocytes.

X Sravan K. Thondam, Christina Daousi, John P. H. Wilding, Jens J. Holst, Gulizar I. Ameen, Chenjing Yang, Catherine Whitmore, Silvia Mora, and Daniel J. Cuthbertson Obesity and Endocrinology Research Group, University Hospital Aintree, Liverpool, United Kingdom; Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool, United Kingdom; Department of Cellular and Molecular Physiology University of Liverpool, Liverpool, United Kingdom; and NovoNordisk Foundation Center for Basic Metabolic Research and Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark

Glucose-dependent insulinotropic polypeptide (GIP) beyond its insulinotropic effects may regulate postprandial lipid metabolism. Whereas the insulinotropic action of GIP is known to be impaired in type 2 diabetes mellitus (T2DM), its adipogenic effect is unknown. We hypothesized that GIP is anabolic in human subcutaneous adipose tissue (SAT) promoting triacylglycerol (TAG) deposition through reesterification of nonesterified fatty acids (NEFA), and this effect may differ according to obesity status or glucose tolerance. Twenty-three subjects categorized into four groups, normoglycemic lean (n = 6), normoglycemic obese (n = 6), obese with impaired glucose regulation (IGR; n = 6), and obese T2DM (n = 5), participated in a double-blind, randomized, crossover study involving a hyperglycemic clamp with a 240-min GIP infusion (2 pmol·kg-1·min-1) or normal saline. Insulin, NEFA, SAT-TAG content, and gene expression of key lipogenic enzymes were determined before and immediately after GIP/saline infusions. GIP lowered NEFA concentrations in the obese T2DM group despite diminished insulinotropic activity (mean NEFA AUC0-4 h ± SE, 41,992 ± 9,843 µmol·l-1·min-1 vs. 71,468 ± 13,605 with placebo, P = 0.039, 95% CI: 0.31-0.95). Additionally, GIP increased SAT-TAG in obese T2DM (1.78 ± 0.4 vs 0.86 ± 0.1-fold with placebo, P = 0.043, 95% CI: 0.1-1.8). Such effect with GIP was not observed in other three groups despite greater insulinotropic activity. Reduction in NEFA concentration with GIP correlated with adipose tissue insulin resistance for all subjects (Pearson, r = 0.56, P = 0.005). There were no significant gene expression changes in key SAT lipid metabolism enzymes. In conclusion, GIP appears to promote fat accretion and thus may exacerbate obesity and insulin resistance in T2DM.

Short chain fatty acids (SCFA) are major products of bacterial fermentation by gut microbiota. Recent literature suggests that microbiota contribute to regulate glucose homeostasis and insulin sensitivity in the host. The purpose of the study was to determine whether the SCFAs acetate, butyrate and propionate impact on glucose and lipid metabolism and insulin signalling in adipose cells in vitro. We cultured differentiated 3T3L1 adipocytes in the presence of acetate, butyrate or propionate for 48 hours. We determined glucose transporter expression and activation of insulin signalling cascades by western blotting. We measured insulin‐stimulated 2‐deoxyglucose uptake and determined total Triglyceride content and expression and activation of Hormone Sensitive lipase (HSL) and Adipose Triglyceride Lipase (ATGL) by western blotting. None of the SCFAs had an effect on Glut1 or Glut4 glucose transporter expression or elicited an effect on basal or insulin‐stimulated glucose uptake. Insulin‐mediated activation of AKT and ERK were similar to that obtained in control untreated cells. While acetate and propionate did not have any effect in total triglyceride accumulation compared to control cells, butyrate treated cells showed lower lipid content compared to untreated cells. Butyrate treated cells showed increased levels of phosphorylated HSL (Ser 660) in basal state (no insulin) whereas the response to insulin was similar to that of untreated cells. We did not detect any changes in the expression levels of HSL, but cells treated with butyrate had increased ATGL levels compared to untreated cells. These data suggests that SCFA do not directly modify glucose uptake, and that butyrate but not other SCFA reduces lipid content by increasing the basal activity of HSL and expression of ATGL in adipocytes.

Adiponectin is an adipokine secreted by white adipocytes involved in regulating insulin sensitivity in peripheral tissues. Secretion of adiponectin in adipocytes relies on the endosomal system, however, the intracellular machinery involved in mediating adiponectin release is unknown. We have previously reported that intracellular adiponectin partially compartmentalizes with rab 5 and rab11, markers for the early/sorting and recycling compartments respectively. Here we have examined the role of several rab11 downstream effector proteins (rab11 FIPs) in regulating adiponectin trafficking and secretion. Overexpression of wild type rab11 FIP1, FIP3 and FIP5 decreased the amount of secreted adiponectin expressed in HEK293 cells, whereas overexpression of rab11 FIP2 or FIP4 had no effect. Furthermore shRNA-mediated depletion of FIP1 enhanced adiponectin release whereas knock down of FIP5 decreased adiponectin secretion. Knock down of FIP3 had no effect. In 3T3L1 adipocytes, endogenous FIP1 co-distributed intracellularly with endogenous adiponectin and FIP1 depletion enhanced adiponectin release without altering insulin-mediated trafficking of the glucose transporter Glut4. While adiponectin receptors internalized with transferrin receptors, there were no differences in transferrin receptor recycling between wild type and FIP1 depleted adipocytes. Consistent with its inhibitory role, FIP1 expression was decreased during adipocyte differentiation, by treatment with thiazolidinediones, and with increased BMI in humans. In contrast, FIP1 expression increased upon exposure of adipocytes to TNFα. In all, our findings identify FIP1 as a novel protein involved in the regulation of adiponectin trafficking and release.