A spatiotemporal proteomic map of human adipogenesis.

White adipocytes function as major energy reservoirs in humans by storing substantial amounts of triglycerides, and their dysfunction is associated with metabolic disorders; however, the mechanisms underlying cellular specialization during adipogenesis remain unknown. Here, we generate a spatiotemporal proteomic atlas of human adipogenesis, which elucidates cellular remodelling as well as the spatial reorganization of metabolic pathways to optimize cells for lipid accumulation and highlights the coordinated regulation of protein localization and abundance during adipocyte formation. We identify compartment-specific regulation of protein levels and localization changes of metabolic enzymes to reprogramme branched-chain amino acids and one-carbon metabolism to provide building blocks and reduction equivalents. Additionally, we identify C19orf12 as a differentiation-induced adipocyte lipid droplet protein that interacts with the translocase of the outer membrane complex of lipid droplet-associated mitochondria and regulates adipocyte lipid storage by determining the capacity of mitochondria to metabolize fatty acids. Overall, our study provides a comprehensive resource for understanding human adipogenesis and for future discoveries in the field.

Exercise-induced crosstalk between immune cells and adipocytes in humans: Role of oncostatin-M.

The discovery of exercise-regulated circulatory factors has fueled interest in organ crosstalk, especially between skeletal muscle and adipose tissue, and the role in mediating beneficial effects of exercise. We studied the adipose tissue transcriptome in men and women with normal glucose tolerance or type 2 diabetes following an acute exercise bout, revealing substantial exercise- and time-dependent changes, with sustained increase in inflammatory genes in type 2 diabetes. We identify oncostatin-M as one of the most upregulated adipose-tissue-secreted factors post-exercise. In cultured human adipocytes, oncostatin-M enhances MAPK signaling and regulates lipolysis. Oncostatin-M expression arises predominantly from adipose tissue immune cell fractions, while the corresponding receptors are expressed in adipocytes. Oncostatin-M expression increases in cultured human Thp1 macrophages following exercise-like stimuli. Our results suggest that immune cells, via secreted factors such as oncostatin-M, mediate a crosstalk between skeletal muscle and adipose tissue during exercise to regulate adipocyte metabolism and adaptation.

CRISPR/Cas9-mediated deletion of adipocyte genes associated with NAFLD alters adipocyte lipid handling and reduces steatosis in hepatocytes in vitro.

Obesity is a major risk factor for the development of nonalcoholic fatty liver disease (NAFLD), and the subcutaneous white adipose tissue (scWAT) is the primary lipid storage depot and regulates lipid fluxes to other organs. Our previous work identified genes upregulated in scWAT of patients with NAFLD: SOCS3, DUSP1, and SIK1. Herein, we knocked down (KD) their expression in human adipose-derived mesenchymal stem cells (hADMSCs) using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology and characterized their phenotype. We found that SOCS3, DUSP1, and SIK1 expression in hADMSC-derived adipocytes was not critical for adipogenesis. However, the metabolic characterization of the cells suggested that the genes played important roles in lipid metabolism. Reduction of SIK1 expression significantly increased both de novo lipogenesis (DNL) and palmitate-induced lipogenesis (PIL). Editing out SOCS3 reduced DNL while increasing isoproterenol-induced lipolysis and insulin-induced palmitate accumulation. Conversely, DUSP1 reduced PIL and DNL. Moreover, RNA-sequencing analysis of edited cells showed that these genes not only altered lipid metabolism but also other biological pathways related to inflammatory processes, in the case of DUSP1, extracellular matrix remodeling for SOCS3, or cellular transport for SIK1. Finally, to evaluate a possible adipocyte-hepatocyte axis, human hepatoma HepG2 cells were cocultured with edited hADMSCs-derived adipocytes in the presence of [3H]-palmitate. All HepG2 cells cultured with DUSP1-, SIK1-, or SOCS3-KD adipocytes decreased [3H]-palmitate accumulation compared with control adipocytes. These results support our hypotheses that SOCS3, DUSP1, and SIK1 regulate multiple aspects of adipocyte function, which may play a role in the progression of obesity-associated comorbidities, such as NAFLD.NEW & NOTEWORTHY Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology successfully edited genomic DNA of human adipose-derived mesenchymal stem cells (hADMSC). SOCS3, SIK1, and DUSP1 regulate adipocyte lipid handling. Silencing SOCS3, SIK1, and DUSP1 expression in hADMSC-derived adipocytes reduces hepatocyte lipid storage in vitro.

An integrated single cell and spatial transcriptomic map of human white adipose tissue.

To date, single-cell studies of human white adipose tissue (WAT) have been based on small cohort sizes and no cellular consensus nomenclature exists. Herein, we performed a comprehensive meta-analysis of publicly available and newly generated single-cell, single-nucleus, and spatial transcriptomic results from human subcutaneous, omental, and perivascular WAT. Our high-resolution map is built on data from ten studies and allowed us to robustly identify >60 subpopulations of adipocytes, fibroblast and adipogenic progenitors, vascular, and immune cells. Using these results, we deconvolved spatial and bulk transcriptomic data from nine additional cohorts to provide spatial and clinical dimensions to the map. This identified cell-cell interactions as well as relationships between specific cell subtypes and insulin resistance, dyslipidemia, adipocyte volume, and lipolysis upon long-term weight changes. Altogether, our meta-map provides a rich resource defining the cellular and microarchitectural landscape of human WAT and describes the associations between specific cell types and metabolic states.

Erratum: DCAF7 regulates cell proliferation through IRS1-FOXO1 signaling.

[This corrects the article DOI: 10.1016/j.isci.2022.105188.].

DCAF7 regulates cell proliferation through IRS1-FOXO1 signaling.

Cell proliferation is dependent on growth factors insulin and IGF1. We sought to identify interactors of IRS1, the most proximal mediator of insulin/IGF1 signaling, that regulate cell proliferation. Using proximity-dependent biotin identification (BioID), we detected 40 proteins displaying proximal interactions with IRS1, including DCAF7 and its interacting partners DYRK1A and DYRK1B. In HepG2 cells, DCAF7 knockdown attenuated cell proliferation by inducing cell cycle arrest at G2. DCAF7 expression was required for insulin-stimulated AKT phosphorylation, and its absence promoted nuclear localization of the transcription factor FOXO1. DCAF7 knockdown induced expression of FOXO1-target genes implicated in G2 cell cycle inhibition, correlating with G2 cell cycle arrest. In Drosophila melanogaster, wing-specific knockdown of DCAF7/wap caused smaller wing size and lower wing cell number; the latter recovered upon double knockdown of wap and dfoxo. We propose that DCAF7 regulates cell proliferation and cell cycle via IRS1-FOXO1 signaling, of relevance to whole organism growth.

Impaired phosphocreatine metabolism in white adipocytes promotes inflammation.

The mechanisms promoting disturbed white adipocyte function in obesity remain largely unclear. Herein, we integrate white adipose tissue (WAT) metabolomic and transcriptomic data from clinical cohorts and find that the WAT phosphocreatine/creatine ratio is increased and creatine kinase-B expression and activity is decreased in the obese state. In human in vitro and murine in vivo models, we demonstrate that decreased phosphocreatine metabolism in white adipocytes alters adenosine monophosphate-activated protein kinase activity via effects on adenosine triphosphate/adenosine diphosphate levels, independently of WAT beigeing. This disturbance promotes a pro-inflammatory profile characterized, in part, by increased chemokine (C-C motif) ligand 2 (CCL2) production. These data suggest that the phosphocreatine/creatine system links cellular energy shuttling with pro-inflammatory responses in human and murine white adipocytes. Our findings provide unexpected perspectives on the mechanisms driving WAT inflammation in obesity and may present avenues to target adipocyte dysfunction.

The Lipid Droplet Knowledge Portal: A resource for systematic analyses of lipid droplet biology.

Lipid droplets (LDs) are organelles of cellular lipid storage with fundamental roles in energy metabolism and cell membrane homeostasis. There has been an explosion of research into the biology of LDs, in part due to their relevance in diseases of lipid storage, such as atherosclerosis, obesity, type 2 diabetes, and hepatic steatosis. Consequently, there is an increasing need for a resource that combines datasets from systematic analyses of LD biology. Here, we integrate high-confidence, systematically generated human, mouse, and fly data from studies on LDs in the framework of an online platform named the "Lipid Droplet Knowledge Portal" (https://lipiddroplet.org/). This scalable and interactive portal includes comprehensive datasets, across a variety of cell types, for LD biology, including transcriptional profiles of induced lipid storage, organellar proteomics, genome-wide screen phenotypes, and ties to human genetics. This resource is a powerful platform that can be utilized to identify determinants of lipid storage.

Communication Between Autophagy and Insulin Action: At the Crux of Insulin Action-Insulin Resistance?

Insulin is a paramount anabolic hormone that promotes energy-storage in adipose tissue, skeletal muscle and liver, and these responses are significantly attenuated in insulin resistance leading to type 2 diabetes. Contrasting with insulin's function, macroautophagy/autophagy is a physiological mechanism geared to the degradation of intracellular components for the purpose of energy production, building-block recycling or tissue remodeling. Given that both insulin action and autophagy are dynamic phenomena susceptible to the influence of nutrient availability, it is perhaps not surprising that there is significant interaction between these two major regulatory mechanisms. This review examines the crosstalk between autophagy and insulin action, with specific focus on dysregulated autophagy as a cause or consequence of insulin resistance.

Complexin-2 redistributes to the membrane of muscle cells in response to insulin and contributes to GLUT4 translocation.

Insulin stimulates glucose uptake in muscle cells by rapidly redistributing vesicles containing GLUT4 glucose transporters from intracellular compartments to the plasma membrane (PM). GLUT4 vesicle fusion requires the formation of SNARE complexes between vesicular VAMP and PM syntaxin4 and SNAP23. SNARE accessory proteins usually regulate vesicle fusion processes. Complexins aide in neuro-secretory vesicle-membrane fusion by stabilizing trans-SNARE complexes but their participation in GLUT4 vesicle fusion is unknown. We report that complexin-2 is expressed and homogeneously distributed in L6 rat skeletal muscle cells. Upon insulin stimulation, a cohort of complexin-2 redistributes to the PM. Complexin-2 knockdown markedly inhibited GLUT4 translocation without affecting proximal insulin signalling of Akt/PKB phosphorylation and actin fiber remodelling. Similarly, complexin-2 overexpression decreased maximal GLUT4 translocation suggesting that the concentration of complexin-2 is finely tuned to vesicle fusion. These findings reveal an insulin-dependent regulation of GLUT4 insertion into the PM involving complexin-2.

Deficiency of the autophagy gene ATG16L1 induces insulin resistance through KLHL9/KLHL13/CUL3-mediated IRS1 degradation.

Connections between deficient autophagy and insulin resistance have emerged, however, the mechanism through which reduced autophagy impairs insulin-signaling remains unknown. We examined mouse embryonic fibroblasts lacking Atg16l1 (ATG16L1 KO mouse embryonic fibroblasts (MEFs)), an essential autophagy gene, and observed deficient insulin and insulin-like growth factor-1 signaling. ATG16L1 KO MEFs displayed reduced protein content of insulin receptor substrate-1 (IRS1), pivotal to insulin signaling, whereas IRS1myc overexpression recovered downstream insulin signaling. Endogenous IRS1 protein content and insulin signaling were restored in ATG16L1 KO mouse embryonic fibroblasts (MEF) upon proteasome inhibition. Through proximity-dependent biotin identification (BioID) and co-immunoprecipitation, we found that Kelch-like proteins KLHL9 and KLHL13, which together form an E3 ubiquitin (Ub) ligase complex with cullin 3 (CUL3), are novel IRS1 interactors. Expression of Klhl9 and Klhl13 was elevated in ATG16L1 KO MEFs and siRNA-mediated knockdown of Klhl9, Klhl13, or Cul3 recovered IRS1 expression. Moreover, Klhl13 and Cul3 knockdown increased insulin signaling. Notably, adipose tissue of high-fat fed mice displayed lower Atg16l1 mRNA expression and IRS1 protein content, and adipose tissue KLHL13 and CUL3 expression positively correlated to body mass index in humans. We propose that ATG16L1 deficiency evokes insulin resistance through induction of Klhl9 and Klhl13, which, in complex with Cul3, promote proteasomal IRS1 degradation.

Resveratrol and Metformin Recover Prefrontal Cortex AMPK Activation in Diet-Induced Obese Mice but Reduce BDNF and Synaptophysin Protein Content.

BACKGROUND: Obesity, insulin resistance, and type 2 diabetes are established risk factors for the development of Alzheimer's disease (AD). Given this connection, two drugs, metformin (MET) and resveratrol (RESV), are considered for the clearance of amyloid-ß peptides through AMPK-mediated activation of autophagy. However, overactivation of AMPK observed in late-stage AD brains and relationships between AMPK and neurogenesis (through mTORC1 inhibition), questions treatment with these drugs. OBJECTIVE: To examine if MET and/or RESV supplementation activates brain AMPK, regulates markers of autophagy, and affects markers of neuronal health/neurogenesis. METHODS: 8-week-old male C57BL/6J mice were fed a low (N = 12; 10% kcal fat; LFD) or high fat diet (N = 40; 60% kcal fat; HFD) for 9 weeks to induce insulin resistance and obesity. HFD mice were then treated with/without MET (250 mg/kg/day), RESV (100 mg/kg/day), or COMBO (MET: 250 mg/kg/day, RESV: 100 mg/kg/day) for 5 weeks. Hippocampus and prefrontal cortex were extracted for western blotting analysis. RESULTS: Cortex AMPK (T172) and raptor (S792, the regulatory subunit of mTORC1) phosphorylation were upregulated following RESV, COMBO treatments. mTOR (S2448) and ULK1 (S555) activation was seen following MET, COMBO and RESV, COMBO treatments, respectively, in the cortex and hippocampus. p62 content was decreased following RESV, COMBO, with LC3 content being increased following RESV treatment in the cortex. Brain derived neurotropic factor (BDNF) was significantly decreased following RESV, COMBO, and synaptophysin following all treatment in the cortex. CONCLUSION: These results demonstrate that while treatments upregulated markers of autophagy in the prefrontal cortex, reductions in neuronal health markers question the efficacy of AMPK as a therapy for AD.

Regulation of Hepatic Follistatin Expression at Rest and during Exercise in Mice.

INTRODUCTION: Follistatin (FST) is a protein with numerous biological roles and was recently identified as an exercise-inducible hepatokine; however, the signals that regulate this are not well understood. The purpose of this study was to delineate potential endocrine factors that may regulate hepatic FST at rest and during exercise. METHODS: This study used four experiments. First, male and female C57BL/6J mice remained sedentary or were subjected to a single bout of exercise at moderate or exhaustive intensity with liver collected immediately post. Second, mice were injected with glucagon (1 mg·kg, 60 min), epinephrine (2 mg·kg, 30 min), glucagon then epinephrine, or saline. Third, mice were pretreated with propranolol (20-60 mg·kg, 30 min) before epinephrine injection. Fourth, glucagon receptor wild type (Gcgr) or knockout (Gcgr) mice were pretreated with saline or propranolol (20 mg·kg, 30 min) and were subjected to a single bout of exhaustive exercise with liver collected immediately post or after 2 h recovery. In all experiments liver FST mRNA expression was measured, and in experiment four FST protein content was measured. RESULTS: A single bout of treadmill exercise performed at an exhaustive but not moderate-intensity increased FST expression, as did injection of glucagon or epinephrine alone and when combined. Pretreatment of mice with propranolol attenuated the epinephrine-induced increase in FST expression. The exercise-induced increase in FST expression was attenuated in Gcgr mice, with no effect of propranolol. Gcgr mice had higher protein content of FST, but there was no effect of exercise or propranolol. CONCLUSIONS: These data suggest that both glucagon and epinephrine regulate hepatic FST expression at rest; however, only glucagon is required for the exercise-induced increase.

Sphingolipid changes do not underlie fatty acid-evoked GLUT4 insulin resistance nor inflammation signals in muscle cells.

Ceramides contribute to obesity-linked insulin resistance and inflammation in vivo, but whether this is a cell-autonomous phenomenon is debated, particularly in muscle, which dictates whole-body glucose uptake. We comprehensively analyzed lipid species produced in response to fatty acids and examined the consequence to insulin resistance and pro-inflammatory pathways. L6 myotubes were incubated with BSA-adsorbed palmitate or palmitoleate in the presence of myriocin, fenretinide, or fumonisin B1. Lipid species were determined by lipidomic analysis. Insulin sensitivity was scored by Akt phosphorylation and glucose transporter 4 (GLUT4) translocation, while pro-inflammatory indices were estimated by IκBα degradation and cytokine expression. Palmitate, but not palmitoleate, had mild effects on Akt phosphorylation but significantly inhibited insulin-stimulated GLUT4 translocation and increased expression of pro-inflammatory cytokines Il6 and Ccl2 Ceramides, hexosylceramides, and sphingosine-1-phosphate significantly heightened by palmitate correlated negatively with insulin sensitivity and positively with pro-inflammatory indices. Inhibition of sphingolipid pathways led to marked changes in cellular lipids, but did not prevent palmitate-induced impairment of insulin-stimulated GLUT4 translocation, suggesting that palmitate-induced accumulation of deleterious lipids and insulin resistance are correlated but independent events in myotubes. We propose that muscle cell-endogenous ceramide production does not evoke insulin resistance and that deleterious effects of ceramides in vivo may arise through ancillary cell communication.

Update on GLUT4 Vesicle Traffic: A Cornerstone of Insulin Action.

Glucose transport is rate limiting for dietary glucose utilization by muscle and fat. The glucose transporter GLUT4 is dynamically sorted and retained intracellularly and redistributes to the plasma membrane (PM) by insulin-regulated vesicular traffic, or 'GLUT4 translocation'. Here we emphasize recent findings in GLUT4 translocation research. The application of total internal reflection fluorescence microscopy (TIRFM) has increased our understanding of insulin-regulated events beneath the PM, such as vesicle tethering and membrane fusion. We describe recent findings on Akt-targeted Rab GTPase-activating proteins (GAPs) (TBC1D1, TBC1D4, TBC1D13) and downstream Rab GTPases (Rab8a, Rab10, Rab13, Rab14, and their effectors) along with the input of Rac1 and actin filaments, molecular motors [myosinVa (MyoVa), myosin1c (Myo1c), myosinIIA (MyoIIA)], and membrane fusion regulators (syntaxin4, munc18c, Doc2b). Collectively these findings reveal novel events in insulin-regulated GLUT4 traffic.

Beneficial effects of combined resveratrol and metformin therapy in treating diet-induced insulin resistance.

The polyphenol compound resveratrol (RSV) has attracted attention due to its reputed beneficial effects on insulin sensitivity. Our lab has previously identified protective effects of RSV against the development of type 2 diabetes in rats. These effects occurred in a manner similar to thiazolidinedione's (TZDs), a class of insulin sensitizing drugs. TZDs are commonly prescribed in combination with metformin (MET) and thus we sought to examine the combined effects of RSV and MET in treating insulin resistance. Male C57BL6 mice were fed a low- (LFD; 10% Kcal from fat) or high-fat diet (HFD; 60% Kcal from fat) for 9 weeks to induce glucose and insulin intolerance. HFD mice were then assigned to control (HFD), MET (231.28 ± 12.24 mg/kg/day), RSV (93.68 ± 3.51 mg/kg/day), or combined (COM; MET 232.01 ± 17.12 mg/kg/day and RSV 92.77 ± 6.92 mg/kg/day) treatment groups. Changes in glucose and insulin tolerance and tissue-specific insulin signaling were measured 4 weeks post-treatment. RSV or MET alone did not have beneficial effects on glucose tolerance, although MET significantly improved insulin tolerance compared to HFD Glucose and insulin tolerance were significantly improved in COM compared to HFD and this was mirrored by enhanced insulin-stimulated AKT phosphorylation in triceps muscle and inguinal subcutaneous adipose tissue in COM compared to HFD mice. Improvements with COM treatment were not explained by differences in body weight, adiposity, or markers of adipose tissue inflammation. In summary, this study provides evidence of beneficial effects of combined RSV and MET therapy in treating impairments in glucose homeostasis.

Reduced ATGL-mediated lipolysis attenuates ß-adrenergic-induced AMPK signaling, but not the induction of PKA-targeted genes, in adipocytes and adipose tissue.

5'-AMP-activated protein kinase (AMPK) is activated as a consequence of lipolysis and has been shown to play a role in regulation of adipose tissue mitochondrial content. Conversely, the inhibition of lipolysis has been reported to potentiate the induction of protein kinase A (PKA)-targeted genes involved in the regulation of oxidative metabolism. The purpose of the current study was to address these apparent discrepancies and to more fully examine the relationship between lipolysis, AMPK, and the ß-adrenergic-mediated regulation of gene expression. In 3T3-L1 adipocytes, the adipose tissue triglyceride lipase (ATGL) inhibitor ATGListatin attenuated the Thr(172) phosphorylation of AMPK by a ß3-adrenergic agonist (CL 316,243) independent of changes in PKA signaling. Similarly, CL 316,243-induced increases in the Thr(172) phosphorylation of AMPK were reduced in adipose tissue from whole body ATGL-deficient mice. Despite reductions in the activation of AMPK, the induction of PKA-targeted genes was intact or, in some cases, increased. Similarly, markers of mitochondrial content and respiration were increased in adipose tissue from ATGL knockout mice independent of changes in the Thr(172) phosphorylation of AMPK. Taken together, our data provide evidence that AMPK is not required for the regulation of adipose tissue oxidative capacity in conditions of reduced fatty acid release.

Sarcolipin knockout mice fed a high-fat diet exhibit altered indices of adipose tissue inflammation and remodeling.

OBJECTIVE: To investigate indices of adipose tissue inflammation and remodeling in high-fat diet (HFD) sarcolipin-knockout (SLN(-) (/-) ) mice. SLN regulates muscle-based nonshivering thermogenesis and is up-regulated with HFD. SLN(-) (/-) mice develop greater diet-induced obesity and glucose intolerance. This is accompanied by increases in circulating catecholamines and fatty acids. Catecholamines and fatty acids play a role in the pathology of adipose tissue inflammation. METHODS: Male mice (wild type and SLN(-) (/-) ) were fed a HFD (42% kcal from fat) for 8 weeks. RESULTS: SLN(-) (/-) mice displayed greater obesity and glucose intolerance. This was accompanied by higher circulating epinephrine and nonesterified fatty acids. Epididymal but not inguinal subcutaneous adipose tissue from SLN(-) (/-) mice displayed higher interleukin-6, suppressor of cytokine signaling 3, interleukin-1ß, and tumor necrosis factor-α mRNA expression, and this was associated with increased markers of macrophage infiltration (F4/80 expression and crown-like structures) and M1 polarization (higher CD11c expression and CD11c/MGL1). Interestingly, this occurred despite SLN(-) (/-) mice having smaller adipocytes. CONCLUSIONS: In conditions of nutrient excess, SLN(-) (/-) mice display depot-specific increases in indices of adipose tissue inflammation and remodeling. This could be a compensatory response to reductions in muscle-based thermogenesis.

Prior exercise training blunts short-term high-fat diet-induced weight gain.

High-fat diets rapidly cause weight gain and glucose intolerance. We sought to determine whether these changes could be mitigated with prior exercise training. Male C57BL/6J mice were exercise-trained by treadmill running (1 h/day, 5 days/wk) for 4 wk. Twenty-four hours after the final bout of exercise, mice were provided with a high-fat diet (HFD; 60% kcal from lard) for 4 days, with no further exercise. In mice fed the HFD prior to exercise training, the results were blunted weight gain, reduced fat mass, and a slight attenuation in glucose intolerance that was mirrored by greater insulin-induced Akt phosphorylation in skeletal muscle compared with sedentary mice fed the HFD. When ad libitum-fed sedentary mice were compared with sedentary high-fat fed mice that were calorie restricted (-30%) to match the weight gain of the previously trained high-fat fed mice, the same attenuated impairments in glucose tolerance were found. Blunted weight gain was associated with a greater capacity to increase energy expenditure in trained compared with sedentary mice when challenged with a HFD. Although mitochondrial enzymes in white adipose tissue and UCP-1 protein content in brown adipose tissue were increased in previously exercised compared with sedentary mice fed a HFD, ex vivo mitochondrial respiration was not increased in either tissue. Our data suggest that prior exercise training attenuates high-fat diet-induced weight gain and glucose intolerance and is associated with a greater ability to increase energy expenditure in response to a high-fat diet.