SENP2 knockdown in human adipocytes reduces glucose metabolism and lipid accumulation, while increases lipid oxidation.

Adipose tissue is one of the main regulative sites for energy metabolism. Excess lipid storage and expansion of white adipose tissue (WAT) is the primary contributor to obesity, a strong predisposing factor for development of insulin resistance. Sentrin-specific protease (SENP) 2 has been shown to play a role in metabolism in murine fat and skeletal muscle cells, and we have previously demonstrated its role in energy metabolism of human skeletal muscle cells. In the present work, we have investigated the impact of SENP2 on fatty acid and glucose metabolism in primary human fat cells by using cultured primary human adipocytes to knock down the SENP2 gene. Glucose uptake and oxidation, as well as accumulation and distribution of oleic acid into complex lipids were decreased, while oleic acid oxidation was increased in SENP2-knockdown cells compared to control adipocytes. Furthermore, lipogenesis was reduced by SENP2-knockdown in adipocytes. Although TAG accumulation relative to total uptake was unchanged, there was increased mRNA expression of metabolically relevant genes such as UCP1 and PPARGC1A and mRNA and proteomic data revealed increased levels of mRNA and proteins related to mitochondrial function by SENP2-knockdown. In conclusion, SENP2 is an important regulator of energy metabolism in primary human adipocytes and its knockdown reduce glucose metabolism and lipid accumulation, while increasing lipid oxidation in human adipocytes.

FGF21 protects against hepatic lipotoxicity and macrophage activation to attenuate fibrogenesis in nonalcoholic steatohepatitis.

Analogues of the hepatokine fibroblast growth factor 21 (FGF21) are in clinical development for type 2 diabetes and nonalcoholic steatohepatitis (NASH) treatment. Although their glucose-lowering and insulin-sensitizing effects have been largely unraveled, the mechanisms by which they alleviate liver injury have only been scarcely addressed. Here, we aimed to unveil the mechanisms underlying the protective effects of FGF21 on NASH using APOE*3-Leiden.CETP mice, a well-established model for human-like metabolic diseases. Liver-specific FGF21 overexpression was achieved in mice, followed by administration of a high-fat high-cholesterol diet for 23 weeks. FGF21 prevented hepatic lipotoxicity, accompanied by activation of thermogenic tissues and attenuation of adipose tissue inflammation, improvement of hyperglycemia and hypertriglyceridemia, and upregulation of hepatic programs involved in fatty acid oxidation and cholesterol removal. Furthermore, FGF21 inhibited hepatic inflammation, as evidenced by reduced Kupffer cell (KC) activation, diminished monocyte infiltration, and lowered accumulation of monocyte-derived macrophages. Moreover, FGF21 decreased lipid- and scar-associated macrophages, which correlated with less hepatic fibrosis as demonstrated by reduced collagen accumulation. Collectively, hepatic FGF21 overexpression limits hepatic lipotoxicity, inflammation, and fibrogenesis. Mechanistically, FGF21 blocks hepatic lipid influx and accumulation through combined endocrine and autocrine signaling, respectively, which prevents KC activation and lowers the presence of lipid- and scar-associated macrophages to inhibit fibrogenesis.

High-calorie modern diets have contributed to growing rates of obesity-linked diseases. One such disease is non-alcoholic steatohepatitis or NASH for short, which affects about 5% of adults in the United States. The livers of people with this condition accumulate fat, become inflamed, and develop scar tissue. People with NASH are also at increased risk of developing liver cancer, type 2 diabetes, and heart disease. Currently, no drugs are available to treat the condition and prevent such severe complications. Previous research has shown the liver produces a stress hormone, called FGF21, in response to fat accumulation. This hormone boosts fat burning and so helps to reduce excess fat in the liver. Drugs that mimic FGF21 have already been developed for type 2 diabetes. But so far, it was unclear if such drugs could also help reduce liver inflammation and scarring in patients with NASH. Liu et al. show that increasing the production of FGF21 in mice with a NASH-like condition reduces fat accumulation, liver inflammation, and scarring. In the experiments, the researchers used gene therapy to ramp up FGF21 production in the livers of mice that develop obesity and a NASH-like condition when fed a high-fat diet for 23 weeks. Increasing FGF21 production prevented the mice from developing obesity while on the high fat diet by making the body burn more fat in the liver and brown fat tissue. The treatment also reduced inflammation and prevented scarring by reducing the number and activity of immune cells in the liver. Increasing the production of the stress hormone FGF21 prevents diet-induced obesity and NASH in mice fed a high-fat diet. More studies are necessary to determine if using gene therapy to increase FGF21 may also cause weight loss and could reverse liver damage in mice that already have NASH. If this approach is effective in mice, it may be tested in humans, a process that may take several years. If human studies are successful, FGF21-boosting therapy might provide a new treatment approach for obesity or NASH.

Elevated Adipocyte Membrane Phospholipid Saturation Does Not Compromise Insulin Signaling.

Increased saturated fatty acid (SFA) levels in membrane phospholipids have been implicated in the development of metabolic disease. Here, we tested the hypothesis that increased SFA content in cell membranes negatively impacts adipocyte insulin signaling. Preadipocyte cell models with elevated SFA levels in phospholipids were generated by disrupting the ADIPOR2 locus, which resulted in a striking twofold increase in SFA-containing phosphatidylcholines and phosphatidylethanolamines, which persisted in differentiated adipocytes. Similar changes in phospholipid composition were observed in white adipose tissues isolated from the ADIPOR2-knockout mice. The SFA levels in phospholipids could be further increased by treating ADIPOR2-deficient cells with palmitic acid and resulted in reduced membrane fluidity and endoplasmic reticulum stress in mouse and human preadipocytes. Strikingly, increased SFA levels in differentiated adipocyte phospholipids had no effect on adipocyte gene expression or insulin signaling in vitro. Similarly, increased adipocyte phospholipid saturation did not impair white adipose tissue function in vivo, even in mice fed a high-saturated fat diet at thermoneutrality. We conclude that increasing SFA levels in adipocyte phospholipids is well tolerated and does not affect adipocyte insulin signaling in vitro and in vivo.

Visible-Light-Induced Tandem Radical Brominative Addition/Cyclization of Activated Alkynes with CBr4 for the Synthesis of 3-Bromocoumarins.

A visible-light-induced tandem radical brominative addition/spiro-cyclization/1,2-ester migration of activated alkynes with CBr4 is developed. This protocol features good functional group tolerance, operational simplicity, and mild reaction conditions without the use of catalysts and external additives, providing easy access to valuable 3-bromocoumarins in generally high yields.

TLCD1 and TLCD2 regulate cellular phosphatidylethanolamine composition and promote the progression of non-alcoholic steatohepatitis.

The fatty acid composition of phosphatidylethanolamine (PE) determines cellular metabolism, oxidative stress, and inflammation. However, our understanding of how cells regulate PE composition is limited. Here, we identify a genetic locus on mouse chromosome 11, containing two poorly characterized genes Tlcd1 and Tlcd2, that strongly influences PE composition. We generated Tlcd1/2 double-knockout (DKO) mice and found that they have reduced levels of hepatic monounsaturated fatty acid (MUFA)-containing PE species. Mechanistically, TLCD1/2 proteins act cell intrinsically to promote the incorporation of MUFAs into PEs. Furthermore, TLCD1/2 interact with the mitochondria in an evolutionarily conserved manner and regulate mitochondrial PE composition. Lastly, we demonstrate the biological relevance of our findings in dietary models of metabolic disease, where Tlcd1/2 DKO mice display attenuated development of non-alcoholic steatohepatitis compared to controls. Overall, we identify TLCD1/2 proteins as key regulators of cellular PE composition, with our findings having broad implications in understanding and treating disease.

Differential In Vitro Effects of SGLT2 Inhibitors on Mitochondrial Oxidative Phosphorylation, Glucose Uptake and Cell Metabolism.

(1) The cardio-reno-metabolic benefits of the SGLT2 inhibitors canagliflozin (cana), dapagliflozin (dapa), ertugliflozin (ertu), and empagliflozin (empa) have been demonstrated, but it remains unclear whether they exert different off-target effects influencing clinical profiles. (2) We aimed to investigate the effects of SGLT2 inhibitors on mitochondrial function, cellular glucose-uptake (GU), and metabolic pathways in human-umbilical-vein endothelial cells (HUVECs). (3) At 100 µM (supra-pharmacological concentration), cana decreased ECAR by 45% and inhibited GU (IC5o: 14 µM). At 100 µM and 10 µM (pharmacological concentration), cana increased the ADP/ATP ratio, whereas dapa and ertu (3, 10 µM, about 10× the pharmacological concentration) showed no effect. Cana (100 µM) decreased the oxygen consumption rate (OCR) by 60%, while dapa decreased it by 7%, and ertu and empa (all 100 µM) had no significant effect. Cana (100 µM) inhibited GLUT1, but did not significantly affect GLUTs' expression levels. Cana (100 µM) treatment reduced glycolysis, elevated the amino acids supplying the tricarboxylic-acid cycle, and significantly increased purine/pyrimidine-pathway metabolites, in contrast to dapa (3 µM) and ertu (10 µM). (4) The results confirmed cana´s inhibition of mitochondrial activity and GU at supra-pharmacological and pharmacological concentrations, whereas the dapa, ertu, and empa did not show effects even at supra-pharmacological concentrations. At supra-pharmacological concentrations, cana (but not dapa or ertu) affected multiple cellular pathways and inhibited GLUT1.

Subcutaneous delivery of FGF21 mRNA therapy reverses obesity, insulin resistance, and hepatic steatosis in diet-induced obese mice.

Fibroblast growth factor 21 (FGF21) is a promising therapeutic agent for treatment of type 2 diabetes (T2D) and non-alcoholic steatohepatitis (NASH). We show that therapeutic levels of FGF21 were achieved following subcutaneous (s.c.) administration of mRNA encoding human FGF21 proteins. The efficacy of mRNA was assessed following 2-weeks repeated s.c. dosing in diet-induced obese (DIO), mice which resulted in marked decreases in body weight, plasma insulin levels, and hepatic steatosis. Pharmacokinetic/pharmacodynamic (PK/PD) modelling of several studies in both lean and DIO mice showed that mRNA encoding human proteins provided improved therapeutic coverage over recombinant dosed proteins in vivo. This study is the first example of s.c. mRNA therapy showing pre-clinical efficacy in a disease-relevant model, thus, showing the potential for this modality in the treatment of chronic diseases, including T2D and NASH.

The SARS-CoV-2 receptor ACE2 is expressed in mouse pericytes but not endothelial cells: Implications for COVID-19 vascular research.

Humanized mouse models and mouse-adapted SARS-CoV-2 virus are increasingly used to study COVID-19 pathogenesis, so it is important to learn where the SARS-CoV-2 receptor ACE2 is expressed. Here we mapped ACE2 expression during mouse postnatal development and in adulthood. Pericytes in the CNS, heart, and pancreas express ACE2 strongly, as do perineurial and adrenal fibroblasts, whereas endothelial cells do not at any location analyzed. In a number of other organs, pericytes do not express ACE2, including in the lung where ACE2 instead is expressed in bronchial epithelium and alveolar type II cells. The onset of ACE2 expression is organ specific: in bronchial epithelium already at birth, in brain pericytes before, and in heart pericytes after postnatal day 10.5. Establishing the vascular localization of ACE2 expression is central to correctly interpret data from modeling COVID-19 in the mouse and may shed light on the cause of vascular COVID-19 complications.

Knockdown of sarcolipin (SLN) impairs substrate utilization in human skeletal muscle cells.

BACKGROUND: Recent studies have highlighted that uncoupling of sarco-/endoplasmic reticulum Ca2+-ATPase (SERCA) by sarcolipin (SLN) increases ATP consumption and contributes to heat liberation. Exploiting this thermogenic mechanism in skeletal muscle may provide an attractive strategy to counteract obesity and associated metabolic disorders. In the present study, we have investigated the role of SLN on substrate metabolism in human skeletal muscle cells. METHODS AND RESULTS: After generation of skeletal muscle cells with stable SLN knockdown (SLN-KD), cell viability, glucose and oleic acid (OA) metabolism, mitochondrial function, as well as gene expressions were determined. Depletion of SLN did not influence cell viability. However, glucose and OA oxidation were diminished in SLN-KD cells compared to control myotubes. Basal respiration measured by respirometry was also observed to be reduced in cells with SLN-KD. The metabolic perturbation in SLN-KD cells was reflected by reduced gene expression levels of peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α) and forkhead box O1 (FOXO1). Furthermore, accumulation of OA was increased in cells with SLN-KD compared to control cells. These effects were accompanied by increased lipid formation and incorporation of OA into complex lipids. Additionally, formation of complex lipids and free fatty acid from de novo lipogenesis with acetate as substrate was enhanced in SLN-KD cells. Detection of lipid droplets using Oil red O staining also showed increased lipid accumulation in SLN-KD cells. CONCLUSIONS: Overall, our study sheds light on the importance of SLN in maintaining metabolic homeostasis in human skeletal muscle. Findings from the current study suggest that therapeutic strategies involving SLN-mediated futile cycling of SERCA might have significant implications in the treatment of obesity and associated metabolic disorders.

Pharmacological treatment with FGF21 strongly improves plasma cholesterol metabolism to reduce atherosclerosis.

AIMS: Fibroblast growth factor (FGF) 21, a key regulator of energy metabolism, is currently evaluated in humans for treatment of type 2 diabetes and non-alcoholic steatohepatitis. However, the effects of FGF21 on cardiovascular benefit, particularly on lipoprotein metabolism in relation to atherogenesis, remain elusive. METHODS AND RESULTS: Here, the role of FGF21 in lipoprotein metabolism in relation to atherosclerosis development was investigated by pharmacological administration of a half-life extended recombinant FGF21 protein to hypercholesterolaemic APOE*3-Leiden.CETP mice, a well-established model mimicking atherosclerosis initiation and development in humans. FGF21 reduced plasma total cholesterol, explained by a reduction in non-HDL-cholesterol. Mechanistically, FGF21 promoted brown adipose tissue (BAT) activation and white adipose tissue (WAT) browning, thereby enhancing the selective uptake of fatty acids from triglyceride-rich lipoproteins into BAT and into browned WAT, consequently accelerating the clearance of the cholesterol-enriched remnants by the liver. In addition, FGF21 reduced body fat, ameliorated glucose tolerance and markedly reduced hepatic steatosis, related to up-regulated hepatic expression of genes involved in fatty acid oxidation and increased hepatic VLDL-triglyceride secretion. Ultimately, FGF21 largely decreased atherosclerotic lesion area, which was mainly explained by the reduction in non-HDL-cholesterol as shown by linear regression analysis, decreased lesion severity, and increased atherosclerotic plaque stability index. CONCLUSION: FGF21 improves hypercholesterolaemia by accelerating triglyceride-rich lipoprotein turnover as a result of activating BAT and browning of WAT, thereby reducing atherosclerotic lesion severity and increasing atherosclerotic lesion stability index. We have thus provided additional support for the clinical use of FGF21 in the treatment of atherosclerotic cardiovascular disease.

Chronic treatment with terbutaline increases glucose and oleic acid oxidation and protein synthesis in cultured human myotubes.

OBJECTIVE: In vivo studies have reported several beneficial metabolic effects of ß-adrenergic receptor agonist administration in skeletal muscle, including increased glucose uptake, fatty acid metabolism, lipolysis and mitochondrial biogenesis. Although these effects have been widely studied in vivo, the in vitro data are limited to mouse and rat cell lines. Therefore, we sought to discover the effects of the ß2-adrenergic receptor agonist terbutaline on metabolism and protein synthesis in human primary skeletal muscle cells. METHODS: Human cultured myotubes were exposed to terbutaline in various concentrations (0.01-30 â€‹µM) for 4 or 96 â€‹h. Thereafter uptake of [14C]deoxy-D-glucose, oxydation of [14C]glucose and [14C]oleic acid were measured. Incorporation of [14C]leucine, gene expression by qPCR and proteomics analyses by mass spectrometry by the STAGE-TIP method were performed after 96 â€‹h exposure to 1 and 10 â€‹µM of terbutaline. RESULTS: The results showed that 4 â€‹h treatment with terbutaline in concentrations up to 1 â€‹µM increased glucose uptake in human myotubes, but also decreased both glucose and oleic acid oxidation along with oleic acid uptake in concentrations of 10-30 â€‹µM. Moreover, administration of terbutaline for 96 â€‹h increased glucose uptake (in terbutaline concentrations up to 1 â€‹µM) and oxidation (1 â€‹µM), as well as oleic acid oxidation (0.1-30 â€‹µM), leucine incorporation into cellular protein (1-10 â€‹µM) and upregulated several pathways related to mitochondrial metabolism (1 â€‹µM). Data are available via ProteomeXchange with identifier PXD024063. CONCLUSION: These results suggest that ß2-adrenergic receptor have direct effects in human skeletal muscle affecting fuel metabolism and net protein synthesis, effects that might be favourable for both type 2 diabetes and muscle wasting disorders.

The small molecule SERCA activator CDN1163 increases energy metabolism in human skeletal muscle cells.

BACKGROUND AND OBJECTIVE: A number of studies have highlighted muscle-specific mechanisms of thermogenesis involving futile cycling of Ca2+ driven by sarco (endo)plasmic reticulum Ca2+-ATPase (SERCA) and generating heat from ATP hydrolysis to be a promising strategy to counteract obesity and metabolic dysfunction. However, to the best of our knowledge, no experimental studies concerning the metabolic effects of pharmacologically targeting SERCA in human skeletal muscle cells have been reported. Thus, in the present study, we aimed to explore the effects of SERCA-activating compound, CDN1163, on energy metabolism in differentiated human skeletal muscle cells (myotubes). METHODS: In this study, we used primary myotube cultures derived from muscle biopsies of the musculus vastus lateralis and musculi interspinales from lean, healthy male donors. Energy metabolism in myotubes was studied using radioactive substrates. Oxygen consumption rate was assessed with the Seahorse XF24 bioanalyzer, whereas metabolic genes and protein expressions were determined by qPCR and immunoblotting, respectively. RESULTS: Both acute (4 â€‹h) and chronic (5 days) treatment of myotubes with CDN1163 showed increased uptake and oxidation of glucose, as well as complete fatty acid oxidation in the presence of carbonyl cyanide 4-(trifluromethoxy)phenylhydrazone (FCCP). These effects were supported by measurement of oxygen consumption rate, in which the oxidative spare capacity and maximal respiration were enhanced after CDN1163-treatment. In addition, chronic treatment with CDN1163 improved cellular uptake of oleic acid (OA) and fatty acid ß-oxidation. The increased OA metabolism was accompanied by enhanced mRNA-expression of carnitine palmitoyl transferase (CPT) 1B, pyruvate dehydrogenase kinase (PDK) 4, as well as increased AMP-activated protein kinase (AMPK)Thr172 phosphorylation. Moreover, following chronic CDN1163 treatment, the expression levels of stearoyl-CoA desaturase (SCD) 1 was decreased together with de novo lipogenesis from acetic acid and formation of diacylglycerol (DAG) from OA. CONCLUSION: Altogether, these results suggest that SERCA activation by CDN1163 enhances energy metabolism in human myotubes, which might be favourable in relation to disorders that are related to metabolic dysfunction such as obesity and type 2 diabetes mellitus.

SENP2 is vital for optimal insulin signaling and insulin-stimulated glycogen synthesis in human skeletal muscle cells.

Sentrin-specific protease (SENP) 2 has been suggested as a possible novel drug target for the treatment of obesity and type 2 diabetes mellitus after observations of a palmitate-induced increase in SENP2 that lead to increased fatty acid oxidation and improved insulin sensitivity in skeletal muscle cells from mice. However, no precedent research has examined the role of SENP2 in human skeletal muscle cells. In the present work, we have investigated the impact of SENP2 on fatty acid and glucose metabolism as well as insulin sensitivity in human skeletal muscle using cultured primary human myotubes. Acute (4 â€‹h) oleic acid oxidation was reduced in SENP2-knockdown (SENP2-KD) cells compared to control cells, with no difference in uptake. After prelabeling (24 â€‹h) with oleic acid, total lipid content and incorporation into triacylglycerol was decreased, while incorporation into other lipids, as well as complete oxidation and ß-oxidation was increased in SENP2-KD cells. Basal glucose uptake (i.e., not under insulin-stimulated conditions) was higher in SENP2-KD cells, whereas oxidation was similar to control myotubes. Further, basal glycogen synthesis was not different in SENP2-KD myotubes, but both insulin-stimulated glycogen synthesis and AktSer473 phosphorylation was completely blunted in SENP2-KD cells. In conclusion, SENP2 plays an important role in fatty acid and glucose metabolism in human myotubes. Interestingly, it also appears to have a pivotal role in regulating myotube insulin sensitivity. Future studies should examine the role of SENP2 in regulation of insulin sensitivity in other tissues and in vivo, defining the potential for SENP2 as a drug target.

Extensive transcription mis-regulation and membrane defects in AdipoR2-deficient cells challenged with saturated fatty acids.

How cells maintain vital membrane lipid homeostasis while obtaining most of their constituent fatty acids from a varied diet remains largely unknown. Here, we used transcriptomics, lipidomics, growth and respiration assays, and membrane property analyses in human HEK293 cells or human umbilical vein endothelial cells (HUVEC) to show that the function of AdipoR2 is to respond to membrane rigidification by regulating many lipid metabolism genes. We also show that AdipoR2-dependent membrane homeostasis is critical for growth and respiration in cells challenged with saturated fatty acids. Additionally, we found that AdipoR2 deficiency causes transcriptome and cell physiological defects similar to those observed in SREBP-deficient cells upon SFA challenge. Finally, we compared several genes considered important for lipid homeostasis, namely AdipoR2, SCD, FADS2, PEMT and ACSL4, and found that AdipoR2 and SCD are the most important among these to prevent membrane rigidification and excess saturation when human cells are challenged with exogenous SFAs. We conclude that AdipoR2-dependent membrane homeostasis is one of the primary mechanisms that protects against exogenous SFAs.

Publisher Correction: Single-cell analysis uncovers fibroblast heterogeneity and criteria for fibroblast and mural cell identification and discrimination.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Single-cell analysis uncovers fibroblast heterogeneity and criteria for fibroblast and mural cell identification and discrimination.

Many important cell types in adult vertebrates have a mesenchymal origin, including fibroblasts and vascular mural cells. Although their biological importance is undisputed, the level of mesenchymal cell heterogeneity within and between organs, while appreciated, has not been analyzed in detail. Here, we compare single-cell transcriptional profiles of fibroblasts and vascular mural cells across four murine muscular organs: heart, skeletal muscle, intestine and bladder. We reveal gene expression signatures that demarcate fibroblasts from mural cells and provide molecular signatures for cell subtype identification. We observe striking inter- and intra-organ heterogeneity amongst the fibroblasts, primarily reflecting differences in the expression of extracellular matrix components. Fibroblast subtypes localize to discrete anatomical positions offering novel predictions about physiological function(s) and regulatory signaling circuits. Our data shed new light on the diversity of poorly defined classes of cells and provide a foundation for improved understanding of their roles in physiological and pathological processes.

Mature Human White Adipocytes Cultured under Membranes Maintain Identity, Function, and Can Transdifferentiate into Brown-like Adipocytes.

White adipose tissue (WAT) is a central factor in the development of type 2 diabetes, but there is a paucity of translational models to study mature adipocytes. We describe a method for the culture of mature white adipocytes under a permeable membrane. Compared to existing culture methods, MAAC (membrane mature adipocyte aggregate cultures) better maintain adipogenic gene expression, do not dedifferentiate, display reduced hypoxia, and remain functional after long-term culture. Subcutaneous and visceral adipocytes cultured as MAAC retain depot-specific gene expression, and adipocytes from both lean and obese patients can be cultured. Importantly, we show that rosiglitazone treatment or PGC1α overexpression in mature white adipocytes induces a brown fat transcriptional program, providing direct evidence that human adipocytes can transdifferentiate into brown-like adipocytes. Together, these data show that MAAC are a versatile tool for studying phenotypic changes of mature adipocytes and provide an improved translational model for drug development.

FOXK1 and FOXK2 regulate aerobic glycolysis.

Adaptation to the environment and extraction of energy are essential for survival. Some species have found niches and specialized in using a particular source of energy, whereas others-including humans and several other mammals-have developed a high degree of flexibility1. A lot is known about the general metabolic fates of different substrates but we still lack a detailed mechanistic understanding of how cells adapt in their use of basic nutrients2. Here we show that the closely related fasting/starvation-induced forkhead transcription factors FOXK1 and FOXK2 induce aerobic glycolysis by upregulating the enzymatic machinery required for this (for example, hexokinase-2, phosphofructokinase, pyruvate kinase, and lactate dehydrogenase), while at the same time suppressing further oxidation of pyruvate in the mitochondria by increasing the activity of pyruvate dehydrogenase kinases 1 and 4. Together with suppression of the catalytic subunit of pyruvate dehydrogenase phosphatase 1 this leads to increased phosphorylation of the E1α regulatory subunit of the pyruvate dehydrogenase complex, which in turn inhibits further oxidation of pyruvate in the mitochondria-instead, pyruvate is reduced to lactate. Suppression of FOXK1 and FOXK2 induce the opposite phenotype. Both in vitro and in vivo experiments, including studies of primary human cells, show how FOXK1 and/or FOXK2 are likely to act as important regulators that reprogram cellular metabolism to induce aerobic glycolysis.

Systems biology reveals uncoupling beyond UCP1 in human white fat-derived beige adipocytes.

Pharmaceutical induction of metabolically active beige adipocytes in the normally energy storing white adipose tissue has potential to reduce obesity. Mitochondrial uncoupling in beige adipocytes, as in brown adipocytes, has been reported to occur via the uncoupling protein 1 (UCP1). However, several previous in vitro characterizations of human beige adipocytes have only measured UCP1 mRNA fold increase, and assumed a direct correlation with metabolic activity. Here, we provide an example of pharmaceutical induction of beige adipocytes, where increased mRNA levels of UCP1 are not translated into increased protein levels, and perform a thorough analysis of this example. We incorporate mRNA and protein levels of UCP1, time-resolved mitochondrial characterizations, and numerous perturbations, and analyze all data with a new fit-for-purpose mathematical model. The systematic analysis challenges the seemingly obvious experimental conclusion, i.e., that UCP1 is not active in the induced cells, and shows that hypothesis testing with iterative modeling and experimental work is needed to sort out the role of UCP1. The analyses demonstrate, for the first time, that the uncoupling capability of human beige adipocytes can be obtained without UCP1 activity. This finding thus opens the door to a new direction in drug discovery that targets obesity and its associated comorbidities. Furthermore, the analysis advances our understanding of how to evaluate UCP1-independent thermogenesis in human beige adipocytes.