Adipocyte gene expression in obesity - insights gained and challenges ahead.

White adipocytes possess extraordinary plasticity with an unparalleled capacity to expand in size with nutritional overload. Several lines of evidence indicate that limitations to this plasticity, as found in both lipodystrophy and obesity, drive several of the comorbidities of these disease, thereby underscoring the need to understand the mechanisms of healthy and unhealthy adipose expansion. Recent single-cell technologies and studies of isolated adipocytes have allowed researchers to gain insight into the molecular mechanisms of adipocyte plasticity. Here, we review current insight into the effect of nutritional overload on white adipocyte gene expression and function. We review the role of adipocyte size and heterogeneity and discuss the challenges and future directions.

Lipolysis regulates major transcriptional programs in brown adipocytes.

ß-Adrenergic signaling is a core regulator of brown adipocyte function stimulating both lipolysis and transcription of thermogenic genes, thereby expanding the capacity for oxidative metabolism. We have used pharmacological inhibitors and a direct activator of lipolysis to acutely modulate the activity of lipases, thereby enabling us to uncover lipolysis-dependent signaling pathways downstream of ß-adrenergic signaling in cultured brown adipocytes. Here we show that induction of lipolysis leads to acute induction of several gene programs and is required for transcriptional regulation by ß-adrenergic signals. Using machine-learning algorithms to infer causal transcription factors, we show that PPARs are key mediators of lipolysis-induced activation of genes involved in lipid metabolism and thermogenesis. Importantly, however, lipolysis also activates the unfolded protein response and regulates the core circadian transcriptional machinery independently of PPARs. Our results demonstrate that lipolysis generates important metabolic signals that exert profound pleiotropic effects on transcription and function of cultured brown adipocytes.

Pyruvate kinase M2 represses thermogenic gene expression in brown adipocytes.

Utilizing the thermogenic capacity of brown adipose tissue is a potential anti-obesity strategy; therefore, the mechanisms controlling expression of thermogenesis-related genes are of interest. Pyruvate kinase (PK) catalyzes the last step of glycolysis and exists as four isoenzymes: PK, liver, PK, red blood cell, PK, muscle (PKM1 and PKM2). PKM2 has both glycolytic and nuclear functions. Here, we report that PKM2 is enriched in brown adipose compared with white adipose tissue. Specific knockdown of PKM2 in mature brown adipocytes demonstrates that silencing of PKM2 does not lead to a decrease in PK activity, but causes a robust upregulation of thermogenic uncoupling protein 1 (Ucp1) and fibroblast growth factor 21 (Fgf21) gene expression. This increase is not mediated by any of the known mechanisms for PKM2-regulated gene expression, thus implying the existence of a novel mechanism for PKM2-dependent effects on gene expression.

Metformin targets brown adipose tissue in vivo and reduces oxygen consumption in vitro.

AIMS: To test the hypothesis that brown adipose tissue (BAT) is a metformin target tissue by investigating in vivo uptake of [11 C]-metformin tracer in mice and studying in vitro effects of metformin on cultured human brown adipocytes. MATERIALS AND METHODS: Tissue-specific uptake of metformin was assessed in mice by PET/CT imaging after injection of [11 C]-metformin. Human brown adipose tissue was obtained from elective neck surgery and metformin transporter expression levels in human and murine BAT were determined by qPCR. Oxygen consumption in metformin-treated human brown adipocyte cell models was assessed by Seahorse XF technology. RESULTS: Injected [11 C]-metformin showed avid uptake in the murine interscapular BAT depot. Metformin exposure in BAT was similar to hepatic exposure. Non-specific inhibition of the organic cation transporter (OCT) protein by cimetidine administration eliminated BAT exposure to metformin, demonstrating OCT-mediated uptake. Gene expression profiles of OCTs in BAT revealed ample OCT3 expression in both human and mouse BAT. Incubation of a human brown adipocyte cell models with metformin reduced cellular oxygen consumption in a dose-dependent manner. CONCLUSION: These results support BAT as a putative metformin target.

GSK3 is a negative regulator of the thermogenic program in brown adipocytes.

Brown adipose tissue is a promising therapeutic target in metabolic disorders due to its ability to dissipate energy and improve systemic insulin sensitivity and glucose homeostasis. ß-Adrenergic stimulation of brown adipocytes leads to an increase in oxygen consumption and induction of a thermogenic gene program that includes uncoupling protein 1 (Ucp1) and fibroblast growth factor 21 (Fgf21). In kinase inhibitor screens, we have identified glycogen synthase kinase 3 (GSK3) as a negative regulator of basal and ß-adrenergically stimulated Fgf21 expression in cultured brown adipocytes. In addition, inhibition of GSK3 also caused increased Ucp1 expression and oxygen consumption. ß-Adrenergic stimulation triggered an inhibitory phosphorylation of GSK3 in a protein kinase A (PKA)-dependent manner. Mechanistically, inhibition of GSK3 activated the mitogen activated protein kinase (MAPK) kinase 3/6-p38 MAPK-activating transcription factor 2 signaling module. In summary, our data describe GSK3 as a novel negative regulator of ß-adrenergic signaling in brown adipocytes.

MCT1 and MCT4 Expression and Lactate Flux Activity Increase During White and Brown Adipogenesis and Impact Adipocyte Metabolism.

Adipose tissue takes up glucose and releases lactate, thereby contributing significantly to systemic glucose and lactate homeostasis. This implies the necessity of upregulation of net acid and lactate flux capacity during adipocyte differentiation and function. However, the regulation of lactate- and acid/base transporters in adipocytes is poorly understood. Here, we tested the hypothesis that adipocyte thermogenesis, browning and differentiation are associated with an upregulation of plasma membrane lactate and acid/base transport capacity that in turn is important for adipocyte metabolism. The mRNA and protein levels of the lactate-H+ transporter MCT1 and the Na+,HCO3- cotransporter NBCe1 were upregulated in mouse interscapular brown and inguinal white adipose tissue upon cold induction of thermogenesis and browning. MCT1, MCT4, and NBCe1 were furthermore strongly upregulated at the mRNA and protein level upon differentiation of cultured pre-adipocytes. Adipocyte differentiation was accompanied by increased plasma membrane lactate flux capacity, which was reduced by MCT inhibition and by MCT1 knockdown. Finally, in differentiated brown adipocytes, glycolysis (assessed as ECAR), and after noradrenergic stimulation also oxidative metabolism (OCR), was decreased by MCT inhibition. We suggest that upregulation of MCT1- and MCT4-mediated lactate flux capacity and NBCe1-mediated HCO3-/pH homeostasis are important for the physiological function of mature adipocytes.

Characterization of immortalized human brown and white pre-adipocyte cell models from a single donor.

Brown adipose tissue with its constituent brown adipocytes is a promising therapeutic target in metabolic disorders due to its ability to dissipate energy and improve systemic insulin sensitivity and glucose homeostasis. The molecular control of brown adipocyte differentiation and function has been extensively studied in mice, but relatively little is known about such regulatory mechanisms in humans, which in part is due to lack of human brown adipose tissue derived cell models. Here, we used retrovirus-mediated overexpression to stably integrate human telomerase reverse transcriptase (TERT) into stromal-vascular cell fractions from deep and superficial human neck adipose tissue biopsies from the same donor. The brown and white pre-adipocyte cell models (TERT-hBA and TERT-hWA, respectively) displayed a stable proliferation rate and differentiation until at least passage 20. Mature TERT-hBA adipocytes expressed higher levels of thermogenic marker genes and displayed a higher maximal respiratory capacity than mature TERT-hWA adipocytes. TERT-hBA adipocytes were UCP1-positive and responded to ß-adrenergic stimulation by activating the PKA-MKK3/6-p38 MAPK signaling module and increasing thermogenic gene expression and oxygen consumption. Mature TERT-hWA adipocytes underwent efficient rosiglitazone-induced 'browning', as demonstrated by strongly increased expression of UCP1 and other brown adipocyte-enriched genes. In summary, the TERT-hBA and TERT-hWA cell models represent useful tools to obtain a better understanding of the molecular control of human brown and white adipocyte differentiation and function as well as of browning of human white adipocytes.