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

Abstract 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

Lipolysis regulates major transcriptional programs in brown adipocytes

β-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 β-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 β-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

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

<div><p>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 β-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.</p></div

Conversion of mature TERT-hWA adipocytes into brown-like adipocytes upon exposure to rosiglitazone.

<p><b>(A)</b> The rosiglitazone-induced browning protocol. <b>(B)</b> Relative mRNA levels of <i>UCP1</i> in mature TERT-hWA adipocytes (day 15, passage 7–14), SGBS and hMADS adipocytes exposed to vehicle or 1 μM rosiglitazone from day 12 to 15. <b>(C)</b> Protein levels of UCP1 in TERT-hWA adipocytes (day 15, passage 13) exposed to vehicle or 1 μM rosiglitazone from day 12 to 15. TFIIB was used as a loading control. <b>(D)</b> Relative mRNA levels of the thermogenesis-related genes <i>EBF2</i>, <i>DIO2</i> and <i>PDK4</i> in mature TERT-hWA adipocytes (day 15, passage 12) exposed to vehicle or 1 μM rosiglitazone from day 12 to 15. <b>(E)</b> Relative mRNA levels of the β-adrenoceptors <i>ADRB1-3</i> in mature TERT-hWA adipocytes (day 15, passage 12) exposed to vehicle or 1 μM rosiglitazone from day 12 to 15. <b>(F)</b> Relative mRNA levels of the mitochondrial markers <i>CPT1B</i>, <i>CS</i> and <i>COXII</i> in mature TERT-hWA adipocytes (day 15, passage 12) exposed to vehicle or 1 μM rosiglitazone from day 12 to 15. In (B) and (D-F), expression levels were normalized to <i>TBP</i> levels. The normalized expression in vehicle-treated cells was set to 1. Data are presented as mean +SEM of one representative experiment done in technical triplicate. Statistical significance was determined by unpaired two-tailed Student’s t-test. *, p < 0.05 versus vehicle-treated cells.</p

Response to β-adrenergic stimulation.

<p><b>(A)</b> Relative mRNA levels of the β-adrenoceptors <i>ADRB1-3</i> in mature TERT-hBA and TERT-hWA adipocytes at passage 10, 15 and 20. Expression levels were normalized to <i>TBP</i> levels. The normalized expression in TERT-hWA adipocytes was set to 1. RT-qPCR data are presented as mean of means +SEM from 5 independent experiments (two experiments in passage 10 and passage 15 and one experiment in passage 20). Statistical significance was determined by paired two-tailed Student’s t-test. *, p < 0.05 versus TERT-hWA. <b>(B)</b> Relative mRNA levels of thermogenic genes in mature TERT-hBA adipocytes (day 12, passage 12) stimulated with 0.1 μM ISO for 6 h. <b>(C)</b> Relative mRNA levels of thermogenic genes in mature TERT-hBA adipocytes (day 12, passage 13) stimulated with 10 μM FSK for 6 h. In (B) and (C), expression levels were normalized to <i>TBP</i> levels. The normalized expression in vehicle-treated cells was set to 1. Data are presented as mean +SEM of one representative experiment done in technical triplicate. Statistical significance was determined by unpaired two-tailed Student’s t-test. *, p < 0.05 versus vehicle-treated cells. <b>(D)</b> UCP1 protein levels in mature TERT-hBA [day 12, passage 10 (P10) and 15 (P15)] stimulated with 0.1 μM ISO for 24 h. TFIIB was used as a loading control. <b>(E)</b> Western blot analysis for phosphorylated adipocyte mediators in mature TERT-hBA adipocytes (day 12, P9) pretreated with 10 μM propranolol or vehicle for 1 h before being stimulated with 10 μM FSK or 0.1 μM ISO for an additional 1 h. <b>(F-G)</b> Representative time course of oxygen consumption and extracellular acidification rates (OCR and ECAR, respectively) in mature TERT-hBA adipocytes (day 12, passage 9) before and after injection of 10 μM ISO or 10 μM FSK. Data are presented as mean +/- SEM of one representative experiment with 9–12 wells per condition. (H) Western blot analysis for phosphorylated HSL in mature TERT-hWA adipocytes (day 12, passage 9) pretreated with 10 μM propranolol or vehicle for 1 h before being stimulated with 10 μM FSK or 0.1 μM ISO for an additional 1 h.</p

Subject and biopsy characterization.

<p><b>(A)</b> Patient information. <b>(B)</b> Relative mRNA levels of the thermogenic genes <i>UCP1</i>, <i>PPARGC1A</i> and <i>EBF2</i> in hBAT and hWAT. <b>(C)</b> Relative mRNA levels of the mitochondrial genes <i>CPT1B</i>, <i>CS</i> and <i>COXII</i> in hBAT and hWAT. In (B) and (C), expression levels were normalized to <i>TBP</i> levels. The normalized expression in hWAT was set to 1, except for <i>UCP1</i> in which hBAT was set to 1. Data represent the mean of a technical duplicate without error bars, since only one patient was included. Statistical analyses were not applied.</p