White-to-brite conversion in human adipocytes promotes metabolic reprogramming towards fatty acid anabolic and catabolic pathways

Abstract

Objective: Fat depots with thermogenic activity have been identified in humans. In mice, the appearance of thermogenic adipocytes within white adipose depots (so-called brown-in-white i.e., brite or beige adipocytes) protects from obesity and insulin resistance. Brite adipocytes may originate from direct conversion of white adipocytes. The purpose of this work was to characterize the metabolism of human brite adipocytes.

Methods: Human multipotent adipose-derived stem cells were differentiated into white adipocytes and then treated with peroxisome proliferator-activated receptor (PPAR)γ or PPARα agonists between day 14 and day 18. Gene expression profiling was determined using DNA microarrays and RT-qPCR. Variations of mRNA levels were confirmed in differentiated human preadipocytes from primary cultures. Fatty acid and glucose metabolism was investigated using radiolabelled tracers, Western blot analyses and assessment of oxygen consumption. Pyruvate dehydrogenase kinase 4 (PDK4) knockdown was achieved using siRNA. In vivo, wild type and PPARα-null mice were treated with a β3-adrenergic receptor agonist (CL316,243) to induce appearance of brite adipocytes in white fat depot. Determination of mRNA and protein levels was performed on inguinal white adipose tissue.

Results: PPAR agonists promote a conversion of white adipocytes into cells displaying a brite molecular pattern. This conversion is associated with transcriptional changes leading to major metabolic adaptations. Fatty acid anabolism i.e., fatty acid esterification into triglycerides, and catabolism i.e., lipolysis and fatty acid oxidation, are increased. Glucose utilization is redirected from oxidation towards glycerol-3-phophate production for triglyceride synthesis. This metabolic shift is dependent on the activation of PDK4 through inactivation of the pyruvate dehydrogenase complex. In vivo, PDK4 expression is markedly induced in wild-type mice in response to CL316,243, while this increase is blunted in PPARα-null mice displaying an impaired britening response.

Conclusions: Conversion of human white fat cells into brite adipocytes results in a major metabolic reprogramming inducing fatty acid anabolic and catabolic pathways. PDK4 redirects glucose from oxidation towards triglyceride synthesis and favors the use of fatty acids as energy source for uncoupling mitochondria.

Keywords: Brite/beige adipocyte; Fatty acid metabolism; Glycerol metabolism; Peroxisome proliferator-activated receptor; Pyruvate dehydrogenase kinase 4.

Graphical abstract

Figure 1

PPARγ and PPARα agonists promote britening of human white adipocytes. (A) Gene expression levels of thermogenic genes. (B) UCP1 protein content. Gene expression levels of (C) brown-, (D) brite- and (E) white-specific markers. (F) PLIN1 protein content. (G) Pictures representative of Oil Red O staining in each condition at day 18. Intracellular triglyceride (TG) content quantified by enzymatic assay. (H) mRNA levels of thermogenic genes in human adipocytes differentiated in primary culture. Data represent mean ± SEM expressed as percentage of control (n = 6–12) for hMADS cells and (n = 2–4) for primary adipocytes. Open bars: control cells (C), full bars: rosiglitazone-treated cells (R), hatched bars: GW7647-treated cells (GW). *: p < 0.05 for R or GW vs. C; **: p < 0.01; ***: p < 0.001.

Figure 2

Britening of white adipocytes induces anabolic and catabolic pathways of fat metabolism. Fatty acid metabolism was investigated in 18 day-differentiated hMADS cells treated or not with rosiglitazone or GW7647 for the last 4 days. (A) Fatty acid (oleate) incorporation into triglycerides (TG). (B) Gene expression level of glycerol kinase (GK). (C) Glycerol incorporation into triglycerides. (D) Glycerol release after stimulation by the β-adrenergic agonist, isoproterenol. (E) ATGL and HSL protein content. (F) Fatty acid (oleate) oxidation. (G) PLIN5 protein content. (H) Basal mitochondrial oxygen consumption rates (OCR) in the presence or not of 50 μM etomoxir. (I) OCR measured after addition of 200 μM oleate. Data represent mean ± SEM expressed as percentage of control (n = 7–12). Open bars: control cells (C), full bars: rosiglitazone-treated cells (R), hatched bars: GW7647-treated cells (GW). *: p < 0.05 for R or GW vs. C; **: p < 0.01; ***: p < 0.001. $$$: p < 0.001 for etomoxir vs. basal condition.

Figure 3

Britening of white adipocytes promotes a shift of glucose metabolism from oxidation towards glycerol production. Glucose metabolism was investigated in 18 day-differentiated hMADS cells treated or not with rosiglitazone or GW7647 for the last 4 days. (A) Glucose transport in the presence or not of 100 nM insulin. (B) Gene expression levels of the glucose transporters GLUT1 and GLUT4 and GLUT1 protein level. (C) Insulin sensitivity estimated by the insulin stimulated-to-basal glucose uptake ratio. (D) Pyruvate uptake, (E) pyruvate oxidation and (F) pyruvate release into the medium in the presence or not of 100 nM insulin. (G) Glyceroneogenesis assessed by incorporation of pyruvic acid into the glycerol moiety of neutral lipids. (H) PCK1 mRNA and protein levels. (I) Lipolysis measured by oleic acid release into the medium after stimulation by the β-adrenergic agonist, isoproterenol. (J) Gene expression levels of the four PDK isoforms. (K) PDK4 and Ser293-phosphorylated PDHE1α protein levels. Data represent mean ± SEM expressed as percentage of control (n = 6–12). Open bars: control cells (C), full bars: rosiglitazone-treated cells (R), hatched bars: GW7647-treated cells (GW). *: p < 0.05 for R or GW vs. C; **: p < 0.01; ***: p < 0.001. $: p < 0.05 for insulin vs. unstimulated condition; $$$: p < 0.001. ##: p < 0.01 for R vs. GW; ###: p < 0.001.

Figure 4

PDK4 knockdown prevents induction of fatty acid oxidation in brite adipocytes. Britening-associated changes were explored in 18 day-differentiated hMADS cells transfected with either a control-or PDK4-targeting siRNA and treated or not with rosiglitazone or GW7647 for the last 4 days. (A) PDK4 mRNA and protein levels. (B) Gene expression levels of UCP1, CIDEA and CITED1. (C) Ser293-phosphorylated PDHE1α protein levels. (D) Pyruvate release into the medium. (E) Glyceroneogenesis assessed by incorporation of pyruvic acid into the glycerol moiety of neutral lipids. (F) Pyruvate oxidation. (G) Fatty acid (oleate) oxidation. Data represent mean ± SEM expressed as percentage of control (n = 6–11). Open bars: siCtrl-treated cells, full bars: siPDK4-treated cells. *: p < 0.05 for R or GW vs. C; **: p < 0.01; ***: p < 0.001. $: p < 0.05 for siPDK4 vs. siCtrl; $$: p < 0.01; $$$: p < 0.001. #: p < 0.05 for R vs. GW; ###: p < 0.001.

Figure 5

PPARα deficiency disrupts WAT britening-induced PDK4 expression in vivo. (A, C, E, G and I) WT and PPARα-null male mice housed at thermoneutrality were treated with CL316,243 (CL, 0.1 mg/kg/d) or vehicle (DMSO) for 10 days. (B, D, F and H) WT and PPARα-null male mice housed at standard temperature were treated with CL316,243 (CL, 1 mg/kg/d) or vehicle (DMSO) for 7 days. Analyses were performed on the inguinal white adipose tissue depot. (A, B) UCP1 mRNA and protein levels. (C–F) Gene expression levels of Cidea, Cpt1b, Cited1 and Tmem26. (G, H) PDK4 mRNA and protein levels. (I) Spearman correlation between UCP1 and PDK4 protein content (n = 23). Data represent mean ± SEM expressed as percentage of vehicle-treated WT mice (n = 7–9 mice per group). Open bars: wild-type mice, hatched bars: PPARα-null mice. NA: not available. *: p < 0.05 for CL vs. DMSO; **: p < 0.01; ***: p < 0.001. $: p < 0.05 for PPARα-null vs. wild type; $$: p < 0.01; $$$: p < 0.001.