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

Barquissau V, Beuzelin D, Pisani DF, Beranger GE, Mairal A, Montagner A, Roussel B, Tavernier G, Marques MA, Moro C, Guillou H, Amri EZ, Langin D - Mol Metab (2016)

Bottom Line: This conversion is associated with transcriptional changes leading to major metabolic adaptations.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.

View Article: PubMed Central - PubMed

Affiliation: INSERM, UMR 1048, Institute of Metabolic and Cardiovascular Diseases, Toulouse, France; University of Toulouse, Paul Sabatier University, France.

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- 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- 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.

No MeSH data available.


Related in: MedlinePlus

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.
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fig4: 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.

Mentions: To directly address the role of PDK4 in the metabolic remodeling associated with britening of hMADS cells, siRNA-mediated knockdown was validated at mRNA and protein levels (Figure 4A). There was no alteration in the expression of UCP1, brown and brite markers and of other PDK isoforms (Figure 4B and Figure S5A,B). Decreased PDK4 expression resulted in lower PDH phosphorylation (Figure 4C). Consequently, pyruvate release from brite adipocytes was decreased to the levels observed in white adipocytes (Figure 4D) and britening-induced glyceroneogenesis was partially inhibited (Figure 4E). Concomitantly, PDK4 knockdown restored pyruvate oxidation in brite fat cells (Figure 4F). Consistent with higher pyruvate oxidation, pyruvate-driven OCR raised by up to 50% over basal in white and PDK4 siRNA-treated brite adipocytes while brite adipocytes did not respond to pyruvate addition (Figure S5C). Contrary to pyruvate oxidation, PDK4 siRNA treatment blunted fatty oxidation in brite adipocytes (Figure 4G). Basal OCR in brite adipocytes, which is highly dependent on fatty acid oxidation (Figure 2H,I), returned to white fat cell level upon PDK4 knockdown (Figure S5D), similarly to what was observed when fatty acid oxidation was prevented by etomoxir (Figure 2H). However, fatty acid oxidation remained higher in PDK4 siRNA-treated brite compared to white adipocytes (Figure 4G), suggesting that other mechanisms such as increased expression of fatty acid metabolism genes (Figure S5A and Table S3) were involved. Fatty acid esterification was not altered by PDK4 knockdown (Figure S5E), demonstrating that PDK4 specifically regulates the oxidative metabolism of fatty acids.


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

Barquissau V, Beuzelin D, Pisani DF, Beranger GE, Mairal A, Montagner A, Roussel B, Tavernier G, Marques MA, Moro C, Guillou H, Amri EZ, Langin D - Mol Metab (2016)

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.
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fig4: 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.
Mentions: To directly address the role of PDK4 in the metabolic remodeling associated with britening of hMADS cells, siRNA-mediated knockdown was validated at mRNA and protein levels (Figure 4A). There was no alteration in the expression of UCP1, brown and brite markers and of other PDK isoforms (Figure 4B and Figure S5A,B). Decreased PDK4 expression resulted in lower PDH phosphorylation (Figure 4C). Consequently, pyruvate release from brite adipocytes was decreased to the levels observed in white adipocytes (Figure 4D) and britening-induced glyceroneogenesis was partially inhibited (Figure 4E). Concomitantly, PDK4 knockdown restored pyruvate oxidation in brite fat cells (Figure 4F). Consistent with higher pyruvate oxidation, pyruvate-driven OCR raised by up to 50% over basal in white and PDK4 siRNA-treated brite adipocytes while brite adipocytes did not respond to pyruvate addition (Figure S5C). Contrary to pyruvate oxidation, PDK4 siRNA treatment blunted fatty oxidation in brite adipocytes (Figure 4G). Basal OCR in brite adipocytes, which is highly dependent on fatty acid oxidation (Figure 2H,I), returned to white fat cell level upon PDK4 knockdown (Figure S5D), similarly to what was observed when fatty acid oxidation was prevented by etomoxir (Figure 2H). However, fatty acid oxidation remained higher in PDK4 siRNA-treated brite compared to white adipocytes (Figure 4G), suggesting that other mechanisms such as increased expression of fatty acid metabolism genes (Figure S5A and Table S3) were involved. Fatty acid esterification was not altered by PDK4 knockdown (Figure S5E), demonstrating that PDK4 specifically regulates the oxidative metabolism of fatty acids.

Bottom Line: This conversion is associated with transcriptional changes leading to major metabolic adaptations.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.

View Article: PubMed Central - PubMed

Affiliation: INSERM, UMR 1048, Institute of Metabolic and Cardiovascular Diseases, Toulouse, France; University of Toulouse, Paul Sabatier University, France.

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- 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- 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.

No MeSH data available.


Related in: MedlinePlus