The mitochondrial pyruvate carrier regulates adipose glucose partitioning in female mice

Abstract

Objective: The mitochondrial pyruvate carrier (MPC) occupies a critical node in intermediary metabolism, prompting interest in its utility as a therapeutic target for the treatment of obesity and cardiometabolic disease. Dysregulated nutrient metabolism in adipose tissue is a prominent feature of obesity pathophysiology, yet the functional role of adipose MPC has not been explored. We investigated whether the MPC shapes the adaptation of adipose tissue to dietary stress in female and male mice.

Methods: The impact of pharmacological and genetic disruption of the MPC on mitochondrial pathways of triglyceride assembly (lipogenesis and glyceroneogenesis) was assessed in 3T3L1 adipocytes and murine adipose explants, combined with analyses of adipose MPC expression in metabolically compromised humans. Whole-body and adipose-specific glucose metabolism were subsequently investigated in male and female mice lacking adipocyte MPC1 (Mpc1^AD-/-) and fed either standard chow, high-fat western style, or high-sucrose lipid restricted diets for 24 weeks, using a combination of radiolabeled tracers and GC/MS metabolomics.

Results: Treatment with UK5099 or siMPC1 impaired the synthesis of lipids and glycerol-3-phosphate from pyruvate and blunted triglyceride accumulation in 3T3L1 adipocytes, whilst MPC expression in human adipose tissue was negatively correlated with indices of whole-body and adipose tissue metabolic dysfunction. Mature adipose explants from Mpc1^AD-/- mice were intrinsically incapable of incorporating pyruvate into triglycerides. In vivo, MPC deletion restricted the incorporation of circulating glucose into adipose triglycerides, but only in female mice fed a zero fat diet, and this associated with sex-specific reductions in tricarboxylic acid cycle pool sizes and compensatory transcriptional changes in lipogenic and glycerol metabolism pathways. However, whole-body adiposity and metabolic health were preserved in Mpc1^AD-/- mice regardless of sex, even under conditions of zero dietary fat.

Conclusions: These findings highlight the greater capacity for mitochondrially driven triglyceride assembly in adipose from female versus male mice and expose a reliance upon MPC-gated metabolism for glucose partitioning in female adipose under conditions of dietary lipid restriction.

Keywords: Adipose; Glyceroneogenesis; Lipogenesis; Mitochondria; Sexual dimorphism.

Figure 1

Mitochondrial pyruvate transport maintains triglyceride storage in 3T3L1 adipocytes. (A–C) Analysis of selected genes from GSE20696 showing mRNA expression during the differentiation of 3T3L1 pre-adipocytes (day 0) to adipocytes (days 2 and day 7), including MPC components (A), the most significantly induced genes (B), and genes involved in mitochondrial pyruvate metabolism (C). Data are expressed as Log base 2 of the fold change from day 0. (D–F) Incorporation of [2–¹⁴C] pyruvate into total lipids (D), or the fatty acyl or glycerol-glyceride fraction of lipids (E – F) following treatment of differentiated 3T3L1 adipocytes with 5 μM UK5099 (D–E) or siRNA against MPC1 or scramble control (F). (G–H) Total triglyceride accumulation in adipocytes treated with 5 μM UK5099 either throughout (G), or at various time points during (H), differentiation. (I) Incorporation of [U–¹⁴C] acetate into total lipids in differentiated 3T3L1 adipocytes treated with or without 5 μM UK5099. (J) mRNA expression of selected genes in 3T3L1 adipocytes treated with DMSO vehicle (VEH) or 5 μM UK5099 throughout differentiation, normalized to 18S control and presented relative to VEH day 0. Data are mean ± SE for at least three experimental replicates (different cell passages). ∗P < 0.05 vs control, ^P < 0.05 vs 10% FBS condition in (D) by paired t-test (E - F and I), one-way ANOVA (H) or 2-way ANOVA (D).

Figure 2

Adipose tissue MPC expression is altered in metabolically compromised mice and humans. (A–B) Normalized, quantified protein expression and representative western blot of mitochondrial pyruvate carrier proteins in subcutaneous adipose tissue from age-matched, chow fed wild type vs genetically obese (ob/ob) male mice (A) and from female and male human subjects with normal glucose tolerance vs impaired fasting glucose plus impaired glucose tolerance (B). (C–H) Pearson correlations between the protein expression of MPC1 (A, D, E) or MPC2 (F, G, H) in subcutaneous adipose tissue and indices of glucose tolerance (C and F), insulin sensitivity (D and G) and adipose insulin resistance (E and H) in human subjects separated by sex. Data are n = 5–8 in each group. ∗P < 0.05, ∗∗∗P < 0.001 vs control group (A and B).

Figure 3

MPC1 knockdown blunts capacity for triglyceride synthesis in mature adipose tissue. (A) Mpc1 floxed allele and Adipoq-Cre recombination was visualized by semiquantitative RT-PCR and MPC complex proteins knockdown efficacy was verified by western blot of adipose lysates. (B) Schematic illustrating major pathways of pyruvate incorporation into adipose triglycerides. (C - D) Ex vivo incorporation of [2–¹⁴C] pyruvate into the fatty acyl (lipogenesis C) or glycerol (glyceroneogenesis D) moieties of total lipids in epididymal adipose explants from male and female Mpc1^AD−/− mice or LoxP^+/+ controls. (E–F) Rates of glycerol release (E) and non-esterified fatty acid re-esterification (F) from epididymal adipose explants from male Mpc1^AD−/− mice or LoxP^+/+ controls under basal (unstimulated) conditions or during treatment with insulin or forskolin plus triacsin C. ∗P < 0.05, ∗∗P < 0.01 for Mpc1^AD−/− vs LoxP^+/+; ^^P < 0.01 for female vs male. Data are mean ± SE for at least five mice per group.

Figure 4

Sex-specific dependencies on the MPC for adipose DNL in vivo. (A - B) In vivo incorporation of intraperitoneally administered [U–¹⁴C] glucose into the fatty acyl moieties of total lipids (lipogenesis) in inguinal (A) and epididymal (B) adipose tissue depots and (C–D) mRNA expression of lipogenic genes in epididymal adipose tissue (eWAT) for male and female Mpc1^AD−/− mice or LoxP^+/+ controls fed either a high fat western-style diet (WD, C) or a zero fat, sucrose enriched diet (ZFD, D) for 24 weeks. ∗P < 0.05 for MPC^AD−/− vs LoxP^+/+; ^###P < 0.001 for ZFD vs WD; ^P < 0.05 for female vs male. Data are mean ± SE for 8-10 mice per group.

Figure 5

Sex-specific dependencies on the MPC for adipose glycerol-3-phosphate synthesis in vivo. (A - D) In vivo incorporation of intraperitoneally administered [U–¹⁴C] glucose into the glycerol moieties of total lipids (glyceroneogenesis A, B, D) or polar metabolites (C) in inguinal adipose (A), epididymal adipose (B - C), or liver (D) for male and female Mpc1^AD−/− mice or LoxP^+/+ controls fed either a high fat western-style diet (WD) or a zero fat, sucrose enriched diet (ZFD) for 24 weeks. (E–F) mRNA expression of genes involved in glycerol metabolism in inguinal (E) or epididymal (F) adipose tissue for male and female Mpc1^AD−/− mice or LoxP^+/+ controls fed a zero fat, sucrose enriched diet (ZFD). ∗∗P < 0.01, ∗∗∗P < 0.001 for Mpc1^AD−/− vs LoxP^+/+; ^#P < 0.05, ^###P < 0.001 main effect of genotype. Data are mean ± SE for 8-10 mice per group.

Figure 6

Decreased adipose TCA cycle pool size in female Mpc1^AD−/− mice fed a lipid restricted diet Polar metabolite abundances in epididymal adipose tissue from male and female Mpc1^AD−/− mice or LoxP^+/+ controls fed a zero fat, sucrose enriched diet (ZFD) determined by gas chromatography mass spectrometry. (A) Summed and (B) individual TCA cycle intermediates, (C) alanine, (D) glutamate, (E) glutamine, (F) branched chain alpha keto acids, (G) the ratio of 3-hydroxybutyrate to alpha keto isovalerate and (H) glycerol-3-phosphate. Abundance data represent relative abundances of metabolites (normalized to labeled valine internal standard) except for alanine and glutamate (pmol/mg tissue). ^P < 0.05, ^^P < 0.01 for females vs males; ∗P < 0.05, ∗∗P < 0.01 main effect of genotype. Data are mean ± SE for 7-8 mice per group.

Figure 7

Whole body lipid and glucose homeostasis are preserved in chow fed Mpc1^AD−/− mice. (A) Total body weight, (B) inguinal and epididymal adipose depot weights, (C) oral glucose tolerance, (D) HOMA-IR index of insulin resistance, (E - F) fasting and CL316,243-stimulated plasma glycerol (E) and non-esterified fatty acids (F) in male and female Mpc1^AD−/− mice or LoxP^+/+ controls fed a standard chow diet. ∗P < 0.05 for MPC^AD−/− vs LoxP^+/+; ^^^P < 0.001 for female vs male. Data are mean ± SE for 8-10 mice per group.

Figure 8

Total adiposity is maintained in Mpc1AD−/− mice fed a lipid restricted diet. (A, D) Percentage body weight gain, (B, E) total fat and lean mass, and (C, F) incremental oral glucose tolerance for male and female MPC^AD−/− mice or LoxP^+/+ controls fed either a zero fat, sucrose enriched diet (A–C) or a high fat western-style diet (D–F) for 24 weeks. ∗P < 0.05 for MPC^AD−/− vs LoxP^+/+. Data are mean ± SE for 8-10 mice per group.