The mitochondrial dicarboxylate carrier prevents hepatic lipotoxicity by inhibiting white adipocyte lipolysis

Abstract

Background & aims: We have previously reported that the mitochondrial dicarboxylate carrier (mDIC [SLC25A10]) is predominantly expressed in the white adipose tissue (WAT) and subject to regulation by metabolic cues. However, the specific physiological functions of mDIC and the reasons for its abundant presence in adipocytes are poorly understood.

Methods: To systemically investigate the impact of mDIC function in adipocytes in vivo, we generated loss- and gain-of-function mouse models, selectively eliminating or overexpressing mDIC in mature adipocytes, respectively.

Results: In in vitro differentiated white adipocytes, mDIC is responsible for succinate transport from the mitochondrial matrix to the cytosol, from where succinate can act on the succinate receptor SUCNR1 and inhibit lipolysis by dampening the cAMP- phosphorylated hormone-sensitive lipase (pHSL) pathway. We eliminated mDIC expression in adipocytes in a doxycycline (dox)-inducible manner (mDIC^iKO) and demonstrated that such a deletion results in enhanced adipocyte lipolysis and promotes high-fat diet (HFD)-induced adipocyte dysfunction, liver lipotoxicity, and systemic insulin resistance. Conversely, in a mouse model with dox-inducible, adipocyte-specific overexpression of mDIC (mDIC^iOE), we observed suppression of adipocyte lipolysis both in vivo and ex vivo. mDIC^iOE mice are potently protected from liver lipotoxicity upon HFD feeding. Furthermore, they show resistance to HFD-induced weight gain and adipose tissue expansion with concomitant improvements in glucose tolerance and insulin sensitivity. Beyond our data in rodents, we found that human WAT SLC25A10 mRNA levels are positively correlated with insulin sensitivity and negatively correlated with intrahepatic triglyceride levels, suggesting a critical role of mDIC in regulating overall metabolic homeostasis in humans as well.

Conclusions: In summary, we highlight that mDIC plays an essential role in governing adipocyte lipolysis and preventing liver lipotoxicity in response to a HFD.

Lay summary: Dysfunctional fat tissue plays an important role in the development of fatty liver disease and liver injury. Our present study identifies a mitochondrial transporter, mDIC, which tightly controls the release of free fatty acids from adipocytes to the liver through the export of succinate from mitochondria. We believe this mDIC-succinate axis could be targeted for the treatment of fatty liver disease.

Keywords: NAFLD; NASH; adipocytes; dicarboxylate carrier; insulin resistance; lipolysis; lipotoxicity; mitochondria; succinate.

Fig. 1.. mDIC is reduced in individuals with insulin resistance and people with NAFLD.

(A-C) mDIC (SLC25A10) expression from human gene expression databases: (A) mDIC levels in sWAT from insulin sensitive (IS, n=5) and insulin resistant (IR, n=4) people, (B) from another study in IS (n=10) and IR (n=9) patients, and (C) from control or diabetic humans (n=5). (D-F) Correlation between sWAT mDIC (SLC25A10) mRNA levels and human insulin sensitivity (D-E, n=23), and intrahepatic triglyceride levels (F, n=46). FFM: fat free mass. (G) mDIC (Slc25a10) mRNA levels in fat tissues from unhealthy and healthy AT (n=3). Fold change (FC) and P-value are shown. Data are presented as mean ± SEM of biologically independent samples. *P<0.05, **P<0.01. Two-tailed Student’s t-test (A-C, G); Pearson correlation analysis for correlation coefficient (r) and two-tailed P-value (D-F).

Fig. 2.. Adipocyte-specific deletion of mDIC impairs succinate transport.

(A) Schematic illustration of the adipocyte-specific, dox-inducible mDIC knockout mouse (mDIC^iKO). (B-C) Validation of mDIC deletion in mDIC^iKO mice: (B) mDIC mRNA levels (n=4 (Control) or n=8 (mDIC^iKO)) and (C) mDIC protein levels in mitochondria (mito) and cytosol (cyto) from sWAT. (D-E) ex vivo mitochondrial respiration in sWAT (D) and in vitro respiration in isolated mitochondria (E, 5 μg proteins). Oxygen consumption rate (OCR) at basal level and post-ADP, Oligomycin (Oligo), FCCP and Rotenone/Antimycin-A injection is shown, n=5. (F-G) TCA cycle intermediate levels in sWAT (F) and succinate levels in different tissues (G), n=4 (Control) or n=8 (mDIC^iKO). (H) Succinate export from mitochondria from control or mDIC^iKO mice, n=6. (I) Extracellular succinate levels from in vitro differentiated adipocytes. 5 μg/mL dox was utilized to induce deletion of mDIC. Percentages relative to control are shown. Data are presented as mean ± SEM of biologically independent samples. *P<0.05, **P<0.01, ***P<0.001. Two-tailed Student’s t-test (B, F-G, I); Two-way ANOVA followed by a Tukey post-test (D-E, H).

Fig. 3.. A lack of mDIC abolishes succinate-driven inhibition of adipocyte lipolysis and leads to exacerbated liver lipotoxicity.

(A) The model of succinate controlling white adipocyte lipolysis and NEFA release. (B) Intracellular cAMP levels in in vitro differentiated adipocytes, n=12. (C) Western blotting image and its quantification (n=2) for pHSL levels in adipocytes treated with 50 μM succinate with or without 10 μM forskolin. (D-E) ex vivo (D, n=6) and in vivo (E, n=5) lipolysis analyses in control and mDIC^iKO mice. (F-M) Control and mDIC^iKO mice were subjected to the following metabolic analyses: (F) Serum profiling (n=3); (G) H&E staining images of liver tissues (bar = 161 μm); (H) Selective inflammation, fibrosis, gluconeogenesis, ER stress, and apoptosis related gene expression in the liver, n=8 (Control) or n=13 (mDIC^iKO); (I) Circulating adiponectin levels (n=3); (J) p-AKT (Ser 473) and total AKT expression in the liver after saline or insulin injection (i.v.) for 5 min (n=3); (K) Relative body weight; (L-M) glucose and insulin levels from (L) OGTT and (M) ITT experiments, n=4 (Control) or n=9 (mDIC^iKO). For all the statistics: data are presented as mean ± SEM of biologically independent samples. *P<0.05; #P<0.05. Two-tailed Student’s t-test (B, F, H-I); Two-way ANOVA followed by a Tukey post-test (C-E, K-M).

Fig. 4.. A sustained lack of mDIC in adipocytes exacerbates hepatic steatosis, steatohepatitis, and fibrosis.

In control or mDIC^iKO mice fed with dox-HFD for 14 weeks: (A) Intrahepatic triglyceride levels; (B) serum AST and ALT levels; (C) NAFLD activity score (NAS) (n=6); (D-E) H&E staining (D) and F4/80 immunofluorescence staining (E) images of liver tissues (bar = 161 μm); (F) Flow cytometry density plot and statistics demonstrating the relative percentage of pro- and anti-inflammatory macrophages in the liver (n=3); (G) Selective inflammation related gene expression in liver macrophages (n=4); (H) Picrosirius red staining images of liver sections (bar = 161 μm). For all the statistics: data are presented as mean ± SEM of biologically independent samples. *P<0.05, **P<0.01, ***P<0.001. Two-tailed Student’s t-test (A-C, F-G).

Fig. 5.. Adipocyte-specific overexpression of mDIC promotes succinate transport.

(A) Schematic illustration of the adipocyte-specific, dox-inducible mDIC overexpression mouse (mDIC^iOE). (B-C) Validation of mDIC enhancement in inducible mDIC^iOE mice: (B) mDIC mRNA levels (n=4 mice) and (C) mDIC protein levels in mitochondria (mito) and cytosol (cyto) from sWAT. (D-E) ex vivo mitochondrial respiration in sWAT (D) and in vitro respiration in isolated mitochondria (E, 5 μg proteins), n=5. (F-G) TCA cycle intermediate levels in sWAT (F) and succinate levels in different tissues (G), n=4. (H) Succinate export from mitochondria from control or mDIC^iOE mice, n=12. (I) Extracellular succinate levels in in vitro differentiated adipocytes, n=12. For all statistics: data are presented as mean ± SEM of biologically independent samples. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Two-tailed Student’s t-test (B, F-G, I); Two-way ANOVA followed by a Tukey post-test (D-E, H).

Fig. 6.. mDIC overexpression restrains adipocyte lipolysis to prevent liver lipotoxicity.

(A) Intracellular cAMP levels from in vitro differentiated adipocytes, n=6. (B-C) Western blotting image (B) and its quantification (C, n=3) for pHSL levels. (D-E) in vitro (D, n=3) and in vivo (E, n=9 (Control) or n=6 (mDIC^iOE)) lipolysis analyses in control and mDIC^iOE mice. (F-M) Control and mDIC^iOE mice were subjected to the following metabolic analyses: (F) Serum profiling (n=4); (G) H&E staining images of liver tissues (bar = 161 μm); (H) Selective inflammation, fibrosis, gluconeogenesis, ER stress, and apoptosis gene expression (n=12); (I) Circulating adiponectin levels (n=4 (Control) or n=6 (mDIC^iOE)); (J) Liver p-AKT (Ser 473) and total AKT expression after saline or insulin injection (n=3); (K) Relative body weight; (L-M) Glucose levels from (L) OGTT and (M) ITT experiments (n=8). For all the statistics: data are presented as mean ± SEM of biologically independent samples. *P<0.05, **P<0.01; #P<0.05, ##P<0.01. Two-tailed Student’s t-test (B, F, H-I); Two-way ANOVA followed by a Tukey post-test (C-E, K-M).

Fig. 7.. A sustained overexpression of mDIC in adipocytes protects against hepatic steatosis, steatohepatitis, and fibrosis.

In control or mDIC^iOE mice fed with dox-HFD for 14 weeks: (A) Intrahepatic triglyceride levels; (B) NAFLD activity score (NAS), n=6; (C-D) H&E staining (C) and F4/80 immunofluorescence staining (D) images of liver tissues (bar = 161 μm); (E) Flow cytometry density plot and statistics (n=3); (F) Selective inflammation gene expression in liver macrophages (n=4); (G) Liver picrosirius red staining images (bar = 161 μm). For all the statistics: data are presented as mean ± SEM of biologically independent samples. *P<0.05, **P<0.01, ***P<0.001. Two-tailed Student’s t-test (A-B, E-F).

Fig. 8.. mDIC controls adipocyte lipolysis in a SUCNR1-dependent manner.

(A) The model of succinate controlling white adipocyte lipolysis and NEFA release dependent on SUCNR1 (GPR91). (B) Western blotting image for SUCNR1 levels in adipocytes transduced with LacZ sgRNA, Sucnr1 sgRNA1, or Sucnr1 sgRNA2 AAVs. (C-D) in vitro lipolysis analysis of control and mDIC^iOE adipocytes transduced with LacZ sgRNA or Sucnr1 sgRNA AAVs (n=6). (E-H) SUCNR1 expression from mouse or human genomic databases: (E) in sWAT from low and high weight gaining mice (n=3); (F) from HFD-challenged mice with or without calorie restriction (CR) (n=12); (G) from control or obese humans (n=6 (control) or n=5 (obese)); and (H) from insulin resistant or insulin sensitive human subjects (n=9 (insulin resistant) or n=10 (insulin sensitive)). For all the statistics: data are presented as mean ± SEM of biologically independent samples. *P<0.05, **P<0.01, ****P<0.0001. Two-tailed Student’s t-test (E-H).