Adipose-specific deletion of TFAM increases mitochondrial oxidation and protects mice against obesity and insulin resistance

Abstract

Obesity and type 2 diabetes are associated with mitochondrial dysfunction in adipose tissue, but the role for adipose tissue mitochondria in the development of these disorders is currently unknown. To understand the impact of adipose tissue mitochondria on whole-body metabolism, we have generated a mouse model with disruption of the mitochondrial transcription factor A (TFAM) specifically in fat. F-TFKO adipose tissue exhibit decreased mtDNA copy number, altered levels of proteins of the electron transport chain, and perturbed mitochondrial function with decreased complex I activity and greater oxygen consumption and uncoupling. As a result, F-TFKO mice exhibit higher energy expenditure and are protected from age- and diet-induced obesity, insulin resistance, and hepatosteatosis, despite a greater food intake. Thus, TFAM deletion in the adipose tissue increases mitochondrial oxidation that has positive metabolic effects, suggesting that regulation of adipose tissue mitochondria may be a potential therapeutic target for the treatment of obesity.

Figure 1. Effects of TFAM Knockout on Mitochondrial Gene and Protein Expression in White and Brown Adipocytes

(A) TFAM expression was analyzed by western blot in isolated mitochondria from BAT and white adipocytes (AF WAT) of Lox and F-TFKO mice at 12 weeks of age. Data are representative of 4 samples/genotype; quantification is shown in the lower panel. TFAM and mitochondrially-encoded genes mRNA levels (B) and mitochondrial DNA copy number (C) were assessed using qPCR in BAT (upper panel) and isolated white adipocytes (AF WAT) (lower panel) from 12-week-old male Lox and F-TFKO mice (n=6/genotype). (C) Mitochondria morphology of Lox and F-TFKO BAT by electron microscopy (picture representative of 5 mice /group). Values are given as mean ± SEM, * p<0.05 versus respective controls.

Figure 2. Ablation of TFAM protects mice from obesity

Body weight of male Lox and F-TFKO mice upon chow diet (CD) (A) or high fat diet (HFD) (B) (n=6/genotype/diet). Representative sections of BAT and WAT from 4 month old Lox and F-TFKO mice on CD (C) and HFD (D) (stained with hematoxylin and eosin. Pictures were taken at 20X). ³H] 2-deoxyglucose uptake (E) and glycerol release (F) for isolated white adipocytes from lox and F-TFKO animals at 12 weeks of age (n=6/genotype). (G) ¹⁴C-Palmitate oxidation by isolated brown adipocytes (BA) and white adipocytes (WA) of 10-12 weeks old lox and F-TFKO mice (n=6/genotype). Values are given as mean ± SEM, * p<0.05.

Figure 3. TFAM deletion in adipose tissue protects from insulin resistance and increases energy expenditure

Glucose level (A) and serum insulin (B) in control Lox and F-TFKO mice upon CD and HFD at 4 months of age upon fed state (n=6-8 animals/genotype/diet). Intraperitoneal glucose (C) and insulin tolerance (D) test on Lox and F-TFKO males mice on CD and HFD at 4 months of age (n=6-8 animals/genotype/diet). Leptin (E) and adiponectin (F) serum levels of Lox and F-TFKO male mice at 4 months of age (n=6-8 animals/genotype). (F) Food intake of two month old Lox and F-TFKO male mice (n=4 animals/genotype). The entire experiment was repeated twice. (G) Oxygen consumption of two month old Lox and F-TFKO male mice (n=4/genotype). The entire experiment was repeated twice. Values are given as mean ± SEM, * p<0.05 and ** p<0.01.

Figure 4. TFAM ablation remodels BAT and WAT mitochondria and increase adipose tissue oxygen consumption

(A) Assessment of proteins in the OXPHOS pathway by western blotting of isolated mitochondria from BAT and white adipocytes (AF WAT) of control and F-TFKO mice (8-10 weeks old). Data are representative of 4 mice per genotype and normalized in FigS9A. F-TFKO and Lox BAT (B) and white adipocyte (C) isolated mitochondria respiratory chain enzyme activities were assessed independently. Complex I (CPLxI), complex II (CPLxII), complexes II and III (CPLxII-III) and complex IV (CPLxIV) (n=6/genotype). (D) Citrate synthase activity in extracts from BAT and WAT of Lox and F-TFKO mice at 4 months of age (n=6/genotype). Oxygen consumption rate (OCR) were determined in a Seahorse X24 Flux Analyzer using pieces (10 mg) from BAT (E) and WAT (F) of Lox and F-TFKO mice under basal (2.5 mM glucose) and pyruvate (10 mM) loaded conditions. Data represent the means ± SEM of the OCRs of 10mg from 6 adipose tissue pieces/tissue/mice from n=6/genotype *p<0.05 and ** p<0.01.

Figure 5. TFAM ablation increase adipose tissue metabolic rate through increased ETC flux and uncoupling

(A) TFAM protein levels in confluent C3H10T1/2 cells were assessed by western blot analysis (upper panel) and mRNA levels assessed by qPCR (lower panel) in the control cell line (ShSc) and 2 independents shTFAM knock down cell lines (shTF1 and shTF2). ATP turnover (B) and proton leak (C) in shTFAM C3H10T1/2 and control cell lines at confluence assessed using a Seahorse Flux Analyzer as described in Methods. (D) State 3 Respiratory rate of F-TFKO and Lox isolated mitochondria from BAT and white adipocyte (AF WAT) in presence of malate/pyruvate (complex I) and ADP from Lox and F-TFKO CD fed male mice at 10-12 weeks of age (n=9/genotype). (E) State 3 Respiratory rate of F-TFKO and Lox isolated mitochondria from BAT and white adipocyte (AF WAT) in presence of Palmitoyl-carnitine/malate (fatty acid) and ADP from Lox and F-TFKO CD fed male mice at 10-12 weeks of age (n=9/genotype). (F) State 3 Respiratory rate of F-TFKO and control isolated mitochondria from BAT and white adipocyte (AF WAT) in presence of Succinate/Glycerol-3-phosphate/rotenone (complex II) and ADP from Lox and F-TFKO CD fed male mice at 10-12 weeks of age (n=9/genotype). (G) Respiratory Control Ratio (RCR) from isolated BAT and white adipocyte (AF WAT) from Lox and F-TFKO CD fed male mice at 10-12 weeks of age in presence of complex II substrate (n=9/genotype). (H) Analysis of membrane potential of isolated mitochondria from BAT and isolated white adipocytes of control (Lox) and F-TFKO male mice based on the accumulation of the TMRE mitochondrial dye by FACS. The data are representative of the basal state and oligomycin treatment and are shown as the medians ± SEM of n=6/genotype. (I) Oxygen consumption rate (OCR) of FACS sorted BAT and white adipocyte mitochondria were measured using a Seahorse Flux Analyzer in presence of succinate/rotenone and ADP. The data are shown from Lox and F-TFKO CD fed male mice at 10-12 weeks of age (n=6/genotype). Values are given as mean ± SEM, * p<0.05 and ** p<0.01.

Figure 6. Adipose tissue metabolic status upon TFAM KO

Lipids peroxidation was measured by TBARS assay (A) and oxidative DNA damage was measured by 8 Hydroxy-2-deoxy guanosine (8-OHdG) (B) from F-TFKO, Lox BAT and WAT of HFD fed mice. The data are represented as fold induction compared to their respective control adipose tissue (n=6/genotype). Set of Acyl carnitine levels specifically upregulated in F-TFKO BAT (C) and WAT (D) were assessed by metabolomic analysis in both fed and 24 h fasting state. Data are shown relative to Lox control fed state (n=5/genotype). Acyl carnitine levels in control and F-TFKO BAT (E) and WAT (F) in fed state were analyzed by metabolomic analysis in fed and 24 h fasting state. Data are shown relative to Lox control fed state (n=5/genotype), *p<0.05 Asterisks indicate a significant difference in all panels (*p<0.05)

Figure 7. Schematic of changes in mitochondria of F-TFKO adipose tissue