Adult-onset obesity is triggered by impaired mitochondrial gene expression

Abstract

Mitochondrial gene expression is essential for energy production; however, an understanding of how it can influence physiology and metabolism is lacking. Several proteins from the pentatricopeptide repeat (PPR) family are essential for the regulation of mitochondrial gene expression, but the functions of the remaining members of this family are poorly understood. We created knockout mice to investigate the role of the PPR domain 1 (PTCD1) protein and show that loss of PTCD1 is embryonic lethal, whereas haploinsufficient, heterozygous mice develop age-induced obesity. The molecular defects and metabolic consequences of mitochondrial protein haploinsufficiency in vivo have not been investigated previously. We show that PTCD1 haploinsufficiency results in increased RNA metabolism, in response to decreased protein synthesis and impaired RNA processing that affect the biogenesis of the respiratory chain, causing mild uncoupling and changes in mitochondrial morphology. We demonstrate that with age, these effects lead to adult-onset obesity that results in liver steatosis and cardiac hypertrophy in response to tissue-specific differential regulation of the mammalian target of rapamycin pathways. Our findings indicate that changes in mitochondrial gene expression have long-term consequences on energy metabolism, providing evidence that haploinsufficiency of PTCD1 can be a major predisposing factor for the development of metabolic syndrome.

Fig. 1. Haploinsufficiency of Ptcd1 affects mt-RNA metabolism.

(A) Photographic representation of size and weight difference between control (Ptcd1^+/+) and heterozygote (Ptcd1^+/−) mice at 10 and 30 weeks of age. (B) Weight (in grams) of control (Ptcd1^+/+, n = 6) and heterozygote (Ptcd1^+/−, n = 6) mice at 5, 10, 15, and 30 weeks of age. Error bars indicate SEM. *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t test. The abundance of unprocessed and mature mitochondrial mRNAs, tRNAs, and rRNAs in livers (C) and hearts (D) of 30-week Ptcd1^+/+ and Ptcd1^+/− mice were analyzed by northern blotting;18S rRNA was used as a loading control. The data are representative of results obtained from at least eight mice from each genotype and three independent biological experiments. (E) Genome browser view of the mean RNA-Seq coverage (log₂ fold change[KO_mean/Ctrl_mean]) in livers and hearts from three Ptcd1^+/+ and three Ptcd1^+/− 30-week-old mice (mean normalized count) showing the region of mt-tRNA^Trp and the downstream effect on the 3′ end processing when PTCD1 is reduced. (F) Genome browser view of the mean RNA-Seq coverage (log₂ fold change[KO_mean/Ctrl_mean]) in livers and hearts from three Ptcd1^+/+ and three Ptcd1^+/− 30-week-old mice (mean normalized count) showing the region of mt-tRNA^Leu(UUR) and the downstream effect on the 3′ end processing when PTCD1 is reduced. (G) mt-RNA junctions were measured in total liver and heart RNA from Ptcd1^+/+ and Ptcd1^+/− 30-week-old mice by qRT-PCR and normalized to 18S rRNA. Error bars indicate SEM. *P < 0.05, Student’s t test.

Fig. 2. PTCD1 reduction causes reduced mitochondrial protein synthesis.

Mitochondrial proteins (25 μg) from liver and heart mitochondria from 30-week-old Ptcd1^+/+ and three Ptcd1^+/− mice were resolved on 4 to 20% SDS-PAGE gels and immunoblotted against antibodies to investigate the steady-state levels of nuclear- and mitochondrial-encoded proteins. Succinate dehydrogenase complex subunit A (SDHA) was used as a loading control. PTCD1 levels are decreased by ~50% in liver (A) and heart (B) mitochondria, indicating its haploinsufficiency. Immunoblots of mitochondrial- and nuclear-encoded OXPHOS proteins in liver (C) and heart (D) mitochondria. Immunoblots of nuclear-encoded mtDNA- and RNA-binding proteins in liver (E) and heart (F) mitochondria. Immunoblots showing the levels of mitochondrial ribosomal proteins in liver (G) and heart (H) mitochondria. Relative abundance of proteins was measured using ImageJ software normalized to the loading control. Error bars indicate SEM. *P < 0.05, **P < 0.01, ^***P < 0.001, Student’s t test. The data are representative of results obtained from at least six mice from each genotype and three independent biological experiments. De novo protein synthesis in liver (I) and heart (J) from Ptcd1^+/+ and three Ptcd1^+/− mice was measured by pulse incorporation of ³⁵S-labeled methionine and cysteine. Equal amounts of mitochondrial protein (50 μg) were separated by SDS-PAGE and visualized by autoradiography. Representative gels from three independent biological experiments are shown. All studies in this figure were performed in 30-week-old mice.

Fig. 3. Reduction of PTCD1 affects the biogenesis of the respiratory chain, causing mild uncoupling and changes in mitochondrial morphology.

Phosphorylating (state 3) and uncoupled respiration in the presence of up to 3 μM FCCP was measured in liver (A) and heart (B) mitochondria from four 30-week-old Ptcd1^+/+ and four Ptcd1^+/− mice using an OROBOROS oxygen electrode using either pyruvate, glutamate, and malate or succinate as substrates in the presence of inhibitors. Isolated liver (C) and heart (D) mitochondria (75 μg) from 30-week-old mice were treated with 1% n-dodecyl-β-d-maltoside and resolved on 4 to 16% BN-PAGE gel. Immunoblotting with the blue native OXPHOS cocktail antibody was used to visualize respiratory complexes. Error bars indicate SEM. *P < 0.05, **P < 0.01, Student’s t test. In-gel activity stains were used for complex V in liver (E) and heart (F) mitochondria. Steady-state levels of OPA1 and OMA-1 in liver (G) and heart (H) mitochondria from at least eight Ptcd1^+/+ and eight Ptcd1^+/− mice were measured by immunoblotting using SDHA as a loading control. Error bars indicate SEM. **P < 0.01, ***P < 0.001, Student’s t test. Mitochondrial morphology and cristae structure in Ptcd1^+/+ and four Ptcd1^+/− livers (I) and hearts (J) were determined using TEM. The data are representative of results obtained from at least three mice from each genotype. Scale bars, 0.25 μm (bottom panels) and 1 μm (top panels). The abundance of MICOS complex proteins was measured in liver (K) and heart (L) mitochondria from at least eight Ptcd1^+/+ and eight Ptcd1^+/− mice using immunoblotting. SDHA was used as a loading control. Error bars indicate SEM. ***P < 0.001, Student’s t test.

Fig. 4. Ptcd1^+/− mice develop liver steatosis and cardiac hypertrophy.

(A) Liver sections cut to 5- or 10-μm thickness were stained with H&E or Oil red O and hematoxylin, respectively, from 30-week-old Ptcd1^+/+ (n = 6) and Ptcd1^+/− (n = 6) mice. (B) Quantitative measurement of Oil red O staining using ImageJ. Values are means ± SEM. *P < 0.05, Student’s t test. (C) ECG parameters for Ptcd1^+/+ (n = 5) and Ptcd1^+/− (n = 5) 40-week-old mice. LVEDD, left ventricular end diastolic diameter; LVESD, left ventricular end systolic diameter; FS, fractional shortening; LVDPW, left ventricular posterior wall in diastole; LVSPW, left ventricular posterior wall in systole; IVDS, intraventricular septum in diastole; IVSS, intraventricular septum in systole; HR, heart rate. Values are means ± SEM. *P < 0.05 compared with Ptcd1^+/+, **P < 0.01, Student’s t test. (D) Heart sections cut to 5-μm thickness were stained with H&E from aged Ptcd1^+/+ (n = 6) and Ptcd1^+/− (n = 6) mice. Scale bars, 100 μm. Enlarged hearts were determined as a measure of heart weight relative to tibia length; *P < 0.05 compared with Ptcd1^+/+, Student’s t test. Photographic representation of size difference between Ptcd1^+/+ and Ptcd1^+/− hearts in adult mice.

Fig. 5. Reduction of PTCD1 causes adult-onset obesity and insulin resistance.

(A) Percentage increase of weight gain from 5 to 30 weeks of age between Ptcd1^+/+ (n = 12) and Ptcd1^+/− (n = 12) mice. *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t test. (B) Weight of intra-abdominal epididymal fat pads in grams for young and adult Ptcd1^+/+ (n = 4) and Ptcd1^+/− (n = 4) mice. (C) Glucose tolerance in 10- and 15-week-old Ptcd1^+/+ (n = 12) and Ptcd1^+/− (n = 12) mice (young mice). (D) Glucose tolerance in 30-week-old Ptcd1^+/+ (n = 12) and Ptcd1^+/− (n = 12) mice (aged mice). (E) Insulin sensitivity in 11- and 16-week-old Ptcd1^+/+ (n = 12) and Ptcd1^+/− (n = 12) mice (young mice). Quantitative values are the area under the curve (AUC) ± SEM. *P < 0.05, Student’s t test. (F) Insulin sensitivity in 30-week-old Ptcd1^+/+ (n = 12) and Ptcd1^+/− (n = 12) mice. Quantitative values are the AUC ± SEM. **P < 0.01, Student’s t test.

Fig. 6. Metabolic hormones, growth factors, and proinflammatory cytokines increase with age in the Ptcd1^+/− mice.

(A) Insulin, triglycerides, cholesterol, IL-6, leptin, and FGF-21 levels were measured in serum obtained from 10-week-old Ptcd1^+/+ (n = 6) and Ptcd1^+/− (n = 6) mice. (B) Insulin, triglycerides, cholesterol, IL-6, leptin, and FGF-21 levels were measured in serum obtained from 30-week-old Ptcd1^+/+ (n = 6) and Ptcd1^+/− (n = 6) mice. Endogenous levels of the SAPK/JNK and its phosphorylated form (Thr¹⁸³/Tyr¹⁸⁵) were determined by immunoblotting of whole liver and heart lysates from 10-week-old (C) or 30-week-old (D) Ptcd1^+/+ and Ptcd1^+/− mice. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as a loading control. Error bars indicate SEM. *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t test.

Fig. 7. The mTOR signaling pathway is differentially regulated in response to mitochondrial dysfunction in Ptcd1^+/− mice.

The mTOR pathway was assessed by immunoblotting using specific antibodies upstream and downstream of mTOR in liver (A) and heart (C) lysates from Ptcd1^+/+ and Ptcd1^+/− 30-week-old mice using GAPDH as a loading control. Immunoblotting was used to measure the abundance of the phosphorylated (Thr¹⁷²) and nonphosphorylated form of AMPKα in liver (B) and heart (D) lysates from 30-week-old Ptcd1^+/+ and Ptcd1^+/− mice using GAPDH as a loading control. Relative abundance of proteins was measured using ImageJ software normalized to the loading control. Error bars indicate SEM. *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t test. The data are representative of results obtained from at least six mice from each genotype and three independent biological experiments.