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. 2024 Apr;67(4):724-737.
doi: 10.1007/s00125-023-06073-5. Epub 2024 Jan 13.

Skeletal muscle TET3 promotes insulin resistance through destabilisation of PGC-1α

Beibei Liu #  1   2 , Di Xie #  1   3 , Xinmei Huang #  1   4 , Sungho Jin  5 , Yangyang Dai  1   6 , Xiaoli Sun  1   7 , Da Li  1   2 , Anton M Bennett  8   9 , Sabrina Diano  5 , Yingqun Huang  10   11
Affiliations

Skeletal muscle TET3 promotes insulin resistance through destabilisation of PGC-1α

Beibei Liu et al. Diabetologia. 2024 Apr.

Abstract

Aim/hypothesis: The peroxisome proliferator-activated receptor-γ coactivator α (PGC-1α) plays a critical role in the maintenance of glucose, lipid and energy homeostasis by orchestrating metabolic programs in multiple tissues in response to environmental cues. In skeletal muscles, PGC-1α dysregulation has been associated with insulin resistance and type 2 diabetes but the underlying mechanisms have remained elusive. This research aims to understand the role of TET3, a member of the ten-eleven translocation (TET) family dioxygenases, in PGC-1α dysregulation in skeletal muscles in obesity and diabetes.

Methods: TET expression levels in skeletal muscles were analysed in humans with or without type 2 diabetes, as well as in mouse models of high-fat diet (HFD)-induced or genetically induced (ob/ob) obesity/diabetes. Muscle-specific Tet3 knockout (mKD) mice were generated to study TET3's role in muscle insulin sensitivity. Genome-wide expression profiling (RNA-seq) of muscle tissues from wild-type (WT) and mKD mice was performed to mine deeper insights into TET3-mediated regulation of muscle insulin sensitivity. The correlation between PGC-1α and TET3 expression levels was investigated using muscle tissues and in vitro-derived myotubes. PGC-1α phosphorylation and degradation were analysed using in vitro assays.

Results: TET3 expression was elevated in skeletal muscles of humans with type 2 diabetes and in HFD-fed and ob/ob mice compared with healthy controls. mKD mice exhibited enhanced glucose tolerance, insulin sensitivity and resilience to HFD-induced insulin resistance. Pathway analysis of RNA-seq identified 'Mitochondrial Function' and 'PPARα Pathway' to be among the top biological processes regulated by TET3. We observed higher PGC-1α levels (~25%) in muscles of mKD mice vs WT mice, and lower PGC-1α protein levels (~25-60%) in HFD-fed or ob/ob mice compared with their control counterparts. In human and murine myotubes, increased PGC-1α levels following TET3 knockdown contributed to improved mitochondrial respiration and insulin sensitivity. TET3 formed a complex with PGC-1α and interfered with its phosphorylation, leading to its destabilisation.

Conclusions/interpretation: Our results demonstrate an essential role for TET3 in the regulation of skeletal muscle insulin sensitivity and suggest that TET3 may be used as a potential therapeutic target for the metabolic syndrome.

Data availability: Sequences are available from the Gene Expression Omnibus ( https://www.ncbi.nlm.nih.gov/geo/ ) with accession number of GSE224042.

Keywords: Diabetes; Insulin resistance; Mitochondria; Obesity; PGC-1α; Skeletal muscle; TET3.

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Figures

Fig. 1
Fig. 1
TET3 expression is increased in muscles of humans with diabetes and muscles of HFD-fed mice. (a) Relative TET1, TET2 and TET3 mRNA levels in muscle tissues from humans with diabetes (age 60.0±4.8 years, BMI 31.8±6.5 kg/m2) or without diabetes (age 59.6±5.0 years; BMI 27.4±5.4 kg/m2) (from the GSE22435 dataset). (b) Relative TET1, TET2 and TET3 mRNA levels in muscle tissues from humans with diabetes (age 51.5±3.6 years, BMI 30.8±2.5 kg/m2) or without diabetes (age 37.8±2.9 years, BMI 25.2±0.8 kg/m2) (from the GSE25462 dataset). (c) Relative TET1, TET2 and TET3 mRNA levels in myocytes from women with diabetes (age 46���63 years, BMI 24–33 kg/m2) or without diabetes (age 41–63 years, BMI 24–35 kg/m2) (from the GSE81965 dataset). (d) qPCR analysis and immunoblot of TET3 in GAS muscles from RC- and HFD-fed mice. The lane of each blot represents an individual mouse; molecular size in kDa is shown. (e) qPCR and immunoblot of TET3 in GAS muscles from control and ob/ob mice at the age of 10 weeks. All data are presented as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001 (two-tailed Student’s t test). Ctrl, control; PS, Ponceau S; T2D, type 2 diabetes
Fig. 2
Fig. 2
Muscle TET3 knockdown enhances insulin sensitivity. (a) qPCR of Tet3 mRNA in GAS from WT and mKD mice at the age of 12 weeks. n=5 mice for each genotype. (b) Immunoblot of TET3 protein in GAS from WT and mKD mice at the age of 12 weeks. Each lane represents an individual mouse, with TET3 protein quantification shown. (c) GTT following 14 h overnight fasting of WT and mKD mice at the age of 10 weeks. n=8 mice in each group. (d–g) Hyperinsulinaemic–euglycaemic clamp studies from WT and mKD mice at the age of 12 weeks. n=8 animals in each group. (h) Per cent body fat of WT (n=8) and mKD (n=10) mice after exposure to HFD for 10 weeks. (i, j) Results of GTT (i) and ITT (j) of WT (n=6) and mKD (n=8) mice after exposure to HFD for 10 weeks. All data are presented as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001 (a, b, f, g and h, two-tailed Student’s t test; c–e and j, two-way ANOVA with Sidak post-test). BAT, brown adipose tissue; EGP, endogenous glucose production; EWAT, epididymis white adipose tissue; GIR, glucose infusion rate; PS, Ponceau S
Fig. 3
Fig. 3
TET3 negatively regulates PGC-1α expression at the post-transcriptional level. (a) qPCR of Pgc1a mRNA in GAS tissues isolated from WT and mKD mice. (b) Immunoblot of PGC-1α in GAS tissues isolated from WT and mKD mice. (c) qPCR of Pgc1a in GAS tissues isolated from RC- and HFD-fed mice. (d) Immunoblot of PGC-1α in GAS tissues isolated from RC- and HFD-fed mice. (e) qPCR of Pgc1a in GAS tissues isolated from age-matched control and ob/ob mice. (f) Immunoblot of PGC-1α in GAS tissues isolated from age-matched control and ob/ob mice. Data are presented as mean ± SEM, n=5 mice in each group. **p<0.01, ***p<0.001 (two-tailed Student’s t test). Ctrl, control
Fig. 4
Fig. 4
PGC-1α is required for TET3-mediated regulation of insulin sensitivity. (a) Immunoblot of TET3 and PGC-1α from human primary myotubes transfected with NT siRNA, TET3 siRNA or TET3 siRNA plus PGC1A siRNA. (b) Mitochondrial respiration of human primary myotubes treated as in (a). n=8 in each group. (c) Glucose uptake of human myotubes transfected with NT siRNA, TET3 siRNA or TET3 siRNA plus PGC1A siRNA as in (a) in the absence (−) or presence (+) of insulin at 100 nmol/l. Results are presented as relative glucose uptake with values in the absence of insulin set as 1. n=3 in each group. (d) Immunoblot of TET3 and PGC-1α from mouse C2C12 myotubes transfected with NT siRNA, Tet3 siRNA or Tet3 siRNA plus Pgc1a siRNA. (e) Mitochondrial respiration of mouse C2C12 myotubes treated as in (d). (f) Glucose uptake of mouse C2C12 myotubes transfected with NT siRNA, Tet3 siRNA or Tet3 siRNA plus Pgc1a siRNA as in (d) in the absence (−) or presence (+) of insulin at 100 nmol/l. n=3 in each group. Data are representative of two independent transfection experiments. *p<0.05, **p<0.01, ***p<0.001 (b and e, two-way ANOVA with Sidak post-test; c and f, two-tailed Student’s t test). FCCP, carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone; OCR, oxygen consumption rate
Fig. 5
Fig. 5
TET3 interacts with and destabilises PGC-1α. (a, b) C2C12 myoblasts were transfected with NT siRNA or Tet3 siRNA. After 48 h, proteins were isolated and TET3 expression was measured by immunoblotting (a). To perform time course analysis, CHX was added at a final concentration of 50 μg/ml and proteins were harvested at the indicated time points, followed by immunoblotting for PGC-1α and GAPDH (b). (c) Quantification of (b); the dotted line indicates the trendline. (d) Schematic of PGC-1α protein domain organisation, showing the activation domain, repression domain, arginine–serine-rich domain (RS) and RNA recognition motif (RRM). Numbers represent amino acids. The blue vertical lines represent phosphorylation at Thr262, Ser265 and Thr298. Not drawn to scale. (e) Mouse GAS tissues were used for immunoprecipitation using preimmune IgG, anti-TET3 or anti-PGC-1α. Representative immunoblots are shown. (f) C2C12 myoblasts were transfected with NT siRNA or Tet3 siRNA as in (a). After 48 h, proteins were isolated and analysed by immunoblotting using antibodies specific for total PGC-1α and PGC-1α phosphorylated at S265 and T298, respectively. (g) Quantification of (f). Data are presented as mean ± SEM. *p<0.05, **p<0.01 (two-tailed Student’s t test). Data are representative of two independent transfection experiments. IB, immunoblotting; IP, immunoprecipitation
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