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, 10 (1), 4720

The Tumor Suppressor TMEM127 Regulates Insulin Sensitivity in a Tissue-Specific Manner

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The Tumor Suppressor TMEM127 Regulates Insulin Sensitivity in a Tissue-Specific Manner

Subramanya Srikantan et al. Nat Commun.

Abstract

Understanding the molecular components of insulin signaling is relevant to effectively manage insulin resistance. We investigated the phenotype of the TMEM127 tumor suppressor gene deficiency in vivo. Whole-body Tmem127 knockout mice have decreased adiposity and maintain insulin sensitivity, low hepatic fat deposition and peripheral glucose clearance after a high-fat diet. Liver-specific and adipose-specific Tmem127 deletion partially overlap global Tmem127 loss: liver Tmem127 promotes hepatic gluconeogenesis and inhibits peripheral glucose uptake, while adipose Tmem127 downregulates adipogenesis and hepatic glucose production. mTORC2 is activated in TMEM127-deficient hepatocytes suggesting that it interacts with TMEM127 to control insulin sensitivity. Murine hepatic Tmem127 expression is increased in insulin-resistant states and is reversed by diet or the insulin sensitizer pioglitazone. Importantly, human liver TMEM127 expression correlates with steatohepatitis and insulin resistance. Our results suggest that besides tumor suppression activities, TMEM127 is a nutrient-sensing component of glucose/lipid homeostasis and may be a target in insulin resistance.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Global deletion of Tmem127 impairs growth and results in low adiposity. a Tmem127 recombinant mouse strategy; b body weight of male wild-type (WT) and Tmem127 knockout (KO) mice (n = 5–10 per genotype) from 6 to 22 months of age; c fat mass/lean mass ratio of male WT and KO mice across age (n = 5–10 per genotype); d core body temperature of adult WT and KO mice in the fed state and after 24 h fasting (n = 16–17 per genotype); e absolute weight of liver, inguinal white adipose tissue (iWAT), epididymal WAT (eWAT), or relative weight of these organs to total body weight in adult WT and KO mice (n = 4–6 per genotype); data were analyzed by Student’s t test. Values are expressed as mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001. Adult mice were 9–12 months of age. See also Supplementary Fig. 1A–K and Supplementary Tables 1 and 2. Source data are provided as a Source Data file
Fig. 2
Fig. 2
Global Tmem127 deficiency disrupts hepatic lipogenesis. a Relative iWAT mRNA expression (log) of the indicated fatty-acid synthesis, storage, oxidation, and transport gene expression in adult WT and KO mice (n = 4–5 per genotype) measured by real-time PCR (RT-PCR) and normalized to TfIIb gene; b western blots of fatty acid synthetase (FASN) and loading control (total AKT) in whole-tissue lysates prepared from iWAT from adult male WT or KO mice (n = 6 per genotype); c hepatic triglyceride content in fed adult WT and KO mice (n = 10–12 per genotype); d relative liver mRNA expression levels (log) of the indicated fatty-acid synthesis, storage, oxidation, and transport gene expression from adult WT and KO mice (n = 4–9 per genotype) measured by RT-PCR. e Western blots of Acetyl-CoA carboxylase (ACC), FASN and loading control (total AKT) in whole-tissue lysates prepared from liver from WT or KO adult male mice (n = 6 per genotype); data were analyzed by Student’s t test. Values are expressed as mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001. Adult mice were 9–12 months of age. See also Supplementary Fig. 2A–C. Source data are provided as a Source Data file
Fig. 3
Fig. 3
Loss of Tmem127 leads to hypoinsulinemia and improved insulin tolerance. a Fasting (6 h) blood glucose of adult WT and Tmem127 KO male mice (n = 8 per genotype); b fasting (6 h) plasma insulin of adult WT and KO male mice (n = 5–6 per genotype); c glucose tolerance test (GTT) and area under the curve (AUC, inset graph) of adult male WT and KO mice (n = 8 per genotype,) after 6-h fasting; d glucose-stimulated insulin secretion (GSIS) of adult male WT and KO mice (n = 4–7 per genotype); e insulin tolerance test (ITT) and AUC (inset) of adult male WT (n = 10) and KO mice (n = 13) after 6-h fasting; f ITT AUC values of young (3–6 months, WT n = 15; KO n = 22) and adult (9–12 months, WT n = 10; KO n = 13 per genotype) male WT and KO mice; g real-time PCR (RT-PCR) hepatic Tmem127 mRNA expression from WT young (3–6 months; n = 7) and adult (9–12 months; n = 8) male mice. Data were analyzed by Student’s t test or two-way ANOVA (in the case of GSIS). Values are expressed as mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001. Adult mice were 9–12-month-old and young mice were 2–6 months old. See also Supplementary Fig. 3A–E. Source data are provided as a Source Data file
Fig. 4
Fig. 4
Tmem127 deletion decreases hepatic gluconeogenesis and increases insulin signaling. a Relative hepatic mRNA expression of the indicated glucose transporter, glycolysis and gluconeogenesis genes from adult WT (n = 8) and KO (n = 9) mice; b pyruvate tolerance test (PTT) and AUC (inset) of adult male WT (n = 8) and KO (n = 9) mice, after 16-h fasting; c ITT and AUC (inset) of adult male WT (n = 6) and KO (n = 11) mice after 16-h fasting; d RT-PCR hepatic G6PC and Pck1 mRNA expression levels from 24 hour fasted (WT n = 3; KO n = 6) vs. fed (WT n = 5; KO n = 9) mice; e western blots of phosphorylated and total Akt proteins in liver whole-cell lysates from adult male, chow-fed WT, or KO mice, graph displays quantification of Akt phosphorylation at serine 473 or threonine 308 normalized by total Akt (n = 6 per genotype); f Rictor pulldown by mTOR immunoprecipitation (IP) in liver from adult male, chow-fed WT, or Tmem127 KO mice, corresponding whole-cell lysates (left panel), graph displays quantification of Rictor/mTOR IP complexes from two independent experiments and four mice per genotype (quantification performed by ImageJ and normalized to mTOR pulldown); g western blot of phosphorylated Ser473 or Thr308 Akt or total Akt from liver (left) or muscle (right) whole cell lysates of adult (> 12 mo) male mice obtained after 6 h of fasting followed by injection of insulin (0.75 units/kg) or vehicle 15 min before harvesting (n= 3 mice per genotype per treatment condition, representative gel shown). Data were analyzed by Student’s t test. Values are expressed as mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001. Adult mice were 9–12 months of age. See also Supplementary Fig. 4A, B. Source data are provided as a Source Data file
Fig. 5
Fig. 5
Tmem127 loss protects against high-fat diet (HFD)-induced hepatic steatosis and insulin resistance. a Weekly body weight of WT and Tmem127 KO male mice on a 60% kcal fat diet for 16 weeks (n = 7–8 per genotype, starting age 5–7 months); b GTT and AUC (inset graph) of HFD-fed adult male WT (n = 8) and KO (n = 10) mice; c fasting (6 h) serum insulin of HFD-fed WT and KO male mice (n = 10 per genotype, 9–11 mo-old); d ITT and AUC (inset) of HFD-fed male WT and KO mice (n = 6 per genotype, 9–11 months); e absolute weight of liver, inguinal white adipose tissue (iWAT), epididymal WAT (eWAT), and respective relative weight of these organs to total body weight in HFD-fed WT (n = 4) and KO (n = 6) mice (9–11 months old); f representative hematoxylin and eosin (HE) sections of liver from HFD-fed WT and KO mice, scale bar is 500 µm; g hepatic triglyceride content in HFD-fed adult WT (n = 6) and KO (n = 7) mice (9–11 mo-old); h relative liver mRNA expression of the indicated fatty-acid synthesis, storage, oxidation, and transport gene expression from HFD-fed male WT and KO mice (n = 6 per genotype, 9–11 mo-old) measured by RT-PCR; i Relative liver mRNA expression of the indicated glucose transporter, glycolysis and gluconeogenesis genes from HFD-fed male WT (n = 5) and KO (n = 6) mice (9–11 mo-old); j relative iWAT mRNA expression of the indicated glucose transporter and glycolysis genes from HFD-fed male WT (n = 4) and KO (n = 7) mice (9–11 mo-old); k relative liver mRNA expression of the indicated lipogenic transcription factor genes from chow or HFD-fed male WT (n = 6 per diet) and KO (n = 9 per diet) mice (9–11 mo-old); Data were analyzed by Student’s t test or analysis of variance (ANOVA). Values are expressed as mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; a = p = 0.06. For 5 K: #comparisons between diet; * comparisons between genotype # or *P < 0.05; ## or **P < 0.01; ###or ***P < 0.001. See also Supplementary Fig. 5A–G. Source data are provided as a Source Data file
Fig. 6
Fig. 6
Tmem127 deletion in liver or adipose tissue leads to opposing glucose metabolism outcomes. a Strategy of generation of liver-specific (LKO) and adipose tissue-specific (AKO) Tmem127 deletion: Tmem127 Flx/Flx mice were crossed with Alb-Cre or Adiponectin-Cre mice, respectively; b western blot of liver or inguinal white adipose tissue (iWAT) lysates of Flx, LKO and AKO mice, alongside controls WT and KO probed with Tmem127 or a loading control antibody; c body weight of male Flx (n = 16), liver-specific Tmem127 KO (LKO, n = 8) and adipose-specific Tmem127 KO (AKO, n = 8) mice (from 2–6 months of age); d body composition of male Flx, LKO and AKO at 3–6 months of age (young; n = 11 LKO, 7 AKO, 7 flx) or 9–12 months old (adult; n = 14 Flx, 5 LKO; 7 AKO). Data are shown as lean/fat mass ratio. e Fasting blood glucose of adult WT and Tmem127 KO male mice (n = 14 LKO, 7 AKO, 16 flx); f fasting serum insulin of adult WT and KO male mice (n = 13 LKO, 7 AKO, 14 flx); g ITT AUC of young (3-6mo) and adult (9–12 mo) male Flx, LKO and AKO mice (number of mice per group is shown in each column); h GTT AUC of young (3–6 mo) and adult (9–12 mo) male Flx, LKO and AKO mice (number of mice per group is shown in each column). Data were analyzed by Student’s t test; *p < 0.05; Values are expressed as mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001. See also Supplementary Fig. 6A–C. Source data are provided as a Source Data file
Fig. 7
Fig. 7
Liver-specific Tmem127 deletion increases hepatic and peripheral insulin sensitivity. a Relative liver, muscle, and iWAT mRNA expression of the indicated glucose transporter and glycolysis genes from Flx (n = 5) and LKO (n = 11) adult mice (9–12 mo old); b western blot of liver and inguinal white adipose tissue lysates from chow-fed LKO and Flx adult male mice, probed with phosphorylated Ser473 and Thr308 Akt and total Akt; c relative liver mRNA expression of the indicated fatty-acid synthesis genes from LKO and AKO adult male mice, represented as ratio over Flx (n = 12 Flx, 11 LKO and 7 AKO, 9–12 mo); d relative liver mRNA expression of the indicated lipogenic transcription factor genes from LKO and AKO adult male mice (n = 11 Flx, 12 LKO and 7 AKO, 9–12 mo). e Real-time (RT) PCR of liver G6pase gene expression in control or AKO 9–12 mo old male mice starved of food for 6 h and re-fed for 3 h (fed) or starved for 24 h (fasted), mice or feed condition (WT n = 4, KO n = 7); f RT-PCR of liver of the indicated fatty acid synthesis genes from chow-fed, adult male Flx (n = 11), and AKO (n = 5) mice; g RT-PCR of liver of the indicated transcription factor genes from adult male Flx (n = 11) and AKO (n = 5) mice; h RT-PCR of inguinal fat (iWAT) Glut4 gene expression from chow-fed, adult male Flx (n = 11) and AKO (n = 5) mice; i western blot of inguinal white adipose tissue lysates from chow-fed LKO and Flx adult male mice (n = 3 per genotype), probed with total ACC and a loading control; j respiratory quotient (RQ) of Flx (n = 5) or AKO (n = 7) chow-fed, adult male mice, data collected for 48 h and sorted by day or night cycle. Data were analyzed by Student’s t test. Values are expressed as mean ± s.e.m. *P < 0.05; **P < 0.01. See also Supplementary Fig. 7A–C. Source data are provided as a Source Data file
Fig. 8
Fig. 8
Effects of TMEM127 deletion in hepatic gluconeogenesis and lipogenesis are cell autonomous. a Western blot of lysates from control (C) or CRISPR-Cas9 mediated- TMEM127 knockout KO HepG2 human hepatic cells treated with or without 100 nm insulin for 10 min, probed with Akt, TMEM127 or a loading control antibody; b real-time (RT) PCR of G6PC (the G6Pase gene) expression in control or TMEM127 KO HepG2 cells deprived of glucose and stimulated with pyruvate 2 mm (three biological repeats); c RT-PCR mRNA expression of the indicated fatty-acid synthesis, transcription factors, and glucose transporter genes from HepG2 control or TMEM127 KO cells (three biological repeats); d RT-PCR of fatty-acid synthesis or transcription factors from primary hepatocytes from Tmem127 WT or KO (Tm-CMV-KO); 12-week old-female mice, n = 3 per genotype). e Western blots of indicated phosphorylated and total proteins from whole-cell lysates of HepG2 TMEM127 KO or control cells with or without Rictor knockdown (Rict) and treated with insulin as indicated. Shown is one of four replicate experiments. Data were analyzed by Student’s t test. Values are expressed as mean ± s.e.m. *P < 0.05; **P < 0.01. See also Supplementary Fig. 8. Source data are provided as a Source Data file
Fig. 9
Fig. 9
Hepatic TMEM127 expression correlates with states of insulin resistance in humans and mice. a Relative liver Tmem127 mRNA expression from adult WT male mice under chow (n = 6), high-fat diet (HFD, n = 6) for 16 weeks or after HFD followed by 10 weeks of chow (HFD > chow, n = 3 per genotype) measured by RT-PCR; b relative liver Tmem127 mRNA expression from adult male Db/db or age-matched heterozygous db mice (n = 6 per genotype); c relative liver Tmem127 mRNA expression of control (n = 8), ob/ob (n=3) or ob/ob (n=3) adult mice treated for 4 weeks with the insulin sensitizer pioglitazone. d Log2-transformed, normalized TMEM127 gene RNAseq read counts in liver biopsies of patients with the indicated status: CTLs = control individuals; NAFLD = nonalcoholic fatty liver disease; NASH = nonalcoholic steatohepatitis; the number of samples per group is indicated below the graph. The statistical test was the negative binomial generalized linear model implemented in DESeq with a likelihood ratio test to compare TMEM127 expression between the groups. The thick horizontal line represents median values, box represents 25th and 75th percentiles, whiskers represent minimum and maximum values, outlier samples are shown as dots; e correlation between TMEM127 mRNA levels shown in d and insulin levels (n = 52). Symbols represent individual patients control samples are shown in green, NAFLD are orange and NASH are red; f correlation between TMEM127 mRNA shown in d and the homeostatic model assessment insulin resistance (HOMA-IR) index. Symbols represent individual patients control samples are shown in green, NAFLD are orange and NASH are red; Spearman’s correlation test was used to test associations between TMEM127 and Insulin levels (shown in e) or HOMA-IR (shown in f). *P < 0.05; **P < 0.01; ***P < 0.001; g working model of Tmem127 function. The upper panel displays a summary of the effects of global loss of Tmem127 on insulin sensitivity and fat deposition under regular diet in young vs. adult mice or after high-fat diet (HFD). Adult knockout (KO) mice show higher hepatic and peripheral insulin sensitivity as well as diminished hepatic fat deposition even after a HFD challenge. The lower panel summarizes our findings in liver- or adipose-specific Tmem127 deficiency. Our data suggest that liver Tmem127 promotes hepatic glucose production and peripheral glucose uptake both by cell autonomous and non-cell autonomous pathways; fat Tmem127 inhibits adipogenesis potential and modulates hepatic gluconeogenesis. Furthermore, it is likely that Tmem127 in other tissues contribute to the metabolic phenotype. See also Supplementary Fig. 9A, B and Supplementary Tables 3 and 4. Source data are provided as a Source Data file

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