TNIK is a conserved regulator of glucose and lipid metabolism in obesity

Abstract

Obesity and type 2 diabetes (T2D) are growing health challenges with unmet treatment needs. Traf2- and NCK-interacting protein kinase (TNIK) is a recently identified obesity- and T2D-associated gene with unknown functions. We show that TNIK governs lipid and glucose homeostasis in Drosophila and mice. Loss of the Drosophila ortholog of TNIK, misshapen, altered the metabolite profiles and impaired de novo lipogenesis in high sugar-fed larvae. Tnik knockout mice exhibited hyperlocomotor activity and were protected against diet-induced fat expansion, insulin resistance, and hepatic steatosis. The improved lipid profile of Tnik knockout mice was accompanied by enhanced skeletal muscle and adipose tissue insulin-stimulated glucose uptake and glucose and lipid handling. Using the T2D Knowledge Portal and the UK Biobank, we observed associations of TNIK variants with blood glucose, HbA1c, body mass index, body fat percentage, and feeding behavior. These results define an untapped paradigm of TNIK-controlled glucose and lipid metabolism.

Fig. 1.. Drosophila misshapen regulates sugar induced metabolism.

(A) Pupation kinetics in control (Tub-GAL4>) and RNA interference (RNAi)–mediated whole-body misshapen (msn) knockdown fed high-protein diet (HPD) or high-sugar diet (HSD) . (B) Total pupation and eclosion of msn RNAi and control animals on HPD and HSD. n = 6 vials (each with 30 larvae) per diet and genotype. dAEL, days after egg laying. (C) Circulating glucose levels in hemolymph of msn RNAi and control prewandering third-instar larvae raised on HPD or HPD with added sugar. n = 12 (each with 10 larvae) per diet and genotype. (D) Experimental outline of sugar induction. First-instar larvae were grown on HPD for 48 hours followed by 16 hours of HPD or HSD feeding. The experimental diets were supplemented with blue food dye to monitor feeding and selection of third-instar fed-state animals for metabolomics and RNA extraction. (E) Principal components analysis (PCA) of the 16 samples analyzed (four samples per diet and genotype). (F) Top 15 up- and down-regulated metabolites (P < 0.05) by sugar in control (top plot) and msn RNAi (bottom plot) animals. For msn RNAi, there were only three significantly up-regulated metabolites. (G) Four-way plot presenting the msn-dependent HSD-regulated metabolites [P<0.05; logFC (fold change) > 0/logFC <0]. (H) Metabolites that were up-regulated by sugar in an msn-dependent manner (P < 0.05). (I) Whole-body expression of msn, FAS, and ACC in control and msn RNAi larvae. n = 4 (five second-instar larvae per sample) per diet and genotype. Statistical significances were calculated using the two-way analysis of variance (ANOVA) in conjunction with Tukey’s multiple comparisons test (B and C) or by one-way ANOVA in conjunction with Dunnett’s multiple comparisons test (I). Data are presented as means ± SD (A to C and I). Metabolomics data (E to H) were analyzed in MetaboAnalyst (see Materials and Methods for further information).

Fig. 2.. Loss of TNIK protects against diet-induced obesity in mice.

(A) TNIK protein content in mouse tissues (n = 4, aged 12 weeks). (B) Experiments at indicated weeks of age before or during 10 weeks of CHOW/high-fat high-sucrose (HFHS) ad libitum diet. CHOW wildtype (WT) littermate female/male (f/m), n = 8/6; HFHS WT f/m, n = 8/7; CHOW Tnik knockout (KO) f/m, n = 8/8; HFHS KO f/m, n = 9/7. (C) Average caloric intake and (D) body weight gain of female mice during 10 week of CHOW/HFHS. Individual P values in order of comparisons: genotype effect within CHOW (blue), *P = 0.0273, *P = 0.0147, *P = 0.0117, (*)P = 0.0587, and (*)P = 0.0989; genotype effect within HFHS (pink), **P = 0.0016, **P = 0.0015, ***P = 0.0009, (*)P = 0.0515, **P = 0.0091,*P = 0.0116, and *P = 0.0325; and diet effect within WT (pink), (#)P = 0.0734, #P = 0.0224, and (#)P = 0.0743. (E) Absolute fat and (F) lean mass of female mice at 4 week of CHOW/HFHS. (G) Representative image of HFHS-fed female mice. (H) Average caloric intake and (I) body weight gain of male mice on 10 weeks of CHOW/HFHS. Individual P values in order of comparisons: genotype effect within CHOW (blue), **P = 0.0047, **P = 0.0020, **P = 0.0011, *P = 0.0114, and *P = 0.0403 and genotype effect within HFHS (pink), **P = 0.0031, *P = 0.0122, **P = 0.0002, *P = 0.0114, *P = 0.0102, *P = 0.0167, and (*)P = 0.0507. (J) Absolute fat and (K) lean mass of male mice at 4 weeks of CHOW/HFHS. (L) Representative image of HFHS-fed male mice. (M) Oxygen consumption (VO₂) and (O) ambulant activity of CHOW-fed male mice (WT/KO, n = 9/12) over 48 hours. (N) Average VO₂ and (P) ambulant activity during the light/dark cycle. Mean values of (Q) energy balance, (R) energy intake, and (S) energy expenditure of CHOW-fed male mice (WT/KO, n = 8/6) over a 2-day period. (T) Bone volume fraction of female and male mice. Data are shown as means ± SEM including individual values where applicable. Statistical significance calculated by two-way ANOVA with Šídák’s multiple comparisons test (C, E, F, H, J, K, and T), two-way ANOVA RM with Šídák’s multiple comparisons test (N and P), mixed-effect analysis with Tukey’s multiple comparisons test (D and I), or by unpaired t test (Q to S). Geno, main genotype effect; diet, main diet effect; X, interaction between genotype and diet.

Fig. 3.. Loss of TNIK protects against diet-induced metabolic dysfunction in mice.

(A) Glucose tolerance of female and male mice at 8 weeks of CHOW/high-fat high sucrose (HFHS). CHOW wildtype (WT) f/m, n = 4/4; HFHS WT f/m, n = 3/3; CHOW Tnik knockout (KO) f/m, n = 6/8; HFHS KO f/m, n = 7/4. Individual P values in order of comparisons: genotype effect within HFHS diet, *P = 0.0225; diet effect within WT genotype, #P = 0.0444. (B) Insulin response before (0 min) and following (20 min) the oral glucose challenge. (C) iAUC of glycemic excursion in response to bolus of glucose [2 g kg⁻¹ body weight (BW)]. (D) Insulin tolerance of female and male mice at 9 weeks of CHOW/HFHS. CHOW WT f/m, n = 7/6; HFHS WT f/m, n = 6/5; CHOW KO f/m, n = 8/5; HFHS KO f/m, n = 8/4. Individual P values in order of comparisons: genotype effect within HFHS diet, *P = 0.0398, *P = 0.0117, **P = 0.0068, and **P = 0.0056 and diet effect within KO genotype, #P = 0.0304 (E) Incremental area over the curve (iAOC) of glycemic excursion in response to a bolus of insulin at 0.3 IU kg⁻¹ BW. (F) Pyruvate tolerance of female and male mice at 7 weeks of CHOW/HFHS. CHOW WT f/m, n = 2/2; HFHS WT f/m, n = 2/3; CHOW KO f/m, n = 2/4; HFHS KO f/m, n = 3/4. Individual P values in order of comparisons: genotype effect within HFHS diet, (*)P = 0.0811, *P = 0.0117, ***P = 0.0009, **P = 0.0098, and *P = 0.0131 and diet effect within KO genotype, (#)P = 0.0965, #P = 0.0284, and (#)P = 0.0547. Data are shown as means + SEM, including individual values where applicable. Statistical significances were calculated using two-way ANOVA RM in conjunction with Tukey’s multiple comparisons test (A) or Šídák’s multiple comparisons test (C, E, and G) or by mixed-effect analysis in conjunction with Tukey’s multiple comparisons test (B, D, and F). Time, main effect of time.

Fig. 4.. TNIK deficiency elevates insulin-stimulated glucose uptake into skeletal muscles by increasing canonical Akt signaling, glucose, and lipid handling.

(A) Glycemic response of female and male chow- or high-fat high-sucrose (HFHS)-fed wildtype (WT) or Tnik knockout (KO) mice at 10 weeks of the diet intervention in response to retro-orbital injection of insulin at 0.3 IU kg⁻¹ body weight (BW). Individual significant P values are provided as the same order of comparisons (left to right): effect of genotype within HFHS diet, ***P = 0.0005, **P = 0.0012, *P = 0.0169, *P = 0.0121, and *P = 0.0289. (B) The effect of Tnik KO and chow or HFHS diet on insulin-stimulated 2DG clearance in gastrocnemius, TA, and soleus. (C) Schematic illustration of pathways analyzed via immunoblotting in gastrocnemius. (D) Immunoblot analyses in gastrocnemius of female and male chow- or HFHS-fed WT or Tnik KO mice during insulin stimulation: glucose-handling proteins (GLUT4, HKII, GS, and PDH E1α) and lipid-handling proteins (CD36). (E) Gastrocnemius muscle glycogen content in female and male Tnik KO or WT mice. Immunoblot analyses in gastrocnemius of female and male chow- or HFHS-fed WT or Tnik KO mice during insulin stimulation: (F) JNK signaling (JNK T183/Y185) and Akt signaling (Akt S473, Akt T308, Akt2, TBC1D4 T642, TBC1D4, FoxO1 S256, and GSK3β S9), (G) OxPhos proteins [NDUFB8 (CI), SDHB (CII), UQCRC2 (CIII), and ATP5A (CV)], and (H) representative blots. Data are shown as means + SEM, including individual values where applicable. Statistical significances were calculated using mixed-effect analysis in conjunction with Tukey’s multiple comparisons test (A) or the two-way ANOVA in conjunction with Tukey’s multiple comparisons test (B) or Šídák’s multiple comparisons test (C and E to G). A.U., arbitrary units.

Fig. 5.. TNIK deficiency elevates insulin-stimulated glucose uptake in WAT via enhanced insulin signaling and prevents diet-induced hepatic steatosis.

The effect of Tnik knockout (KO) and chow or high-fat high-sucrose (HFHS) diet on insulin-stimulated (A) 2DG clearance in gWAT; (B) content of proteins involved in Akt signaling (Akt S473, Akt T308, Akt2, FoxO1 S256, and GSK3β S9) and glucose handling (GLUT4 and PDH E1α); (C) representative blots; (D) mRNA levels of gWAT adipokines [adiponectin (adpn) and leptin (Lep)] and inflammatory cytokines (IL6 and TNFa); (E) plasma FGF-21 concentration; (F) content of proteins representative of gWAT lipid uptake (CD36) and lipogenesis (FAS and SCD1); (G) liver TG levels; (H) liver CD36 protein content; (I) liver FAS protein content; (J) representative blots; and (K) mRNA levels representative of liver lipid uptake (CD36 and Vldr), lipogenesis (Srebp1, Fas, Scd1, and Acc), lipid oxidation (Pdk4 and Acadl), and gluconeogenesis (Pepck, G6P, and Pcx). Data are shown as means + SEM, including individual values where applicable. Statistical significances were calculated using two-way ANOVA in conjunction with Šídák’s multiple comparisons test (A, B, D, E, G, H, I, and K) or Tukey’s multiple comparisons test (F). Data are shown as means + SEM, including individual values where applicable.

Fig. 6.. TNIK variants correlate with T2D-related traits.

(A) Bottom-line meta-analyzed association between variants of TNIK and metabolic traits extracted from T2DKP (https://t2d.hugeamp.org). (B) Association between TNIK pLOF carrier status and poor appetite and overeating, body fat percentage, and blood glucose in UK Biobank participants calculated from linear regression models of association. Point estimates are shown as bars. (C) Illustration of findings obtained in the current study. The illustration was created using BioRender.