Trib1 deficiency causes brown adipose respiratory chain depletion and mitochondrial disorder

Abstract

Tribbles homolog 1 (TRIB1) belongs to the Tribbles family of pseudokinases, which plays a key role in tumorigenesis and inflammation. Although genome-wide analysis shows that TRIB1 expression is highly correlated with blood lipid levels, the relationship between TRIB1 and adipose tissue metabolism remains unclear. Accordingly, the aim of the present study was to explore the role of TRIB1 on mitochondrial function in the brown adipose tissue (BAT). Trib1-knockout mice were established using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology. The metabolic function of the BAT was induced by a β3-adrenoceptor agonist and the energy metabolism function of mitochondria in the BAT of mice was evaluated. Trib1-knockout mice exhibited obesity and impaired BAT thermogenesis. In particular, Trib1 knockout reduced the ability of the BAT to maintain body temperature, inhibited β3-adrenoceptor agonist-induced thermogenesis, and accelerated lipid accumulation in the liver and adipose tissues. In addition, Trib1 knockout reduced mitochondrial respiratory chain complex III activity, produced an imbalance between mitochondrial fusion and fission, caused mitochondrial structural damage and dysfunction, and affected heat production and lipid metabolism in the BAT. Conversely, overexpression of Trib1 in 3T3-L1 adipocytes increased the number of mitochondria and improved respiratory function. These findings support the role of Trib1 in regulating the mitochondrial respiratory chain and mitochondrial dynamics by affecting mitochondrial function and thermogenesis in the BAT.

Fig. 1. After adrenaline stimulation, the expression of Trib1 in brown adipose increased.

a The mRNA level of Trib1 in a variety of organs and tissues of normal C57BL/6J mice. b, c Lipid drop oil red staining and mRNA level of Trib1 in 3T3-L1 cells during adipogenesis. Scale bar: 100 μm. d, e H&E-stained and lipid droplet area statistics of BAT and iWAT sections obtained from the C57BL/6J mice treated with cold exposure or CL316243 (β3-adrenoceptor agonist). Scale bar: 100 μm. f–h After CL316243 treatment, the mRNA levels of Trib1 and thermogenesis-related genes in BAT. i, j After CL316243 treatment, TRIB1 and thermogenic protein in BAT. BAT, brown adipose tissue; iWAT, inguinal white adipocyte tissue; eWAT, epididymal white adipocyte. In the bar figure, each data represents mean ± SEM (n = 6). One-way ANOVA multiple comparisons with Tukey’s test, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 over control group.

Fig. 2. Decreased thermogenesis in Trib1-knockout mice.

a Representative adipose tissue image of Trib1-knockout mice. Body composition (b) and ratio of tissue weight-to-bodyweight (c) of Trib1 KO and WT mice treated with CL316243 or not. Representative hematoxylin and eosin (H&E) staining image (d) and adipocyte area (e) of BAT, iWAT, and eWAT from Trib1 KO and WT mice treated with CL316243 or not. Scale bar: 100 μm. Representative hematoxylin and eosin (H&E) staining and oil red O staining image (f), and red staining area (g) of liver tissue from Trib1 KO and WT mice treated with CL316243 or not. Scale bar: 100 μm. BAT, brown adipose tissue; iWAT, inguinal white adipocyte tissue; eWAT, epididymal white adipocyte. In the bar figure, each data represents mean ± SEM (n = 5). Indicated comparisons were made using Student’s paired t-test, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 over Trib1 WT mice; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 over Trib1 WT mice treated with CL316243.

Fig. 3. Mitochondrial structural damage of brown adipose in Trib1-knockout mice.

Serum LDL (a), HDL (b), TC (c), insulin (d), leptin (e), and adiponectin (f) levels in Trib1-wild-type and knockout mice. Energy expenditure was evaluated by measurement of oxygen consumption (g) and carbon dioxide release (h) in Trib1 KO and WT mice treated with CL316243 or not. After 8 h of cold exposure, the rectal temperature (i) of Trib1 KO and WT mice treated with CL316243 or not. j The mRNA level of thermogenesis-related genes in BAT from Trib1 KO and WT mice treated with CL316243 or not. TEM images of BAT (k) and iWAT (l) from Trib1-knockout mice treated with CL316243 or not. Black arrows indicated abnormal mitochondria. Scale bar: 0.5 μm. LDL, low-density lipoprotein; HDL, high-density lipoprotein; TC, total cholesterol; UCP1, uncoupling protein 1; Prdm16, PR/SET domain 16; BAT, brown adipose tissue; iWAT, inguinal white adipocyte tissue. In the bar figure, each data represents mean ± SEM (n = 5). Indicated comparisons were made using Student’s paired t-test, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 over Trib1 WT mice; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 over Trib1 WT mice treated with CL316243.

Fig. 4. Trib1 knockout leads to the decrease and dysfunction of mitochondrial assembly protein in mice.

a The Venn diagram of RNA-seq indicated the number of differentially expressed genes between Trib1-knockout and wild-type mice, respectively (n = 3). b The volcano plot of RNA-seq showing up- and downregulated genes between Trib1-knockout and wild-type mice. c Kyoto Encyclopedia of Genes and Genomes analysis of downregulated pathway in Trib1-knockout mice and wild-type mice. Heat map of thermogenesis (d), oxidative phosphorylation (e), fatty acid metabolism (f), and insulin signaling pathway (g) gene clusters in Trib1-knockout and wild-type mice. Red and blue represent upregulation and downregulation expression, respectively. h The final elution (CoIP) samples were analyzed by Western blot and stained with Coomassie brilliant blue. The specific bands were used for mass spectrometry analysis. i The final elution (CoIP) samples were analyzed by Western blot and incubated with uqcrc2 primary antibody.

Fig. 5. Trib1 knockout results in decreased ATP production and disruption of mitochondrial homeostasis.

a, b Western blots of mitochondrial electron transport chain complexes (complexes I–V) in brown adipose of Trib1-knockout and wild-type mice, and the results of optical density analyses. c The mRNA level of brown adipose respiratory chain complex in Trib1-knockout and wild-type mice. d Mitochondrial complex III activity of brown adipose in Trib1 KO and WT mice treated with CL316243 or not. e ATP level of brown adipose in Trib1-knockout and wild-type mice. f, g Expression of mitochondrial fusion and fission proteins in brown adipose tissue of Trib1-knockout and wild-type mice, and the results of optical density analyses. h Mitochondrial fusion and fission gene mRNA level in Trib1-knockout and wild-type mice. i Mitochondrial copy number and fatty acid oxidation gene mRNA level of brown adipose in Trib1-knockout and wild-type mice. DRP1, dynamin-related protein 1; FIS1, fission protein 1; Mfn1, mitofusin 1; Mfn2, mitofusin 2; OPA1, optic atrophy 1; TFAM, transcription factor; CPT1α, carnitine palmitoyltransferase 1 A; MCAD, medium-chain acyl-CoA dehydrogenases; LCAD, long-chain acyl-CoA dehydrogenases. In the bar figure, each data represents mean ± SEM (n = 5). Indicated comparisons were made using Student’s paired t-test, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 over Trib1 WT mice; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 over Trib1 WT mice treated with CL316243.

Fig. 6. Overexpression of Trib1 reduces lipid droplet accumulation in 3T3-L1 cells and increases respiratory metabolism.

a After overexpression of Trib1, 3T3-L1 cells were stained with oil red staining and lipid droplets under natural light. Oil red scale bar: 400 μm; Lipid droplets under natural light scale bar: 1000 μm/400 μm. b The Venn diagram of RNA-seq indicated the number of differentially expressed genes between 3T3-L1 cells control group and Trib1 overexpressing group, respectively (n = 3). c The volcano plot of RNA-seq showing up- and downregulated genes between 3T3-L1 cells control group and Trib1 overexpressing group. d Heat map of thermogenesis gene clusters in 3T3-L1 cells control group and Trib1 overexpressing group. Red and blue represent upregulation and downregulation expression, respectively. e Kyoto Encyclopedia of Genes and Genomes analysis of upregulated pathway in 3T3-L1 cells control group and Trib1 overexpressing group. f Cells were stained with MitoTracker and Hoechst 33342. Scale bar: 200 μm. g, h Seahorse XF24 mitochondrial stress analyses for OCR in 3T3-L1 cells treated by overexpression of Trib1 (Trib1-OE), Uqcrc2 (Uqcrc2-OE), and inhibitor (Trib1-OE + AA). OE, overexpression; AA, antimycin A. Each data represents mean ± SEM (n = 6). One-way ANOVA multiple comparisons with Tukey’s test, ^*P < 0.05, ^**P < 0.01, ^***P < 0.01over control group.