Fig 2: Skeletal muscle–specific ATGL deletion and its impact on lipid homeostasis in SMAKO mice. A: The LoxP-modified ATGL construct. B: ATGL mRNA expression relative to 18S control gene by quantitative PCR in muscle and nonmuscle tissues with endogenous ATGL expression in quadriceps arbitrarily set to 1 (♀, 10 weeks, chow, fasted 12 h; n = 5 to 6/group). Percent decrease in ATGL mRNA expression in SMAKO relative to WT mice for select muscle tissues is shown in the table (bottom). C: ATGL protein expression relative to Ran GTPase (RAN) control in skeletal versus cardiac muscle (♀, 10 weeks, chow, fasted 12 h, gastrocnemius and heart; n = 5 to 6/group). D: TAG hydrolase activity at baseline and in the presence of the HSL-specific inhibitor 76-0079, the ATGL-specific activator CGI-58, or HSL inhibitor plus CGI-58 (♂, 28 weeks, chow, fasted 12 h, red gastrocnemius; n = 6/group). E: Skeletal muscle histology of control (left), SMAKO (middle), and GAKO (right) mice including general morphology by H&E staining (top) and IMTG content by ORO staining (bottom) (♂, 28 weeks, chow, fasted 12 h, gastrocnemius-plantaris-soleus [GPS] complex). F: IMTG content by ORO staining using quantitative IF (♂, 28 weeks, fasted 12 h, GPS complex, average of four muscle areas each; n = 4/group). Type 2B fibers of chow-fed WT mice are arbitrarily set to 1. G: Intramyocellular DAG, ceramide, and FA-CoA content using biochemical analysis of whole muscle (♂, 28 weeks, fasted 12 h, quadriceps; n = 3 to 4/group). For overall effects having P < 0.05: D, diet; F, fiber type; G, genotype; T, treatment (with HSL-inhibitor or CGI-58). For specific comparisons having P < 0.05: #for effect of diet; *for effect of genotype; and @for effect of treatment. AU, arbitrary units; BAC, bacterial artificial chromosome; BAT, brown adipose tissue; D, diet; EDL, extensor digitorum longus; ES, embryonic stem; Gas, gastrocnemius; Hrt, heart; PGAT, perigonadal adipose tissue; Quad, quadriceps; Sol, soleus; TA, tibialis anterior.

Fig 3: Hnscr knockdown in adipocytes decreases lipolysis. (A) The mRNA levels of Hnscr in primary iWAT adipocytes treated with si-Hnscr and si-NC (n = 6). (B) The mRNA levels of genes related to lipogenesis, fat uptake, and β-oxidation in primary iWAT adipocytes treated with si-Hnscr and si-NC (n = 6). (C) The mRNA levels of UCP1 in primary iWAT adipocytes treated with si-Hnscr and si-NC (n = 6). (D) The mRNA levels of genes related to lipolysis in primary iWAT adipocytes treated with si-Hnscr and si-NC (n = 6). (E, F) Protein levels of ATGL and HSL phosphorylation in primary iWAT adipocytes treated with Ad-Hnscr and Ad-scramble (n = 6). (G) Cell glycerol level. Statistical significance was calculated by 2-tailed Student's t test or 2-way ANOVA (*P < .05, **P < .01, ***P < .001).

Fig 4: Inhibition of purine biosynthesis in differentiating cells potentiates AMPK and disrupts lipogenesis.A, schematic of AMPK regulation of lipid metabolism. B, Western blot analysis of pAMPK and AMPK after 6MP and MIZ treatment in primary SVF cells differentiated into adipocytes for 6 days. Data shown are from two biological replicates. C, quantification of band intensity from (B). Statistical significance was determined using one-way ANOVA multiple comparisons test. Error bars indicate mean ± SD, ∗∗p ≤ 0.01 and ∗∗∗p ≤ 0.001. D, 1-14C glucose incorporation into lipid fraction analyzed from primary SVF cells differentiated for 6 days and treated with vehicle, 6MP, or MIZ. Statistical significance was determined using one-way ANOVA multiple comparisons test. Error bars indicate mean ± SD, ∗∗p ≤ 0.01. E, Western blot analysis of pAMPK, AMPK, pACC, ACC, pHSL, HSL, pATGL, and ATGL in primary SVF cells after 6 days of differentiation and treatment with indicated drugs. Data shown are from two biological replicates. F, gene expression profile in primary SVF cells differentiated for 6 days and treated with vehicle, 6MP, or MIZ. Statistical significance was determined using one-way ANOVA multiple comparisons test. All data are representative of 2 to 3 independent experiments. 6MP, 6-mercaptopurine; ACC, acetyl CoA carboxylase 1; AMPK, AMP-activated protein kinase; ATGL, adipose triglyceride lipase; MIZ, mizoribine.

Fig 5: Loss of SRA increases the hepatic ATGL expression.(a) RT-qPCR analysis of mRNA expression of FFA oxidation, lipogenesis and VLDL metabolism-related genes in livers of male SRAKO (KO, n = 8) or WT (n = 8) littermates (20 weeks of age) fed with normal chow. (b) Hepatic mRNA level of ATGL in SRAKO (KO, n = 8) or WT (n = 8) littermates (20 weeks of age) fed with normal chow or HFD. The mRNA levels of genes were normalized to 36B4 expression. The data are presented as the mean ± SE and expressed as fold-change relative to the level of WT-Chow. *p < 0.05. (c) Liver lysates from male SRAKO (KO) or WT littermates (20 weeks of age) under different diet conditions were immunoblotted with anti-ATGL and anti-β-actin antibodies.