Adipose triglyceride lipase deficiency causes tissue-specific changes in insulin signaling

Abstract

Triacylglycerol accumulation in insulin target tissues is associated with insulin resistance. Paradoxically, mice with global targeted deletion of adipose triglyceride lipase (ATGL), the rate-limiting enzyme in triacylglycerol hydrolysis, display improved glucose tolerance and insulin sensitivity despite triacylglycerol accumulation in multiple tissues. To determine the molecular mechanisms for this phenotype, ATGL-deficient (ATGL(-/-)) and wild-type mice were injected with saline or insulin (10 units/kg, intraperitoneally), and then phosphorylation and activities of key insulin-signaling proteins were determined in insulin target tissues (liver, adipose tissue, and muscle). Insulin signaling and/or glucose transport was also evaluated in isolated adipocytes and skeletal muscle ex vivo. In ATGL(-/-) mice, insulin-stimulated phosphatidylinositol 3-kinase and Akt activities as well as phosphorylation of critical residues of IRS1 (Tyr(P)-612) and Akt (Ser(P)-473) were increased in skeletal muscle in vivo. Insulin-stimulated phosphatidylinositol 3-kinase activity and total insulin receptor and insulin receptor substrate 1, but not other parameters, were also increased in white adipose tissue in vivo. In contrast, in vivo measures of insulin signaling were decreased in brown adipose tissue and liver. Interestingly, the enhanced components of insulin signaling identified in skeletal muscle and white adipose tissue in vivo and their expected downstream effects on glucose transport were not present ex vivo. ATGL deficiency altered intramyocellular lipids as well as serum factors known to influence insulin sensitivity. Thus, skeletal muscle, rather than other tissues, primarily contributes to enhanced insulin sensitivity in ATGL(-/-) mice in vivo despite triacylglycerol accumulation, and both local and systemic factors contribute to tissue-specific effects of global ATGL deficiency on insulin action.

FIGURE 1.

Body weight, body composition, glucose tolerance, and insulin sensitivity. A, body weight of male (M) and female (F) WT and ATGL^−/− mice (n = 16–41 per group). B, total body fat mass, and C, lean mass of WT and ATGL^−/− mice as assessed by EchoMRI (n = 3–17 per group). D, GTT, plasma glucose in 7–8-h fasted male mice at 8–10 weeks of age following intraperitoneal administration of 1.5 g of glucose/kg of body weight (n = 8–18 per group). E, serum insulin following administration of 1.5 g of glucose/kg of body weight in a separate cohort of 7–8 week-old mice of mixed gender (n = 4–5 per group). Experimental conditions and blood glucose curves were comparable with the GTT shown in D. F, ITT, plasma glucose in 7–8-h fasted male mice at 7–9 weeks of age following intraperitoneal administration of 0.75 units of insulin/kg of body weight (n = 8–18 per group). For both ITT and GTT, the areas under the curve are shown (D and F, insets). Data are expressed as mean ± S.E. *, p < 0.05; **, p < 0.01; ***, p < 0.001 for effect of genotype as determined by unpaired two-tailed Student's t test.

FIGURE 2.

Blood glucose, serum lipids, and liver glycogen. A, plasma glucose during fasting in 6-10-week-old female mice (n = 10–18 per group). B, serum NEFAs, and C, TAGs in 7–10-week-old mice following a 6-h fast (n = 8 per group). D, liver glycogen in ad libitum-fed and 6-h-fasted, 7-14-week-old mice of mixed gender (n = 3–4 per group). Data are expressed as means ± S. E. *, p < 0.05; **, p < 0.01; ***, p < 0.001 for effect of genotype or feeding status as determined by unpaired two-tailed Student's t test.

FIGURE 3.

Liver insulin signaling in vivo. Male mice at 10–12 weeks of age were fasted for 6 h, injected intraperitoneally with saline or insulin at 10 units/kg of body weight, and sacrificed 10 min thereafter. A and B, insulin-stimulated site-specific phosphorylation of IR, IRS1, and Akt as assessed by immunoblotting analysis (n = 3–5 per group). For quantification, phosphoproteins were normalized to the corresponding total proteins. Ran GTPase served as loading control. C, IRS1-associated PI3K activity; D, IRS2-associated PI3K activity; and E, Akt activity (n = 3–7 per group). IP, immunoprecipitation. Data are expressed as mean ± S.E. *, p < 0.05 for effect of genotype as determined by unpaired two-tailed Student's t test (B) or one-way ANOVA (C–E).

FIGURE 4.

WAT insulin signaling in vivo. A–E, for insulin signaling in WAT in vivo, male mice at 10–12 weeks of age were fasted for 6 h, injected intraperitoneally with saline or insulin at 10 units/kg of body weight, and sacrificed 10 min thereafter. A and B, insulin-stimulated site-specific phosphorylation of IR, IRS1, and Akt as assessed by immunoblotting analysis (n = 3–5 per group). For quantification, phosphoproteins were normalized to the corresponding total proteins. GAPDH served as loading control. A and C, expression of total IR and IRS1 relative GAPDH control. D, IRS1-associated PI3K activity; and E, Akt activity (n = 3–7 per group). Data are expressed as mean ± S.E. *, p < 0.05 for effect of genotype as determined by unpaired two-tailed Student's t test (B and C) or one-way ANOVA (D and E).

FIGURE 5.

Glucose transport in isolated white adipocytes. For glucose transport into isolated adipocytes ex vivo, adipocytes were isolated from perigonadal WAT of 6- to 8-week-old male mice following a 6-h fast (n = 3–8 per group) and then assayed for uptake of [U-¹⁴C]glucose in the presence of 0, 0.2, 0.3, 0.4, 0.6, 1, 5, or 100 nm insulin. A, lipid content per adipocyte. B, basal glucose transport in the absence of insulin. C, absolute values for the effect of insulin on glucose transport. D, transformed values for the effect of insulin on glucose transport after correcting for differences in basal glucose transport. Data are expressed as mean ± S.E. Curves were generated by nonlinear regression with no constraints (C and D). **, p < 0.01 for effect of genotype as determined by unpaired two-tailed Student's t test (A and B).

FIGURE 6.

Skeletal muscle insulin signaling in vivo. Male mice at 10–12 weeks of age were fasted for 6 h, injected intraperitoneally with saline or insulin at 10 units/kg of body weight, and sacrificed 10 min thereafter. A and B, insulin-stimulated site-specific phosphorylation of the IR, IRS1, and Akt as assessed by immunoblotting analysis (n = 3–5 per group). For quantification, phosphoproteins were normalized to total proteins. Ran GTPase served as loading control. C, IRS1-associated PI3K activity, and D, Akt activity (n = 3–7 per group). E, Glut4 protein expression relative to Ran GTPase control as determined by Western blot analysis. Data from gastrocnemius muscle are shown. Data are expressed as mean ± S.E. *, p < 0.05; **, p < 0.01 for effect of genotype as determined by unpaired two-tailed Student's t test (B and E) or one-way ANOVA (C and D).

FIGURE 7.

Skeletal muscle insulin signaling and glucose transport ex vivo. A–D, for analysis of insulin signaling in skeletal muscle ex vivo, soleus muscles were dissected from 6- to 10-week-old female mice following a 6-h fast and incubated in the presence or absence of 33 nm insulin. A and B, insulin-stimulated site-specific phosphorylation of the IR, IRS1, and Akt as assessed by immunoblotting analysis (n = 3–5 per group). For quantification, phosphoproteins were normalized to total proteins. Ran GTPase served as loading control. C, IRS1-associated PI3K activity; and D, Akt activity (n = 3–9 per group). E, for analysis of glucose uptake into skeletal muscle ex vivo, soleus muscles were dissected from 9- to 13-week-old female mice following a 6-h fast (n = 9–11 per group) and assayed for uptake of 2-[³H]deoxyglucose in presence or absence of 33 nm insulin. Similar results were observed for EDL. Data are expressed as mean ± S.E. *, p < 0.05 for effect of genotype as determined by unpaired two-tailed Student's t test (B) or one-way ANOVA (C–E).

FIGURE 8.

Local and systemic factors contributing to skeletal muscle insulin action. Lipid analysis was performed in skeletal muscle of 7–9-week-old mice following a 6-h fast. A, Oil Red O staining for neutral lipids in gastrocnemius of ATGL^−/− and WT mice. B, TAG concentrations in soleus muscle of female mice (n = 4 per group). C, DAG; and D, ceramide contents in soleus muscle of mice of mixed gender (n = 3–5 per group). E, long chain FA-CoA concentrations in soleus muscle of male mice (n = 6). All lipid levels were normalized to tissue wet weight. F, gene expression analysis in skeletal muscle of 8-week-old female mice following a 6-h fast (n = 8–12). G, serum RBP4 in 7–10-week-old mice following a 6-h fast (8–16 per group) determined by immunoblot analysis. Data are expressed as mean ± S.E. *, p < 0.05; **, p < 0.01; ***, p < 0.001 for effect of genotype as determined by unpaired two-tailed Student's t test.