Complementary roles of IRS-1 and IRS-2 in the hepatic regulation of metabolism

Abstract

Hepatic insulin resistance is a critical component in the development of type 2 diabetes mellitus. In many cases, insulin resistance in liver is associated with reduced expression of both major insulin receptor substrate (IRS) proteins, IRS-1 and IRS-2. To investigate the specific functions of IRS-1 and IRS-2 in regulating liver function in vivo, we developed an adenovirus-mediated RNA interference technique in which short hairpin RNAs (shRNAs) are used to knock down IRS-1, IRS-2, or both, by 70-80% in livers of WT mice. The knockdown of IRS-1 resulted in an upregulation of the gluconeogenic enzymes glucose-6 phosphatase and phosphoenolpyruvate carboxykinase, as well as a marked increase in hepatic nuclear factor-4 alpha. Decreased IRS-1 was also associated with a decrease in glucokinase expression and a trend toward increased blood glucose, whereas knockdown of IRS-2 resulted in the upregulation of lipogenic enzymes SREBP-1c and fatty acid synthase, as well as increased hepatic lipid accumulation. The concomitant injection of IRS-1 and IRS-2 adenoviral shRNAs resulted in systemic insulin resistance, glucose intolerance, and hepatic steatosis. The alterations in the dual-knockdown mice were associated with defective Akt activation and Foxo1 phosphorylation. Taken together, our results demonstrate that hepatic IRS-1 and IRS-2 have complementary roles in the control of hepatic metabolism, with IRS-1 more closely linked to glucose homeostasis and IRS-2 more closely linked to lipid metabolism.

Figure 1

RNAi adenoviruses cause substantial and specific knockdown of IRS-1 and IRS-2 in liver. (A) Western blots for IRS-1, IRS-2, and actin in liver lysates from mice treated with siGFP, IRS1U6, IRS2U6, or both IRS1U6 and IRS2U6 (1+2). Livers were collected from mice after an overnight fast, and proteins were extracted and processed as described in Methods. Each lane represents liver lysates from a different mouse. (B) Muscle, fat, kidney, and brain lysates of the mice represented in A were subjected to immunoblots for IRS-1 and IRS-2. (C) Quantitative RT-PCR analysis of IRS-1 and IRS-2 mRNA levels in livers 5 days after treatment with siGFP, IRS1U6, IRS2U6, or both viruses simultaneously (n = 6). Total RNA was extracted from livers and made into cDNA, and primers specific to IRS-1 and IRS-2 were used to determine levels of expression, as described previously (58). (D) Time course of effect of RNAi adenovirus. Liver lysates were made from mice 1, 3, 5, 7, or 14 days after viral injection and blotted for IRS-1 and IRS-2 proteins levels. Day 0 liver lysates were obtained from age-matched mice injected with saline. These results are representative of 3 independent experiments.

Figure 2

Dual knockdown of IRS-1 and IRS-2 results in defects in glucose homeostasis. Fasting blood glucose (A) and insulin levels (B) as well as random-fed blood glucose (C) and insulin levels (D) of mice treated with RNAi adenovirus. Glucose and insulin levels are plotted as the mean ± SEM (n = 10–20 mice per treatment group in both fasted and fed state). (E) Insulin tolerance tests (1 U/kg, i.p.) were performed 5 days after adenovirus injection. Results represent blood glucose concentration as a percentage of starting value at time point 0 and are expressed as mean ± SEM (n = 8). (F) Results from E expressed as area under ITT curves. AUC, area under the curve. (G) Glucose tolerance tests (2 g/kg, i.p.) were performed on mice following a 16-hour fast 5 days after adenovirus injection. Blood samples were collected and glucose measured at the times indicated. (H) Results shown in G expressed as area under GTT curves. Open circles, siGFP; closed squares, IRS1U6; open diamonds, IRS2U6; closed circles, both IRS1U6 and IRS2U6. *P < 0.05 vs. siGFP; **P < 0.01 vs. siGFP; ^#P = 0.06 vs. siGFP.

Figure 3

Hypomorphic expression of IRS-1 and IRS-2 causes defects in PI3K activation. (A) IRS-1 immunoprecipitates blotted with 4G10, an anti-PY antibody (top panel). PI3K activity was also measured from these IRS-1 immunoprecipitates (bottom panel, n = 4). (B) As in A, but with IRS-2 immunoprecipitates. (C) PI3K activity in PY-immunoprecipitates (bottom panel), and a representative phosphatidylinositol-(3, 4, 5) trisphosphate (PIP₃) level (top). Bars represent mean ± SEM (n = 4). *P < 0.05 vs. siGFP treatment; **P < 0.01 vs. siGFP.

Figure 4

Significant defects in Akt activation and downstream targets of Akt in mice treated with IRS1U6 and IRS2U6. (A) Insulin-stimulated liver lysates from mice treated with RNAi adenovirus blotted with anti-phospho Akt (S473) antibody (top panel). Blots were then stripped and reprobed for total Akt (bottom panel). The blot is representative of 3 independent experiments. (B) Akt kinase activity was measured in Akt immunoprecipitates using Crosstide as a substrate (56). Bars represent mean ± SEM (n = 3). *P < 0.01 dual treatment vs. siGFP. (C) Insulin-stimulated liver lysates from mice treated with RNAi adenovirus blotted with antibodies against phospho-Foxo1 (S256) or phopsho-MAPK (T202/Y204). The blots for the phosphorylated proteins were then stripped and reprobed with antibodies for total Foxo1 or total MAPK. These Western blots are representative of 3 independent experiments.

Figure 5

Decreased leptin and increased FFA levels in IRS-knockdown mice. (A) Serum adiponectin levels of random-fed mice treated with RNAi adenovirus. (B) Serum leptin levels of random-fed mice treated with RNAi adenovirus. (C) Serum free fatty acid levels in random-fed knockdown mice. Bars represent mean ± SEM (n = 5). *P < 0.05 vs. single treatment vs. siGFP treatment; **P < 0.01 vs. siGFP treatment.

Figure 6

Downregulated IRS protein expression in liver results in changes in expression of gluconeogenic genes and Gck. The relative mRNA levels of indicated genes in the livers of fasting mice treated with RNAi adenovirus, as measured by quantitative RT-PCR: (A) PEPCK, (B) G6Pase, (C) F-1,6BP, (D) Gck, and (E) HNF-4α. Vertical axes represent the fold change in mRNA levels compared with siGFP treatment. The bars represent the fold change in expression of each gene relative to the mean expression in siGFP-treated controls ± SEM (n = 8). *P < 0.05 vs. siGFP treatment; **P < 0.01 vs. siGFP.

Figure 7

Decreased IRS-2 and hyperinsulinemia combine to cause increased lipogenic gene expression and activation of downstream LXR genes. The relative mRNA levels of lipogenic genes in the livers of fasting mice treated with RNAi adenovirus, as measured by RT-PCR: (A) SREBP-1c, (B) FAS, (C) LXR, (D) ABCA1, (E) ABCG8, and (F) CYP7A1. The vertical axes represent the fold change in mRNA levels compared with siGFP treatment. The bars represent the fold change in expression of each gene relative to the mean expression in siGFP-treated controls ± SEM (n = 8). *P < 0.05 vs. siGFP treatment; **P < 0.01 vs. siGFP.

Figure 8

Dual knockdown of hepatic IRS-1 and IRS-2 causes dyslipidemia, increased liver TG content, and hepatic steatosis. (A) Dual knockdown causes increased serum TG levels. Each bar represents the mean serum TG content ± SEM (n = 6). (B) Increased TG accumulation in the livers of mice treated with IRS1U6 and IRS2U6. Each bar represents the mean value of measured liver TG content per gram liver ± SEM (n = 6). (C) H&E staining of livers from fasted mice, 5 days after treatment with RNAi adenovirus. *P < 0.05 vs. siGFP treatment. Magnification, ×400.