Altered glucose homeostasis in mice with liver-specific deletion of Src homology phosphatase 2

Abstract

The Src homology 2 domain-containing protein-tyrosine phosphatase Shp2 has been implicated in a variety of growth factor signaling pathways, but its role in insulin signaling has remained unresolved. In vitro studies suggest that Shp2 is both a negative and positive regulator of insulin signaling, although its physiological function in a number of peripheral insulin-responsive tissues remains unknown. To address the metabolic role of Shp2 in the liver, we generated mice with either chronic or acute hepatic Shp2 deletion using tissue-specific Cre-LoxP and adenoviral Cre approaches, respectively. We then analyzed insulin sensitivity, glucose tolerance, and insulin signaling in liver-specific Shp2-deficient and control mice. Mice with chronic Shp2 deletion exhibited improved insulin sensitivity and increased glucose tolerance compared with controls. Acute Shp2 deletion yielded comparable results, indicating that the observed metabolic effects are directly caused by the lack of Shp2 in the liver. These findings correlated with, and were most likely caused by, direct dephosphorylation of insulin receptor substrate (IRS)1/2 in the liver, accompanied by increased PI3K/Akt signaling. In contrast, insulin-induced ERK activation was dramatically attenuated, yet there was no effect on the putative ERK site on IRS1 (Ser(612)) or on S6 kinase 1 activity. These studies show that Shp2 is a negative regulator of hepatic insulin action, and its deletion enhances the activation of PI3K/Akt pathway downstream of the insulin receptor.

FIGURE 1.

Liver-specific Shp2 deletion. A and B, immunoblots of Shp2 expression in peripheral insulin-responsive tissues isolated from Shp2^fl/fl (fl/fl, control), Alb-Cre Shp2^+/− (HET), and Alb-Cre Shp2^−/− (LSHKO) mice at 20 weeks of age. Liver lysates were also probed for PTP1B (bottom panel). Note that compared with control mice, hepatic Shp2 protein expression was ∼50 and ∼15% in HET and LSHKO mice, respectively, whereas Shp2 expression in muscle and fat was unaffected. C, immunoblot analysis of Shp2 expression in liver lysates of old (23 months) control and LSHKO mice on chow diet. D, hematoxylin and eosin-stained liver sections from 20-week-old (top panels) and 23-month-old (lower panels) control and LSHKO mice.

FIGURE 2.

Improved insulin sensitivity and glucose tolerance in mice with hepatic Shp2 deletion. A, ITTs on Shp2^fl/fl (n = 8) and LSHKO (n = 7) mice on chow diet at 12 weeks of age (0.65 milliunit of insulin/g of body weight). B, data from A expressed as percentage change of initial value. C, GTTs on Shp2^fl/fl (n = 8) and LSHKO (n = 8) mice on chow diet at 9 weeks of age (1.1 mg/g of body weight). D, insulin concentrations at basal and 15 and 30 min during GTT. E, ITTs on Shp2^fl/fl (n = 8) and LSHKO (n = 7) mice on chow diet at 17 months of age (0.85 milliunit of insulin/g of body weight). F, data from E expressed as percentage change of initial value. Data were analyzed by repeated-measures ANOVA and Tukey-Kramer honestly significant difference test (*, p ≤ 0.05; **, p ≤ 0.01).

FIGURE 3.

Improved glucose tolerance in mice with acute hepatic Shp2 deletion. Shp2^fl/fl male mice were injected with Ad-Cre (n = 13) or Ad-LacZ (n = 7) (control). A, Shp2 levels determined in liver lysates from Ad-LacZ- and Ad-Cre-injected mice. B, GTT in mice before viral injection. The underlined, italic portion of the mouse genotype indicates the type of virus that was subsequently injected. C, GTT on the same cohort of mice 3 weeks after adenovirus injection (Shp2 deletion). D and E, mRNA expression of PEPCK and G6Pase (D) and SREBP1c and FAS (E) measured by quantitative real-time PCR and normalized against GAPDH, in livers of Ad-Cre- and Ad-LacZ-injected mice. GTT data were analyzed by repeated-measures ANOVA and Tukey-Kramer honestly significant difference test, and RT-PCR data were analyzed using two-tailed Student's t test (*, p < 0.05; **, p < 0.01).

FIGURE 4.

Enhanced insulin signaling in LSHKO mice. Mice were injected intraperitoneally with saline or insulin (10 milliunits/g) and then killed 5 or 10 min after injection, as indicated. Bar graphs represent normalized data (arbitrary units) pooled from three independent experiments from control (n = 8) and LSHKO (n = 8) mice. A, overall tyrosyl phosphorylation in liver lysates from control and LSHKO mice is shown. The blot was probed with anti-ERK antibodies as a control for loading. B, lysates were immunoprecipitated (IP) with IR antibodies, immunoblotted with anti-phosphotyrosine antibodies, and then stripped and reprobed for IR to control for loading. Lysates were also immunoblotted using IR Tyr¹¹⁶²/Tyr¹¹⁶³ phospho-specific antibodies (bottom panel). C, IRS1 was immunoprecipitated from liver lysates, then immunoblotted with anti-phosphotyrosine antibodies, phospho-specific antibodies for Tyr⁶⁰⁸ and Ser³⁰⁷, and IRS1 antibodies (bottom panel). D, IRS1 immunoprecipitates were probed using antibodies against the p85 regulatory subunit of PI3K. E, IRS2 was immunoprecipitated, immunoblotted with anti-phosphotyrosine antibodies, and then reprobed with IRS2 antibodies. All blots were scanned and quantified using FluorChem 9900 (B–E, right panels). Statistical analysis was performed using two-tailed Student's t test (**, p ≤ 0.01; *, p ≤ 0.05).

FIGURE 5.

Enhanced Akt and attenuated ERK phosphorylation in LSHKO mice. A and B, total cell lysates were immunoblotted for p-Akt (A) and p-ERK (B) and the corresponding total proteins. C, IRS1 was immunoprecipitated, immunoblotted with phospho-specific antibodies for Ser⁶¹², and then reprobed with IRS1 antibodies. D and E, total cell lysates also were immunoblotted for pS6K1 (Thr³⁸⁹) (D) and pS6 (Ser²³⁵/Ser²³⁶) (E). All blots were scanned and quantified using FluorChem 9900 (A–E, right panels). Statistical analysis was performed using two-tailed Student's t test (**, p ≤ 0.01; *, p ≤ 0.05).

FIGURE 6.

Proposed model for regulation of insulin signaling by hepatic Shp2. Following insulin stimulation, activated IR phosphorylates IRS1 on multiple tyrosyl sites and leads to the recruitment and activation of signal relay molecules including PI3K and Shp2. Shp2 binds to the C-terminal tyrosyl residues of IRS1 and dephosphorylates p85 binding site(s), thus decreasing PI3K binding to IRS1 and attenuating downstream signaling, namely Akt.