Integrin α1-null mice exhibit improved fatty liver when fed a high fat diet despite severe hepatic insulin resistance - PubMed (original) (raw)

Integrin α1-null mice exhibit improved fatty liver when fed a high fat diet despite severe hepatic insulin resistance

Ashley S Williams et al. J Biol Chem. 2015.

Abstract

Hepatic insulin resistance is associated with increased collagen. Integrin α1β1 is a collagen-binding receptor expressed on hepatocytes. Here, we show that expression of the α1 subunit is increased in hepatocytes isolated from high fat (HF)-fed mice. To determine whether the integrin α1 subunit protects against impairments in hepatic glucose metabolism, we analyzed glucose tolerance and insulin sensitivity in HF-fed integrin α1-null (itga1(-/-)) and wild-type (itga1(+/+)) littermates. Using the insulin clamp, we found that insulin-stimulated hepatic glucose production was suppressed by ∼50% in HF-fed itga1(+/+) mice. In contrast, it was not suppressed in HF-fed itga1(-/-) mice, indicating severe hepatic insulin resistance. This was associated with decreased hepatic insulin signaling in HF-fed itga1(-/-) mice. Interestingly, hepatic triglyceride and diglyceride contents were normalized to chow-fed levels in HF-fed itga1(-/-) mice. This indicates that hepatic steatosis is dissociated from insulin resistance in HF-fed itga1(-/-) mice. The decrease in hepatic lipid accumulation in HF-fed itga1(-/-) mice was associated with altered free fatty acid metabolism. These studies establish a role for integrin signaling in facilitating hepatic insulin action while promoting lipid accumulation in mice challenged with a HF diet.

Keywords: Extracellular Matrix; Insulin Resistance; Integrin; Lipid Metabolism; Liver Metabolism.

© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

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Figures

FIGURE 1.

FIGURE 1.

Integrin α1 protein expression is increased in hepatocytes isolated from HF-fed mice. Shown are the results of Western blot analysis for integrin α1 protein expression in isolated hepatocytes from chow- and HF-fed mice (n = 5/group). Integrin α1 protein expression was normalized to β-actin. Data are presented as means ± S.E. (n = 4–5). *, p < 0.05, chow-fed versus HF-fed. A.U., arbitrary units.

FIGURE 2.

FIGURE 2.

Collagen I protein expression is increased in integrin α1-null mice. A, Western blot analysis of collagen I (Col I) performed on liver homogenates from basal 5-h fasted mice (n = 4/group). Integrated intensities were obtained using ImageJ software, and collagen I protein expression was normalized to β-actin. A.U., arbitrary units. Data are presented as means ± S.E. §, p < 0.05 compared with chow-fed itga1+/+ mice. B, representative images from immunohistochemical staining of collagen I in livers from basal 5-h fasted mice (n = 5–7/group).

FIGURE 3.

FIGURE 3.

Integrin α1β1 protects against diet-induced hepatic insulin resistance. GTTs were performed on chow- and HF-fed itga1+/+ and _itga1_−/− mice. Arterial glucose (A and C) and insulin (B and D) levels were measured during the GTTs (n = 5–7/group). Arterial glucose levels (E) and glucose infusion rates (F) were measured during the hyperinsulinemic-euglycemic (insulin) clamp (n = 5–6/group). Mice were fasted for 5 h prior to the start of the insulin clamp. Blood glucose levels were maintained between 150 and 160 mg/dl during steady state (80–120 min). Glucose (50%) was infused to maintain euglycemia. Endogenous glucose production (EndoRa; G) and whole-body disappearance rates (Rd; H) were determined during the steady-state period of the insulin clamp. Data are presented as means ± S.E. *, p < 0.05 compared with the basal measurement in HF-fed itga1+/+ mice; §, p < 0.05 compared with HF-fed itga1+/+ mice during the insulin clamp.

FIGURE 4.

FIGURE 4.

Integrin α1β1 facilitates hepatic insulin action in HF-fed mice. Western blot analysis was performed on liver homogenates from basal 5-h fasted and 5-h fasted insulin-clamped mice. A, immunoprecipitation (IP) of the insulin receptor (IR) was performed on liver homogenates after the insulin clamp prior to an immunoblot (IB) for phosphotyrosine (pY; n = 4/group). Insulin receptor phosphorylation was determined as the ratio of phosphotyrosine to total insulin receptor. B, representative blots of liver insulin signaling after the insulin clamp (n = 4–5/group). C, mRNA was extracted from both basal 5-h fasted and 5-h fasted insulin-clamped livers. qPCR was performed to determine the gene expression of gluconeogenic genes G6pc and Pepck. D, FAK and FAK phosphorylation was determined in liver homogenates from basal 5-h fasted mice (n = 4/group). FAK phosphorylation was quantified as the ratio of phospho (p)-FAK to FAK. The percent increase in FAK phosphorylation was calculated relative to wild-type mice. Integrated intensities were obtained using Odyssey and ImageJ software. Data are presented as means ± S.E. §, p < 0.05 compared with HF-fed itga1+/+ mice; *, p < 0.05 compared with chow-fed itga1+/+ mice; ▵, p < 0.05 compared with chow-fed _itga1_−/− mice; ‡, p < 0.05 compared with basal 5-h fasted mice. A.U., arbitrary units.

FIGURE 5.

FIGURE 5.

Integrin α1β1 promotes hepatic lipid accumulation. A, Oil Red O staining of liver neutral lipid droplets. Liver TG (B) and DAG (C) content was quantified in 5-h fasted mice (n = 5/group). Data are presented as means ± S.E. *, p < 0.05 compared with chow-fed itga1+/+ mice; §, p < 0.05 compared with HF-fed itga1+/+ mice.

FIGURE 6.

FIGURE 6.

Effect of integrin α1 deletion on TG metabolism. A, circulating plasma TGs in basal 5-h fasted mice. B, hepatic TG secretion was determined using tyloxapol to block VLDL-TG clearance from the circulation. C, quantification of TG secretion rates. D, mRNA was extracted from basal 5-h fasted livers, and qPCR was used to determine lipogenic gene expression. Data are presented as means ± S.E. *, p < 0.05 compared with chow-fed itga1+/+ mice; §, p < 0.05 compared with HF-fed itga1+/+ mice (n = 5–8/group).

FIGURE 7.

FIGURE 7.

Effect of integrin α1β1 on FFA metabolism. A, arterial non-esterified FFAs (NEFAs) in the basal 5-h fasted state and during the insulin clamp. B, expression of several genes implicated in the regulation of fatty acid metabolism. mRNA was extracted from 5-h fasted livers, and qPCR was performed to determine gene expression (n = 5–6/group). C and D, high resolution respirometry was performed on mechanically permeabilized liver pieces from 5-h fasted mice. Data are presented as means ± S.E. *, p < 0.05 compared with chow-fed itga1+/+ mice; §, p < 0.05 compared with HF-fed itga1+/+ mice. CSA, citrate synthase activity.

FIGURE 8.

FIGURE 8.

Model whereby integrin α1β1 protects against severe hepatic insulin resistance while promoting TG accumulation. Integrin α1 protein expression increases when wild-type mice are fed a HF diet. This leads to increased integrin α1β1 cell signaling upon collagen binding. Upon insulin stimulation, the combination of both insulin and integrin α1β1 signaling leads to the phosphorylation and subsequent activation of IRS1 and Akt. This results in the partial suppression of hepatic glucose output. Circulating FFAs are taken up by the liver and utilized primarily for the synthesis of DAG and TG, while some may be shunted toward the mitochondria for mitochondrial (mt) respiration. In contrast, when integrin α1-null mice are fed a HF diet and insulin levels are high, the absence of integrin signaling leads to decreased IRS1 and Akt activation. This results in no suppression of hepatic glucose output. Circulating FFAs are lower, and the available FFAs are utilized primarily by the mitochondria for mitochondrial respiration. This results in decreased DAG and TG levels.

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