Pathogenesis of selective insulin resistance in isolated hepatocytes

Abstract

The development of insulin resistance (IR) in the liver is a key pathophysiologic event in the development of type 2 diabetes. Although insulin loses its ability to suppress glucose production, it largely retains its capacity to drive lipogenesis. This selective IR results in the characteristic hyperglycemia and dyslipidemia of type 2 diabetes. The delineation of two branched pathways of insulin receptor (InsR) signaling to glucose versus triglyceride production, one through FoxO and the other through SREBP-1c, provides a mechanism to account for this pathophysiological abnormality. We tested the complementary hypothesis that selective IR arises due to different intrinsic sensitivities of glucose production versus de novo lipogenesis to insulin as a result of cell-autonomous down-regulation of InsR number in response to chronic hyperinsulinemia. We demonstrate in mouse primary hepatocytes that chronic hyperinsulinemia abrogates insulin's inhibition of glucose production, but not its stimulation of de novo lipogenesis. Using a competitive inhibitor of InsR, we show that there is a 4-fold difference between levels of InsR inhibition required to cause resistance of glucose production versus lipogenesis to the actions of insulin. Our data support a parsimonious model in which differential InsR activation underlies the selective IR of glucose production relative to lipogenesis, but both processes require signaling through Akt1/2.

Keywords: FOXO; atherosclerosis; diabetes; gluconeogenesis; glucose metabolism; insulin receptor; insulin resistance; lipid metabolism; lipogenesis; lipoprotein metabolism; liver.

FIGURE 1.

Chronic hyperinsulinemia induces downregulation of the insulin receptor. A, linear regression analysis of liver InsRβ protein level as a function of the logarithm of the circulating insulin level in adult Insr^+/− mice. AU, arbitrary units. B, InsRβ protein was quantified by densitometric analysis of the Western blot shown in B, in which the number of each lane corresponds to the same numbered point in A. C, Western blot analysis of InsR signaling in hepatocytes following chronic insulin treatment. Each lane contains equal amounts of protein pooled from three independent experiments. The densitometric quantification of InsR protein relative to Akt is labeled above each lane. pT308, phospho-Thr-308; pS473, phospho-Ser-473; pS9, phospho-Ser-9; pS2448, phospho-Ser-S2448; p, phosphorylated; un-p, unphosphorylated; Gck, glucokinase; pre-Rx, pretreatment.

FIGURE 2.

Metabolic effects of chronic insulin treatment. A–C, relative glucose production (A) and gene expression (B and C) in control or CHI-treated primary hepatocytes incubated for 5 h with vehicle, cAMP, or cAMP and insulin. Data are normalized to vehicle-treated control. AU, arbitrary units. D–G, relative de novo lipogenesis (D) and gene expression (E–G) in control or CHI-treated primary hepatocytes incubated for 5 h with vehicle or 10 nm insulin. Data are normalized to vehicle-treated control and represent mean ± S.E. of at least three independent experiments performed in triplicate. *, p < 0.05, **, p < 0.01, ****, p < 0.0001 by Tukey's post hoc analysis following two-way analysis of variance. #, p < 0.05, ##, p < 0.01 relative to control by two-tailed, unpaired Student's t test.

FIGURE 3.

Insulin sensitivity of glucose and lipid metabolism. A, peptide-based InsR antagonism Western blots of InsR signaling components in response to treatment with 10 nm insulin and/or the indicated concentrations of S961. Each lane represents pooled samples of equivalent amounts of lysate from three independent experiments. pT308, phospho-Thr-308; pS473, phospho-Ser-473; pS9, phospho-Ser-9; pS2448, phospho-Ser-S2448; p, phosphorylated; un-p, unphosphorylated. B, graphical representation of densitometric analysis of data in A. IC₅₀ is calculated as the log₁₀ of the S961 concentration at which each dose-response curve intercepts 50% of maximal insulin response. C–E, relative glucose production (C) and gene expression (D and E) in primary hepatocytes incubated for cAMP with or without insulin and the indicated concentrations of S961. AU, arbitrary units. F, relative de novo lipogenesis in control or S961-treated primary hepatocytes incubated for 5 h with vehicle or 10 nm insulin. G, percent maximal insulin response of glucose production and de novo lipogenesis as a function of S961 concentration. Curves are calculated from data in panels C and F. IC₅₀ is calculated as log₁₀ of the S961 concentration at which each dose-response curve intercepts 50% maximal insulin response. Data in A–E are normalized to insulin-/S961-untreated control and represent mean ± S.E. of at least three independent experiments performed in triplicate. *, p < 0.05, **, p < 0.01, ***, p < 0.001, ****, p < 0.0001 relative to vehicle-treated control and £, p < 0.05, ££, p < 0.01 relative to insulin-treated control by Tukey's post hoc analysis following one-way analysis of variance.

FIGURE 4.

Effect of Akt inhibition on glucose production and lipogenesis. A, Western blots of InsR cascade components in response to treatment with insulin and/or 5 μm Akti-1/2. Blots are representative of three independent experiments. pT308, phospho-Thr-308; pS9, phospho-Ser-9; pS2448, phospho-Ser-S2448; p, phosphorylated; un-p, unphosphorylated. B, relative glucose production in primary hepatocytes pretreated with the indicated concentrations of Akti-1/2 or vehicle and then incubated for 5 h with cAMP with or without 10 nm insulin. AU, arbitrary units. C and D, gene expression in primary hepatocytes pretreated with 5 μm Akti-1/2 or vehicle and incubated for 5 h with cAMP with or without 1 nm insulin. E, relative de novo lipogenesis in primary hepatocytes pretreated with the indicated concentrations of Akti-1/2 or vehicle and incubated for 5 h with or without 10 nm insulin. Data are mean ± S.E. of at least two independent experiments performed in triplicate. For B and E, *, p < 0.05, **, p < 0.01, ***, p < 0.001 relative to vehicle-treated control by Dunnett's post hoc analysis following one-way analysis of variance. For C and D, **, p < 0.01, ***, p < 0.001, ****, p < 0.0001 for designated comparison by Tukey's post hoc analysis following two-way analysis of variance. F, schematic illustrating the degree of intact InsR and Akt signaling to de novo lipogenesis and glucose production in the healthy state, selective IR, and complete IR.