Hepatic insulin resistance is sufficient to produce dyslipidemia and susceptibility to atherosclerosis

Abstract

Insulin resistance plays a central role in the development of the metabolic syndrome, but how it relates to cardiovascular disease remains controversial. Liver insulin receptor knockout (LIRKO) mice have pure hepatic insulin resistance. On a standard chow diet, LIRKO mice have a proatherogenic lipoprotein profile with reduced high-density lipoprotein (HDL) cholesterol and very low-density lipoprotein (VLDL) particles that are markedly enriched in cholesterol. This is due to increased secretion and decreased clearance of apolipoprotein B-containing lipoproteins, coupled with decreased triglyceride secretion secondary to increased expression of Pgc-1 beta (Ppargc-1b), which promotes VLDL secretion, but decreased expression of Srebp-1c (Srebf1), Srebp-2 (Srebf2), and their targets, the lipogenic enzymes and the LDL receptor. Within 12 weeks on an atherogenic diet, LIRKO mice show marked hypercholesterolemia, and 100% of LIRKO mice, but 0% of controls, develop severe atherosclerosis. Thus, insulin resistance at the level of the liver is sufficient to produce the dyslipidemia and increased risk of atherosclerosis associated with the metabolic syndrome.

Figure 1. FPLC profiles of plasma lipoproteins from Lox and LIRKO mice

Serum was obtained from six month old mice on a normal chow diet, after a 4.5 hour fast, and (A) total cholesterol or (B) triglycerides was measured (n=5-6, p=0.04). Serum was subjected to FPLC analysis, and (C) cholesterol and (D) triglycerides were measured in each of the eluted fractions. Data are presented as the average of 2-4 mice per genotype. Similar results were obtained in two other experiments. (E) Apolipoprotein levels were examined by immunoblotting whole serum (left) or serum subjected to fractionation by FPLC. FPLC samples were prepared by pooling equal volumes of each fraction from three mice. In this and all other figures, error bars represent the SEM.

Figure 2. Insulin resistance alters gene expression

(A) Expression of the PGC and SREBP expression was measured in the livers of two-month old, non-fasted mice on a chow diet by real time PCR analysis or immunoblotting of liver extracts (PGC-1) or nuclear extracts (SREBP-1). SREBP-1 protein was also measured in two-month old mice after fasting and re-feeding (n=4-8, *p<0.05,). (B) Real time PCR analysis of cDNA prepared from the livers of two to four month old non-fasted chow fed mice. See text for gene names. (C) Two-month old Lox and LIRKO mice were gavaged with 40 mg/kg LXR agonist (T090137) or vehicle every 24 hours for two doses, and sacrificed four hours after the second dose, in the non-fasted state. Real time PCR was performed on cDNA prepared from these livers (n=5-8, *p<0.05, **p<0.005). (D) Hepatocytes were isolated from two to three month old Lox and LIRKO mice, and cultured overnight in the presence of 100 nM insulin, and either 5 μM LXR agonist or vehicle. RNA was extracted and subjected to real time PCR analysis. (n=4-6, *p<0.05)

Figure 3. VLDL metabolism

A-C) Two- to three- month old chow-fed Lox and LIRKO mice were injected with Triton WR1339 after a six hour fast. Serum triglycerides were measured at 0, 90 and 180 minutes (n=5-7, p ≤ 0.01 at each time point). After 180 minutes, serum was collected and equal volumes were pooled, and subjected to ultracentrifugation to obtain the VLDL fraction. VLDL triglycerides (A) and cholesterol (B) were measured. (D, E) apoB secretion and VLDL clearance were measured in four-month old mice after ten weeks on the Western diet. (D) The rate of apoB synthesis was measured as the amount of radiolabeled apoB100 and apoB48 present in the serum one hour after the injection of Triton WR1339 and [³⁵S]methionine, normalized to the amount present in the Lox control (n=4-5, *p<0.01) (E) Fractional clearance of LDL apoB was determined by measuring plasma radioactivity over 24 hrs after injection of ¹²⁵I –labeled mouse LDL. Results are expressed as the percentage of LDL radioactivity remaining in plasma at each time point (n=3). Inset shows LDL receptor immunoblot of liver extracts prepared from these mice.

Figure 4. Dyslipidemia and Atherosclerosis on the Atherogenic Diet

A-D) Serum was obtained from four month old mice fed the atherogenic diet for two months, after a six hour fast. Total cholesterol (A) and triglycerides (B) were measured in the serum. Equal amounts of serum were pooled from two mice of each genotype, and subjected to FPLC fractionation. Cholesterol (C) and triglycerides (D) were measured in each fraction. Data are presented as an average of three samples per genotype. E-F) Lox and LIRKO mice were placed on an atherogenic diet at two months of age, and sacrificed three to four months later, at which time the aorta was examined for the presence of atherosclerosis (E) Quantitation of the plaque area in Lox and LIRKO mice (*p=0.01, using a Mann-Whitney U-test). (F) Gross dissected anatomy in situ (left); sections through aortic sinus and the aortic valve stained with hematoxylin and eosin (middle); and entire aorta with adventitial fat carefully removed, flat mounted “en face” and stained for fat with Sudan IV (right).