The role of endothelial insulin signaling in the regulation of vascular tone and insulin resistance

Abstract

Insulin receptors (IRs) on vascular endothelial cells have been suggested to participate in insulin-regulated glucose homeostasis. To directly address the role of insulin action in endothelial function, we have generated a vascular endothelial cell IR knockout (VENIRKO) mouse using the Cre-loxP system. Cultured endothelium of VENIRKO mice exhibited complete rearrangement of the IR gene and a more than 95% decrease in IR mRNA. VENIRKO mice were born at the expected Mendelian ratio, grew normally, were fertile, and exhibited normal patterns of vasculature in the retina and other tissues. Glucose homeostasis under basal condition was comparable in VENIRKO mice. Both eNOS and endothelin-1 mRNA levels, however, were reduced by approximately 30-60% in endothelial cells, aorta, and heart, while vascular EGF expression was maintained at normal levels. Arterial pressure tended to be lower in VENIRKO mice on both low- and high-salt diets, and on a low-salt diet VENIRKO mice showed insulin resistance. Thus, inactivation of the IR on endothelial cell has no major consequences on vascular development or glucose homeostasis under basal conditions, but alters expression of vasoactive mediators and may play a role in maintaining vascular tone and regulation of insulin sensitivity to dietary salt intake.

Figure 1

Recombination of the IR gene and expression of the IR mRNA in endothelial cells of VENIRKO mice. (a) Schematic representation of the IR/lox allele showing the position of the primers and the recombined IR knockout allele indicating the deletion of exon 4. (b) The PCR product from control mice corresponds to the 266 bp from the amplification between primers 1 and 2 of the endogenous IR gene allele. The PCR product from VENIRKO mice corresponds to the 220 bp from the amplification between primers 2 and 3. (c) Real-time quantitative RT-PCR analysis to determine the IR expression in RNA extracts from primary cultures of control and VENIRKO mice. The expression level of IR in VENIRKO endothelial cells (lane 3 and 4) were reduced to 3–5% of control (lane 1 and 2). *P < 0.01 for differences between control and VENIRKO mice.

Figure 2

Endothelial mediator gene expression in control and VENIRKO endothelial cells. (a) Northern blot analysis of eNOS in heart and aorta. Total RNA (20 μg) from heart and aorta of control and VENIRKO mice (n = 6 each) was fractionated and hybridized to cDNA probes for eNOS and 36B4 mRNA. (b) Real-time quantitative RT-PCR analysis to examine the VEGF, eNOS, and ET-1 in RNA extracts from primary cultures. Two independent sets of endothelial cells were prepared from control and two from VENIRKO mice. Each RNA sample was analyzed by real-time quantitative RT-PCR on three independent experiments. *P < 0.05 for differences between control and VENIRKO mice.

Figure 3

Flat-mount retinas. Microphotographs (×100) of flat-mount retinas prepared from 17-day-old control (a and b) and VENIRKO (c and d) mice, which had been perfused with fluorescein-conjugated dextran. Microphotographs in a and c show small arteries and branches of the central artery of the retina. Microphotographs in b and d show the deep capillary bed of the same areas shown in a and c.

Figure 4

Evaluation of time course of insulin action in control and VENIRKO mice by euglycemic-hyperinsulinemic clamp. Insulin was administered intravenously at time 0 as a bolus (40 mU/kg) and constant infusion (4 mU/kg/min) for the rest of the experiment. Euglycemia was maintained by glucose infusion. (a) Glucose infusion rates (mg/kg/min) in control (filled circle, n = 3) and VENIRKO (open circle, n = 3). (b) Blood glucose concentrations during the clamp.

Figure 5

GTTs and ITTs at 2 and 6 months of age. (a)GTT at 2 (left) and 6 (right) months of age in control (filled circle, n = 55 and 53), heterozygous (open square, n = 30 and 29), and VENIRKO (open circle, n = 29 and 27) mice. (b) ITT at 2 (left) and 6 (right) months of age in control (n = 67 and 38), heterozygous (n = 30 and 25), and VENIRKO (n = 30 and 17) mice. *P < 0.05 and ^†P < 0.01 for differences between control and VENIRKO mice.

Figure 6

BP and HR under basal conditions and low and high NaCl intake. (a) SBP, MBP, DBP, and HR in males (left) and females (right) control (black bars) and VENIRKO (white bars) mice. *P < 0.05 for differences between mean values in control (n = 20 males and n = 11 females) and VENIRKO (n = 13 males and n = 5 females). (b) SBP, MBP, DBP, and HR under low- and high-salt diet in male control (filled circle, n = 12) and VENIRKO (open circle, n = 12) mice. *P < 0.05 for differences in mean values between control and VENIRKO mice.

Figure 7

ITTs under dietetic low and high NaCl intake. ITT in control (filled circle, n = 7) and VENIRKO (open circle, n = 6) mice. Under low-salt diet (a) and high-salt diet (b). *P < 0.05 for differences in mean values between control and VENIRKO mice.