Chronic Insulin Infusion Down-Regulates Circulating and Urinary Nitric Oxide (NO) Levels Despite Molecular Changes in the Kidney Predicting Greater Endothelial NO Synthase Activity in Mice

Abstract

Insulin therapy is often needed to overcome insulin receptor resistance in type 2 diabetes; however, the impact of providing additional insulin to already hyperinsulinemic subjects is not clear. We infused male TALLYHO/Jng (TH) mice (insulin resistant) with insulin (50 U/kg·bw/d) or vehicle (control) by osmotic minipump for 14 days. One group of insulin-infused mice was switched to 4% NaCl diet (high-sodium diet, HSD) in the second week. Blood chemistry revealed a significantly higher anion gap and blood sodium concentrations with insulin infusion, i.e., relative metabolic acidosis. Systolic BP and heart rate were slightly (~5 mm Hg) higher in insulin-infused versus control mice. HSD resulted in a modest and transient rise in mean arterial blood pressure (BP), relative to control or insulin-infused, normal-NaCl-fed mice. In kidney, insulin infusion: (1) increased total and phosphorylated (serine-1177) endothelial nitric oxide synthase (eNOS) band densities; (2) reduced band density of the uncoupled form of eNOS; and (3) increased renal homogenate nitric oxide synthase (NOS) activity. Despite this, plasma and urine levels of nitrates plus nitrites (NOx) fell with insulin infusion, by day 14 (40⁻50%) suggesting worsening of resistance. Overall, insulin infusion ramps up the cellular means in kidney to increase vasodilatory and natriuretic NO, but in the long term may be associated with worsening of insulin receptor resistance.

Keywords: blood pressure; heart rate; hypertension; metabolic syndrome; oxidative stress.

Figure 1

Blood pressure—Daily mean (A) systolic and (B) diastolic blood pressures in control (vehicle infused, 1% NaCl diet), insulin infused (fed 1% NaCl all study) and insulin infused plus high-NaCl diet (4%, high NaCl second week,) treated mice; (C) light-to-dark period mean arterial pressure (MAP) ratio on various days; (D) delta (change) in mean arterial pressure (MAP) in various periods; (mean ± sem, n = 4–7/group); * indicates a significant difference from control and † from insulin-treated, by multiple comparisons testing following a significant one-way analysis of variance (ANOVA, p < 0.05).

Figure 2

Heart rate—(A) Daily average heart rate (HR) in control (vehicle infused, 1% NaCl diet), insulin infused (fed 1% NaCl all study) and insulin infused plus high-NaCl diet (4%, HS) treated mice; in various periods (B) light-to-dark period HR ratio on various days (mean ± sem, n = 4–7/group); * indicates a significant difference from control, by multiple comparisons testing following a significant one-way ANOVA (p < 0.05).

Figure 3

Body and kidney weights—(A) Final mean body weights in the three groups; (B) final kidney weights (average right and left kidneys); * indicates a significant (p < 0.05) difference between groups (mean ± sem, n = 11–12/group).

Figure 4

Plasma parameters—(A) Insulin concentration; (B) nitrates plus nitrites (NOx) concentration; * indicates a significant (p < 0.05) difference between groups (mean ± sem, n = 7–8/group).

Figure 5

Urine NOx activity—(A) Urine NOx (24-h collection) during baseline and days 1, 7, and 14; (B) cortex NOS activity; (C) Medulla NOS activity (combined inner medulla and inner stripe of the outer medulla); (D) cortex arginase activity; (mean ± sem, n = 8–12/group for (A–C) and 3–4/group for (D)). * indicates a significant difference between groups (p < 0.05); ‡ indicates a significant difference from baseline, and † between identified days, by two-way repeated measures (RM) ANOVA followed by multiple comparison’s testing.

Figure 6

Western blotting for endothelial nitric oxide synthase (eNOS) protein—(A) Representative Western blots of cortex homogenates. Separate blots were probed for p-S1177-eNOS, p-Y657-eNOS, and total eNOS. Equal amounts of protein were loaded in each lane. Summaries of band densities (normalized to β-actin) for (B) p-S1177-eNOS, p-Y657-eNOS, total eNOS; (C) Representative western blots of outer medulla homogenates. Summaries of band densities (normalized to β-actin) for (D) p-S1177-eNOS, p-Y657-eNOS, total eNOS; ∗ indicates a significant difference between groups by one-way ANOVA (mean ± sem; n = 8–12/group for statistics).

Figure 7

Western blot of eNOS from unreduced samples—(A) Representative Western blot for eNOS run on cortex homogenates solubilized with Laemmli without dithiotrietol (DTT) and run under non-reducing conditions. Arrows indicate coupled (active, 280 kDa) and uncoupled (140 kDa) bands. (B) Band densities summary; * indicates a significant difference between groups by one-way ANOVA (p < 0.05, mean ± sem; n = 8/group for statistics).