Obesity, type 2 diabetes, and impaired insulin-stimulated blood flow: role of skeletal muscle NO synthase and endothelin-1

Abstract

Increased endothelin-1 (ET-1) and reduced endothelial nitric oxide phosphorylation (peNOS) are hypothesized to reduce insulin-stimulated blood flow in type 2 diabetes (T2D), but studies examining these links in humans are limited. We sought to assess basal and insulin-stimulated endothelial signaling proteins (ET-1 and peNOS) in skeletal muscle from T2D patients. Ten obese T2D [glucose disposal rate (GDR): 6.6 ± 1.6 mg·kg lean body mass (LBM)^-1·min^-1] and 11 lean insulin-sensitive subjects (Lean GDR: 12.9 ± 1.2 mg·kg LBM^-1·min^-1) underwent a hyperinsulinemic-euglycemic clamp with vastus lateralis biopsies taken before and 60 min into the clamp. Basal biopsies were also taken in 11 medication-naïve, obese, non-T2D subjects. ET-1, peNOS (Ser1177), and eNOS protein and mRNA were measured from skeletal muscle samples containing native microvessels. Femoral artery blood flow was assessed by duplex Doppler ultrasound. Insulin-stimulated blood flow was reduced in obese T2D (Lean: +50.7 ± 6.5% baseline, T2D: +20.8 ± 5.2% baseline, P < 0.05). peNOS/eNOS content was higher in Lean under basal conditions and, although not increased by insulin, remained higher in Lean during the insulin clamp than in obese T2D (P < 0.05). ET-1 mRNA and peptide were 2.25 ± 0.50- and 1.52 ± 0.11-fold higher in obese T2D compared with Lean at baseline, and ET-1 peptide remained 2.02 ± 1.9-fold elevated in obese T2D after insulin infusion (P < 0.05) but did not increase with insulin in either group (P > 0.05). Obese non-T2D subjects tended to also display elevated basal ET-1 (P = 0.06). In summary, higher basal skeletal muscle expression of ET-1 and reduced peNOS/eNOS may contribute to a reduced insulin-stimulated leg blood flow response in obese T2D patients.

New & noteworthy: Although impairments in endothelial signaling are hypothesized to reduce insulin-stimulated blood flow in type 2 diabetes (T2D), human studies examining these links are limited. We provide the first measures of nitric oxide synthase and endothelin-1 expression from skeletal muscle tissue containing native microvessels in individuals with and without T2D before and during insulin stimulation. Higher basal skeletal muscle expression of endothelin-1 and reduced endothelial nitric oxide phosphorylation (peNOS)/eNOS may contribute to reduced insulin-stimulated blood flow in obese T2D patients.

Keywords: blood flow; endothelial signaling proteins; insulin resistance.

Fig. 1.

Serum glucose (A) and insulin (B) levels during the hyperinsulinemic-euglycemic clamp in Lean individuals and obese individuals with type 2 diabetes (T2D). Percent change in femoral artery blood flow from basal to 45 min of insulin stimulation in the Lean individuals and obese individuals with T2D (C). Black circles represent the Lean individuals and the open circles represent the obese T2D individuals. All samples were derived at the same time and processed in parallel. Values are means ± SE. *P < 0.05 from Lean.

Fig. 2.

Protein kinase B (AKT; A), phosphorylation of AKT (pAKT; B), endothelial nitric oxide synthase (eNOS; C), phosphorylation of eNOS (peNOS) (D), and endothelin-1 (ET-1) (E) peptide at basal and following 60 min of insulin stimulation in the Lean individuals and obese individuals with type 2 diabetes (T2D). Representative blots are shown for each protein measured (F). Black bars represent the Lean individuals and open bars represent the obese T2D individuals. All samples were derived at the same time and processed in parallel. Values are means ± SE; a.u., arbitrary units.

Fig. 3.

NOS3 mRNA expression (A) and pre-pro endothelin-1 (ET-1) mRNA expression (B) at basal and following 60 min of insulin stimulation in the Lean individuals and obese individuals with type 2 diabetes (T2D). Black bars represent the Lean individuals and open bars represent the obese T2D individuals. All samples were derived at the same time and processed in parallel. Values are means ± SE.

Fig. 4.

Protein kinase B (AKT; A), phosphorylation of AKT (pAKT; B), endothelial nitric oxide synthase (eNOS; C), phosphorylation of eNOS (peNOS; D), and endothelin-1 (ET-1; E) at rest in the Lean individuals, obese type 2 diabetes (T2D) individuals, and obese individuals without T2D. Representative blots for each protein measured (F). All samples were derived at the same time and processed in parallel. Values are means ± SE.