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Inorganic nitrate supplementation enhances skeletal muscle insulin sensitivity in nNOS null mice

Physiological concentrations of nitric oxide (NO) are essential for normal skeletal muscle and endothelial function. People with type 2 diabetes have decreased skeletal muscle neuronal NO synthase (nNOS) expression. NO can also be formed from nitrite (NO₂) and nitrate (NO₃), which is found in high quantities in green leafy vegetables. Therefore, NO₂ and NO₃ can provide an alternative source of NO which may be of great importance. Several studies have recently shown NO₃ supplementation improves disturbed glucose metabolism in endothelial NOS (eNOS) null mice (Carlström et al., 2010) and exhibit anti-obesity and anti-diabetic effects in ob/ob mice, C57BL/6J mice and rats (Roberts et al., 2015). Long term (17 months) dietary nitrate supplementation in C57BL/6J mice improves fasting insulin and insulin response (Hezel et al., 2015).

In our study, dietary supplementation of NO₃ at 1mM (Carlström et al., 2010) in drinking water was given for 14 weeks to male and female nNOS null mice which are insulin resistant (Huang et al., 1993; Shankar et al., 2000). IpGTT, IpITT, ex vivo muscle insulin stimulation and muscle mitochondria function were examined. After 14 weeks, overnight-fasted mice were anesthetised with pentobarbital sodium (60 mg/kg intraperitoneal) and both extensor digitorum longus (EDL) and soleus muscles were rapidly dissected for insulin-stimulated glucose uptake ex vivo, gastrocnemius muscles were dissected for mitochondria respiration analysis and brown and white fat, liver, tibialis anterior (TA) muscles and serum were collected for further analysis. We found chronic nitrate treatment lowered overnight fasting glucose levels in males (4.2 mM vs 4.9 mM, P=0.02) and improved glucose tolerance in females (P=0.0001-0.02). In both genders, NO₃ treatment enhanced EDL skeletal muscle insulin-stimulated glucose uptake (5.3-fold increase in NO₃ treatment group compared with 3.2 fold increase in vehicle group, P≤0.01). There was no effect of NO₃ treatment on insulin-stimulated glucose uptake in the soleus. Chronic nitrate treatment had no effect on body weight, caloric intake, fat composition or skeletal muscle mitochondria function in any group.

In conclusion, these results suggest that nitrate supplementation may increase flux through the nitrate–nitrite–NO pathway which partly compensates for a lack of skeletal muscle endogenous NO generation by nNOS.

Carlström M, Larsen FJ, Nyström T, Hezel M, Borniquel S, Weitzberg E, Lundberg JO. (2010). Dietary inorganic nitrate reverses features of metabolic syndrome in endothelial nitric oxide synthase-deficient mice. Proc Natl Acad Sci USA 107, 17716-20.

Roberts LD, Ashmore T, Kotwica AO, Murfitt SA, Fernandez BO, Feelisch M, Murray AJ, Griffin JL. (2015) Inorganic nitrate promotes the browning of white adipose tissue through the nitrate-nitrite-nitric oxide pathway. Diabetes 64, 471–484.

Hezel MP, Liu M, Schiffer TA, Larsen FJ, Checa A, Wheelock CE, Carlström M, Lundberg JO, Weitzberg E. (2015) Effects of long-term dietary nitrate supplementation in mice. Redox Biol 29, 234-242.

Huang PL, Dawson TM, Bredt DS, Snyder SH, Fishman MC. (1993) Targeted disruption of the neuronal nitric oxide synthase gene. Cell 75, 1273–1286.

Shankar RR, Wu Y, Shen HQ, Zhu JS, Baron AD. (2000). Mice with gene disruption of both endothelial and neuronal nitric oxide synthase exhibit insulin resistance. Diabetes 49, 684-687.