Dietary nitrate ameliorates metabolic syndrome in mice

I became interested in nitrate because early studies have been consistently showing that it may improve exercise performance by lowering the oxygen cost of exercise, and this research is in humans.  Reading through other literature, it seems there is a paradigm shift occurring from the thought of nitrate as undesirable and toxic toward an understanding of an under-appreciated metabolic role by this bioactive molecule from food.  After all, it can be found in high amounts in some vegetables.

A recent animal study by Carlström et al. pins nitrate as a very important molecule central to metabolic homeostasis (open access here).

The authors discuss research supporting a defect in nitric oxide (NO) synthesis and bioavailability as a potential link between symptoms of metabolic syndrome (insulin resistance, dyslipidemia, hypertension, weight gain, etc).  They then highlight the recent work showing that nitrate is a substrate for NO synthesis (nitrate->nitrite->NO; for basic metabolism see this post), as well as being a long understood oxidation product of NO metabolism.

In this study, the investigators performed several experiments within:

Experiment 1: Because eNOS gene disruption is known to cause metabolic syndrome in mice and humans (the major NO production pathway, up to 70%), mice deficient in eNOS were given nitrate acutely (intraperitoneally) and chronically (dietary via sodium nitrate) to verify if plasma and tissue levels of nitrite and nitrosylation products increased (the latter suggests some cGMP- a messenger mediating vasodilation- independent mechanisms). They did acutely but only at the tissue level chronically.  The dose of sodium nitrate was 0.1 mmol/kg/day to make up for the eNOS deficiency (which consequent to diminishing NO production, slows endogenous nitrate production); this is equivalent to 100-300 grams of nitrate rich vegetables.

The amount of nitrate in vegetables can be very inconsistent because of a number of environmental factors, but here is a table of nitrate rich foods I pulled one of the previous posts:

nitratecontent

(Hord et al. 2009)

Experiment 2: The authors again fed eNOS deficient mice nitrate, and in drinking water, as well as a control eNOS deficient group plain water to see if metabolic dysfunctions could be ameliorated.  After 7 weeks, the nitrate fed group had reduced body weight, visceral fat, and triglycerides compared to the control group.  Nitrate nearly corrected glucose intolerance compared to the disturbed untreated group, and it had no effect on young wild-type or neuronal NO deficient mice (these were done to test if oxidative stress may be involved, see below).  It also lowered average arterial blood pressure in the nitrate group.  Interestingly, mitochondrial biogenesis appeared not to mediate these effects, as a number of relevant measures were similar between the 2 groups.

Since NO has a very short half life (seconds), its effects are short lived. The authors propose the following (emphasis mine):

The dose of dietary nitrate was chosen only to just replace what is being generated by eNOS under normal conditions (19). The fact that this very modest amount had such profound biological effects supports the intriguing possibility that endogenous nitrate levels are already sufficient to affect cellular processes. Thus, in addition to the second-by-second regulation of vascular tone by eNOS-derived NO, its oxidized end-product nitrate may serve as a long-lived reservoir for NO-like bioactivity in tissues. This result would be mechanistically similar to the earlier proposed role of S-nitrosothiols (23) or nitrite (10) as stable carriers and transducers of NO-like bioactivity in blood.

Also:

Although nitrite is clearly an intermediate in bioactivation of nitrate (10, 16, 24), the terminal effector may be one of several related bioactive nitrogen oxide species, including NO (10), S-nitrosothiols, and nitrated fatty acids (25).

I’ve never heard of nitrated fatty acids- that last reference is an interesting study administering nitrated fatty acids to ob/ob mice which lowered insulin and glucose in without increasing weight gain.  This is significant because the nitrated fatty acids are PPARgamma agonists, but have more selective properties on PPARgamma than thiazolidinediones which bind to PPARgamma but do often cause weight gain.  A new drug class is emerging it appears.

So we don’t know exactly how this works yet- the relative contributions of the products of nitrate metabolism, such as bioactive nitrogen oxide species (like NO), S-nitrosothiols, and nitrated fatty acids need to be teased apart.

The authors address a lack of effect on mitochondrial measures from nitrate, since an effect on mitochondria would be assumed as a strong link to insulin resistance, by suggesting oxygen consumption, substrate oxidation, and/or ROS generation may be affected. The latter is supported by the wild-type and nNOS deficient experiments which shouldn’t experience exaggerated oxidative stress like the eNOS knockouts.

Finally, their concluding paragraph is of interest:

The present findings are highly relevant from a nutritional perspective as well, as the amount of nitrate used is readily achievable via a normal diet. Recent studies show that the same dose of nitrate used here (0.1 mmol·kg−1·d−1) is sufficient for induction of NO-like bioactivity in humans, including a robust reduction in blood pressure, inhibition of platelet aggregation, and improvement of endothelial function (15, 16). Epidemiological data clearly suggest that a diet rich in vegetables protects against cardiovascular disease and development of type 2 diabetes (39, 40). Interestingly, in a recent metanalysis on fruit and vegetable intake and incidence of type 2 diabetes, green leafy vegetables were specifically identified to be beneficial (41). Long-term intervention studies in humans are warranted to explore if such protective effects are related to the high nitrate content of this food group. If the findings presented here are applicable in humans, the current view of inorganic nitrate as an unwanted toxic residue in the food chain may have to be revised.

Thought provoking indeed!

Would people with metabolic dysfunction necessarily have to eat 100s of grams of nitrate rich vegetables to increase endogenous nitrate/nitrite? Nope- it should be noted that exercise elevates nitrite and cGMP in patients with metabolic syndrome, calorie restriction increases eNOS expression and cGMP concentrations (and eNOS may mediate many of the life extension effects), and serum nitrite, nitrate, and plasma cGMP increase in weight loss trials.  This suggests that there is overlap here (exercise, calorie restriction, and signaling from many constituents from plants often have considerable overlap).  These variables needs to be considered in studies.

Reference

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. Proceedings of the National Academy of Sciences of the United States of America, 107 (41), 17716-20 PMID: 20876122

  • Herb

    So now it seems clear – or at clearer – why both celery and beets have been shown to lower blood pressure. More nitrate equals more NO equals lower BP.

    • http://www.recomp.com Colby

      Yes- a number of studies have found that isolated nitrate or foods rich in nitrate (like beetroot juice) consistently lower BP