In metabolic disorders associated with atherosclerosis, NO synthesis and/or stability is reduced [5, 6, 14]. To determine if NO bioavailability may modulate the response to a high fat diet, we assessed serum and genetic markers of metabolism in mice with decreased (eNOS ko) as well as increased (DDAH) NO bioavailability.
We found that differing basal levels of NO synthetic capacity influence the response to a HFD as assessed by glucose and adiponectin levels; the angiogenic response; and adipose (BAT and WAT) gene expression. The data suggest that in aggregate, NO activity is protective against some of the metabolic perturbations induced by a high fat diet.
Diet-induced insulin resistance
Epidemiological, clinical and basic research studies have demonstrated that a high fat diet induces insulin resistance. Most studies suggest that increased dietary fat causes whole-body and regional (liver, adipose tissue, muscle) insulin resistance in both animals and humans. Vessby et al. documented that insulin sensitivity was impaired by 10% in healthy individuals who receive an isoenergetic diet containing a high content of saturated fatty acids for 3 months. A change in the composition of the dietary fatty acids, ie. decreasing saturated fatty acid and increasing monounsaturated fatty acid content, improved insulin sensitivity . Substituting 11% of the saturated fatty acids with short-chain omega-3 fatty acids (alpha-linolenic acid, 18:3 omega 3) prevented insulin resistance induced by a saturated-fat diet in rats .
Duplain  and Sydow  using glucose clamp studies reported insulin resistance in eNOS ko mice and increased insulin sensitivity in DDAH transgenic mice. It has been reported that elevated plasma levels of ADMA are associated with insulin resistance, micro/macrovascular diabetic complications, and may predict cardiovascular events in type 2 diabetic patients. In turn, Lu et al. documented that some genetic variations in DDAH1 could contribute to higher risk of type 2 diabetes independently of plasma ADMA levels.
For example, SNP (single nucleotide polymorphism) rs1241321 in DDAH1 was found to be associated with a higher type 2 diabetes risk independently of plasma ADMA levels. AA genotype at rs1241321 appeared to be more insulin-sensitive in comparison to AG/GG individuals. Thus, the DDAH1 gene could play an important role in the pathogenesis of type 2 diabetes .
In our studies in order to access insulin resistance we focused on the fasting levels of glucose and insulin. Our work confirms that a high fat diet rich in saturated fatty acids induces insulin resistance, which was observed in all groups. However, the DDAH animals were resistant to the diet-induced increase in glucose levels observed in the control animals. This was despite a greater weight gain in the DDAH transgenic animals in response to the high fat diet (see discussion below). Previous studies by Tanaka  demonstrated higher NO level in DDAH mice thus lower glucose levels in the DDAH transgenic mice reflect the fact that NO is known to increase glucose transport, in part by increasing the translocation to the cell surface of Glut 4, the active transporter of glucose . In skeletal muscle from eNOS ko mice, which according to Kanetsuna studies present lower NO levels , there is diminished insulin-stimulated glucose uptake, indicating that insulin activation of NO may contribute to the stimulation of glucose transport .
Furthermore, the DDAH transgenic animals exhibited higher adiponectin levels. Adiponectin is an adipocytokine that increases glucose uptake, reduces gluconeogenesis and lipogenesis, and enhances β-oxidation of fat, by activating AMPK and PPARα . A characteristic feature of individuals with diabetes mellitus or insulin resistance is a decrease of adiponectin levels . There appears to be a reciprocal relationship between adiponectin and NO. Adiponectin deficient mice exhibit impaired endothelium-dependent vasodilation . This is likely due to the fact that adiponectin increases the stability of eNOS mRNA and half-life, enhances the association of eNOS with Hsp90 and stimulates the phosphorylation of eNOS, which together lead to increased NO production [32, 33]. Adiponectin may also prevent NO degradation by reducing the production of superoxide anion by endothelial cells . On the other hand, NO appears to positively regulate adiponectin levels. Chronic administration of the NOS inhibitor L-NAME to Sprague-Dawley rats reduces plasma adiponectin levels . Conversely, chronic enhancement of NO synthesis (the DDAH transgenic mouse) is associated with resistance to diet-induced reduction in adiponectin levels (the current study).
High fat diet and angiogenesis
Endothelial NOS activity plays a critical role in angiogenesis and endothelial function. Survival, proliferation and migration of endothelial cells, and their secretion of angiogenic factors, are dependent upon NO [36, 37]. The angiogenic response to ischemia after femoral artery ligation is impaired in eNOS deficient mice, whereas it is enhanced in eNOS overexpressing mice . Similarly, after femoral artery ligation, chronic infusion of ADMA reduces capillary density and perfusion, whereas DDAH overexpression (associated with lower endogenous ADMA levels) increases angiogenic response and improves endothelial function .
One of the diseases associated with impaired endothelium-dependent NO mediated vasodilation (endothelial dysfunction) is congestive heart failure (CHF). Coronary endothelial dysfunction in CHF may be due to decreased DDAH, which in consequence, leads to a higher ADMA level . Moreover, Riccioni et al. showed that patients who received pharmacological treatment for acute congestive HF (diuretics, digoxin, ACE-inhibitors or angiotensin receptor blockers, and nitroglicerin), have significantly higher plasma ADMA levels after pharmacological treatment in comparison to pre-treatment. Such results suggest that acute renal impairment function and the modulation of NOS determine plasma ADMA levels after therapy .
The current study concentrated on the effect of NO availability on angiogenesis in mice. To achieve this aim, another model of angiogenesis, in response to subcutaneous administration of matrigel, was used. Consistent with previous studies, we observed that the vascularity of the matrigel plug was increased in the DDAH transgenic mice, as manifested by a higher number of vessel-like structures (with and without lumen) as well as a greater number of PECAM1 positive cells. Since angiogenesis is crucial for development and growth of all organs (including adipose), NOS activity may in part explain the discordance in body weight gain with the HFD in the different groups. By comparison to the control mice, the HFD induced a greater weight gain in the DDAH mice, whereas in the eNOS-/- animals it induced less weight gain. This observation is consistent with a previous report that eNOS deficient female mice are smaller than wild type ones .
We were surprised to find that angiogenesis in response to the matrigel plug was not reduced in the eNOS-/- mice. It is possible that the matrigel itself, containing a number of angiogenic cytokines including bFGF  potently recruits other angiogenic mechanisms, including inflammatory cells carrying iNOS . It is also possible that in the setting of a HFD, the concomitant inflammation and oxidative stress contribute to the angiogenic stimulus.
Angiogenesis, lipogenesis and metabolism
Adipose tissue is highly vascularized, and each adipocyte is nourished by an extensive capillary network. Adipose tissue may be considered as the largest endocrine gland, as it produces several systemically active adipokines (eg. leptin, adiponectin, resistin) as well as VEGF, angiopoietins, HGF, IGF-1, angiogenin, IL-6, TNF-α and fatty acid metabolites. Many of these may promote the inflammation and angiogenesis response associated with adipose tissue accumulation . It is also documented that adipose tissue endothelial cells promote preadipocyte differentiation . Vascularity of adipose tissue is, therefore, critical for development, maturation, plasticity and functions of adipose tissue as a metabolic and an endocrine organ [47, 48]. Thus, it may seem counterintuitive that, if angiogenesis promotes lipogenesis, then the DDAH animals (which have an enhanced angiogenic response) have a normalized metabolic state. However, DDAH overexpression may have additional effects to offset any metabolic effects of angiogenesis-enhanced lipogenesis. For example, the reduction in ADMA would be anticipated to reduce inflammation. Previous studies have shown that endothelium-derived NO is a potent anti-inflammatory molecule, suppressing the expression of chemokines and adhesion molecules mediating immune cell infiltration . In this regard, there are data indicating that the number of immune cells in adipose tissue is related to insulin resistance . Alternatively, it could be that reductions in ADMA and increased NOS activity could have a direct effect on adipose gene expression. In this respect we observed intriguing differences between the DDAH and eNOS-/- mice in adipose gene expression.
NOS activity and adipose gene expression in response to diet
As shown in Table 2 the expression of markers characteristic for differentiated adipocytes, such as Fabp4, Ucp1, Lpl or Lipe, were downregulated in DDAH animals (ie. with higher NO bioavailability). By contrast, we observed upregulation of genes associated with lipogenesis in eNOS deficient mice. In the adipose tissue of these animals genes regulating adipogenesis (Cebpa, Cebpb, Ppadb, Foxo1, Nr2f1, Mef2d, Ncor, Tgfb, Gdf10, Ucp1, Glut4) as well as fatty acid (Fasn, Acly) and triglyceride (Dgat2, Agpat2) synthesis were upregulated. By comparison to the controls, genes concerning fatty acid oxidation were downregulated in both groups. The studies suggest that increased NO availability promotes changes in adipocyte gene expression, but not in adipogenesis. This may explain why, despite greater angiogenic capacity, the DDAH transgenic mice do not have greater adipogenesis.
Our microarray analysis of gene expression in WAT revealed that a number of genes involved in protection from oxidative stress such as Fos, So d or Gstt were upregulated in DDAH mice. This might reflect a compensatory response to nitrosative stress that may be induced by increased concentrations of the free radical NO . Alternatively, this could reflect direct effects on gene expression by NO, as cGMP can increase the expression of superoxide dismutase 2 .
The main purpose of adipose tissue (especially WAT) is storage of high energy compounds . In addition to regulating vascular tone, nitric oxide plays a crucial role in regulating metabolism of this tissue. It has been shown that in adipocytes, hepatocytes and myocytes nitric oxide activates glucose uptake as well as mitochondrial-biogenesis and catabolism (mitochondrial oxidation of glucose and FFA) . These effects are observed in response to physiological concentrations of NO, endogenously produced by nNOS and eNOS . These studies suggest a catabolic role for NO. The recent discovered mitochondrial NOS (mtNOS) may also play a role, as it maintains a low level of NO generation in healthy tissue (0.004-4.0 nmol NO min-1 mg-1 protein). The mtNOS has significant effects on cellular turnover, metabolism and survival [11, 12].