Steven Murfitt

Inorganic Nitrate Promotes the Browning of White Adipose Tissue Through the Nitrate-Nitrite-Nitric Oxide Pathway

Inorganic nitrate was once considered an oxidation end product of nitric oxide metabolism with little biological activity. However, recent studies have demonstrated that dietary nitrate can modulate mitochondrial function in man and is effective in reversing features of the metabolic syndrome in mice. Using a combined histological, metabolomics, and transcriptional and protein analysis approach, we mechanistically defined that nitrate not only increases the expression of thermogenic genes in brown adipose tissue but also induces the expression of brown adipocyte–specific genes and proteins in white adipose tissue, substantially increasing oxygen consumption and fatty acid β-oxidation in adipocytes. Nitrate induces these phenotypic changes through a mechanism distinct from known physiological small molecule activators of browning, the recently identified nitrate-nitrite-nitric oxide pathway. The nitrate-induced browning effect was enhanced in hypoxia, a serious comorbidity affecting white adipose tissue in obese individuals, and corrected impaired brown adipocyte–specific gene expression in white adipose tissue in a murine model of obesity. Because resulting beige/brite cells exhibit antiobesity and antidiabetic effects, nitrate may be an effective means of inducing the browning response in adipose tissue to treat the metabolic syndrome.

Nitrate enhances skeletal muscle fatty acid oxidation via a nitric oxide-cGMP-PPAR-mediated mechanism

BackgroundInsulin sensitivity in skeletal muscle is associated with metabolic flexibility, including a high capacity to increase fatty acid (FA) oxidation in response to increased lipid supply. Lipid overload, however, can result in incomplete FA oxidation and accumulation of potentially harmful intermediates where mitochondrial tricarboxylic acid cycle capacity cannot keep pace with rates of β-oxidation. Enhancement of muscle FA oxidation in combination with mitochondrial biogenesis is therefore emerging as a strategy to treat metabolic disease. Dietary inorganic nitrate was recently shown to reverse aspects of the metabolic syndrome in rodents by as yet incompletely defined mechanisms.ResultsHerein, we report that nitrate enhances skeletal muscle FA oxidation in rodents in a dose-dependent manner. We show that nitrate induces FA oxidation through a soluble guanylate cyclase (sGC)/cGMP-mediated PPARβ/δ- and PPARα-dependent mechanism. Enhanced PPARβ/δ and PPARα expression and DNA binding induces expression of FA oxidation enzymes, increasing muscle carnitine and lowering tissue malonyl-CoA concentrations, thereby supporting intra-mitochondrial pathways of FA oxidation and enhancing mitochondrial respiration. At higher doses, nitrate induces mitochondrial biogenesis, further increasing FA oxidation and lowering long-chain FA concentrations. Meanwhile, nitrate did not affect mitochondrial FA oxidation in PPARα−/− mice. In C2C12 myotubes, nitrate increased expression of the PPARα targets Cpt1b, Acadl, Hadh and Ucp3, and enhanced oxidative phosphorylation rates with palmitoyl-carnitine; however, these changes in gene expression and respiration were prevented by inhibition of either sGC or protein kinase G. Elevation of cGMP, via the inhibition of phosphodiesterase 5 by sildenafil, also increased expression of Cpt1b, Acadl and Ucp3, as well as CPT1B protein levels, and further enhanced the effect of nitrate supplementation.ConclusionsNitrate may therefore be effective in the treatment of metabolic disease by inducing FA oxidation in muscle.

Adipose tissue fatty acid chain length and mono-unsaturation increases with obesity and insulin resistance

The non-essential fatty acids, C18:1n9, C16:0, C16:1n7, C18:0 and C18:1n7 account for over 75% of fatty acids in white adipose (WAT) triacylglycerol (TAG). The relative composition of these fatty acids (FA) is influenced by the desaturases, SCD1-4 and the elongase, ELOVL6. In knock-out models, loss of SCD1 or ELOVL6 results in reduced Δ9 desaturated and reduced 18-carbon non-essential FA respectively. Both Elovl6 KO and SCD1 KO mice exhibit improved insulin sensitivity. Here we describe the relationship between WAT TAG composition in obese mouse models and obese humans stratified for insulin resistance. In mouse models with increasing obesity and insulin resistance, there was an increase in scWAT Δ9 desaturated FAs (SCD ratio) and FAs with 18-carbons (Elovl6 ratio) in mice. Data from mouse models discordant for obesity and insulin resistance (AKT2 KO, Adiponectin aP2-transgenic), suggested that scWAT TAG Elovl6 ratio was associated with insulin sensitivity, whereas SCD1 ratio was associated with fat mass. In humans, a greater SCD1 and Elovl6 ratio was found in metabolically more harmful visceral adipose tissue when compared to subcutaneous adipose tissue.

Mechanistic insights revealed by lipid profiling in monogenic insulin resistance syndromes

BackgroundEvidence from several recent metabolomic studies suggests that increased concentrations of triacylglycerols with shorter (14–16 carbon atoms), saturated fatty acids are associated with insulin resistance and the risk of type 2 diabetes. Although causality cannot be inferred from association studies, patients in whom the primary cause of insulin resistance can be genetically defined offer unique opportunities to address this challenge.MethodsWe compared metabolite profiles in patients with congenital lipodystrophy or loss-of-function insulin resistance (INSR gene) mutations with healthy controls.ResultsThe absence of significant differences in triacylglycerol species in the INSR group suggest that changes previously observed in epidemiological studies are not purely a consequence of insulin resistance. The presence of triacylglycerols with lower carbon numbers and high saturation in patients with lipodystrophy suggests that these metabolite changes may be associated with primary adipose tissue dysfunction. The observed pattern of triacylglycerol species is indicative of increased de novo lipogenesis in the liver. To test this we investigated the distribution of these triacylglycerols in lipoprotein fractions using size exclusion chromatography prior to mass spectrometry. This associated these triacylglycerols with very low-density lipoprotein particles, and hence release of triacylglycerols into the blood from the liver. To test further the hepatic origin of these triacylglycerols we induced de novo lipogenesis in the mouse, comparing ob/ob and wild-type mice on a chow or high fat diet, confirming that de novo lipogenesis induced an increase in relatively shorter, more saturated fatty acids.ConclusionsOverall, these studies highlight hepatic de novo lipogenesis in the pathogenesis of metabolic dyslipidaemia in states where energy intake exceeds the capacity of adipose tissue.

Comparisons of metabolic and physiological changes in rats following short term oral dosing with pesticides commonly found in food

¹H Nuclear Magnetic Resonance spectroscopy has been used to profile urinary metabolites in male Fischer F344 rats in order to assess the metabolic changes induced by oral exposure to two benzimidazole fungicides (carbendazim and thiabendazole) and two bipyridyllium herbicides (chlormequat and mepiquat). Exposure levels were selected to be lower than those expected to cause overt signs of toxicity. We then compared the sensitivity of the metabolomics approach to more traditional methods of toxicity assessment such as the measurement of growth and organ weights. Separate, acute exposure experiments were conducted for each pesticide to identify potential metabolic markers of exposure across four doses (and a control). Growth, organ weights and feeding/drinking rates were not significantly affected by any compounds at any dose levels tested. In contrast, metabolic responses were detected within 8 and 24h for chlormequat and mepiquat, and after 24h for carbendazim and thiabendazole. These results demonstrate the potential for the use of metabolomics in food toxicity testing.

Inorganic nitrate mimics exercise-stimulated muscular fiber-type switching and myokine and GABA release

Exercise is an effective intervention for the prevention and treatment of type 2 diabetes. Skeletal muscle combines multiple signals that contribute to the beneficial effects of exercise on cardiometabolic health. Inorganic nitrate increases exercise efficiency, tolerance, and performance. The transcriptional regulator peroxisome proliferator–activated receptor γ coactivator 1α (PGC1α) coordinates the exercise-stimulated skeletal muscle fiber-type switch from glycolytic fast-twitch (type IIb) to oxidative slow-twitch (type I) and intermediate (type IIa) fibers, an effect reversed in insulin resistance and diabetes. We found that nitrate induces PGC1α expression and a switch toward type I and IIa fibers in rat muscle and myotubes in vitro. Nitrate induces the release of exercise/PGC1α-dependent myokine FNDC5/irisin and β-aminoisobutyric acid from myotubes and muscle in rats and humans. Both exercise and nitrate stimulated PGC1α-mediated γ-aminobutyric acid (GABA) secretion from muscle. Circulating GABA concentrations were increased in exercising mice and nitrate-treated rats and humans; thus, GABA may function as an exercise/PGC1α-mediated myokine-like small molecule. Moreover, nitrate increased circulating growth hormone levels in humans and rodents. Nitrate induces physiological responses that mimic exercise training and may underlie the beneficial effects of this metabolite on exercise and cardiometabolic health.

Metabolomic Analysis of Akt1-Mediated Muscle Hypertrophy in Models of Diet-Induced Obesity and Age-Related Fat Accumulation

Akt1 is a serine/threonine kinase that promotes cell growth and survival. Previously, Akt1 activation in a double transgenic (DTG) mouse model fed a high-fat/high-sucrose (HF/HS) diet was found to promote type IIb muscle growth and to lead to a significant reduction in obesity. Here, we have used metabolomics to examine the metabolic perturbations in blood serum and liver and gastrocnemius tissues of the DTG mice. Multivariate statistics highlighted consistent metabolic changes in gastrocnemius muscle following Akt1 activation, which included significant reductions of serine and histidine-containing dipeptides (anserine and carnosine), in addition to increased concentrations of phosphorylated sugars. In addition, Akt1-mediated regression in obesity could be associated with increased glycolysis in gastrocnemius muscle as well as increased gluconeogenesis, glycogenolysis, and ketogenesis in the liver. In old DTG animals, Akt1 activation was found to improve glucose metabolism and confer a beneficial effect in the regression of age-related fat accumulation. This study identifies metabolic changes induced by Akt1-mediated muscle growth and demonstrates a cross-talk between distant organs that leads to a regression of fat mass. The current findings indicate that agents that promote Akt1 induction in muscle have utility in the regression of obesity.

Metabolomics and Lipidomics Study of Mouse Models of Type 1 Diabetes Highlights Divergent Metabolism in Purine and Tryptophan Metabolism Prior to Disease Onset

With the increase in incidence of type 1 diabetes (T1DM), there is an urgent need to understand the early molecular and metabolic alterations that accompany the autoimmune disease. This is not least because in murine models early intervention can prevent the development of disease. We have applied a liquid chromatography (LC-) and gas chromatography (GC-) mass spectrometry (MS) metabolomics and lipidomics analysis of blood plasma and pancreas tissue to follow the progression of disease in three models related to autoimmune diabetes: the nonobese diabetic (NOD) mouse, susceptible to the development of autoimmune diabetes, and the NOD-E (transgenic NOD mice that express the I-E heterodimer of the major histocompatibility complex II) and NOD-severe combined immunodeficiency (SCID) mouse strains, two models protected from the development of diabetes. All three analyses highlighted the metabolic differences between the NOD-SCID mouse and the other two strains, regardless of diabetic status indicating that NOD-SCID mice are poor controls for metabolic changes in NOD mice. By comparing NOD and NOD-E mice, we show the development of T1DM in NOD mice is associated with changes in lipid, purine, and tryptophan metabolism, including an increase in kynurenic acid and a decrease in lysophospholipids, metabolites previously associated with inflammation.

Erratum: Adipose tissue fatty acid chain length and mono-unsaturation increases with obesity and insulin resistance (Scientific Reports (2015) 5:18366 DOI:10.1038/srep18366)

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