Metabolic patterns in insulin-sensitive male hypogonadism

Abstract

Male hypogonadism is a disorder characterised by low levels of the hormone testosterone. At beginning subjects with low levels of testosterone do not show insulin resistance (insulin-sensitive patients), which develops over time (insulin-resistance patients). To analyse the metabolic alterations mainly related to decreased testosterone, we performed metabolomics investigations on the plasma of males with hypogonadism who showed normal insulin levels. Plasma from patients with low testosterone (<8 nmol/l) and homeostatic model assessment for insulin-resistance-index (HOMAi) < 2.5, as well as matched controls, was analysed by UHPLC and mass spectrometry. Then metabolites were then subjected to multivariate statistical analysis and grouped by metabolic pathways. Glycolysis was not altered, as expected for the presence of insulin activity, but imbalances in several other pathways were found, such as the pentose phosphate pathway (PPP), glycerol shuttle, malate shuttle, Krebs cycle (TCA) and lipid metabolism. The PPP was significantly upregulated. Moreover, while the first steps of the Krebs cycle were downregulated, 2-oxoglutarate was replenished via glutaminolysis. Since glutaminolysis leads to an activation of the malate aspartate cycle, greater amounts of NADH and ATP with respect to the control were recorded. The activation of the glycerol shuttle was also recorded, with consequent lower triglyceride production and downregulation of beta-oxidation. This explained the moderately increased dyslipidaemia, as well as the mild increase in body mass index (BMI) observed in insulin-sensitive hypogonadism. Finally, a significant decrease in carnosine was recorded, explaining the muscle weakness commonly observed.

Fig. 1. Metabolic Set Enrichment Analysis (MSEA) showing the most altered metabolites revealed in the plasma of hypogonadal men.

Colour intensity (white to red) reflects increasing statistical significance, while the circle diameter covaries with pathway impact. a The graph was obtained by plotting on the y-axis the −log of p-values from pathway enrichment analysis and on the x-axis the pathway impact values derived from pathway topology analysis. Metabolic Pathway Analysis (MetPA). All the matched pathways are displayed as circles. The colour and size of each circle are based on the p-value and pathway impact value, respectively. b The graph was obtained by plotting on the y-axis the −log of p-values from the pathway enrichment analysis and on the x-axis the pathway impact values derived from the pathway topology analysis

Fig. 2. Altereted pathways in hypogonadic IS patients.

Intermediates of glycolysis (a). Intermediates of the pentose phosphate pathway (b). Total amount of PPP metabolites in the plasma appears to be increased with respect to the control. Intermediates of the glycerol shuttle (c). The columns represent the mean ± SD (n = 15) metabolite concentration over hypogonadal plasma. *p < 0.05, **p < 0.01, against hypogonadal men

Fig. 3. Intermediates of TCA measured in the plasma of hypogonadal patients, revealing that only the first part of this metabolic pathway was reduced.

Acyl-carnitine, fundamental to the transport of fatty acids into mitochondria, was less highly produced from acetyl-CoA (a). A portion of acetyl-CoA was converted into mevalonic acid and then into cholesterol (b). The replenishment of TCA intermediates was due of glutaminolysis (c) a stepwise process by which glutamine is converted into glutamate, which is in turn transformed into α-ketoglutarate and aspartate, which are converted to oxaloacetate. Metabolites are expressed as the mean ± SD (n = 15) concentration over hypogonadal plasma. *p < 0.05, **p < 0.01, against hypogonadal men

Fig. 4. Energy metabolism.

The replenishment of TCA intermediates through glutaminolysis leads to activation of the malate aspartate cycle, increased of NADH and ATP, and decreased AMP and NAD (a, b). Increased glutathione disulphide as a marker of oxidative stress (c). Metabolites are expressed as the mean ± SD (n = 15) concentration over hypogonadal plasma. *p < 0.05, against hypogonadal men

Fig. 5. Amino acids metabolism.

Amino acids involved in the malate-aspartate shuttle (a). Decreases branched-chain amino acids (BCAA) leucine, isoleucine and valine (b). Plasma amino acids that were significantly increased (c). Metabolites are expressed as the mean ± SD (n = 15) concentration over hypogonadal plasma. *p < 0.05, **p < 0.01, against hypogonadal men

Fig. 6. Metabolism of carnosine production from β-alanine.

The columns represent mean ± SD (n = 15) concentration over hypogonadal plasma. *p < 0.05, against hypogonadal men