Declining muscle NAD+ in a hyperandrogenism PCOS mouse model: Possible role in metabolic dysregulation

Abstract

Polycystic ovary syndrome (PCOS) is a common endocrine disorder, defined by reproductive and endocrine abnormalities, with metabolic dysregulation including obesity, insulin resistance and hepatic steatosis. Recently, it was found that skeletal muscle insulin sensitivity could be improved in obese, post-menopausal, pre-diabetic women through treatment with nicotinamide mononucleotide (NMN), a precursor to the prominent redox cofactor nicotinamide adenine dinucleotide (NAD⁺). Given that PCOS patients have a similar endocrine profile to these patients, we hypothesised that declining NAD levels in muscle might play a role in the pathogenesis of the metabolic syndrome associated with PCOS, and that this could be normalized through NMN treatment. Here, we tested the impact of NMN treatment on the metabolic syndrome of the dihydrotestosterone (DHT) induced mouse model of PCOS. We observed lower NAD levels in the muscle of PCOS mice, which was normalized by NMN treatment. PCOS mice were hyperinsulinaemic, resulting in increased adiposity and hepatic lipid deposition. Strikingly, NMN treatment completely normalized these aspects of metabolic dysfunction. We propose that addressing the decline in skeletal muscle NAD levels associated with PCOS can normalize insulin sensitivity, preventing compensatory hyperinsulinaemia, which drives obesity and hepatic lipid deposition, though we cannot discount an impact of NMN on other tissues to mediate these effects. These findings support further investigation into NMN treatment as a new therapy for normalizing the aberrant metabolic features of PCOS.

Keywords: Animal model; Hyperandrogenism; NMN; Polycystic ovary syndrome (PCOS).

Figure 1

Experimental design and validation.A. For this study, PCOS was induced in female mice by subcutaneous insertion of a dihydrotestosterone (DHT) implant in a peripubertal mice for 12 weeks. Control mice were implanted with a blank (empty) pellet. Mice were treated for 8 weeks with NMN (2 g/L) via drinking water or regular water from week 12. Food intake, estrous cycling, fasted blood glucose and insulin and glucose tolerance test (GTT) were assessed before collection of serum and tissues following 8 full weeks of NMN administration. B Number of completed cycles in a 12-day period, showing DHT-induced acyclicity in PCOS females and NMN had no influence on the number of completed cycles, n = 6–7 mice per experimental group. Data expressed as the mean ± SEM. C, Estrous cycle pattern in representative females. P, proestrus; E, estrus; M, metestrus; D, diestrus. D, Number of corpora lutea per ovary, showing DHT-induced anovulation in PCOS mice and there was no significant effect of NMN, n = 4 ovaries per experimental group. Data expressed as the mean ± SEM. E, Histological sections of representative ovaries from Control and DHT-induced PCOS mice treated with and without the NMN. Star, corpora lutea.

Figure 2

PCOS depletes the muscle NAD ⁺ metabolome. Levels of NAD⁺, NADH and nicotinamide (NAM) in muscle (A–C) and liver (D–F) of PCOS mice treated with or without NMN. These metabolites form part of the recycling pathway for NAD⁺ synthesis, shown in G. Metabolites measured by mass spectrometry and normalised to tissue weight. n = 6–8 per group and data expressed as mean ± SD. Analysed by one-way ANOVA with a post-hoc Dunnett's multiple comparison test, p-value results as indicated on graphs.

Figure 3

NMN ameliorates DHT-induced hyperinuslinaemia and obesity.A. Fasting blood insulin levels are increased with DHT treatment and reduced by NMN co-treatment, despite no change in B pancreatic islet area, as assessed by immunohistochemistry, with C representative images shown. D, Fasting blood glucose levels were obtained during glucose tolerance test (GTT), with E incremental area under the curve (iAUC) of GTTs shown. These fasting values were used to calculate F, homeostatic measure of insulin resistance (HOMA-IR), showing a significant increase in HOMA-IR in PCOS mice, but a partial amelioration in PCOS + NMN females. DHT treatment increased G body weight, with no impact of NMN treatment on H food intake in PCOS + NMN mice before and after treatment. PCOS mice were obese, with I increased fat pad weights in inguinal, parametrial, mesenteric, retroperitoneal and brown fat-pad weights, however these were reduced in animals co-treated with NMN. J, Histomorphometric assessment of adipocyte diameter in parametrial fat pads, with K, representative histology shown. Increased adipocyte dimeter was matched by F decreased serum levels of adiponectin. n = 6–7 per experimental group and data are expressed as the mean ± SD. Analysed by one-way ANOVA with a post-hoc Dunnett's multiple comparison test, p-value results as indicated on graphs. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Figure 4

Resolution of hepatic steatosis with NMN treatment. Liver sections were subject to oil red O staining to assess hepatic lipid deposition, as assessed by A, area of oil red O staining in each group, with B representative sections shown. The pattern of oil red O staining was consistent with measurements of C liver triglyceride content, showing a DHT-induced increase in PCOS mice, and a beneficial impact of NMN administration. Unlike liver, serum levels of D cholesterol and E triglycerides showed no differences between experiment groups. n = 6–7 per experimental group and data are expressed as the mean ± SEM. Analysed by one-way ANOVA with a post-hoc Dunnett's multiple comparison test, p-value results as indicated on graphs. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)