Introduction:

Deficiency of nicotinamide nucleotide transhydrogenase (NNT), a mitochondrial enzyme involved in antioxidant defense, impairs glucose tolerance in male mice [1]. However, females generally show lower levels of oxidative stress than males [2], whereas aging is associated with progressively increased oxidative stress [3], suggesting possible sex- and age-specific effects of NNT deletion. In addition, while skeletal muscle is the primary site for glucose disposal, it remains unknown if NNT deficiency directly impairs muscle function.

Aims:

This study aimed to investigate whether the effects of NNT deficiency on glucose metabolism and muscle function are sex-dependent across the lifespan.

Methods:

All animal handling procedures were conducted in accordance with the EU Directive 2010/63/EU and were approved by the Animal Welfare Committee of Wageningen University (2020.W-0019.004). Female and male congenic BL6JRcc.BL6J-Nnt^C57BL/6J/Wuhap (Nnt^Δ) mice and B6JRcc(B6J)-Nnt⁺/Wuhap (Nnt^WT) mice were utilized for experiments at 3, 6, 12, 18, and 24 months of age (n=13-15 per experimental group). Body weight, body composition, and food intake were measured. Oral glucose tolerance test (OGTT) were performed to assess glucose handling, and glucose and insulin levels were measured via a glucose meter and insulin ELISA kits, respectively. The Rotarod and inverted mesh hanging test (IMHT) was performed to assess motor coordination/flexibility and muscle strength/endurance, respectively. All data are presented as means ± SD. Data at each age point were analyzed using two-way ANOVA followed by Tukey’s post hoc test, with genotype and sex as fixed factors. Statistical significance was set at P<0.05.

Results:

Female mice exhibited significantly lower body weight and lean mass compared to male mice at different ages (P<0.0007). Glucose area under the curve (AUC) during the OGTT was lower in females compared to males at 3 months (1365.2±178.0 female vs. 1605.3±77.0 mmol/L·min male, P=0.039) and 12 months (1534.8±236.2 female vs. 1796.3±176.5 mmol/L·min male, P=0.013), indicating greater glucose tolerance. However, this protective effect in females disappeared at older ages (18, 24 months, P>0.224). Serum insulin AUC and basal insulin resistance (HOMA-IR) were lower in females compared to males at both younger (3 months, 54.4-61.2% lower, P<0.0001) and older ages (12, 18, 24 months, 35.3-73.6% lower in females vs. males, P<0.026). Nevertheless, NNT deficiency impaired glucose tolerance, an effect that occurred in both males and females at all ages tested (P<0.039). With respect to muscle function, females outperformed males in motor coordination on the Rotarod (P<0.001 to P=0.066) and muscle endurance during the IMHT (P<0.001 to P=0.063). NNT deficiency did not affect motor coordination (P>0.069 to P=0.745). However, muscle strength and endurance were lower in mice lacking NNT at 3 months (17370±2820 Nnt^WT vs. 12394±3398 sec·g Nnt^Δ, P=0.009), 18 months (1322±324 Nnt^WT vs. 865±11 sec·g Nnt^Δ, P=0.02), and 24 months (1341±405 Nnt^WT vs. 867±98 sec·g Nnt^Δ, P=0.015), independently of sex.

Conclusions:

Females display better glucose tolerance, insulin sensitivity, and muscle endurance compared to males. Despite these sex-driven differences, NNT deficiency drives glucose intolerance in both males and females across all ages, and impairs muscle function during early adulthood and old age.