Reduced dietary intake of micronutrients with antioxidant properties negatively impacts muscle health in aged mice

Abstract

Background: Inadequate intake of micronutrients with antioxidant properties is common among older adults and has been associated with higher risk of frailty, adverse functional outcome, and impaired muscle health. However, a causal relationship is less well known. The aim was to determine in old mice the impact of reduced dietary intake of vitamins A/E/B6/B12/folate, selenium, and zinc on muscle mass, oxidative capacity, strength, and physical activity (PA) over time.

Methods: Twenty-one-month-old male mice were fed either AIN-93-M (control) or a diet low in micronutrients with antioxidant properties (=LOWOX-B: 50% of mouse recommended daily intake of vitamins A, E, B6, and B12, folate, selenium, and zinc) for 4 months. Muscle mass, grip strength, physical activity (PA), and general oxidative status were assessed. Moreover, muscle fatigue was measured of m. extensor digitorum longus (EDL) during an ex vivo moderate exercise protocol. Effects on oxidative capacity [succinate dehydrogenase (SDH) activity], muscle fibre type, number, and fibre cross-sectional area (fCSA) were assessed on m. plantaris (PL) using histochemistry.

Results: After 2 months on the diet, bodyweight of LOWOX-B mice was lower compared with control (P < 0.0001), mainly due to lower fat mass (P < 0.0001), without significant differences in food intake. After 4 months, oxidative status of LOWOX-B mice was lower, demonstrated by decreased vitamin E plasma levels (P < 0.05) and increased liver malondialdehyde levels (P = 0.018). PA was lower in LOWOX-B mice (P < 0.001 vs. control). Muscle mass was not affected, although PL-fCSA was decreased (~16%; P = 0.028 vs. control). SDH activity and muscle fibre type distribution remained unaffected. In LOWOX-B mice, EDL force production was decreased by 49.7% at lower stimulation frequencies (P = 0.038), and fatigue resistance was diminished (P = 0.023) compared with control.

Conclusions: Reduced dietary intake of vitamins A, E, B6, and B12, folate, selenium, and zinc resulted in a lower oxidative capacity and has major impact on muscle health as shown by decreased force production and PA, without effects on muscle mass. The reduced fCSA in combination with similar SDH activity per fibre might explain the reduced oxidative capacity resulting in the increased fatigue after exercise in LOWOX-B mice.

Keywords: Antioxidants; Function; Muscle quality; Nutrition; Strength.

Figure 1

Experimental set‐up. accl, acclimatization period; mo, months; LOWOX‐B, group of mice with diet low in specific micronutrients with antioxidant properties; DA, daily activity measurements; GS, in vivo muscle grip strength measurements; DEXA, body composition analyses; MPS, muscle protein synthesis analysis; MPB, muscle protein breakdown; O/N, overnight.

Figure 2

Biomarkers of nutritional and oxidative status at start (t = 0) and after 4 months of LOWOX‐B diet. (A) Vitamin A plasma levels, (B) homocysteine plasma levels, (C) vitamin E plasma levels, (D) hepatic total GSH levels, and (E) hepatic MDA levels. At start n = 11, ctrl n = 6, and LOWOX‐B n = 10. Different characters indicate statistically significant differences (mixed model with post hoc SIDAK, P < 0.05).

Figure 3

Effects of age and LOWOX‐B diet on body composition and physical strength. (A) Bodyweight development, (B) lean mass, (C) fat mass, (D) absolute maximal forelimb strength, with a trend (P = 0.073) to decreased strength after 4 months of the LOWOX‐B diet, and (E) mean total daily physical activity. At start n = 11, ctrl n = 6 and LOWOX‐B n = 10. *Statistically significant age effect, #statistically significant diet effect at indicated time point (mixed model with post hoc SIDAK, P < 0.05).

Figure 4

Ex vivo muscle function: effects of 4 month LOWOX‐B diet on force–frequency of EDL muscle. (A) Maximal force production, (B) contraction velocity, and (C) relaxation velocity. Ctrl n = 6 and LOWOX‐B n = 10. @Overall statistically significant diet effect, #statistically significant diet effect at indicated time point (mixed model adjusted for growth curve analysis with post hoc SIDAK, P < 0.05) and ns not significant.

Figure 5

Ex vivo muscle function: effects of 4 month LOWOX‐B diet on exercise performance. (A) Maximal rate of force production, (B) maximal force production during first 25 contractions of exercise protocol, (C) contraction velocity development, (D) relaxation velocity development, and (E) fatigue index. Ctrl n = 6 and LOWOX‐B n = 10. Different characters indicate a statistically significant diet effect (mixed model with post hoc SIDAK, P < 0.05).

Figure 6

Ex vivo muscle protein synthesis (MPS) and breakdown (MPB). (A) MPS in GM muscles and (B) MPB in buffer of GM muscle, (C) atrogin/MAFbx EDL gene expression and (D) MuRF1 EDL gene expression at 25 months of age after 4 month diet. Ctrl n = 6 and LOWOX‐B n = 10. MPS was measured during incubation with neutral buffer (basal state) and after addition of leucine (anabolic buffer). Different characters indicate a statistically significant difference (mixed model with post hoc SIDAK, P < 0.05).

Figure 7

Effects of 4 month LOWOX‐B diet on PL muscle fibre‐type distribution, fibre CSA, and SDH activity. (A–F) Typical examples of PL muscle cross sections with double immunofluorescent labelled muscle fibres for either myosin heavy chain types IIA and IIX (A and D; green type IIA and blue type IIX) or types I and IIB (B and E, green type I and blue type IIB) or stained for SDH activity (C and F). The basal lamina, intramuscular connective tissue, and aponeuroses were stained red by wheat germ agglutinin. Note that around the distal, aponeurosis is the high oxidative region with predominantly type IIA, IIAX, IIX, and IIB muscle fibres. On the outer side, is the low oxidative (glycolytic) region with predominantly type IIX and IIB muscle fibres. (G) Muscle fibre‐type distributions. (H) Mean CSA for the different muscle fibre types. (I) SDH activity in the different muscle fibres types. (J) Spatially integrated SDH activity (i.e. product of muscle fibre CSA and SDH activity). Ctrl: n = 8 and LOWOX‐B: n = 7. Scale bar indicates 300 μm. *Statistically significant difference (P < 0.05).