SRT2183 and SRT1720, but not Resveratrol, Inhibit Osteoclast Formation and Resorption in the Presence or Absence of Sirt1.

Background: Osteoclastic bone resorption markedly increases with aging, leading to osteoporosis characterized by weak and fragile bones. Mice exhibit greater bone resorption and poor bone mass when Sirt1 is removed from their osteoclasts. Here we investigated the ex vivo impacts of putative Sirt1 activators, Resveratrol (RSV), SRT2183, and SRT1720, on osteoclast formation and activity in primary mouse bone marrow cells (BMCs) derived from wild-type (WT) and osteoclast specific Sirt1 knockout (OC-Sirt1KO) mice and in the RAW264.7 mouse macrophage cell line. Results: We found that SRT2183 and SRT1720 inhibit the formation of osteoclasts and actin belts in BMCs and RAW264.7 cells, whereas RSV does not. We also observed that the OC-Sirt1KO mice exhibited less bone mineral density, and the BMCs harvested from these mice yielded more osteoclasts than BMCs harvested from littermate controls. Interestingly, both SRT2183 and SRT1720 reduced osteoclast and actin belt formation in BMCs from OC-Sirt1KO mice. SRT2183 and SRT1720 also significantly disrupted actin belts of mature osteoclasts generated from BMCs of WT mice, within 3 and 6 hours of administration, respectively. Furthermore, these compounds inhibited the resorption activity of mature osteoclasts, while RSV did not. Conclusion: Our findings suggest SRT2183 and SRT1720 impede bone resorption by disrupting actin belts of mature osteoclasts, inhibit actin belt formation, and inhibit osteoclastogenesis even in the absence of Sirt1. Thus, the mechanism of action of these compounds appears to extend beyond Sirt1 activation and possibly pave the way for potential new therapies in alleviating osteoporosis associated bone loss.

Short-term nicotinamide riboside treatment improves muscle quality and function in mice and increases cellular energetics and differentiating capacity of myogenic progenitors.

OBJECTIVES: Nicotinamide adenine dinucleotide (NAD+), an essential cofactor for mitochondrial function, declines with aging, which may lead to impaired physical performance. Nicotinamide riboside (NR), a NAD+ precursor, restores cellular NAD+ levels. The aim of this study was to examine the effects of short-term NR supplementation on physical performance in middle-aged mice and the effects on mouse and human muscle stem cells. METHODS: We treated 15-mo-old male C57BL/6J mice with NR at 300 mg·kg·d-1 (NR3), 600 mg·kg·d-1 (NR6), or placebo (PLB), n = 8 per group, and assessed changes in physical performance, muscle histology, and NAD+ content after 4 wk of treatment. RESULTS: NR increased total NAD+ in muscle tissue (NR3 P = 0.01; NR6 P = 0.004, both versus PLB), enhanced treadmill endurance and open-field activity, and prevented decline in grip strength. Histologic analysis revealed NR-treated mice exhibited enlarged slow-twitch fibers (NR6 versus PLB P = 0.014; NR3 P = 0.16) and a trend toward more slow fibers (NR3 P = 0.14; NR6 P = 0.22). We next carried out experiments to characterize NR effects on mitochondrial activity and cellular energetics in vitro. We observed that NR boosted basal and maximal cellular aerobic and anaerobic respiration in both mouse and human myoblasts and human myotubes. Additionally, NR treatment improved the differentiating capacity of myoblasts and increased myotube size and fusion index upon stimulation of these progenitors to form multinucleated myotubes. CONCLUSION: These findings support a role for NR in improving cellular energetics and functional capacity in mice, which support the translation of this work into clinical settings as a strategy for improving and/or maintaining health span during aging.

Pinpointing a Role for Vitamin D in Frailty: A Time for Animal Models?

Frailty is a condition marked by greater susceptibility to adverse outcomes, including disability and mortality, which affects up to 50% of those 80 years of age and older. Concurrently, serum vitamin D insufficiency and deficiency, for which as many as 70% of older adults may be at risk, potentially play an important role in frailty onset and progression. Large population driven studies have uncovered associations between low serum vitamin D levels and higher incidence of frailty. However, attempts to apply vitamin D therapeutically to treat and/or prevent frailty have not yielded consistent support for benefits. Given the complexity and inconsistency arising from human studies involving vitamin D, our research group has recently published on animal models of vitamin D insufficiency. Combining our model with the emerging development of animal frailty assessment, we identified that higher than standard levels of vitamin D supplementation may delay frailty in mice. In this viewpoint article, we will discuss current knowledge regarding the importance of vitamin D in frailty progression, the emerging significance of animal models in addressing these relationships, and the future for pre-clinical and clinical research.

Vitamin D Insufficiency Reduces Grip Strength, Grip Endurance and Increases Frailty in Aged C57Bl/6J Mice.

Low 25-OH serum vitamin D (VitD) is pervasive in older adults and linked to functional decline and progression of frailty. We have previously shown that chronic VitD insufficiency in "middle-aged" mice results in impaired anaerobic exercise capacity, decreased lean mass, and increased adiposity. Here, we examine if VitD insufficiency results in similar deficits and greater frailty progression in old-aged (24 to 28 months of age) mice. Similar to what we report in younger mice, older mice exhibit a rapid and sustained response in serum 25-OH VitD levels to differential supplementation, including insufficient (125 IU/kg chow), sufficient (1000 IU/kg chow), and hypersufficient (8000 IU/kg chow) groups. During the 4-month time course, mice were assessed for body composition (DEXA), physical performance, and frailty using a Fried physical phenotype-based assessment tool. The 125 IU mice exhibited worse grip strength (p = 0.002) and inverted grip hang time (p = 0.003) at endpoint and the 8000 IU mice transiently displayed greater rotarod performance after 3 months (p = 0.012), yet other aspects including treadmill performance and gait speed were unaffected. However, 125 and 1000 IU mice exhibited greater frailty compared to baseline (p = 0.001 and p = 0.038, respectively), whereas 8000 IU mice did not (p = 0.341). These data indicate targeting higher serum 25-OH vitamin D levels may attenuate frailty progression during aging.

High intensity interval training improves physical performance in aged female mice: A comparison of mouse frailty assessment tools.

Frailty syndrome increases the risk for disability and mortality, and is a major health concern amidst the geriatric shift in the population. High intensity interval training (HIIT), which couples bursts of vigorous activity interspersed with active recovery intervals, shows promise for the treatment of frailty. Here we compare and contrast five Fried physical phenotype and one deficit accumulation based mouse frailty assessment tools for identifying the impacts of HIIT on frailty and predicting functional capacity, underlying pathology, and survival in aged female mice. Our data reveal a 10-minute HIIT regimen administered 3-days-a-week for 8-weeks increased treadmill endurance, gait speed and maintained grip strength. One frailty tool identified a benefit of HIIT for frailty, but many were trending suggesting HIIT was beneficial for physical performance in these mice, but the 8-week timeframe may have been insufficient to induce frailty benefits. Finally, most frailty tools distinguished between surviving or non-surviving mice, whereas half correlated with functional capacity measured by nest building ability, and none correlated with underlying pathology. In summary, this study supports the ongoing development of mouse assessment tools as useful instruments for frailty research.

AP-1 and Mitf interact with NFATc1 to stimulate cathepsin K promoter activity in osteoclast precursors.

Cathepsin K (CTSK) is a secreted protease that plays an essential role in osteoclastic bone resorption and osteoporotic bone loss. We have previously shown that activator protein 1 (AP-1) stimulates CTSK promoter activity and that proximal nuclear factor of activated T cells cytoplasmic 1 (NFATc1)-binding sites play a major role in the stimulation of CTSK gene expression by receptor activator of NFκB ligand (RANKL). In the present study, we have extended these observations and further dissected the effects of transcription factors involved in the regulation of CTSK gene expression. Our aim was to investigate the cooperative interplay among transcription factors AP-1, microphthalmia-associated transcription factor (Mitf), and NFATc1, and the consequent regulatory effects on CTSK transcription. Experiments were carried out in RAW 264.7 cells, which can be readily differentiated to osteoclasts upon RANKL stimulation. Our data show that AP-1, Mitf, and NFATc1 are capable of independently stimulating CTSK promoter activity. A combination of any two factors further enhances CTSK promoter activity, with the combination of AP-1 (c-fos/c-jun) and NFATc1 inducing the largest increase. We further identify a synergistic effect when all three factors cooperate intimately at the proximal promoter region, yielding maximal transcriptional upregulation of the CTSK promoter. RANKL induces temporal localization of AP-1 and NFATc1 to the CTSK promoter. These results suggest that the interaction of multiple transcription factors mediate a maximal response to RANKL-induced CTSK gene expression.

Short Session High Intensity Interval Training and Treadmill Assessment in Aged Mice.

High intensity interval training (HIIT) is emerging as a therapeutic approach to prevent, delay, or ameliorate frailty. In particular short session HIIT, with regimens less than or equal to 10 min is of particular interest as several human studies feature routines as short as a few minutes a couple times a week. However, there is a paucity of animal studies that model the impacts of short session HIIT. Here, we describe a methodology for an individually tailored and progressive short session HIIT regimen of 10 min given 3 days a week for aged mice using an inclined treadmill. Our methodology also includes protocols for treadmill assessment. Mice are initially acclimatized to the treadmill and then given baseline flat and uphill treadmill assessments. Exercise sessions begin with a 3 min warm-up, then three intervals of 1 min at a fast pace, followed by 1 min at an active recovery pace. Following these intervals, the mice are given a final segment that starts at the fast pace and accelerates for 1 min. The HIIT protocol is individually tailored as the speed and intensity for each mouse are determined based upon initial anaerobic assessment scores. Additionally, we detail the conditions for increasing or decreasing the intensity for individual mice depending on performance. Finally, intensity is increased for all mice every two weeks. We previously reported in this protocol enhanced physical performance in aged male mice and here show it also increases treadmill performance in aged female mice. Advantages of our protocol include low administration time (about 15 min per 6 mice, 3 days a week), strategy for individualizing for mice to better model prescribed exercise, and a modular design that allows for the addition or removal of the number and length of intervals to titrate exercise benefits.

High Intensity Interval Training Improves Physical Performance and Frailty in Aged Mice.

Sarcopenia and frailty are highly prevalent in older individuals, increasing the risk of disability and loss of independence. High intensity interval training (HIIT) may provide a robust intervention for both sarcopenia and frailty by achieving both strength and endurance benefits with lower time commitments than other exercise regimens. To better understand the impacts of HIIT during aging, we compared 24-month-old C57BL/6J sedentary mice with those that were administered 10-minute uphill treadmill HIIT sessions three times per week over 16 weeks. Baseline and end point assessments included body composition, physical performance, and frailty based on criteria from the Fried physical frailty scale. HIIT-trained mice demonstrated dramatic improvement in grip strength (HIIT 10.9% vs -3.9% in sedentary mice), treadmill endurance (32.6% vs -2.0%), and gait speed (107.0% vs 39.0%). Muscles from HIIT mice also exhibited greater mass, larger fiber size, and an increase in mitochondrial biomass. Furthermore, HIIT exercise led to a dramatic reduction in frailty scores in five of six mice that were frail or prefrail at baseline, with four ultimately becoming nonfrail. The uphill treadmill HIIT exercise sessions were well tolerated by aged mice and led to performance gains, improvement in underlying muscle physiology, and reduction in frailty.