Type 2 diabetes (T2D) incidence in adolescents is rising and may interfere with peak bone mass acquisition. We tested the effects of early-onset T2D on bone mass, microarchitecture, and strength in the TALLYHO/JngJ mouse, which develops T2D by 8 weeks of age. We assessed metabolism and skeletal acquisition in male TALLYHO/JngJ and SWR/J controls (n = 8-10/group) from 4 weeks to 8 and 17 weeks of age. Tallyho mice were obese; had an approximately 2-fold higher leptin and percentage body fat; and had lower bone mineral density vs SWR at all time points (P < .03 for all). Tallyho had severe deficits in distal femur trabecular bone volume fraction (-54%), trabecular number (-27%), and connectivity density (-82%) (P < .01 for all). Bone formation was higher in Tallyho mice at 8 weeks but lower by 17 weeks of age vs SWR despite similar numbers of osteoblasts. Bone marrow adiposity was 7- to 50-fold higher in Tallyho vs SWR. In vitro, primary bone marrow stromal cell differentiation into osteoblast and adipocyte lineages was similar in SWR and Tallyho, suggesting skeletal deficits were not due to intrinsic defects in Tallyho bone-forming cells. These data suggest the Tallyho mouse might be a useful model to study the skeletal effects of adolescent T2D.

The effects of obesity on bone metabolism are complex, and may be mediated by consumption of a high fat diet and/or by obesity-induced metabolic dysregulation. To test the hypothesis that both high fat (HF) diet and diet-induced metabolic disease independently decrease skeletal acquisition, we compared effects of HF diet on bone mass and microarchitecture in two mouse strains: diet-induced obesity (DIO)-susceptible C57BL/6J (B6) and DIO-resistant FVB/NJ (FVB). At 3 wks of age we weaned 120 female FVB and B6 mice onto normal (N, 10% Kcal/fat) or HF diet (45% Kcal/fat) and euthanized them at 6, 12 and 20 weeks of age (N = 10/grp). Outcomes included body mass; percent fat and whole-body bone mineral density (WBBMD, g/cm2) via DXA; cortical and trabecular bone architecture at the midshaft and distal femur via μCT; and marrow adiposity via histomorphometry. In FVB HF, body mass, percent body fat, WBBMD and marrow adiposity did not differ vs. N, but trabecular bone mass was lower at 6 wks of age only (p < 0.05), cortical bone geometric properties were lower at 12 wks only, and bone strength was lower at 20 wks of age only in HF vs. N (p < 0.05). In contrast, B6 HF had higher body mass, percent body fat, and leptin vs. N. B6 HF also had higher WBBMD (p < 0.05) at 9 and 12 wks of age but lower distal femur trabecular bone mass at 12 wks of age, and lower body mass-adjusted cortical bone properties at 20 wks of age compared to N (p < 0.05). Marrow adiposity was also markedly higher in B6 HF vs. N. Overall, HF diet negatively affected bone mass in both strains, but was more deleterious to trabecular bone microarchitecture and marrow adiposity in B6 than in FVB mice. These data suggest that in addition to fat consumption itself, the metabolic response to high fat diet independently alters skeletal acquisition in obesity.

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Starvation induces low bone mass and high bone marrow adiposity in humans, but the underlying mechanisms are poorly understood. The adipokine leptin falls in starvation, suggesting hypoleptinemia may be a link between negative energy balance, bone marrow fat accumulation and impaired skeletal acquisition. If so, treating mice with leptin during caloric restriction (CR) should reduce marrow adipose tissue (MAT) and improve bone mass. To test this hypothesis, female C57Bl/6J mice were fed a 30% calorie restricted (CR) or normal (N) diet from 5–10 weeks of age, with daily injections of vehicle (VEH), 1 mg/kg leptin (LEP1), or 2 mg/kg leptin (LEP2) (N=6–8/group). Outcomes included body mass, %body fat and whole body bone mineral density (BMD) via pDXA, cortical and trabecular microarchitecture via μCT, and MAT volume via μCT of osmium tetroxide-stained bones. Overall, CR mice had lower body mass, %body fat, BMD, and cortical bone area fraction, but more connected trabeculae, vs. N mice (p<0.05 for all). Most significantly, while MAT was elevated in CR vs. N overall, leptin treatment blunted MAT formation in CR mice by 50% vs. vehicle (p<0.05 for both leptin doses). CR LEP2 mice weighed less vs. CR VEH mice at 9–10 wks of age (p<0.05), but leptin treatment did not affect %body fat, BMD or bone microarchitecture within either diet. These data demonstrate that once daily leptin bolus during CR inhibits bone marrow adipose expansion without affecting bone mass acquisition, suggesting leptin has distinct effects on starvation-induced bone marrow fat formation and skeletal acquisition.

Bone receives mechanical stimulation from two primary sources, muscle contractions and external gravitational loading, but the relative contribution of each source to skeletal health is not fully understood. Understanding the most effective loading for maintaining bone health has important clinical implications for prescribing physical activity for the treatment or prevention of osteoporosis. Therefore, we investigated the relative effects of muscle paralysis and reduced gravitational loading on changes in muscle mass, bone mineral density and microarchitecture. Adult female C57Bl/6J mice (n=10/group) underwent one of the following: unilateral botulinum toxin (BTX) injection of the hindlimb, hindlimb unloading (HLU), both unilateral BTX injection and HLU, or no intervention. BTX and HLU each led to significant muscle and bone loss. The effect of BTX was diminished when combined with HLU, though generally the leg that received the combined intervention (HLU + BTX) had the most detrimental changes in bone and muscle. We found an indirect effect of BTX affecting the uninjected (contralateral) leg that led to significant decreases in bone mineral density and deficits in muscle mass and bone architecture relative to the untreated controls; the magnitude of this indirect BTX effect was comparable to the direct effect of BTX treatment and HLU. Thus, while it was difficult to definitively conclude whether muscle forces or external gravitational loading contribute more to bone maintenance, it appears that BTX-induced muscle paralysis is more detrimental to muscle and bone than hindlimb unloading.