Arachidonic acid exacerbates diet-induced obesity and reduces bone mineral content without impacting bone strength in growing male rats.

Long-chain polyunsaturated fatty acids modulate bone mass and adipocyte metabolism. Arachidonic acid (AA, C20:4 n-6) is elevated in obesity and postulated to stimulate bone resorption. This study aimed to determine the effect of AA on bone mass, quality, and adiposity in diet-induced obesity during growth. Male Sprague-Dawley rats (n=42, 4-week) were randomized into groups fed a control diet (CTRL, AIN-93G), high-fat diet (HFD, 35% kcal fat) or HFD + AA (1% w/w diet) for 6 weeks. Body composition, bone mineral density and microarchitecture were measured using dual-energy X-ray absorptiometry and micro-computed tomography. Red blood cell fatty acid profile was measured with gas chromatography. Group differences were evaluated using repeated measures two-way analysis of variance with Tukey-Kramer post hoc testing. Total energy intake did not differ among diet groups. At week 6, HFD + AA had significantly greater body fat % (12%), body weight (6%) and serum leptin concentrations (125%) than CTRL, whereas visceral fat (mass and %, assessed with micro-computed tomography) was increased in both HFD and HFD + AA groups. HFD + AA showed reduced whole body bone mineral content and femur mid-diaphyseal cortical bone cross-sectional area than HFD and CTRL, without impairment in bone strength. Contrarily, HFD + AA had greater femur metaphyseal trabecular vBMD (35%) and bone volume fraction (5%) compared to controls. Inclusion of AA elevated leptin concentrations in male rats. The early manifestations of diet-induced obesity on bone mass were accelerated with AA. Studies of longer duration are needed to clarify the effect of AA on peak bone mass following growth cessation.

Lean body mass accretion is elevated in response to dietary vitamin D: A dose-response study in female weanling rats.

Vitamin D status positively relates to lean body mass in infants. This study tested the effect of vitamin D on body composition and growth-related hormones. It was hypothesized that low vitamin D status programs for higher fat mass accretion. Female weanling Sprague-Dawley rats (4 weeks; n = 6/diet) were randomized to AIN-93G diets with modified vitamin D contents for 8 weeks: group 1 (1 IU vitamin D3/g diet), group 2 (2 IU vitamin D3/g diet), and group 3 (4 IU vitamin D3/g diet). At week 0, 4, and 8 of study, measurements included: serum 25(OH)D3, IGF-1, IGFBP3, leptin, and whole body composition assessed with DXA. Differences among groups were tested using mixed model ANOVA with Tukey's post hoc t-tests. No differences were observed in baseline body composition and biomarkers, nor did body weight and food intake differ over the study. At week 8, serum 25(OH)D3 in group 3 was higher (P < .0001) compared to groups 1 and 2. At 8 weeks, lean mass (P < .05) and lean mass accretion (P < .05) were significantly higher in groups 2 and 3 compared to group 1. Serum IGF-1 concentration declined over time (P < .001) with smaller declines at week 8 in group 3 (P < .05). Serum IGFBP3 concentration was lower at week 4 in group 2 compared to groups 1 and 3. Serum leptin concentration and fat mass were not affected by diet. These results suggested that the achievement of higher vitamin D status may support a lean body phenotype without altering weight gain.

Dietary docosahexaenoic acid contributes to increased bone mineral accretion and strength in young female Sprague-Dawley rats.

Growing evidence suggests that docosahexaenoic acid (DHA: 22:6n-3) enhances bone mineral content (BMC) and bone mineral density (BMD) in adulthood and during aging, however the effects during and after sexual maturation are unclear. The aim of this study was to examine the dose-response of BMC, BMD and microarchitectural properties of bone to dietary DHA in healthy growing female rats during acquisition of peak bone mass (PBM). Female Sprague-Dawley rats (n = 12/diet) were randomized to receive a control diet (AIN-93 M, 60 g soybean oil/kg diet) or an experimental diet containing 0.1, 0.4, 0.8 and 1.2% DHA (w/w of total diet) for 10 weeks. Dietary DHA increased the whole body, lumbar spine and long bone BMC compared to the control, in addition to higher aBMD and also BMD. Additionally, an increase in cortical bone microarchitecture parameters of lumbar spine as well as peak force were observed in dietary DHA diet groups. Dietary DHA contributes to PBM when consumed during and after sexual maturation, however higher doses of DHA do not provide further benefits.

Docosahexaenoic Acid at 0.4% of Dietary Weight Enhances Lean Mass in Young Female Sprague-Dawley Rats.

BACKGROUND: Docosahexaenoic acid (DHA; 22:6n-3) is an n-3 (ω-3) fatty acid known for beneficial effects on body composition. OBJECTIVE: The objective of the study was to test the dose response of lean and fat mass to DHA in healthy growing female rats. METHODS: Female Sprague-Dawley rats (7 wk at baseline; n = 12/diet) were randomly assigned to receive a control (AIN-93M; 60 g soybean oil/kg diet) or experimental diet for 10 wk. Experimental diets contained 0.1%, 0.4%, 0.8%, or 1.2% DHA (wt:wt of total diet). Imaging for whole-body and abdominal composition was conducted using dual-energy X-ray absorptiometry and microcomputed tomography, respectively, at weeks 0, 5, and 10. Fatty acid profiles of several tissues were analyzed using gas chromatography. Serum leptin, C-reactive protein, and plasma insulin-like growth factor I concentrations were measured at each time point using immunoassays. Data were tested using Pearson's correlations and mixed-model ANOVA. RESULTS: No differences were observed in weight at baseline or food intake throughout the study. Overall, a 6% increase (P < 0.05) in whole-body and abdominal lean mass was observed in the 0.4%-DHA diet group compared with the control diet group. Moreover, the abdominal visceral fat mass was 31.4% lower in rats in the 0.4%-DHA than in the 1.2%-DHA diet group (P < 0.001). Rats in the 1.2%-DHA diet group showed greater percent differences in whole-body (32.5% and 40.6% higher) and in abdominal (33.9% and 49.4% higher) fat mass relative to the 0.1%- and 0.4%-DHA diet groups, respectively (P < 0.01). Accordingly, serum leptin concentration was lower in the 0.1%-DHA (38.2%) and 0.4%-DHA (43.8%) diet groups (P < 0.01) than in the 1.2%-DHA diet group and positively related to whole-body fat mass (r = 0.91, P < 0.0001). CONCLUSION: Dietary DHA at 0.4% of dietary weight effectively enhances lean mass and proportionally reduces fat mass in growing female rats.

Vitamin D Status and Immune Health Outcomes in a Cross-Sectional Study and a Randomized Trial of Healthy Young Children.

In young children, the relationship between vitamin D and biomarkers of immune function is not well elucidated. The objective was to investigate relationships between vitamin D and immune function in young children. Data were from a cross-sectional study (study 1) of healthy children 1.8⁻5.9 years (n = 457) and a 12 weeks trial using vitamin D fortified foods (study 2) in healthy 1.8⁻8.7 years old (n = 77) in Montreal, Canada. Vitamin D status and ex vivo immune function were assessed. In study 1 (male: n = 242; 53%), plasma IL-6, TNFα and CRP were significantly higher (p < 0.05) in children with 25-hydroxyvitamin D (25(OH)D) ≥ 75 nmol/L compared to.

Vitamin D status and functional health outcomes in children aged 2-8 y: a 6-mo vitamin D randomized controlled trial.

Background: Most Canadian children do not meet the recommended dietary intake for vitamin D. Objectives: The aims were to test how much vitamin D from food is needed to maintain a healthy serum 25-hydroxyvitamin D3 [25(OH)D3] status from fall to spring in young children and to examine musculoskeletal outcomes. Design: Healthy children aged 2-8 y (n = 51) living in Montreal, Canada, were randomly assigned to 1 of 2 dietary vitamin D groups (control or intervention to reach 400 IU/d by using vitamin D-fortified foods) for 6 mo, starting October 2014. At baseline and at 3 and 6 mo, anthropometric characteristics, vitamin D metabolites (liquid chromatography-tandem mass spectrometry), and bone biomarkers (IDS-iSYS, Immunodiagnositc Systems; Liaison; Diasorin) were measured and physical activity and food intakes surveyed. At baseline and at 6 mo, bone outcomes and body composition (dual-energy X-ray absorptiometry) were measured. Cross-sectional images of distal tibia geometry and muscle density were conducted with the use of peripheral quantitative computed tomography scans at 6 mo. Results: At baseline, participants were aged 5.2 ± 1.9 (mean ± SD) y and had a body mass index z score of 0.65 ± 0.12; 53% of participants were boys. There were no differences between groups in baseline serum 25(OH)D3 (66.4 ± 13.6 nmol/L) or vitamin D intake (225 ± 74 IU/d). Median (IQR) compliance was 96% (89-99%) for yogurt and 84% (71-97%) for cheese. At 3 mo, serum 25(OH)D3 was higher in the intervention group (P < 0.05) but was not different between groups by 6 mo. Although lean mass accretion was higher in the intervention group (P < 0.05), no differences in muscle density or bone outcomes were observed. Conclusions: The consumption of 400 IU vitamin D/d from fall to spring did not maintain serum 25(OH)D3 concentration or improve bone outcomes. Further work with lean mass accretion as the primary outcome is needed to confirm if vitamin D enhances lean accretion in healthy young children. This trial was registered at www.clinicaltrials.gov as NCT02387892.

DHA and EPA in red blood cell membranes are associated with dietary intakes of omega-3-rich fish in healthy children.

Omega-3 long-chain polyunsaturated fatty acids (n-3 LCPUFA) are important in child development. The primary objective of this study was to investigate the associations between dietary intakes of n-3 LCPUFA and red blood cell (RBC) n-3 LCPUFA in young children. Healthy children, (2-8y) underwent RBC fatty acid profiling. Dietary intakes were parent-reported over 6 mo using three 24h dietary intake assessments and three 30 d food frequency questionnaires (FFQ). Participants (n = 49, 5.6 ± 1.9y), were 59% male, and had a body mass index (BMI) z-score of 0.65 ± 0.84. Dietary n-3 LCPUFA intakes were not different over time. RBC docosahexaenoic acid (DHA) positively correlated with average DHA from the 24h recalls. RBC DHA and eicosapentaenoic acid (EPA) positively correlated with average n-3 LCPUFA-rich fish intake from the FFQ. RBC appear to reflect long-term stable intakes of n-3 LCPUFA during growth in healthy young children.

Aerobic Endurance Training Does Not Protect Bone Against Poorly Controlled Type 1 Diabetes in Young Adult Rats.

Streptozotocin (STZ)-induced type 1 diabetes mellitus (T1DM) decreases trabecular bone volume and bone strength in rodents. The current study investigated the potential protective effects of aerobic endurance training (AET) on bone in STZ-induced T1DM young adult rats. Sixty-four 8-week-old male Sprague-Dawley rats were randomly divided into 4 groups of 16: control non-T1DM sedentary (CS) and exercised (CX), T1DM sedentary (DS) and exercised (DX). Blood glucose was maintained at 9-15 mmol/L using subcutaneously implanted insulin pellets (Linplant, Linshin Canada, Inc.). AET was performed at ~75-85% VO2max for 1 h/day, 5 day/week for 10 weeks. Areal and volumetric bone mineral density (aBMD and vBMD; excised femur) were measured using dual-energy X-ray absorptiometry (DXA; QDR 4500A) and micro computed tomography (µCT; Aloka). Bone strength was tested using a 3-point bending test (Instron 5544 Load Frame). Two-way ANOVA was used to test for T1DM and exercise differences followed by Tukey's HSD tests for interaction effects; significance was set at P < 0.05. T1DM had lower body weight (18.0%), aBMD (8.6%), cortical vBMD (1.6%), trabecular vBMD (2.1%), maximum load at break (22.2%), and increased elastic modulus (11.3%) vs. control (P < 0.001). Exercise in T1DM further decreased body weight (4.7%) vs. sedentary (P = 0.043) and maximum extension during the bending test that demonstrated DX was increased (7.3%) vs. CX (P = 0.033). There were no other beneficial effects of exercise on bone. These results suggest that 10 weeks of AET in rats do not have protective effects on bone in the short term and that T1DM rats have compromised bone health.

The C-3α Epimer of 25-Hydroxycholecalciferol from Endogenous and Exogenous Sources Supports Normal Growth and Bone Mineral Density in Weanling Rats.

BACKGROUND: The C-3α epimer of 25-hydroxycholecalciferol [3-epi-25(OH)D3] is elevated in infants. OBJECTIVES: We tested whether increasing cholecalciferol intake results in a dose-response in plasma 3-epi-25(OH)D3 We also examined bone and mineral metabolism in response to 3-epi-25(OH)D3 treatment. METHODS: Sprague Dawley rats (4 wk old) were randomly assigned (n = 6/group of each sex) to AIN-93G diets with cholecalciferol at 1 (control), 2, or 4 IU/g diet for objective 1 and to diets with 3-epi-25(OH)D3 at 0.5 or 1 IU/g diet or 25-hydroxycholecalciferol [25(OH)D3] at 0.5 IU/g diet for objective 2 for 8 wk. Measurements at weeks 0, 4, and 8 included body weight and length, plasma vitamin D metabolites, bone biomarkers, and bone mineral density determined by using dual-energy X-ray absorptiometry. Lumbar vertebra 3 (L3) geometry and volumetric bone mineral density (vBMD) were measured using microcomputed tomography. Differences between groups were identified for males and females separately. RESULTS: Weight and food intake were not different between groups. Elevated plasma 3-epi-25(OH)D3 was observed only in females in the 4 IU cholecalciferol/g diet group (mean ± SD: 24.7 ± 17.1 ng/mL), compared with the control group (5.3 ± 1.4 ng/mL; P = 0.001). By week 8, both male and female rats in the 3-epi-25(OH)D3 groups had >87% greater plasma 3-epi-25(OH)D3 concentrations relative to the 25(OH)D3 reference group (P < 0.0001). At week 8 in males only, parathyroid hormone was significantly lower (P = 0.019) in both 3-epi-25(OH)D3 groups than in the 25(OH)D3 group, and L3 total vBMD was higher (P = 0.004) in the 0.5 IU 3-epi-25(OH)D3 group than in the 25(OH)D3 group. CONCLUSIONS: Endogenously generated 3-epi-25(OH)D3 is more prominent in female than in male rats. Exogenous 3-epi-25(OH)D3 was as effective as 25(OH)D3 in supporting bone mineral accretion in both sexes. It thus appears that 3-epi-25(OH)D3 has biological activity and should be further explored.

Conjugated linoleic acid mitigates testosterone-related changes in body composition in male guinea pigs.

We hypothesize that conjugated linoleic acid (CLA) may be effective in preventing the changes in total and regional body composition and increases in interleukin (IL) 6 that occur as a result of hypogonadism. Male guinea pigs (n = 40, 70- to 72-week retired breeders) were block randomized by weight into 4 groups: (1) sham surgery (SHAM)/control (CTRL) diet, (2) SHAM/conjugated linoleic acid (CLA) diet (1%), (3) orchidectomy (ORX)/CTRL diet, and (4) ORX/CLA diet. Dual-energy x-ray absorptiometry scans were performed at baseline and week 16 to assess body composition. Serum IL-6 was analyzed using an enzyme-linked immune sorbent assay. Fatty acids (FAs) from visceral and subcutaneous adipose tissue were analyzed using gas chromatography. In ORX/CTRL guinea pigs, percent total body fat increased by 6.1%, and percent lean mass decreased by 6.7% over the 16-week treatment period, whereas no changes were observed for either parameter in ORX/CLA guinea pigs. Guinea pigs fed the CLA diet gained less percent total, upper, and lower body fat than those fed the CTRL diet regardless of surgical treatment. Regional adipose tissue FA composition was reflective of dietary FAs. Serum IL-6 concentrations were not different among groups. In this study, we observed that, in male guinea pigs, hypogonadism resulted in increased fat mass and decreased lean mass. In addition, CLA was effective in reducing gains in body fat and maintaining lean mass in both hypogonadal and intact guinea pigs.

Dietary vitamin D dose-response in healthy children 2 to 8 y of age: a 12-wk randomized controlled trial using fortified foods.

BACKGROUND: Vitamin D is fundamental for bone health. A high proportion of Canadian 2- to 8-y-olds do not meet the Estimated Average Requirement (EAR) of 400 IU/d. OBJECTIVE: The objective was to determine whether vitamin D intakes consistent with the EAR or Recommended Dietary Allowance (RDA), through fortification of additional dairy products, would result in higher vitamin D status in young children. DESIGN: Participants aged 2-8 y (n = 77; Montreal, Canada) were randomly assigned to 1 of 3 dietary vitamin D targets (control; EAR: 400 IU/d; or RDA: 600 IU/d) for 12 wk (January to April 2014). Anthropometric measurements, demographic characteristics, dietary intakes, fasting serum parathyroid hormone, 25-hydroxyvitamin D [25(OH)D], and ionized calcium were compared by using mixed-model ANOVA. RESULTS: Participants' mean ± SD age was 5.1 ± 1.9 y; 54.5% were boys with body mass index z scores of 0.50 ± 0.85. Compliance was 85% overall. No differences were observed in baseline dietary vitamin D intakes or serum 25(OH)D. At 12 wk, the EAR and RDA groups had significantly higher vitamin D intakes [median (IQR): control, 227 (184-305) IU/d; EAR, 410 (363-516) IU/d; and RDA, 554 (493-653) IU/d; P < 0.05] and serum 25(OH)D concentrations (control: 55.8 ± 12.3 nmol/L; EAR: 64.1 ± 10.0 nmol/L; and RDA: 63.7 ± 12.4 nmol/L; P < 0.05) than the control group. Ninety-six percent of children in the EAR and RDA groups and 67% of the control group had 25(OH)D concentrations ≥50 nmol/L. CONCLUSION: Increasing the vitamin D intakes of young children through fortification of alternative dairy products results in significantly higher serum concentrations of 25(OH)D and a significantly greater proportion of children with serum 25(OH)D ≥50 nmol/L during periods of minimal ultraviolet B radiation exposure. This trial was registered at clinicaltrials.gov as NCT02097160 and had Health Canada Temporary Marketing Authorization Letters for both products (TM-13-0432 and TM-13-0433).

Dual-energy X-ray absorptiometry, peripheral quantitative computed tomography, and micro-computed tomography techniques are discordant for bone density and geometry measurements in the guinea pig.

This study aims to examine agreement among bone mineral content (BMC) and density (BMD) estimates obtained using dual-energy X-ray absorptiometry (DXA), peripheral quantitative computed tomography (pQCT), and micro-computed tomography (µCT) against high-resolution µCT and bone ash of the guinea pig femur. Middle-aged (n = 40, 86 weeks) male guinea pigs underwent in vivo followed by ex vivo DXA (Hologic QDR 4500A) scanning for intact and excised femur BMC and areal density. To assess bone architecture and strength, excised femurs were scanned on pQCT (Stratec XCT 2000L) as well as on two µCT scanners (LaTheta LCT-200; Skyscan 1174), followed by three-point bending test. Reproducibility was determined using triplicate scans; and agreement assessed using Bland-Altman plots with reference methods being high-resolution µCT (Skyscan) for BMD and bone ashing for BMC. All techniques showed satisfactory ex vivo precision (CV 0.05-4.3 %). However, bias compared to the reference method was highest (207.5 %) in trabecular bone volume fraction (BV/TV) measured by LaTheta, and unacceptable in most total femur and cortical bone measurements. Volumetric BMD (vBMD) and BV/TV derived by LaTheta and pQCT at the distal metaphysis were biased from the Skyscan by an average of 49.3 and 207.5 %, respectively. Variability of vBMD, BV/TV and cross-sectional area at the diaphysis ranged from -5.5 to 30.8 %. LaTheta best quantified total femur BMC with an upper bias of 3.3 %. The observed differences among imaging techniques can be attributable to inherent dissimilarity in construction design, calibration, segmentation and scanning resolution used. These bone imaging tools are precise but are not comparable, at least when assessing guinea pig bones.

The generation of C-3α epimer of 25-hydroxyvitamin D and its biological effects on bone mineral density in adult rodents.

The source and function of C-3α epimer of 25(OH)D (C-3 epimer) is unknown. The objectives were to (1) establish if increasing doses of vitamin D (VD) results in a proportionate dose-response in C-3 epimer; and (2) determine the biological response of bone to C-3 epimer treatment. Sprague Dawley rats (12 weeks, n = 36 female, n = 36 male) were randomized to control AIN93-M diet (1 IU VD3/g diet) or an experimental diet for 8 weeks containing VD3 at 2 or 4 IU/g diet, C-3 epimer at 0.5 or 1 IU/g diet or 25(OH)D (0.5 IU/g diet). BW and food consumption were measured weekly. Blood was sampled at week 0, 4, and 8 for assessment of VD metabolites and bone metabolism biomarkers. DXA (week 0, 4, and 8) and in vivo micro CT (µCT) (week 0 and 8) were performed in vivo plus ex vivo µCT imaging and bone biomechanics. Dietary intake and anthropometry did not differ among diet groups. The dose-response of VD generated significantly elevated C-3 epimer only in females with concentrations of 4 IU VD diet group [mean 84.6 (62.5) nmol/L] exceeding control [mean 21.4 (18.5) nmol/L, p = 0.005]. Both sexes in the 25(OH)D group did not show significant increases in C-3 epimer, whereas 0.5 and 1 IU epimer groups exceeded 100 nmol/L of C-3 epimer by 8 weeks. These data suggest C-3 epimer is endogenously generated with higher intakes of VD. Endogenous and exogenous C-3 epimer accumulates in serum without impact upon bone health outcomes in a healthy young adult model over 8 weeks.

Orchidectomy-induced alterations in volumetric bone density, cortical porosity and strength of femur are attenuated by dietary conjugated linoleic acid in aged guinea pigs.

Age-related osteoporosis and sarcopenia are ascribed in part to reductions in anabolic hormones. Dietary conjugated linoleic acid (CLA) improves lean and bone mass, but its impact during androgen deficiency is not known. This study tested if CLA would attenuate the effects of orchidectomy (ORX)-induced losses of bone and lean tissue. Male guinea pigs (n=40; 70-72 weeks), were randomized into four groups: (1) SHAM+Control diet, (2) SHAM+CLA diet, (3) ORX+Control diet, (4) ORX+CLA diet. Baseline blood sampling and dual-energy X-ray absorptiometry (DXA) scans were conducted, followed by surgery 4 days later with the test diets started 7 days after baseline sampling. Serial blood sampling and DXA scans were repeated 2, 4, 8 and 16 weeks on the test diets. Body composition and areal BMD (aBMD) of whole body, lumbar spine, femur and tibia were measured using DXA. At week 16, muscle protein fractional synthesis rate (FSR), volumetric BMD (vBMD), microarchitecture and bone strength were assessed. Body weight declined after SHAM and ORX surgery, with slower recovery in the ORX group. Dietary CLA did not affect weight or lean mass, but attenuated gains in fat mass. Lean mass was stable in SHAM and reduced in ORX by 2 weeks with whole body and femur bone mineral content (BMC) reduced by 4 weeks; CLA did not alter BMC. By week 16 ORX groups had lower free testosterone and myofibrillar FSR, yet higher cortisol, osteocalcin and ionized calcium with no alterations due to CLA. ORX+Control had higher prostaglandin E2 (PGE2) and total alkaline phosphatase compared to SHAM+Control whereas ORX+CLA were not different from SHAM groups. Femur metaphyseal vBMD was reduced in ORX+CTRL with the reduction attenuated by CLA. Femur cortical thickness (Ct.Th.) and biomechanical strength were reduced and cortical porosity (Ct.Po.) elevated by ORX and attenuated by CLA. This androgen deficient model with a sarcopenic-osteoporotic phenotype similar to aging men responded to dietary CLA with significant benefits to femur density and strength.

The role of glycosyl phosphatidyl inositol (GPI)-anchored cell surface proteins in T-cell activation.

Glycosylphosphatidylinositol (GPI)-anchored cell surface proteins are widely expressed in tissues, including cells of immunohematopoietic origin. Cross-linking of GPI-linked proteins on T lymphocytes, such as Thy-1 (CD90), Ly-6 A/E, CD48, CD59 and others, induces T-cell mitogenesis. Similar to cross-linking with T-cell receptor (TcR)-specific antibodies, ligation of GPI-anchored proteins induces an intracellular flux of calcium, an up-regulation of activation-associated cell surface proteins and the elaboration of growth-promoting lymphokines. These events are dependent on p56(lck)-mediated tyrosine phosphorylation of substrates. GPI-linked proteins are constitutively clustered in sphingolipid-rich membrane domains. Actin-driven rearrangements of the cytoskeleton are probably responsible for the physical approximation of TcR and GPI-anchored proteins in mature immunological synapses. Functionally, GPI-linked proteins can supplant for signal I and productively collaborate with CD28 to fully activate T cells.