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Department of Human Nutritional Sciences, University of Manitoba, Winnipeg, MB, R3T 2N2 Canada
3To whom correspondence should be addressed. E-mail: hweiler{at}cc.umanitoba.ca.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • bone • obesity • hyperinsulinemia • Type 2 diabetes mellitus • PUFA
Childhood obesity is increasingly referred to as an epidemic. The prevalence of obesity among Canadian youth, 7–13 y old, tripled between 1981 and 1996 (1). Childhood obesity is a precursor to poor health (2) and is commonly quoted as a risk factor for the development of heart disease, metabolic syndrome, and diabetes mellitus (DM)4 in adults (2). Obesity and its associated insulin resistance are major risk factors for the development of Type 2 DM (3). Recently Type 2 DM has evolved in children (4). There is also increasing evidence that children who are overweight or obese have less mineralized bones after correction for bone size (5,6), and they fracture more often than nonobese children (6–8). No reports exist in children with Type 2 DM concerning bone metabolism and the development of peak bone mass. In adults, hyperinsulinemia was suggested to be an osteogenic factor and to be responsible for the increased bone mineral density (BMD) in women, but not men, with Type 2 DM (9). Obesity is a prevailing feature of Type 2 DM; in adults without DM, obesity is associated with an increased bone mass and a reduced incidence of osteoporosis (10,11). In theory, adults with DM who are obese may have higher bone mass. However, decreased or unaltered BMD was reported in Type 2 DM [reviewed in (12)], and elevated bone resorption was reported in postmenopausal women with Type 2 DM (13). Thus, it is critical to learn of the interactions among obesity, hyperinsulinemia, and bone in childhood and early adulthood because this is the period in which peak bone mass is set (14).
Dietary (n-3) PUFA are important in the primary management of Type 2 DM through improvements in lipid metabolism (15,16). They may also offset the adverse affects of Type 2 DM on bone metabolism. Our laboratory reported recently that consumption of dietary (n-3) PUFA from fish oil, high in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) for 5 wk enhanced bone mass in healthy Sprague-Dawley rats, but to a lesser extent in those with streptozotocin-induced Type 1 DM (17). There are numerous reports that demonstrate positive effects of dietary (n-3) PUFA on bone mass over similar time frames in healthy animals (18,19), but standardization of the total PUFA across diets has not been forthcoming. For example, in our previous study, the total dietary PUFA was 61.6% in the soybean oil–based control diet group compared with 46.4% of dietary fat in the fish oil diet group, representing a 25% reduction in PUFA (17). Similarly, in the study of Watkins et al. (18), total PUFA was 
68% in the control and reduced to 25% of the dietary fatty acids (FA) after fish oil was added to the fat blend. These reductions in total PUFA are important because both (n-6) and (n-3) PUFA are precursors for the prostaglandins E2 (PGE2) and E3 (PGE3), respectively. Both are essential to bone metabolism, and alteration in the precursor pool affects PGE metabolism. Modification of dietary PUFA leads to parallel alterations in bone FA (20–22) and PG metabolism (19–21). PGE2 exhibits biphasic effects on bone formation, stimulating bone formation at low concentrations, but inhibiting it at high concentrations (23). PGE2 is synthesized from arachidonic acid (AA) within the osteoblast cell (24); however, the amount required for the optimum development of bone is not known. PGE3 also mediates bone cell function and is synthesized from EPA. However, AA is more readily synthesized to PGE2 than EPA is to PGE3 (24). To date, the studies in which (n-3) dietary PUFA were shown to elevate bone mass also showed reduced PGE2, but it is not entirely clear whether the reduction is due to altered total precursor pool or the type of FA precursor.
The primary objective of this study was to determine the effects of dietary 18-carbon (n-6) PUFA (linoleic acid: LA, 18:2), 18-carbon (n-3) PUFA (
-linolenic acid: ALA, 18:3) and long chain (n-3) PUFA (EPA: 20:5, DHA: 22:6), while keeping the proportion of total PUFA constant, on bone mass and biomarkers of bone metabolism in an obese and hyperinsulinemic state. The Zucker fatty model was selected because the fa/fa phenotype is characterized by hyperinsulinemia (25), but is relatively normoglycemic or very slightly hyperglycemic (26–28), thus removing hyperglycemia as a confounding variable. The fa/fa Zucker rat is also characterized by elevated body weight and fat mass, and initially similar long bone mass (29–31), which appears to decline between 3 and 6 mo of age (31). It has been suggested as a good model for bone metabolism in juvenile obesity (30). Determining the benefits of dietary (n-3) PUFA on bone mass is important because similar dietary interventions are used experimentally to control hyperlipidemia in the diabetic state (14,15). This study assessed the benefit of (n-3) PUFA to bone early in the disease course. After establishing the benefits early in the disease course, exploring the possibility of maintaining bone mass during established diabetes would be an important next step.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Each diet was nutritionally adequate and was based on the AIN-93G formulation (33), with each diet having 100 g fat/kg diet instead of the recommended 70 g/kg. The test diets (Table 1) were a flaxseed oil mixture (FXO) diet, a menhaden oil mixture (MO) diet, and the SO diet. The FXO diet consisted primarily of flaxseed oil (60 g/kg); the remaining oils were safflower oil (10 g/kg), coconut oil (2 g/kg), and canola oil (1 g/kg). The MO diet consisted of menhaden oil (70 g/kg) and safflower oil (30 g/kg). The SO diet consisted of safflower oil (70 g/kg), coconut oil (20 g/kg), and canola oil (10 g/kg). To ensure that the balance among the FA categories was similar among the diets, the test oils were analyzed by GC (34). Lipids were extracted with 2:1 chloroform:methanol and were methylated with 3 mol/L methanolic hydrochloric acid (Supelco) for 1 h at 80°C. Fatty acid esters were separated on a DB-225 capillary column (30 m x 0.25 mm i.d. with 0.25 µm film thickness) using a Varian Star 3400 Gas Chromatography System with a flame ionization detector. All diets had similar SFA, monounsaturated fatty acid (MUFA), and PUFA totals (Table 1). The selection of safflower oil, compared with soybean oil, as the base of the control diet provided the majority of PUFA as the (n-6) series.
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Body and tail length. Growth was assessed using body length and tail length. Body length was measured from nose tip to the anus and tail length was measured from the anus to the tail tip. Both measurements were done with the rat placed on its back and measured to the nearest 0.1 cm with a ruler.
Bone mineral and biochemistry. After the rats were killed, right femurs were excised and cleaned of soft tissue. Femur measurements (thickness of diaphysis, head, proximal epiphysis, and knee joint) were obtained using a caliper to the nearest 0.01 mm as described by Reichling et al. (36). Femurs were then measured for bone area (BA), BMC, and BMD in situ using dual-energy X-ray absorptiometry (DXA: 4500A, Hologic; small animal software option). Femurs were placed in a plastic water bath with 2 cm of water above the bone and aligned in an anterior-posterior position. DXA was shown to be a simple, accurate and precise technique for measuring BMC and BMD in isolated small animal bones (37). Only water should be used for determination of BMC and BMD by DXA (37). The CV for triplicate assessment of BA, BMC, and BMD of excised rat femurs by this DXA were 0.74, 1.03, and 0.64%, respectively. Femurs were dried in an oven (85°C) for 48 h and then measured for dry weight.
Femur calcium and phosphorus were determined after wet ashing of femurs using concentrated nitric acid at room temperature for 48 h (38). The digest was diluted to obtain a final concentration of 5% (v:v) nitric acid in deionized water and then measured using inductively coupled plasma optical emission spectroscopy (Varian Liberty 200, Varian Canada). Mineral content was expressed per dry weight of the femur.
Osteoblast activity (bone formation) was determined by measuring plasma osteocalcin using an ELISA (Rat-Mid osteocalcin, Osteometer BioTech A/S). Osteoclast activity (bone resorption) was determined by measuring bone-related plasma degradation products of C-terminal telopeptides of type 1 collagen using an ELISA (RatLaps, Osteometer BioTech A/S). For osteocalcin and C-terminal telopeptides of type I collagen, the calculated percentage of agreement was >80%.
Bone organ culture was performed on left femur diaphysis (0.2 g), obtained immediately after the rats were killed, as described by Dekel et al. (39). Femur sections were incubated in HBSS (Sigma Chemical) for 2 h at 37°C in a shaking water bath, followed by the removal of bone and rapid freezing of solution. Samples were stored at –20°C until duplicate analysis of PGE2 by ELISA (R&D Systems) and corrected to the weight of the femur segment studied. To minimize interference of the HBSS with the alkaline phosphatase enzyme, standards were reconstituted using this solution rather than the buffer provided with the kit. In addition, the antibody cross-reacts with PGE1 (70%) and PGE3 (16.3%). For PGE2, the CV was <15%.
Statistics. Differences among the groups were detected by 2-way ANOVA. Where appropriate, post hoc analysis was conducted to detect differences among dietary treatments using Tukey’s test. Data are expressed as means ± SEM. Differences with P < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The fa/fa rats had femurs that were lighter and shorter than femurs of lean rats (Table 2); there were no diet effects on femur weight (P = 0.1194) or length (P = 0.1676). The fa/fa rats also had lower femoral head and proximal epiphysis widths, but not diaphysis or knee joint widths (Table 2). Diet had a main effect on diaphysis width (P < 0.0001); the group fed fish oil had greater width than the control group (MO 0.383 ± 0.004 vs. SO 0.365 ± 0.005 cm, P = 0.019), but the FXO group was intermediate (0.375 ± 0.003 cm). Femoral head, proximal epiphysis, or knee joint widths did not differ due to diet (data not shown). The fa/fa rats also had lower femoral BA and BMC than lean rats (Fig. 1a,b), but BMD was not affected by genotype (Fig. 1c). There were no diet effects for femur BA, BMC, and BMD (Fig. 1). Femur calcium and phosphorous concentration did not differ, regardless of genotype (Table 2). Femur calcium (MO 6.09 ± 0.05, SO 5.99 ± 0.06, FXO 6.09 ± 0.06 mmol/g) and femur phosphorus (MO 4.26 ± 0.04, SO 4.19 ± 0.05, FXO 4.26 ± 0.05 mmol/g) did not differ among the diet groups.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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It is notable in the fa/fa rats that the thickness of the femoral head and proximal epiphysis were reduced relative to the lean controls. This is a novel observation and suggests that with time, fracture risk in these regions may be elevated as bone loss occurs with advancing age (48). Forearm fractures are more common in obese children than in children of healthy body weight (6,8). Whether the fracture rates in obese children are linked to hyperinsulinemia has not been reported. The present study suggests that hyperinsulinemia might be part of the mechanism because dietary minerals and other dietary constituents were consistent among the fa/fa and lean rats. Based on adults with Type 2 DM, it is possible as well that as hyperglycemia ensues, the fracture rate might become a pressing issue over time. For example, Forsen et al. (49) found an increased fracture rate in 50- to 74-y-old women with Type 2 DM and had the disease for >5 y, whereas women with a shorter disease duration and men did not have an increased fracture rate. Thus, fracture risk might soon be added to the sequelae of Type 2 DM in children.
Femoral BMD was not altered in fa/fa Zucker rats when dietary treatments were initiated at a young age and bone mass assessed when they were young adults. In humans, measurement of BMD of the vertebrae or femoral neck using DXA or dual photon absorptiometry showed either no differences between subjects with Type 2 DM and controls (50,51) or increased values (9,10,12,13,52), whereas low BMD was seldom found (53). These studies were conducted in subjects after they had attained peak bone mass. For children and young adults who have not yet attained peak bone mass, hyperinsulinemia and obesity may negatively affect bone formation. This is relevant because bone mass attained early in life is the most important determinant of lifelong bone health (54). Those with the highest peak bone mass after adolescence have the greatest protective advantage against bone loss later in life (54).
Insulin is regarded as a systemic regulator of bone formation (51). Hyperinsulinemia is thought to enhance the mitogenic and anabolic actions of insulin in bone (12). The already elevated BMD in patients just diagnosed with diabetes (12,55,56) supports this thesis. In premenopausal women with Type 2 DM, bone resorption was elevated without a reduction in BMD (51). However, if increased bone resorption persists in the long term, a negative effect on bone mass may be expected after menopause (41). In advanced Type 2 DM, osteocalcin concentrations were also reduced (57–59). Both increased bone resorption and reduced bone formation would lead to reduced BMD, which has been observed in people with poorly controlled Type 2 DM (60–62). Negative calcium balance is also observed with hyperglycemia (62). In individuals with Type 2 DM and poor metabolic control, low BMD could be due to glucosuria with hypercalciuria and mild secondary hyperparathyroidism (45). The rats used in this study had normal glucose levels (data not shown) as well as biomarkers of bone metabolism. Therefore, if hyperglycemia is responsible for changes in bone formation and resorption markers in humans with Type 2 DM, it makes sense that these markers were not altered in fa/fa and lean Zucker rats because hyperglycemia was not a factor.
In an earlier study, PGE2 release was reduced and bone mass was enhanced over 5 wk in rats fed a MO diet (17) similar to that used in the current study. Although the reduction of the (n-6):(n-3) FA ratio using (n-3) PUFA reduced ex vivo PGE2 release from bone in the current study, it did not affect bone mass during the 9-wk study. Release of less PGE2 from the tibia after a reduction in the (n-6):(n-3) dietary PUFA ratio (to <2.6) was also observed in chicks (21) and rats (18) in association with enhanced bone formation rates. We did not conduct histomorphometry, but our result of enhanced diaphysis width with consumption of the MO diet concurs with the data of Watkins et al. (18) for bone formation rate. Because no differences were detected in the biomarkers of bone formation and resorption in the Zucker rats, it is possible that plasma osteocalcin and C-terminal telopeptides of type I collagen are not sensitive enough to reflect formation rates in the diaphysis alone. In another study, the activity of bone-specific alkaline phosphatase and formation rate were higher in rats fed diets with high amounts of (n-3) FAs and a (n-6):(n-3) ratio of 1.2, but osteocalcin was not affected (18). The diet was similar to the MO diet in this study, with a (n-6):(n-3) ratio of 1 and both were designed using MO (EPA and DHA).Whether the dietary interventions affected bone architecture will require further study because in Zucker rats, regional BMD is not reduced despite modified trabecular architecture (29).
One of the strengths of this research is that the proportions of total PUFA, MUFA, and SFA in our diets were relatively constant. The only other study to our knowledge in which SFA and MUFA were held constant, although the (n-6):(n-3) ratio was reduced from 9.0:1 to 4.5:1, also did not demonstrate altered bone metabolism or mass (63). In other studies in which semipurified AA and DHA were added to piglet formula without changing the dietary MUFA, SFA, and PUFA, PGE2 was unaltered, but bone mass was higher (22,64,65). This suggests that PGE2 and LCPUFA have different effects on bone. Lucia et al. (66) demonstrated this using PGE2 injections vs. dietary LCPUFA; PGE2 injections enhanced bone formation and dietary LCPUFA reduced bone resorption. Other groups also observed reduced bone resorption in rats with the addition of combinations of (n-6) and (n-3) PUFA and LCPUFA (67). The common features in the studies in which bone mass was enhanced were that the studies were short in duration, ranging from 2 wk in pigs (22,64,65) to 5 or 6 wk in rats (67), and that all were initiated at weanling age or younger. Longer-term studies have not been conducted, and inception after weaning has not been well studied in healthy rats. Thus, the current study adds important new information that dietary (n-3) PUFA supplemented between 6 and 15 wk of age does not benefit bone mass in Zucker rats. The lack of effect in these young adult rats is similar to a study in healthy women by Bassey et al. (68) in which dietary PUFA supplementation did not affect whole body bone mass. Reduced release of PGE2 in the rats, however, may become critical when bone loss begins with advanced aging. Bone loss was reduced by feeding fish oils (69) or a 
-linolenic acid (GLA) + EPA diester (70) to ovariectomized rodents. Further, proximal femur BMD was improved by dietary GLA + EPA + DHA in women 80 y of age (71).
In summary, this study demonstrates that reduction in PGE2 during slow growth does not benefit long bone mass as measured using DXA. The fa/fa Zucker rat is a good model for early diabetic manifestations in bone before the onset of hyperglycemia. Based on morphometry, the fa/fa Zucker rat has reduced long bone mass compared with controls and could be at higher risk of proximal femoral fracture with time. Whether children or young adults with obesity and/or hyperinsulemia are also at risk requires investigation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Funded by grants from the Natural Sciences and Engineering Research Council of Canada (H.A.W.); and the Flax Council of Canada and Canada-Manitoba Agri-food Research and Development Initiative (C.G.T.). The Children’s Hospital Foundation of Manitoba purchased and maintains the densitometer research facilities of the Manitoba Institute of Child Health used in this study. H.A.W. is in receipt of a New Investigator Salary Award from the Canadian Institutes of Health Research. R.C.M. was supported by a University of Manitoba fellowship and a Natural Sciences and Engineering Research Council of Canada Scholarship. M.E.G. was supported by a Natural Sciences and Engineering Research Council of Canada Scholarship.
4 Abbreviations used: AA, arachidonic acid; ALA, -linolenic acid; BA, bone area; BMC, bone mineral content; BMD, bone mineral density; DHA, docosahexaenoic acid; DM, diabetes mellitus; DXA, dual energy X-ray absorptiometry; EPA, eicosapentaenoic acid; FA, fatty acid; FXO, flaxseed oil; GLA, -linolenic acid; IGF-1, insulin-like growth factor-1; LA, linoleic acid; LCPUFA, long-chain PUFA; MO, menhaden oil; PG, prostaglandin; SO, safflower oil.
Manuscript received 5 August 2004. Initial review completed 7 September 2004. Revision accepted 28 December 2004.
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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