Weight gain model in prepubertal rats: prediction and phenotyping of obesity-prone animals at normal body weight.

OBJECTIVE: Male Sprague-Dawley rats maintained from birth on a high-fat diet were examined to determine whether a specific measure before puberty can identify and allow one to characterize prepubertal rats at normal weight with high vs low risk for adult obesity. MATERIALS AND METHODS: Measures from weaning (day 21) to around puberty (day 45) were taken of weight gain, absolute body weight and daily energy intake on a high-fat diet and related to the amount of body fat accumulated at maturity (80-100 days of age). Rats identified by a specific prepubertal measure as obesity-prone (OP) vs obesity-resistant (OR) were then characterized before and after puberty. RESULTS: Prepubertal weight gain from days 30 to 35 of age was the strongest and earliest positive correlate of ultimate body fat accrual in adult rats. The highest (8-10 g/day) compared to lowest (5-7 g/day) weight-gain scores identified accurately and reproducibly distinct OP and OR subgroups at day 35 that became obese or remained lean, respectively, as adults. The OP rats with rapid prepubertal weight gain and 50% greater adiposity at maturity (day 100) exhibited the expected phenotype of already-obese rats. These included elevated levels of leptin, insulin, triglycerides and glucose, increased galanin (GAL) peptide levels in the paraventricular nucleus (PVN) and reduced neuropeptide Y (NPY) levels in the arcuate nucleus (ARC). Before puberty (day 35), the OP rats with normal fat pad weights, energy intake and endocrine profile similar to OR rats exhibited these disturbances characteristic of obese rats. They had decreased capacity for fat oxidation in muscle, increased GAL expression in PVN and reduced expression of NPY and agouti-related protein in ARC. CONCLUSION: Prepubertal weight gain can identify OP rats on day 35 when they have minimal body fat but exhibit specific metabolic and neurochemical disturbances expected to promote obesity and characteristics of already-obese adult rats.

Model for predicting and phenotyping at normal weight the long-term propensity for obesity in Sprague-Dawley rats.

Tests were conducted to determine whether weight gain or nutrient intake measures during the first week of exposure to a macronutrient diet can accurately predict an animal's long-term propensity towards obesity. In multiple groups of normal-weight Sprague-Dawley rats (n=35-70/group), daily weight gain during the first 5 days on a high-fat diet (45-60% fat) was found to be strongly, positively correlated (r=+0.71 to r=+0.82) with accumulated body fat in 4 dissected depots after 4-6 weeks on the diet. This measure consistently identified obesity-prone (OP) rats which, relative to the obesity-resistant (OR) rats, were only slightly heavier (+15 g, 4%) and hyperphagic (+9 kcal, 8%) after 5 days but markedly heavier (+70g) with up to 2-fold greater fat mass after several weeks on the diet. Other dietary conditions and measures revealed weaker relationships to ultimate body fat accrual. The OP rats identified by their 5-day weight-gain score exhibited at this early stage clear disturbances characteristic of markedly obese rats. These included elevated leptin, insulin, triglycerides and glucose, along with increased lipoprotein lipase activity (LPL) in adipose tissue and galanin expression in the paraventricular nucleus. Most notable were significant reductions in muscle of LPL activity and ratio of beta-hydroxyacyl-CoA dehydrogenase to citrate synthase activity, indicating a decline in lipid transport and capacity of muscle to metabolize lipids. By occurring early with initial weight gain, these hypothalamic and metabolic disturbances in OP rats, favoring fat storage in adipose tissue over fat oxidation in muscle, may have causal relationships to long-term accumulation of body fat.

Different forms of obesity as a function of diet composition.

OBJECTIVE: To characterize the phenotype of obesity on a high-carbohydrate diet (HCD) as compared to a high-fat diet (HFD) or moderate-fat diet (MFD). METHODS AND PROCEDURES: In four experiments, adult Sprague-Dawley rats (275-300 g) were maintained for several weeks on a: (1) HFD with 50% fat; (2) balanced MFD with 25% fat; or (3) HCD with 10% fat/65% carbohydrate. Then, based on the amount of body fat accumulated in four dissected fat pads, the animals were subgrouped as lean (lowest tertile) or obese (highest tertile) and characterized with multiple measures. RESULTS: The obese rats of these diet groups, with 70-80% greater body fat than the lean animals, exhibited elevated levels of leptin and insulin and increased activity of lipoprotein lipase in adipose tissue (aLPL), with no change in muscle LPL. Characteristics common to the obese rats on the HFD or MFD, but not seen on the HCD, were hyperphagia, elevated circulating levels of triglycerides (TG), nonesterified fatty acids (NEFA) and glucose, and a significant increase in beta-hydroxyacyl-CoA dehydrogenase (HADH) activity in muscle, reflecting its greater capacity to metabolize fat. This was accompanied by a significant increase in expression of the peptide, galanin (GAL), in the paraventricular nucleus (PVN), as measured by in situ hybridization and real-time quantitative PCR, and also in GAL peptide immunoreactivity. These measures of GAL were consistently, positively correlated with circulating TG levels and also with HADH activity in muscle. In contrast to these fat-associated changes, rats that became obese on an HCD maintained normal caloric intake and levels of TG, NEFA, and glucose. They also showed no change in PVN GAL mRNA or peptide. Instead, they exhibited a significant reduction in HADH activity compared to the lean animals, along with increased activity of phosphofructokinase in muscle, a key enzyme in glycolysis. CONCLUSION: Specific characteristics of obesity, including expression of hypothalamic peptides, are dependent upon diet composition. Whereas obesity on an HFD is associated with hyperphagia and elevated lipids, fat metabolism in muscle, and fat-stimulated peptides such as GAL, obesity on an HCD with a similar increase in body fat shows none of these characteristics and instead exhibits a metabolic pattern in muscle that favors carbohydrate over fat oxidation. These results suggest the existence of multiple forms of obesity with different underlying mechanisms that are diet dependent.

PVN galanin increases fat storage and promotes obesity by causing muscle to utilize carbohydrate more than fat.

To understand the function of the feeding-stimulatory peptide, galanin (GAL), in eating and body weight regulation, the present experiments tested the effects of both acute and chronic injections of this peptide into the paraventricular nucleus (PVN) of rats. With food absent during the test, acute injection of GAL (300 pmol/0.3 microl) significantly increased phosphofructokinase activity in muscle, suggesting enhanced capacity to metabolize carbohydrate, and reduced circulating glucose levels. It also decreased beta-hydroxyacyl-CoA dehydrogenase activity in muscle, indicating reduced fat oxidation, while increasing circulating non-esterified fatty acids (NEFA) and lipoprotein lipase activity in adipose tissue (aLPL). Chronic PVN injections of GAL (300 pmol/0.3 microl/injection) versus saline over 7-10 days significantly stimulated daily caloric intake and increased the weight of four dissected fat depots by 30-40%. These effects, accompanied by elevated levels of leptin, triglycerides, NEFA and aLPL activity, were evident only in rats on a diet with at least 35% fat. Thus, by favoring carbohydrate over fat metabolism in muscle and reversing hyperglycemia, PVN GAL may have a function in counteracting the metabolic disturbances induced by a high-fat diet. As a consequence of these actions, GAL can promote the partitioning of lipids away from oxidation in muscle towards storage in adipose tissue.

A self-correcting indirect calorimeter system for the measurement of energy balance in small animals.

Indirect calorimetry involves measurement of CO(2) produced and O(2) consumed by an organism. These measurements are then used to calculate energy output, metabolic rate (MR), and respiratory quotient (RQ), a relative assessment of carbohydrate and lipid oxidation. By far the most difficult aspect of indirect calorimetry is measurement of O(2). Moreover, the abundance of O(2) (20.95%) relative to CO(2) (0.03%) in ambient conditions dictates that measurement errors of O(2) have greater implications on calculations of MR and RQ. Because compressed air is not feasible for use with animals in long-term experiments, changes in ambient conditions are nearly unavoidable. A self-correcting indirect calorimetry system was designed and constructed utilizing differential O(2) and CO(2) analyzers and a blank cage to monitor ambient conditions periodically. The system was validated by changing ambient O(2) and CO(2) concentrations by infusing N(2) into the system during a test butane burn. MR and RQ were largely unaffected by these changes in ambient conditions, and inclusion of a blank cage in the system accounted for slight calibration offsets. MR and RQ were measured in mice (n = 95) with and without correction for any small changes in ambient conditions measured in the blank cage. Coefficients of variation for MR and RQ were significantly decreased by taking into account ambient conditions measured in the blank cage (P < 0.001), which resulted in a 2.3% increase in precision for measurement of MR. This system will be used to more accurately assess long-term measurements of energy balance in the many murine models of leanness and obesity to gain better insights into pathophysiology and treatment of human obesity.

Fat oxidation, lipolysis, and free fatty acid cycling in obesity-prone and obesity-resistant rats.

Defects in fat metabolism may contribute to the development of obesity, but what these defects are and where they occur in the feeding/fasting cycle are unknown. In the present study, basal fat metabolism was characterized using a high-fat diet (HFD)-induced model of obesity development. Male rats consumed a HFD (45% fat, 35% carbohydrate) ad libitum for either 1 or 5 wk (HFD1 or HFD5). After 1 wk on the HFD, rats were separated on the basis of body weight gain into obesity-prone (OP, > or =48 g) or obesity-resistant (OR,

Effects of high fat or high sucrose diets on rat femora mechanical and compositional properties.

Diets high in fat and/or sucrose decrease whole bone mechanical properties and mineralization. This study examines the impact on rat bone mechanical properties and composition of age and diets (a) low in fat, (b) high in sucrose, and (c) high in fat. Sprague-Dawley rats aged 3 weeks (weanling [W]; n = 42), 8 weeks (young [Y]; n = 42), 16 weeks (teenage [T]; n = 39) and 56 weeks (old [O]; n = 40) were randomly assigned to groups: low fat, high sucrose and high fat with n = 12-16 per group. All animals were fed a purified low-fat, high starch diet for two weeks, and fed a low fat (STD), high sucrose (HSD), or high-fat (HFD) for five additional weeks. After sacrifice, the femurs were harvested and non-osseous tissue was removed. The bones were dried at 25 degrees C for 48 hours. Length and the periosteal minimum and maximum diameter (D-min and D-max) at the mid-diaphysis of the femurs were measured with Vernier calipers. The femurs were rehydrated and tested via three-point flexure. Bones were weighed after drying at 105 degrees C (48 hours; Dry-M) and 800 degrees C (24 hours; Ash-M). Percent mineralization (%Min) was calculated as Ash-M/Dry-M X 100%. Length, D-min and D-max, Dry-M and Ash-M all significantly (p < 0.05) increased with age (W < Y < T < O) within each group. %Min and stiffness were significantly greater in [O] than in the younger femurs. No significant results were seen in any age group due to varying diet. These results indicate that five weeks of high fat or high sucrose diet feeding does not affect whole bone size, composition or mechanical properties. Whether a longer dietary period or different diet composition would elicit changes requires further study.

Developmental stage modifies diet-induced peripheral insulin resistance in rats.

In the present study, the effects of age and diet on glucose disappearance and tissue-specific glucose uptake (R'g) were examined under basal or hyperinsulinemic, euglycemic conditions in male Sprague-Dawley rats. Rats were equicalorically fed either a high-starch diet (68% of kcal), high-fat diet (HFD; 45% of kcal), or high-sucrose diet (68% of kcal), beginning at either 5 (W; weanling), 10 (Y; young), 18 (M; mature), or 58 wk (O; older) of age for 5 wks (n = 6-9. group(-1) x diet(-1)). Body weight gain was not significantly different among dietary groups within a given age. Significant (P< 0.05) age effects were observed on basal and clamp free fatty acid concentrations. Significant diet effects were observed on basal and clamp triglyceride concentrations. There were significant diet and age effects on basal skeletal muscle R'g. This interaction was primarily due to an age-associated increase in basal R'g microg x g(-1). min(-1)) in HFD (gastrocnemius R'g: 0.9+/-0.2 in W, 1.1+/-0.2 in Y, 1.8+/-0.2 in M, 2.5+/-0.2 in O). Both age and diet significantly decreased insulin-stimulated muscle R'g. However, whereas age-associated reductions in both glucose-6-phosphate concentration and glycogen synthase activity were observed, significant diet effects were observed on glucose-6-phosphate concentrations only. Age significantly reduced basal and clamp adipose tissue R'g when expressed per gram of tissue but significantly increased R'g when expressed per total fat pad mass. These data suggest that diet-induced changes in peripheral glucose metabolism are modulated by age.

Adipose tissue-derived tumor necrosis factor activity correlates with fat cell size but not insulin action in aging rats.

Adipose tissue-derived tumor necrosis factor (AT-TNF) protein and messenger RNA (mRNA) has been shown to correlate with insulin resistance in some studies. However, in a study using different aged Fischer 344 rats, AT-TNF activity correlated more strongly with cell size than with fasting plasma insulin. The present study was undertaken to more carefully examine the relationship among AT-TNF, adipose cell size, and insulin action using more precise measures of insulin action. Basal and hyperinsulinemic, euglycemic clamps were performed in male Sprague Dawley rats at four different ages (8, 13, 21, and 61 weeks old). [3-(3)H]glucose and 2-deoxy-D-[1-(14)C]glucose were used to assess glucose kinetics and tissue-specific glucose uptake. Because TNF activity represents the summation of TNF synthesis, secretion, and the amount of soluble inhibitors present, TNF activity was measured using a bioassay, in addition to measuring TNF protein and mRNA levels. AT-TNF activity increased significantly with age, as did the glucose infusion rate, a measure of whole body insulin resistance. However, AT-TNF activity did not correlate with any parameter of insulin action measured during the hyperinsulinemic, euglycemic clamps. In epididymal fat, AT-TNF activity correlated with: glucose infusion rate: r = -0.50, P = 0.17; rate of appearance: r = -0.19, P = 0.35; rate of disappearance: r = 0.08, P = 0.69. As was noted before, AT-TNF activity correlated well with fat cell size (r = 0.76, P < 0.001 in epididymal fat; r = 0.58, P = 0.007 in SUB fat). These data suggest that although AT-TNF activity and insulin resistance increase with age, the two are not functionally related. These data do not eliminate the potential role of nonadipose TNF in the regulation of insulin action.

Menhaden oil prevents but does not reverse sucrose-induced insulin resistance in rats.

Although fish oil supplementation may prevent the onset of diet-induced insulin resistance in rats, it appears to worsen glycemic control in humans with existing insulin resistance. In the present study, the euglycemic, hyperinsulinemic (4x basal) clamp technique with [3-3H]glucose and 2-deoxy-[1-14C]glucose was used to directly compare the ability of fish oil to prevent and reverse sucrose-induced insulin resistance. In study 1 (prevention study), male Wistar rats were fed a purified high-starch diet (68% of total energy), high-sucrose diet (68% of total energy), or high-sucrose diet in which 6% of the fat content was replaced by menhaden oil for 5 wk. In study 2 (reversal study), animals were fed the high-starch or high-sucrose diets for 5 wk and then the sucrose animals were assigned to one of the following groups for an additional 5 wk: high starch, high sucrose, or high sucrose with 6% menhaden oil. Rats fed the high-starch diet for 10 wk served as controls. In study 3 (2nd reversal study), animals followed a similar diet protocol as in study 2; however, the reversal period was extended to 15 wk. In study 1, the presence of the fish oil in the high-sucrose diet prevented the development of insulin resistance. Glucose infusion rates (GIR, mg.kg-1.min-1) were 17.0 +/- 0.9 in starch, 10.6 +/- 1.7 in sucrose, and 15.1 +/- 1.5 in sucrose with fish oil animals. However, in study 2, this same diet was unable to reverse sucrose-induced insulin resistance (GIR, 16.7 +/- 1.4 in starch, 7.1 +/- 1.5 in sucrose, and 4.8 +/- 0.9 in sucrose with fish oil animals). Sucrose-induced insulin resistance was reversed in rats that were switched back to the starch diet (GIR, 18.6 +/- 3.0). Results from study 3 were similar to those observed in study 2. In summary, fish oil was effective in preventing diet-induced insulin resistance but not able to reverse it. A preexisting insulin-resistant environment interferes with the positive effects of menhaden oil on insulin action.

Fat and energy balance.

In summary, an imbalance between energy intake and energy expenditure can explain approximately 80% of the variance in body weight gain in this dietary model of obesity. Several metabolic variables appear to contribute to differences in energy balance. A high RQ and an inappropriate suppression of glucose production by insulin appear to be linked to the increase in energy intake that occurs when obesity-prone rats are provided with the high-fat diet. In addition, early tissue enzymatic differences in obesity-prone versus obesity-resistant rats may contribute to differences in energy expenditure and/or to differences in nutrient partitioning. In this dietary model, susceptibility to dietary obesity involves a metabolic environment that includes a high RQ and a reduced ability of insulin to suppress glucose appearance (FIG. 9). However, this environment does not lead to obesity nor to a measurable difference in body weight gain when the susceptible rats are eating a low-fat diet. The high-fat diet is a necessary catalyst for the observed variability in body weight gain and the development of obesity. As a catalyst, the high-fat diet results in an imbalance between energy intake and energy expenditure in some, but not all, rats. This imbalance interacts with the permissive metabolic environment (tissue enzymatic profile favoring carbohydrate utilization and lipid storage) to produce obesity on the high-fat diet. Later, in the HFD feeding period, the rate of weight gain is not significantly different between OP and OR rats, although net fat accumulation remains greater in the former group. It is interesting that this later period is characterized by a reduction in the difference in both RQ and energy intake between OP and OR rats. Thus, during the later stages of HFD feeding, the discrepancy in both energy balance and nutrient balance between OP and OR rats is reduced. This dietary model of obesity is relevant to human obesity. While the prevalence of obesity is high, the majority of people are not obese. The high prevalence of obesity may be due to environmental catalysts that interact with inherent behavioral and metabolic characteristics that favor nutrient retention. Resistance to obesity can be achieved by avoiding these environmental catalysts, by having inherent characteristics that prevent nutrient retention, or both. Our work suggests that the complete understanding of obesity will require not only the identification and functional significance of the genes that determine the inherent capacity of the behavioral and metabolic systems, but also the role of environmental catalysts in determining where and how these systems operate.

Female rats do not develop sucrose-induced insulin resistance.

In male rats, 2 wk of high-sucrose feeding results in insulin resistance and hypertriglyceridemia [Pagliassotti, M.J., P.A. Prach, T.A. Koppenhafer, and D.A. Pan. Am. J. Physiol. 271 (Regulatory Integrative Comp. Physiol. 40): R1319-R1326, 1996]. The present study aimed to determine if female rats also become insulin resistant and hypertriglyceridemic in response to high-sucrose feeding. Female Wistar rats (7 wk old) were fed either a high-sucrose diet (68% energy) (SU) or a high-starch diet (68% energy) (ST) for 3, 5, or 8 wk. In each animal, glucose kinetics were measured using [3-(3)H]glucose under basal and hyperinsulinemic conditions (insulin infusion 4.0 mU.kg-1.min-1). Body weight and basal glucose kinetics were not different between diet groups at 3, 5, or 8 wk. Glucose infusion rate (mg.kg-1.min-1) was not different between groups (3 wk: 17.7 +/- 1.6 ST, 16.6 +/- 0.9 SU; 5 wk: 16.1 +/- 0.9 ST, 15.1 +/- 2.0 SU; 8 wk: 18.3 +/- 1.9 ST, 16.1 +/- 1.5 SU). Clamp rate of glucose appearance (mg.kg-1.min-1) was also not different between diet groups (3 wk: 4.0 +/- 1.6 ST, 3.6 +/- 1.4 SU; 5 wk: 2.6 +/- 1.0 ST, 2.3 +/- 1.14 SU; 8 wk: 5.9 +/- 1.8 ST, 7.7 +/- 1.2 SU). No difference was observed in plasma and tissue triglycerides or tissue glycogen between sucrose- and starch-fed animals. We therefore conclude that female rats, in contrast to males, do not develop sucrose-induced insulin resistance and hypertriglyceridemia.

Reduced insulin suppression of glucose appearance is related to susceptibility to dietary obesity in rats.

To examine the relationship between insulin action and body weight regulation in male rats, the following studies were performed. In study 1, rats (n = 31) were fed a low-fat diet (LFD) for 4 wk, and then glucose kinetics were estimated under basal and hyperinsulinemic conditions using the glucose clamp. After clamps, these same rats were placed on a high-fat diet (HFD) for 5 wk. In study 2, rats (n = 30) were fed an LFD for 3 wk and then a high-sucrose diet for 1 wk to produce selective hepatic insulin resistance. Clamps were then performed, and after clamps, these same rats were placed on an HFD for 5 wk. In study 3, rats (n = 30) were fed an LFD for 1 wk and then a high-sucrose diet for 3 wk to produce widespread insulin resistance. Clamps were then performed, and after clamps, these same rats were placed on an HFD for 5 wk. The rate of glucose appearance (R(a)) during the hyperinsulinemic clamps was the only pre-HFD variable that correlated (r = 0.49, P < 0.01 in study 1; r = 0.51, P < 0.001 in study 2) with weight gain on the HFD. Clamp R(a) also correlated with energy intake on the HFD in study 1 (r = 0.64, P < 0.001) and study 2 (r = 0.59, P < 0.001). Clamp R(a) and energy intake on the HFD accounted for similar portions of the variance in body weight gain on the HFD. Weight gain and fat-pad mass were increased (P < 0.05) in study 2 compared with study 1. In study 3, pre-HFD glucose kinetics were not correlated with energy intake or weight gain on the HFD. Widespread insulin resistance did not significantly reduce the rate of weight gain on the HFD. Thus insulin action on R(a) can influence body weight gain on an HFD. The effects of R(a) on body weight gain appear to be mediated via effects on energy intake. Selective hepatic insulin resistance can increase body weight gain on an HFD, but widespread insulin resistance does not significantly reduce HFD-induced weight gain.

Contribution of energy intake and tissue enzymatic profile to body weight gain in high-fat-fed rats.

The purpose of the current study was to examine the enzymatic profile [phosphofructokinase (PFK), beta-hydroxyacyl-CoA dehydrogenase (HADH), and citrate synthase (CS)] in gastrocnemius muscle, heart, and liver in rats allowed ad libitum access to a high-fat diet (HFD, 45% of kcal from corn oil). Male Wistar rats were fed a low-fat diet (LFD, 12% of kcal from corn oil) for a 2-wk baseline period after which some continued on the LFD and others were placed on the HFD. After 1 wk on the HFD, rats were categorized as obesity-resistant (OR), -intermediate (OI), or -prone (OP) on the basis of body weight gain (OR, lower tertile; OI, middle tertile; OP, upper tertile). At 1, 2, and 5 wk, rats from each group were killed (n = 9-14 from each group/time point) after a 24-h fast. At the end of the 5-wk dietary period, weight gain was 114.8 +/- 4.3 in LFD, 125.2 +/- 3.7 in OR, 147.1 +/- 4.1 in OI, and 173.7 +/- 3.5 g in OP rats (OP > OI > OR, LFD; P < 0.001). Energy intake was highly correlated with weight gain on the HFD at each time point (r > or = 0.72, P < 0.001). After 1 wk on the HFD, significant correlations between the ratio of PFK/HADH (an indication of the relative capacity for glycolysis vs. beta-oxidation, r = 0.4, P = 0.03) and HADH/CS (an indication of the capacity for beta-oxidation relative to total oxidative capacity, r = -0.56, P = 0.001) in the gastrocnemius muscle and weight gain were observed. At week 2, significant correlations between these ratios and weight gain were observed in the gastrocnemius, liver, and heart. In contrast, these ratios were not significantly correlated with weight gain at 5 wk. These results suggest that rats most susceptible to weight gain or a HFD are characterized by a continuous increase in energy intake (explaining approximately 50% of the variance in weight gain) and an early tissue enzymatic profile that favors carbohydrate over fat use.

Contribution of dietary and loading changes to the effects of suspension on mouse femora.

The present study assessed the contributions of feeding changes and unloading to the overall measured effects of 2-wk hindlimb (Tail) suspension on the mouse femora. Feeding changes were addressed by considering the effects of matched feeding among suspended and control mice. The effects of hind limb unloading were considered by comparing suspended mice to mice equipped identically (though not suspended) and matched-fed. The feeding and unloading aspects of suspension appear to cause distinctly differing effects on the stereotypic modeling of the femora. Matched-feeding was accompanied by increased resorption surface in comparison to suspended mice, while unloading led to reduced bone formation at the mid-diaphysis of the femora. Reduced mineral content was observed in the bones of suspended mice when compared to the other mice groups, but without increased resorption surface. Thus, the unloading aspects of the antiorthostatic suspension protocol apparently causes reduced formation and mineralization in the femur.

The role of sex and genotype on antiorthostatic suspension effects on the mouse peripheral skeleton.

Previous antiorthostatic suspension studies have used a single sex and strain of rat or mouse. Nonetheless, broadly similar effects of suspension on the two species indicates a generalized effect of suspension not attributed to specific genetic, behavioral, or sex-linked etiology. In order to directly test genetic and sex-linked factors, the effects of suspension on the appendicular bone of male and female BALB-CJ, C57BL-6J, and DBA-2J mice were compared. These genotypes were selected based on their widely different developmental and behavioral characteristics as well as on past research involving a heterogeneous strain derived from these strains. The effects of suspension on the geometric, mechanical, and material properties of the femora, humeri, and tibiae were determined. Among the bone types, the femora were most significantly affected by suspension. The effects of suspension were similar in nature in male and female mice aged 1.7 months. Strain-dependent suspension effects may be indicative of bone developmental differences in the strains at the age chosen.