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Article

Bones, Muscles and Visceral Organs of Protein-Malnourished Rats (Rattus norvegicus) Grow More Slowly but for Longer Durations to Reach Normal Final Size

Department of Biological Sciences, University of Cincinnati, Cincinnati, OH 45221-0006

¹To whom correspondence should be addressed.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Starting at weaning (22 d), Sprague-Dawley rats were fed either a control diet high in protein (CT, 24% protein) or an isocaloric low protein diet (LPT, 4% protein) to determine how protein malnutrition alters the rate and timing of limb bone growth. Length and width measurements were taken from longitudinal radiographs to provide complete growth trajectories of both treatments. Data collection continued until rats reached adult size, which varied among diet-sex groups. The rats were then killed and five muscles and eight organs were weighed. A nonlinear Gompertz model was then fit to each trajectory for 13 skeletal measurements, producing parameters that described the rate and timing of growth for each rat, the unit of analysis. Parameter differences due to diet, sex and litter were tested by using a mixed-model, three-way ANOVA. For most measurements, the LPT rats were not significantly smaller than the CT rats, for the model’s prediction of final size. Bone length was significantly less affected than width. The instantaneous initial growth rate, maximum rate of growth and rate of growth decay were significantly higher in the control rats for all measurements. The rats fed the low protein diet grew for significantly longer periods of time. For all muscles and most organs relative to body size, there was no difference between rats fed the two diets. The exceptions, eyes and brains, were proportionally larger in the LPT rats, suggesting that these organs receive nutritional priority during growth. For the systems in this study, structures that grow or have the potential for extended growth are less affected by the nutritional insult.

KEY WORDS: • protein malnutrition • skeletal growth • organ growth • rats • Gompertz

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Despite having a genetic basis, ultimate skeletal size is influenced by environmental stimuli such as nutrition (Dammrich 1991 , Loveridge and Noble 1994 , Prader et al. 1963 ). Protein malnutrition is known to have a significant and measurable effect on the rate and timing of growth (Golden 1994 , Malcom 1979 ). Many studies demonstrate that insufficient levels of dietary protein inhibit growth and therefore increase the time necessary to reach full adult size (Cameron and Eshelman 1996 , Dammrich 1991 , Edozien and Switzer 1978 , Prader et al. 1963 , Yayha and Millward 1994 ). Reviews by Golden (1994) and Martorell et al. (1994) point out that skeletal height as well as bone maturity are delayed to similar extents and the growth period is prolonged in malnourished children; hence, the capacity for full recovery remains viable. For example, Keet et al. (1971) showed that malnourished children from Cape Town continued to increase in height when normal children reach an asymptote at 17 y.

Different parts of long bones respond differently to nutritional insult. Even under extreme conditions marked by the cessation of body weight gain, the body maintains priority for longitudinal skeletal growth (McCance 1960 , Stewart et al. 1975 , Widdowson and McCance, 1963 ). This increase in bone length continues at the expense of having thinner, less dense diaphyses. Yayha and Millward (1994) report that the epiphyseal cartilage width in the tibia of rats was more sensitive to protein deprivation than bone length and that tibia epiphysis length was least affected.

Although many of these studies document the effects of protein malnutrition on the growth rate of limb bones (Stewart et al. 1975 , Yayha and Millward 1994 ), none contain the complete growth trajectories of malnourished and control individuals to compare differences in the rates and timing of developmental events. It is not known how protein malnutrition alters growth patterns throughout ontogeny to produce morphological variations and whether these differences are manifested in final size.

As part of a larger study (Miller and German 1999 ), this paper investigates the effect of protein malnutrition on limb bone growth using longitudinal data to compare individual growth trajectories. These data can better explain differences in final size between malnourished and control individuals, which may result from differences in the rate and timing of growth. These data can also show which bones are more sensitive to protein deficiency and whether males and females respond differently.

	MATERIALS AND METHODS

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Animals and diets.

The data used in this study were collected as part of a larger project (Miller and German 1999 ). Rattus norvegicus breeders were obtained from a colony of Zivic Miller:Sprague-Dawley strain of rats at the University of Cincinnati. The two treatments used in this study were a control diet (CT) containing 24% protein and an isocaloric low protein diet (LPT) with 4% protein (Dyets, Bethlehem, PA). The sex of each rat was determined at weaning and rats were randomly assigned to one of the diets yielding four groups, i.e., male control, female control, male experimental and female experimental. Miller and German (1999) showed that the LPT rats not only consume less protein at any age, but also less protein per gram body weight than the CT rats. The males had a significantly greater body weight than females, and body weight continued to increase for a significantly (P < 0.01) longer period (Table 1 .).

Data collection.

Isoflurane gas (Anaquest, Liberty Corner, NJ) at 2–3.5%/L O₂ was administered for 5 min through an Ohio Compact Anesthesia Machine to lightly anesthetize the rats. Once sedated, the rats were positioned on a film cassette for X-raying using a Bennett Mammography Machine (Bennett X-ray, Copiague, NY) set for 0.25 s at 75 mA and 44–47 kV. Both dorsoventral and lateral radiographs were taken of each individual rat on Kodak MRM-1 diagnostic film (Kodak, Rochester, NY). Radiograph frequency varied with the age of the individual rat. From age 22–48 d, rats were X-rayed three times a week when growth was increasing rapidly. As growth slowed, the frequency was reduced to two times a week (49–76 d), then to one time a week (77–118 d), and finally to once every 2 wk (119–180 d). After radiography was completed, the rats awoke from sedation within a few minutes and resumed normal activity. Fiorello and German (1997) have shown no adverse growth effects resulting from radiography.

Length and width measurements of the limb bones were recorded using a Numonics AccuGrid Digitizing tablet (Numonics, Montgomeryville, PA; accuracy of 0.127 mm) and the program Digit (written by Dave Hertweck, Department of Biological Sciences, University of Cincinnati). All measurements were recorded from the right side of the body to reduce interlimb variation. Radiographs displaying poor alignment or resolution for a particular measurement were not included. Although past studies indicate that measurements from radiographs are not equal to those made with calipers on bones, the differences were not systematically biased (unpublished data). The value of using longitudinal data rather than cross-sectional data is one of the reasons for the design of this study (German and Meyers 1989 , German and Stewart 2000 , Stewart and German 1999 ). A total of 35 repeatable and homologous landmarks were digitized to give two-dimensional distances (mm) for each long bone. Table 2 gives descriptions of the measurements recorded.

Statistical analysis.

Our previous results from studying these individual rats (Miller and German 1999 ) suggested that the significant effect of malnutrition is the variation in the growth trajectories, rather than the endpoints of growth. Thus, models that can characterize the trajectories are important to this analysis. Because mammalian growth is nonlinear and characterized by a sigmoidal pattern, many workers have used one member of the family of logistic curves, such as logistic, Gompertz or Putter-Bertalanffy to model growth (Cothran et al. 1985 , Eisen 1976 , Gille et al. 1996 , Laird et al. 1965 ). Although these models differ slightly in their parameters when applied to empirical data, their differences are usually subtle and not biologically important (German and Meyers 1989 , Jolicoeur and Pirlot 1988 , Laird et al. 1965 ). The most overriding concern is a model whose parameters have biological interpretations, with results that are comparable to existing data. Thus, the Gompertz model was chosen to calculate estimates of several parameters because the parameters are interpretable, they have proved to be good fits in the past and the results from this study will be comparable to others containing similar data (German et al. 1994 , Lightfoot and German 1998 , Maunz and German 1996 , Miller and German, 1999 ).

The NONLIN module of SYSTAT (Wilkonson 1997 ) was used to analyze the data obtained from two algebraically equivalent forms of the Gompertz equation:^EQUATION

(1)

(2)

where y represents the measurement taken and t equals time in days. The base of the natural logarithm is represented by e. A is the asymptote or final size of y. It is the end product of the growth trajectory. The remaining parameters (w, I, k, b and T_f) are descriptions of the growth trajectory. Parameter w is the initial size of the measurement at t = 0, I measures the instantaneous initial growth rate at t = 0 and k is the rate of exponential growth decay. The parameter b has little biological importance, describing initial growth (Laird et al. 1965 ). Values for A, b and k were obtained from nonlinear regressions performed in SYSTAT using ^{Eq. (1)} , whereas values for w and I were calculated using the following relationships:^{EQUATION EQUATION}

(3)

(4)

Growth duration, T_f, can also be calculated from these equations. It represents the time at which each measurement is increasing at only 5% of its maximum growth rate (R_m) or essentially when growth stops. R_m is calculated by taking the first derivative of the Gompertz equation ^{(Equation 5)} , which gives the rate of growth over time.

(5)

Gompertz parameters were obtained for the limb bone measurements of all 37 rats; thus, the unit of analysis was the individual rat. Parameters for radius and ulna width as well as all distal widths were not obtained because of the nonsigmoidal nature of these curves. A three-way ANOVA was then used to test for differences among these parameters for sex, diet and litter. Interaction between sex and diet was also tested. Litter was included to account for any differences in growth due to litter; however, it was seldom significant. The amount of variation explained by the model was determined by the corrected R² values. High R² values for both treatments were essential in making growth comparisons between the diets using the same model (Klingenberg 1998 ). Groups were significantly different if the P-value was <0.01.

Linear regressions were fit to plots of length vs. width for each individual rat to test for allometric or scaling differences between the diets; thus, the individual rat was once again the unit of analysis. To test whether variation in shape differed as a result of diet, slopes were calculated for the following measurements: humerus length vs. proximal width; humerus length vs. deltoid tuberosity width; humerus length vs. diaphysis width; humerus length vs. distal width; radius length vs. diaphysis width; ulna length vs. diaphysis width; femur length vs. proximal epiphysis width; femur length vs. proximal width; femur length vs. diaphysis width; femur length vs. distal width; tibia length vs. proximal width; tibia length vs. diaphysis length; and tibia length vs. distal width. These are standard allometric coefficients used to estimate biomechanical scaling of long bones (McMahon 1973 and 1975 ). Analysis of covariance was then used to test for significant differences among the slopes for the four diet-sex groups. Statistically different slopes meant that the proportional change in length to width over time was different among the groups being compared. No difference in slope meant that the groups were growing identically and their bones would be similar sizes provided the intercepts were not significantly different.

The organ data were analyzed with a complete three-way, fixed-factor ANOVA using sex, diet, and litter as crossed, fixed factors. Litter was not a significant factor for these data. Data on muscle and organ size were scaled by dividing by final body weight. Previous work (Miller and German 1999 , Stewart and German 1999 ) suggested that there are significant differences between males and females, as well as small but significant differences in final size between rats fed the CT and those fed the LPT. We were interested in testing whether diet effect on organ and muscle size went beyond its effect on body size. The only variable analyzed separately for each sex was gonad weight. Growth and function of the gonads is not equivalent in the same sense as the other organs included in this study.

	RESULTS

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Sexual dimorphism.

The Gompertz model gave an accurate growth estimate for the limb bone data (Fig. 1 ). For all measurements, the mean corrected R² was 0.928. The minimum corrected R² was 0.803 for proximal tibia width, whereas the maximum corrected R² was 0.982 for femur length.

View larger version (19K): [in this window] [in a new window]   Figure 1. The Gompertz model fit to femur length vs. time for one male rat fed the control diet and one male rat fed the low protein diet as an example of how well the model fit the data.

Dietary effects.

Longitudinal limb bone data collected for the CT rats produced sigmoidal growth trajectories for all 13 skeletal measurements, whereas the longitudinal limb bone data for the LPT rats produced more linear growth trajectories (Fig. 2 ). Therefore, it was more difficult to obtain a good empirical fit using the Gompertz model for the LPT group. The mean corrected R² was slightly lower for the LPT curves (R² = 0.915) compared with the CT (R² = 0.942). The initial size, w, should not be significantly different between the two groups because the rats were selected randomly for the two diets after weaning. Nevertheless, the LPT rats had larger initial sizes in half of the measurements recorded. Statistical tests of the actual initial sizes showed no difference between the dietary groups (P > 0.01). These two results suggest that the Gompertz model was not estimating the initial size accurately due to the linear nature of the LPT curves.

View larger version (17K): [in this window] [in a new window]   Figure 2. The fitted Gompertz model for humerus length for the averages of male and female rats fed the control and low protein diets. The lower panel is the first derivative for each of the four models, measuring growth rate over time. The control rats had a substantially higher initial growth rate. However, this initial growth rate for the control rats then drastically decayed as growth continued, resulting in the low protein–fed rats having a higher rate of growth at 60 d. This difference in growth rate continued until growth ultimately stopped at 300 d or later.

View larger version (13K): [in this window] [in a new window]   Figure 3. The fitted Gompertz model of ulna length vs. time in male rats fed the control and low protein diets.. The low protein–fed rats achieved greater final length estimates for the radius and ulna by growing for significantly longer periods of time with a higher rate of growth. The lower panel is the first derivative showing that the low protein–fed rats had a higher rate of growth at 80 d, which continued until 400 d.

In contrast to the final size estimates obtained from the Gompertz equation, significant differences (P < 0.01) occurred in all of the actual final measurements between CT and LPT males (Table 4 ). For the 13 skeletal measurements, the CT male rats had significantly (P < 0.01) greater final lengths. These differences were on average 5.7%, ranging from 0.032–0.108. The differences in width were greater in size averaging 12.7% and ranging from 0.076–0.158. When comparing actual final size differences between the CT and LPT females, the number of significant differences decreases to 8 of the 13 measurements. The LPT females ultimately reached final sizes similar to those of the CT females for five measurements, four of which were length measurements. The average difference in length between the CT females and the LPT females was 1.7%, ranging from 0.002–0.034. Like the males, the average difference in bone width for females increased to 7.5% with a range of 0.047–0.134.

There were few instances in which the males and females reacted differently to the low levels of dietary protein. Therefore, variation in growth can be explained by two factors, sex and diet, implying that the growth response to the low protein diet was the same for males and females. The parameter with the most interactions was T_f, with a significant difference in humerus diaphysis width and femur diaphysis width. For humerus diaphysis width and femur diaphysis width, the effect of low dietary protein on growth duration resulted in a higher degree of sexual dimorphism between the LPT males and females.

Allometry and scaling.

During ontogeny, some scaling differences could arise between the two diets and sexes due to variations in the growth parameters. Only two scaling differences were observed between the sexes, whereas several differences occurred between diets. The males had a significantly faster slope for tibia length vs. tibia diaphysis width and a marginally steeper slope for radius length vs. radius width. When comparing differences between the diets, the CT rats had significantly greater slopes than the LPT rats for 6 of the 13 scaling measurements, meaning that for any increase in width, the CT increased in length at a faster rate. Therefore, the LPT rats had shorter but proportionately wider bones. For all of the remaining measurements except radius width, there were no significant scaling differences in slopes between the diets, meaning that the LPT and CT rats grew proportionately larger at the same rate. For radius length vs. radius width, the LPT rats grew longer at a significantly faster rate than the CT rats. There was only one interaction between sex and diet for proximal femur width, indicating that the majority of scaling differences were attributable to differences in sex or diet.

Muscles and organs.

Although the weight of muscles in males was larger absolutely than that of females for both dietary groups (Table 5 ), there were no significant differences due to sex, diet, or interaction between sex and diet, in the weights of muscles relative to body weight (P > 0.10) and only one marginal difference due to diet in the masseter muscle (P = 0.07, = 0.01). Thus, all differences in muscle size were a function of differences in body weight.

The results for eye and brain weight are more complex. In both cases, females had proportionately larger eyes (P < 0.01) and brains (P < 0.01) than males. There was also a significant diet effect for both eyes (P < 0.01) and brain (P < 0.01) when they were scaled to body size. The significant diet effect showed that the LPT rats were proportionately larger than the CT rats for both eyes and brain relative to body size. Using the raw data, i.e., without scaling for body size, the diet effect was not significant for either organ (P = 0.20 brain; P = 0.28 eyes). Thus for each sex, the eyes and brain were absolutely the same size, but the LPT rats were slightly smaller in body weight.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
These results, as did those in the related project (Miller and German, 1999 ), differ from earlier studies (Elias and Samonds 1977 , Pucciarelli 1980 and 1981 , Samonds and Hegsted 1978 , Stewart et al. 1975 ). Although both Edozien and Switzer (1978) and Yayha and Millward (1994) found that rates of growth slowed in animals fed lower levels of protein, they did not follow growth long enough to determine ultimate adult size of malnourished individuals. Furthermore, none of these researchers looked at the asymptotes of growth models, which in our data predicted no difference in final size. Although differences in final length and width of the limb bones between the LPT and CT rats were significant, similar to the results for the craniofacial skeleton (Miller and German 1999 ), they have only marginal biological importance. It is entirely possible that had we followed the LPT rats for a longer period of time, even this difference of a few percentage points would have disappeared.

The differences we measured in the growth of lengths between LPT rats and CT rats were not the same as those in the growth of widths. For final size in both sexes, the lengths were closer between the two treatments than were the widths. However, looking at the allometric results for scaling over time, the relative length to width slope, the CT rats had larger scaling coefficients, indicating for a given increase in width, the length of the bones of the CT rats increased faster than those of the LPT rats. Because the two treatments end up at approximately the same length and the rates of growth for both widths and lengths were significantly lower in the LPT rats, the duration of growth for the lengths of bones must have been relatively longer than for the widths (Table 3) .

These results on length vs. width suggest a hypothesis for the dietary effect of the specifics of limb bone growth. After the primary center of ossification forms in the diaphysis of the long bone, the perichondral template is ossified and becomes the periostium of the long bone. Ossification forms a girdle of bone around the diaphysis, and lengthening of the bone occurs at the diaphyseal-epiphyseal junctions at the ends of the bones. Thus the width of the bone is determined earlier (although remodeling can occur) than the length, which continues to exhibit primary growth until epiphyseal fusion occurs (Larsen 1997 ). It is possible that a longer duration of growth in LPT rats is due to a developmental delay in epiphyseal fusion that is not possible in the earlier diaphyseal ossification.

The pattern of size differences among organs is also a function of the timing of growth. The lack of differences between LPT and CT rats for muscles per body size likely reflects the fact that muscles continue growing even after final ossification of the limb bones. Structures that could continue to grow, either skeletal or muscular, did so. Significant size differences due to diet in the eyes and brains, with the LPT rats being relatively larger than the CT rats, are probably subject to different timing and growth considerations. We propose two, not mutually exclusive explanations. First, the LPT rats were not nutritionally compromised until after weaning, and much of the growth of these structures occurs prenatally. Eyes, as is true of the brain, are characterized by "excessive early enlargement" (Marsh and Vannier 1985 ) and attainment of adult size far in advance of most other adult structures. Second, it is likely that the brain and eyes have developmental priority (Larsen 1997 ) and that if the body had limited resources, it would devote more to the brain. Thus, even during growth of rats fed the low protein diet, the brains and eyes would be less affected than other organs.

Given the data in this paper, it is difficult to sort out the relative importance of these two explanations. However, we have some preliminary data to support the second hypothesis (Reichling 2000 ). Offspring of malnourished dams, a second generation of LPT rats, were killed at weaning and their organs weighed. These pups, subjected to the effect of malnutrition through early development, had patterns of brain scaling when corrected for body weight similar to the data described here. The LPT pups had relatively larger brains per gram body than the CT pups

The overall conclusion, given the different systems we have examined, is that structures that grow or have the potential for extended growth are less affected by the nutritional insult, thus lengths of bone relative to widths, and the cranial widths. In general, structures with less flexibility in the duration of growth, e.g., bone widths or various aspects of the mandible will end up smaller in the malnourished animals (Miller and German 1999 ). The exceptions to this latter conclusion appear to be the elements of the neural system we examined, which seem to maintain developmental priority. Although rats fed the low protein diet grew more slowly, cessation of growth was sufficiently delayed for them to achieve near adult size for several different physiological systems.

	ACKNOWLEDGMENTS

 
We thank James Fortman, Andrew Lammers, Jeff Miller, John Ranker, Scott Stewart and Samuel Outten for their assistance in data gathering and analysis.

Manuscript received January 10, 2000. Initial review completed February 28, 2000. Revision accepted April 7, 2000.

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