The Journal of Nutrition Vol. 128 No. 3 March 1998, pp. 520-524

Expression of Mutant Bovine Growth Hormone Genes in Mice Perturbs Age-Related Nutrient Utilization Patterns^1,2,3,4

Nancy D. Turner^{*, 5}, Joanne R. Knapp, F. Michael Byers^*, and John J. Kopchick^**

^* Texas A&M University, Department of Animal Science and Faculty of Nutrition, College Station, TX 77843-2471;  University of Vermont, Animal and Food Sciences Department, Burlington, VT 05401; and ^** Edison Biotechnology Institute, Ohio University, Athens, OH 45701-2979

	ABSTRACT

Abstract Introduction Methods Results Discussion References

Three lines of transgenic mice expressing mutant bovine growth hormone (bGH) genes and displaying small (G119K), near normal (M11) or large (M4) phenotypes and nontransgenic control (NTC) mice were used to determine GH-associated, age-specific changes in empty body composition. The single amino acid substitution in G119K mice reduced the quantities (P < 0.001) and early rates (P < 0.05) of deposition for water, protein and ash but resulted in similar quantities of fat as the NTC mice. The change in relative quantities of empty body components indicated the G119K analogue altered nutrient partitioning, basal metabolism and (or) nutrient availability to effect the differential observed in body composition. The two amino acid substitutions in the bGH gene expressed by the M11 mice caused only a small change in phenotype, but age-related changes in the accretion of protein, fat and ash indicated these mice were not mature by 68 d of age. The bGH analogue produced by the M4 mice resulted in a doubling (P < 0.001) of body weight in comparison with the NTC mice, a result of the increasing (P < 0.001) rate of weight gain. Empty body component gain of the M4 mice also indicated they had not yet matured by 68 d of age. The G119K and M4 mutant forms of bGH altered rates and composition of growth, possibly through redirection of tissue nutrient utilization, modification of nutrient metabolism, and(or) nutrient availability.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Growth hormone (GH)⁶ is a 191 amino acid hormone that regulates not only essential animal growth functions (Isaksson et al. 1985) but also metabolism of lipids, protein, minerals and carbohydrates (Goodman 1978, Goodman et al. 1986, Kostyo and Nutting 1973, Swislocki 1968). The ability to regulate this multitude of biological effects is potentially the result of either putative tissue-specific GH receptor subtypes (Press 1988, Smal et al. 1987) or of multiple active domains in the GH molecule (Kostyo 1986, Salem 1988). Through site-directed mutagenesis of the bGH gene, Chen et al. (1991a and 1991b) demonstrated that specific amino acid substitutions results in bGH analogues that are either GH antagonists or agonists. The resulting animal phenotypes are a dwarf-type mouse, a mouse that is similar to a wild-type mouse, or a mouse that is similar to one expressing a normal bGH gene.

Studying body composition can lead to an understanding of how metabolic and hormonal (e.g., GH) mediators work throughout the life cycle (Roubenoff 1997), and how hormonal mediators may impinge upon metabolism. Although much is known of how the mutant bGH analogues expressed in the transgenic mice generated by Chen et al. (1991a and 1991b) affect phenotypic growth, little is known of how body composition changes throughout the life cycle. Because of the differences in degree of agonist and antagonist activities of the mutant bGH analogues produced by these mice, a comparison of body composition among these mouse lines would permit an assessment of how GH affects tissue-specific and age-related regulation of nutrient deposition. Therefore, the objectives of this study were to determine the composition of mice expressing one of the three mutant bGH genes for 40 d after weaning and to determine age-specific patterns of nutrient deposition in response to the mutant bGH analogues.

	MATERIALS AND METHODS

Abstract Introduction Methods Results Discussion References

Three lines of the bGH transgenic mice generated by Chen et al. (1991a and 1991b) that display either small (G119K), near normal (M11) or large (M4) phenotypes were used. The M4 mice produce a bGH analogue that contains leucine at position 117 instead of glutamate (Chen et al. 1991b). M11 transgenic mice express a mutant bGH gene that results in two mutations; leucine at position 121 is replaced by proline and glutamate at position 126 is replaced by glycine (Chen et al. 1991a). Transgenic G119K mice produce a bGH analogue in which glycine at position 119 is replaced by lysine. This molecule was the first GH antagonist to be reported (Chen et al. 1991b).

All animal procedures used in this experiment were approved by Ohio University's Animal Care and Use Committee. Mice were generated by breeding a single transgenic male mouse expressing one of the specific mutations with nontransgenic C57BL/6 female mice. Because a single transgenic male expressing each of the mutations was used for breeding, the number of insertions in all offspring were uniform (Knapp et al. 1994). Nontransgenic litter mates from each of the groups served as controls (NTC). Pups were evaluated by Southern and slot blot analyses for presence of the mutated bGH genes (Knapp et al. 1994, Sambrook et al. 1989). The bGH genes are linked to the mouse metallothionein promoter, producing elevated basal expression without activation by a heavy-metal inducer. Thus relatively constant levels of the bGH analogue and insulin-like growth factor-I (IGF-I) are maintained in the transgenic mice (Knapp et al. 1994).

Mice were housed in a temperature controlled room (25°C) with 14 h of light exposure (on from 0500 h). Because of gender differences in growth rate and body size, only male mice were used. Mice were weaned at 28 d of age and randomly assigned to an age for killing (28, 38, 48, 58 and 68 d). Mice were housed individually and permitted free access to water and a nonpurified diet (Purina mouse chow No. 5020, Richmond, IN) (PMI 1996) containing 52% carbohydrate, 20% protein, 9% fat, 3% fiber, and minerals and vitamins for adequate growth of the mouse parent lines (Connell et al. 1981). Chemical analyses were performed using AOAC (1990) procedures on aliquots of the diet used for this experiment to verify its composition.

At the assigned ages, mice were decapitated, viscera removed and the gastrointestinal tract emptied. Livers were removed for other analyses and were not returned to the empty bodies. The empty bodies, composed of carcass and viscera (without liver), were ground using liquid nitrogen, and aliquots analyzed for dry matter, ash, protein and ether extract (AOAC 1990).

Quantities of empty body protein, fat and ash for each group were calculated from empty body weight and percentage protein, fat and ash. The quantities were fitted with nonlinear functions vs. age to describe transgene effects on composition as well as the regulation of rates and composition of growth (SAS Institute Inc. 1985). From the first derivative of these functions, daily rates of tissue deposition were derived for all groups at selected ages over the study. Results from the regression models are presented in figures along with the variance in slope (Sy) calculated from the model mean squares error so differences among genetic lines can be determined (SAS Institute Inc. 1985). The variance for rates of tissue deposition were calculated with the output from the covariance matrix of the quantity regressions. All composition data reported within this paper will be based upon the empty body weight, however, for brevity the term empty body will not usually be used.

	RESULTS

Abstract Introduction Methods Results Discussion References

Quantities of components.  Empty body weight of G119K mice was lower (P < 0.0001) at all ages compared to the other lines (Fig. 1). The NTC and M11 mice had similar weights from 28 to ~50 d of age. However, at 68 d of age, the weight of M11 mice was 4.5 g greater than for the NTC mice. Weight of the M4 mice was greater (P < 0.0001) than all other groups throughout the experiment. At 68 d of age the weight of M4 mice was 1.7-fold greater than that of the G119K mice.

View larger version (34K): [in this window] [in a new window]   Fig 1. Quantities of empty body components in 28- to 68-d-old mice expressing one of three mutant bGH genes (G119K, M11, M4) or nontransgenic control mice (NTC). Values were calculated from regression equations that included age·transgene and age2·transgene interactions. The number of observations was 15 for NTC and G119K and 12 for M11 and M4 mice. The Sy = 0.45, 0.26, 0.14, 0.24 and 0.025 for empty body weight, water, protein, fat and ash, respectively.

Empty body water paralleled the response for weight in each of the four lines. Protein increased from 2.74 g at 28 d of age to 4.56 g at 68 d of age for the NTC mice. The quantity of protein was lower (P < 0.002) for the G119K mice than any other line between 28 and 68 d of age. Protein weight of G119K mice at 68 d of age (2.73 g) was less (P < 0.002) than the quantity in the other three groups at 28 d of age (Fig. 1). The quantity of protein in the M11 and M4 mice at 28 d of age was similar to that in the NTC mice, yet by 68 d of age, M11 and M4 mice had 1.00 and 2.62 g more (P < 0.002) protein than the NTC mice.

Fat weight for NTC mice increased from 1.53 g at 28 d of age to 2.70 g at 68 d of age. Although the G119K mice had gained only 76% of the weight, the NTC mice had gained by 68 d of age, they deposited 64% more (P < 0.002) fat than the NTC mice (Fig. 1). Thus even though overall growth was impeded in the G119K mice, deposition of adipose tissue proceeded uninhibited by the G119K mutant bGH analogue. Whereas weights of fat in M11 and NTC mice were similar at 28 d of age, by 68 d of age the M11 mice had 1.28 g more (P < 0.002) fat than the NTC mice. At 28 d of age, the M4 mice had only 0.26 or 0.41 g more fat than the NTC or G119K mice (Fig. 1). By 68 d of age, the M4 mice had 236% more (P < 0.001) fat than the NTC mice.

The quantity of ash in NTC and M11 did not differ until the end of the experiment (Fig. 1). At 38 d of age, ash weight in M4 mice was greater (P < 0.001) than in NTC mice. The amount of ash deposited by M11 and M4 mice by 68 d of age was 84 and 160% more than was deposited by 28 d of age, whereas G119K mice had stored only 60% more ash during the 48-d period.

Rates of component gain.  The rate of weight gain declined with age in all mice, except for M4 mice in which weight gain increased from 516 mg/d at 28 d of age to 673 mg/d at 68 d of age (Fig. 2). Even though the initial rate of weight gain in G119K mice was lower than the rate observed in NTC mice, the rate of gain was similar in these two groups from ~48 to 68 d of age (Fig. 2). Water gain mirrored weight, except that the rate was essentially constant in the M4 mice (281-276 mg/d range). The rates of protein gain declined as the mice in all four groups aged (Fig. 2). We found that the NTC and G119K mice essentially had stopped depositing protein between 58 and 68 d of age, whereas the M11 and M4 mice were still depositing relatively large amounts of protein each day (45.6 and 82.5 mg/d, respectively).

View larger version (40K): [in this window] [in a new window]   Fig 2. Rates of empty body component gain in 28- to 68-d-old mice expressing one of three mutant bGH genes (G119K, M11, M4) or nontransgenic control mice (NTC). Values were calculated as the first derivative of the weight data regression equations, which included age·transgene and age2·transgene interactions. The number of observations was 15 for NTC and G119K and 12 for M11 and M4 mice. The Sy = 17.6, 10.3, 3.5, 4.2 and 0.61 for rate of empty body weight, water, protein, fat and ash, respectively.

The quantity of fat in weight gain increased as the mice aged (Fig. 2). The quantities of fat gain exhibited by the four mouse lines were quite divergent, ranging from 18.4 mg at 28 d of age for the NTC mice to 277.6 mg/d for the M4 mice at 68 d of age. The rate of fat gain for M4 mice at 28 d of age was 3.7-fold greater (P < 0.05) than the NTC mice, and by 68 d of age, the rate of fat gain was 5.9-fold greater (P < 0.05) in the M4 mice than in NTC. The G119K and M11 transgenic mouse lines had greater (P < 0.05) rates of fat gain than the NTC mice by 38 and 28 d of age, respectively.

Rates of ash gain were not similar for NTC and M11 mice by 38 d of age (Fig 2). However, the decline in rate of ash gain was greater (P < 0.05) with age in the NTC mice. The rate of ash gain for the G119K mice was less than in the NTC mice throughout the experiment. Rate of ash gain declined only 3.2 mg/d during the 40 d growing period in the M4 mice from their initial rate of 21.5 mg/d at 28 d of age.

Percentage of component gain.  The percentage of water, protein, and ash in gain declined with age, and the percentage of fat in gain increased with age (Fig. 3). However, the three mutant bGH analogues obviously had an impact on the age at which changes in tissue accretion occurred as well as the degree to which the relative composition of tissue accretion changed.

View larger version (32K): [in this window] [in a new window]   Fig 3. Percentage of empty body components in gain in 28- to 68-d-old mice expressing one of three mutant bGH genes (G119K, M11, M4) or nontransgenic control mice (NTC). The number of observations was 15 for NTC and G119K and 12 for M11 and M4 mice. The Sy = 0.78, 1.18, 2.15 and 0.27 for fractional rate of empty body water, protein, fat and ash, respectively.

Percentage of water in gain was less in G119K mice from the onset of the experiment. However, the percentage of water in weight gain declined less rapidly in the G119K line than in the other three lines (Fig. 3), resulting in similar relative quantities of water being deposited in G119K and M4 mice at 40 d of age. The G119K, M4 and NTC mice had similar relative quantities of water at 66 d of age (Fig. 3).

The percentage of protein in weight gain of NTC mice was parallel to that for the M11 and M4 mice until 50 d of age, when the percentage of protein in gain began to rapidly decline in the NTC mice (Fig. 3). In contrast, the percentage of protein in gain, although declining, was linear and positive for M11 and M4 mice until the experiment was terminated. By 48 d of age, the G119K mice were exhibiting a rapid decrease in the percentage of protein in gain from 28 d of age, reaching zero at 62 d of age and becoming negative thereafter. The negative values resulted from more protein being degraded than synthesized and deposited in the G119K mice, indicating these mice were in negative protein balance at that time.

For all mice, percentages of fat in gain increased with age (Fig. 3). The initial percentage of fat in gain was greater (P < 0.05) in M4 mice than in NTC, but because of the slower rate of increase in the M4 line, the percentage fat in gain in these two lines of mice was similar by 64 d of age. By 68 d of age, fat was >72% of weight gain (P < 0.05) in the G119K mice but was only 53, 35 and 41% for the NTC, M11 and M4 mice, respectively.

The percentage of ash in weight gain was small in all the lines (Fig. 3). The rapid decrease in ash in gain in G119K mice resulted in their accreting less (P < 0.05) ash as a percentage of weight gain than all other lines by 52 d of age. The percentage of ash deposited in M4 mice declined by only 2.09 percentage units between 28 and 68 d of age. In contrast, the M11 mice exhibited a reduction of 3.76 percentage units during the same time.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

Composition.  The two amino acid substitutions in the M11 bGH analogue caused only a small increase in the weight of empty body components relative to the NTC mice. The bGH analogue produced by the M4 mice resulted in an ~1.5-fold increase in IGF-I concentrations (Knapp et al. 1994), but these mice exhibited nearly a twofold increase in weight in comparison to the NTC mice. Empty body components were still increasing or only starting to plateau by 68 d of age in the M4 mice. The G119K mice had reduced quantities of components other than fat when compared with the other three mouse lines at all ages. The depression in weight resulting from the G119K bGH analogue was apparent when the mice were weaned at 28 d of age. Hikida et al. (1995) suggested that the differences in muscle mass of these three lines may have been established in utero. Pantaleon et al. (1997) subsequently has documented that GH receptors and GH are expressed in preimplantation mouse embryos, further supporting the opportunity for the G119K mutation to have affected fetal, as well as neonatal growth of these mice.

Maturation.  Bauman et al. (1982) stated the role of GH was to redirect nutrients toward a specified target or process, and the nature of the target or process was dependent on an animals' developmental stage. Normally GH is involved primarily in promoting skeletal and lean tissue growth before puberty (Pell and Bates 1990), yet as the animal matures, muscle growth is no longer a priority for nutrient utilization. The decline in percentages of water, protein and ash gain and increase in percentage of fat gain in the NTC and G119K mice between 50 and 60 d of age indicated these animals had matured physiologically. The divergence in weight, water, protein and ash between NTC and M11 mice at ~48 d of age results from the normal decrease in growth that occurs with achieving adult body size and reduction in endogenous GH secretion (Ho and Hoffman 1993). Because the amount of the M11 bGH analogue secreted continued throughout the experiment (Knapp et al. 1994), growth continues in contrast to the NTC mice, which experience an age-related decline in GH secretion (Ho and Hoffman 1993). Although the percentages of protein and ash gain observed in the M4 and M11 mice were decreasing, the rates had yet to plateau, which indicates that these mouse lines were not mature before this experiment was terminated.

Nutrient utilization.  Expression of the G119K bGH gene did not change the percentage of viscera (14.8%) compared with NTC (14.7%), yet the percent composed of liver was less (P < 0.01) in G119K mice (4.04%) relative to NTC mice (4.41%) (Knapp et al. 1994). The M11 and M4 gene mutations increased (P < 0.01) the percentage of liver (7.08 and 7.58%) and viscera (15.9 and 15.7%) in comparison with NTC mice (Knapp et al. 1994). However, the G119K mutation did increase feed efficiency (20.5%) in comparison with that of NTC, M11 and M4 mice (15.9, 13.1 and 13.7%, respectively) (Knapp et al. 1994). Even though the G119K mice were consuming more feed as a percentage of weight, they could not assimilate the consumed nutrients into protein or deposit the mineral. The data of Bird et al. (1994) indicated that glucose absorption from the small intestine was altered in their line of GH transgenic mice. This would suggest the G119K bGH mutation either redirected nutrient utilization, changed metabolic requirements for maintenance or changed nutrient absorptive capacity of the G119K mice.

When nutrient intake is limiting, the major role of GH is an inhibition of lipogenesis and/or stimulation of lipolysis to provide energy (Pell and Bates 1990). Solomon et al. (1994) found that bGH transgenic pigs had reduced carcass fat content in comparison with control siblings. Gopinath and Etherton (1989) demonstrated that continuous administration of GH to pigs reduces insulin sensitivity of tissues. Insulin resistance in muscle can lead to obesity through dyslipidemia (Moller et al. 1996). However, the obesity observed in the G119K mice does not appear to result from insulin resistance as blood glucose concentrations do not differ from those of NTC mice, and it was not the result of an inability to secrete the analogue because conformational changes resulting from this mutation did not alter its secretion (Knapp et al. 1994). Chen et al. (1991b) concluded G119K bGH molecules were a competitive antagonist of endogenous GH because the structure prevented receptor dimerization, a requirement for GH action (Argetsinger and Carter-Su 1996; de Vos et al. 1992).

The capacity for muscle protein deposition is dependent on amino acid availability and regulation of actin and myosin gene expression. GH administration to critical care patients reduces liver ureagenesis and excretion, which increases nitrogen retention and balance (Pacitti et al. 1992), in part due to a reduction in Vmax of amino acid transporters (Inoue et al. 1993). By reducing amino acid utilization for ureagenesis, amino acids are spared for skeletal muscle protein synthesis. The apparent reduction in hepatic responsiveness to the G119K bGH analogue (Knapp et al. 1994) may alter the use of amino acids for urea synthesis, which would reduce the amino acids available for protein synthesis. This could explain the observed negative percentage of protein in empty body gain exhibited by the G119K mouse after 62 d of age.

In summary, the bGH mutant genes tested in this experiment resulted in mice with different phenotypes, which grew at different rates, and reprioritized nutrient partitioning, which then altered body composition and affected the age of maturity. Further research with G119K and M4 mice is warranted to determine the potential mechanisms whereby these bGH analogues might affect nutrient absorption, maintenance requirements, nutrient partitioning to specific tissues or if the G119K gene mutation alters hepatic or extrahepatic amino acid utilization. In addition, work must be conducted with animals that are both younger and older than those used in this experiment to determine the onset of changes in body composition initiated by tissue responsiveness to the mutant bGH analogues and to determine at what age maturity is reached in the M11 and M4 mice.

	FOOTNOTES

Manuscript received 22 September 1997. Initial reviews completed 28 October 1997. Revision accepted 26 November 1997.

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