Nutritional growth retardation or nutritional dwarfing (ND) denotes a linear growth deterioration accompanied by inadequate weight gain 1. It has been associated with short stature since growth deceleration represents an adaptive response to suboptimal nutrition. In the United States, ND is frequently associated with self imposed dieting due to fear of obesity and/or hypercholesterolemia, extreme exercise and eating disorders, among many other reasons 234. In contrast to more severe forms of malnutrition observed worldwide, ND is manifested by a height-for-age deficit while both weight-for-height and standard biochemical nutritional indexes remain within normal limits 1. Several reports have also demonstrated reduced Insulin-like Growth Factor-1 (IGF-I) level and decreased erythrocyte Na⁺K⁺-ATPase activity during suboptimal energy intake 56789. The degree of energy restriction portrays one of the most important factors related with growth hormone (GH) action. Consequently, elevated GH level along with reduced IGF-I, Insulin-like Growth Factor Binding Protein-3 (IGFBP-3) and Insulin levels have been described in both human and animal research as a consequence of starvation 101112131415161718. Furthermore, attenuated spontaneous GH secretion has also been reported in patients with ND during puberty 19. However, utilizing an animal model of suboptimal nutrition, it has been reported that GH effects are feasible during mild energy restriction by administering exogenous GHi 2021.

Although adequate energy intake plays a major role affecting growth, micronutrients like Zn have also demonstrated to be essential regulators of growth and GH action 222324. Profound Zn deficiency with adequate energy intake has been frequently associated with growth failure and delayed sexual maturation, affecting cell division, DNA, RNA and protein synthesis 2526. Zinc deficiency also reduces the level of liver GH receptors, serum IGF-I, GH binding proteins and both mRNA's for GH receptors and IGF-I 2426. Furthermore, binding of Zn in cultured rat hepatocytes with a Zn chelator DTPA (diethylenetriaminepenta-acetic acid) did not reduce mRNAs for IGF-I, GH receptors or GH binding proteins while metallothionein gene expression was strongly inhibited. Therefore, the decline in IGF-I associated with in vivo Zn deficiency did not appear to be due to changes in extracellular Zn concentration at the hepatocyte level 27. Often Zn deficiency is associated with reduced energy intake 2829 and the effects of each one of these nutritional alterations alone, or in combination with each other is not known. Furthermore, the effects of GHi administration in rats with experimentally induced suboptimal energy with and without decreased Zn intakes have not been elucidated.

In this paper we report the effects of exogenous GHi administration in an animal model of suboptimal nutrition where energy and/or Zn intake was decreased by 30% of requirements 52021. Our goal was to determine if exogenous GHi administration would attenuate the detrimental effects of mild restrictions of energy and Zn individually or in combination with each other. Furthermore, we hypothesized that energy expenditure and physical activity would be reduced with mild nutrient restrictions. This was determined utilizing a new Enhanced Metabolic Testing Activity Chamber.

The experimental animal model of suboptimal nutrition allowed the independent assessment of suboptimal intake of specific nutrients and the effects of administered exogenous GHi. At the same time we were able to determine the effects of administered GHi under these conditions. In this study different conditions of suboptimal nutrition in rats were assessed by limiting dietary energy and/or zinc intakes to 70% of ad-libitum. This was possible by utilizing purified diets which allowed us to provide a precise amount of all nutrients, including Zn, which might not have been possible with commercial rat chows. Furthermore, this also permits determination of the effects of rhGH administration on 24-hour energy expenditure and on the index of physical activity utilizing an established rodent model of suboptimal nutrition, which has never been studied before. The data showed that exogenous recombinant human growth hormone enhanced body weight gain and increased serum IGF-I levels in energy restricted rats. Furthermore, serum IGFBP-3 levels were also increased during simultaneous suboptimal energy and zinc restriction. However, suboptimal zinc restriction alone did not interfere with body weight gain or exacerbate any of the detrimental effects of specific suboptimal energy restriction. Moreover, GHi treatment reduced the amount of total body water in energy restricted rats. Finally, a reduction in energy expenditure and the index of physical activity were shown during suboptimal energy intake and Zn restriction however, GHi treatment did not enhance these parameters. The rat's physical activity was not restricted in any way. The slightly smaller space provided by the Nalgene metabolic cage might have potentially reduced the rat's spontaneous physical activity, however this appeared not to affect the results. All rats had metabolic measurements under the same conditions.

Decreased body gain was previously reported during mild suboptimal energy intake in rats fed at 60 and 80% of ad-libitum energy intake 5. These findings showed that a 40% reduction in energy intake reduced weight gain by 61% of control levels over a four week period. When considering all of the rats fed the reduced energy diet in the present study, regardless of treatment or Zn levels, a 30% reduction in energy intake resulted in a 40% reduction in body weight gain in comparison to all control animals.

It was previously found that GHi given to rats fed a restricted diet (60% energy) resulted in increased cumulative weight gain when compared to their NSS controls, while IGF-I and IGFBP-3 levels were elevated 20. Our results confirm that GHi enhanced body weight gain by >18% when rats were fed suboptimally (reduction by 30% in dietary energy). Body weight differences were mainly associated to energy restriction. No body weight differences were evident in Zn restricted rats as long as the energy intake was appropriate.

Increments in IGF-I and IGFBP-3 serum concentrations were clearly linked to the effects of GHi in energy restricted rats. However, our data demonstrated no differences in serum IGF-I and IGFBP-3 concentrations with mild isolated Zn restriction. Thus, reduced Zn intake influenced the action of GHi in IGF-I and IGFBP-3 serum concentrations only when the energy intake was simultaneously restricted, suggesting no role for the suboptimal intake of Zn. It is possible that reducing Zn to only 70% of requirements, with adequate energy, is not enough to cause any significant effects on the hormonal parameters studied. It may be necessary to further reduce dietary Zn before an effect on IGF-I or IGFBP-3 is observed. For example, Zn deficiency in rats fed adequate energy grew poorly and showed reduced serum IGF-I, GH receptor numbers and GH binding proteins 26. Furthermore, gene expression for both IGF-I and GH receptors in rats were reduced when fed a Zn deficient, adequate energy diet 24.

Altogether, recombinant human growth hormone therapy reduces carbohydrate utilization shifting all metabolic loads to lipid metabolism 3031. Moreover, physical activity, normal growth, dietary intake 31 and GH itself have an essential role in body composition 32333435. For instance, the administration of GHi, promoting lipid utilization, reduces total body fat. In our study our partially energy restricted animals showed a slight increase in body weight gain with GHi treatment. Moreover, we found a slight reduction in the respiratory quotient with GHi administration in those rats on energy restricted diets. This suggests that GHi treatment shifts the metabolic load to lipogenesis, as has been found in a previous study 3137. Furthermore, GHi action was not affected by restricted Zn intake as long as the energy intake was not restricted.

The amount of GH administered may have variable effects on body composition in rats. For example, investigators in two previous studies found minimal changes in body fat and fat-free mass in rats fed ad-libitum and administered 0.05 or 0.10 mg/100 g body weight of GHi daily 2021. This is suggestive that under ad-libitum or mild energy restriction GHi treatment at or below 0.10 mg/100 g body weight will not change the amount of fat-free mass relative to body fat. It is possible that a higher dosage of GHi will elicit these effects. For example, body fat decreased significantly in similarly fed rats when administered 0.35 mg/100 g body weight of GHi daily 37. Moreover, the whole body carcass analysis methodology may not be sensitive enough to detect subtle changes in body fat in rats at the lower dosages of GHi used in this and in similar studies 2021. This is further evidenced by the lack of changes in energy expenditure and physical activity index observed in rats fed either ad-libitum or energy restricted diets and treated with 0.1 mg/100 g body weight of GHi.

In this study energy restriction resulted in a reduction of 24-hour energy expenditure and physical activity index in rats. Furthermore, these changes were not affected by GHi treatment. This is suggestive that metabolic adaptations occurred due to suboptimal energy intake. Others have found similar metabolic and biochemical changes associated with suboptimal energy intake. For example, erythrocyte NaK-ATPase was reduced in children diagnosed with nutritional dwarfing, a form of suboptimal nutrition that exists for a prolonged period of time 8. Similar reductions in erythrocyte NaK-ATPase were also found in rats fed energy restricted diets 5. Moreover, metabolic rate and physical activity were reduced when rats were fed energy restricted diets 43. The results of all these studies 58202137 suggests that metabolic adaptations occur beginning with mild energy restriction.

Growth deceleration, subsequent growth failure and short stature are the most remarkable consequences of persistent suboptimal nutrition 12345. Although adequate energy intake has a preponderant role affecting growth, many other micronutrients play a meaningful role in growth regulation and growth hormone action. Furthermore, extensive animal and human research have already established the association between energy restriction, severe Zn deficiency and growth failure. Therefore, elevated growth hormone levels, along with reduced IGF-I, IGFBP-3 and insulin levels, have been described in severe energy and Zn restrictions 1011121314151617182223242526.

Malnourished humans have increased, whereas rats have decreased serum levels of growth hormone 38. Decreased growth-hormone levels in starved rats may be caused by increased somatotropin-releasing inhibitory hormone or by reduced stimulation of hypothalamic somatotroph cells by growth-hormone-releasing hormone 39. Furthermore, serum leptin has been found to play a role in the decline of growth hormone in rats 40. However, serum insulin growth factor-1 (IGF-I) production in starved rats remains sensitive to growth hormone levels 41. In previous studies we have validated the rat model for testing various treatments during suboptimal nutrition 52021.

Growth retardation is the most predominant finding in many of the conventional Zn deficiency studies 242526272829. However, limited information exists concerning the effects of suboptimal Zn intake 42 and no one has investigated the effects of GHi administration during experimental suboptimal Zn restriction. Thus, our study showed GHi therapy promoting body weight gain despite suboptimal Zn intake. Consequently, in contrast to most of these studies involving severe Zn deficiency, our data suggest a secondary role of Zn when Zn intake was only restricted to 70% of daily requirements while maintaining adequate energy intake. In addition to weight gain, other GHi metabolic effects were also obtained, such as increases in both IGF-I and IGFBP-3 levels, during Zn restriction if associated with appropriate energy intake.

Our study results give us an idea of the role of Zn in growth during mild malnutrition, which is not clearly understood. The assessment of Zn status is hampered by the lack of a single sensitive and specific biochemical factor 43. Currently, the clinical method to assess Zn status in children is to measure increase growth velocity in response to Zn supplementation. Controversy exists between studies, where positive effects of Zn supplementation have been found against no effects of supplementation. No effects were found in adolescents with ND and in sub Saharan infants and young children 5612131415 while malnourished infants and children showed increased growth 678. Researchers have suggested that these differences may be attributed to the severity of Zn deficiency, to the dose, frequency of dosing and age of the infants. It has also been observed that in non-stunted infants, Zn supplementation results in increased growth compared with the control group, but with a less pronounced effect. Therefore, researchers have concluded that beneficial effects of Zn supplementation on growth are related to the degree of stunting and of Zn deficiency. Furthermore, it was suggested that Zn supplementation halted the stunting process, due to improved appetite and decreased episodes of gastrointestinal and respiratory disease, in stunted infants in rural Ethiopia 44.

The amount and method of administration of growth hormone, along with the type of feeding regime might cause differences in anabolic responses. For example, healthy normally fed rats injected with 20 micrograms daily of recombinant human growth hormone at the sight of an induced fracture for 10 days showed reduced healing times without changes in bodyweight 45. In another study, 15 day slow release recombinant human growth hormone laminar implants elicited the same anabolic effects in rats as the daily injectable form but without the associated problems such as renal toxicity and stress at the injection site 46. Furthermore, a single injection of microencapsulated slow release recombinant human growth hormone in immunosuppressed Hpx rats showed greater long term pharmacological effects such as increased growth rate and IGF-I levels in comparison with daily injections for 35 days 47. Moreover, there were no differences in the anabolic effects of either rodent or human growth hormone administration in rats 48. In our study we utilized GHi was injected daily just under the skin (SC) at the back of the neck. The half-life of this type of growth hormone is 124 minutes when utilized in rodents 49. We have conducted three prior studies of suboptimal nutrition in our laboratory 52021 utilizing the same type of growth hormone. We always observed increased growth and body weight gain without any apparent health problems. It is possible that the short half life of this product prevented any toxic effects in our rats.

Our results demonstrate that beneficial effects of GHi are obtained during mild energy restriction. Moreover, mild Zn restriction does not have negative effects on body weight gain. Growth hormone therapy does promote body weight gain despite mild energy and Zn restrictions without any apparent effects on metabolic rate and physical activity. Finally, no additive effects between Zn and energy are observed during mild combined restriction of energy and Zn intakes.

ANOVA = Analysis of Variance

BF = Body Fat

FFM = Fat-free mass

GH = Growth hormone

Hg = Mercury

IGF-I = Insulin-like Growth Factor-1

IGFBP-3 = Insulin-like Growth Factor Binding Protein-3

NSS = Normal Saline Solution

ND = Nutritional dwarfing

PSI = Pounds per square inch

rhGH = recombinant human growth hormone

GHi = exogenous immunoreactive growth hormone

Na⁺K⁺-ATPase = Sodium-potassium ATP transporter enzyme

TBW = Total body water

Zn = Zinc

BW = Body weight

SD = Standard deviation

24-h EE = Twenty-four hour energy expenditure

RQ = Respiratory quotient

The author(s) declare that they have no competing interests.

Dr. Russell Rising has contributed to the design of the experiment and conducted the data analysis. Furthermore, he either participated in some of the actual data acquisition or supervised pediatric research fellows in this regard. He also assisted in the preparation of the small grants necessary for funding of this project. Finally, he also assisted in the writing and editing of this manuscript.

Dr. Fima Lifshitz contributed to the preparation of the manuscript and assisted with data analysis. He also edited some of the grant proposals necessary for the financial support of this study. Both authors were involved in the final editing of this manuscript.