Effects of GH deficiency and GH replacement on inter-male aggressiveness in mice.

OBJECTIVE: Growth hormone (GH) has been suggested to influence aggressive behavior in several species, but no data are presently available in GH-deficient (GHD) animals. The aim of this study was to elucidate the effects of GHD on aggressive behavior in a mouse model of isolated GHD due to removal of the GHRH gene (GHRH knock out, GHRHKO), and to evaluate the effects of GH replacement. DESIGN: We studied two groups of adult male mice: Ten GH-sufficient animals heterozygous for GHRHKO allele (HTZ), and 30 GHRHKO animals. Behavior was measured by scoring several aggression parameters after isolation, when the animal was challenged against an intruder both in neutral and home cage. Animals were then re-studied after the GHRHKO mice were left untreated (control, Ctrl), or were treated for 2 weeks with daily subcutaneous recombinant GH or with vehicle (Veh). Blood samples were collected before and after GH or Veh treatment, and assayed for serum IGF-I and testosterone. RESULTS: The GHRHKO mice showed significantly reduced aggressiveness compared to HTZ animals. GH (but not Veh) administration normalized isolation-induced aggressive behavior in GHRHKO mice, despite lack of full serum IGF-I normalization. No difference was noted in serum testosterone levels among all groups at any of the time points. CONCLUSIONS: These findings show that GHD reduces aggressive behavior in GHRHKO mice, that GH replacement normalizes aggressiveness, and that this behavior change is not related to an increase in serum testosterone.

Partial rescue of growth failure in growth hormone (GH)-deficient mice by a single injection of a double-stranded adeno-associated viral vector expressing the GH gene driven by a muscle-specific regulatory cassette.

Growth hormone (GH) deficiency (GHD) causes somatic growth impairment. GH has a short half-life and therefore it must be administered by daily subcutaneous injections. Adeno-associated viral (AAV) vectors have been used to deliver genes to animals, and double-stranded AAV (dsAAV) vectors provide widespread and stable transgene expression. In the present study we tested whether an intramuscular injection of dsAAV vector expressing GH under the control of a muscle creatine kinase regulatory cassette would ensure sufficient systemic GH delivery in conjunction with muscle-specific expression. Virus-injected GHD mice showed a significant (p < 0.05) increase in body length and body weight, without reaching full normalization, and significant (p < 0.05) reduction in absolute and relative visceral fat. Quantitative RT-PCR showed preferential GH expression in skeletal muscles that was confirmed by qualitative fluorescence analysis in mice injected with a similar virus expressing green fluorescent protein. The present study shows that systemic GH delivery to GHD animals is possible via a single intramuscular injection of dsAAV carrying a muscle-specific GH-expressing regulatory cassette.

A single injection of double-stranded adeno-associated viral vector expressing GH normalizes growth in GH-deficient mice.

GH is secreted by the somatotropic cells of the pituitary gland, and its deficiency (GHD) impairs longitudinal growth. Due to its short half-life, GH therapy needs administration of GH injections daily. Adeno-associated viral vectors (AAV) can deliver gene therapy to animals with possible future applications in humans. The new generation of double-stranded AAV vectors (dsAAV) provides widespread, strong, and stable transgene expression without toxicity and immune response. To determine whether such a new system could be used to deliver GH to a mouse model of isolated GHD due to ablation of the GHRH knock-out gene (GHRHKO), we have created AAV viral particles containing mouse GH cDNA driven by a cytomegalovirus promoter (dsAAV8-CMV-GH), and tested them in male GHRHKO mice. GHRHKO animals received either a single (low dose) or two (high dose) i.p. injections of dsAAV8-CMV-GH (1x10(11) particles) at the 10th and 11th days of age, or a placebo injection, and were followed up to the 6th or 24th week of life. A single dsAAV8-GH injection caused body length and weight normalization. At week 6, serum GH was higher in mice receiving both virus doses compared with controls, while it was normal at week 24. Serum IGF-1 increased in both virus-treated groups, and it was normal at 24 weeks. GH mRNA expression was detected in liver, skeletal, and heart muscle of virus-injected animals. These data show that normalization of longitudinal growth can be reached in GHD mice using a single injection of a double-stranded adeno-associated virus expressing GH.

Vitamin A deficiency does not influence longitudinal growth in mice.

OBJECTIVE: Growth hormone (GH) stimulates longitudinal growth, directly and indirectly, through the mediation of circulating and locally produced insulin-like growth factor 1 (IGF-1). The exact role of individual vitamins in modulating GH secretion or action, and somatic growth in general, is still not completely understood. It has been suggested that vitamin A (VA) and its physiologic active metabolite retinoic acid influence longitudinal growth by promoting the differentiation of pituitary cells toward GH-secreting cells and by stimulating secretion of GH. Accordingly, epidemiologic studies have shown that short children have lower VA intake than do children with normal stature. METHODS: To determine whether VA deficiency causes impairment of GH secretion, we have investigated the effect of a severely VA-deficient diet on growth in mice. Ten male mice born from mothers fed with VA-deficient diet since conception were maintained on a VA-deficient diet until the end of week 8 of life. Ten male mice born from mothers fed with a VA-sufficient diet and receiving a normal diet after weaning served as the control group. At the end of the study, we measured animals' weight and length, body composition, tibia and femur lengths, liver retinol and retinyl esters storages, serum IGF-1, serum thyroxine, serum GH, and pituitary GH mRNA levels. RESULTS: Despite evidence of significant VA deficiency, we observed no effect on longitudinal growth or changes in pituitary GH mRNA, serum thyroxine, serum GH, or serum IGF-1 levels. CONCLUSION: VA deficiency does not negatively affect longitudinal growth and pituitary GH content and action in mice.

Heterozygosity for a mutation in the growth hormone-releasing hormone receptor gene does not influence adult stature, but affects body composition.

CONTEXT: Biallelic mutations in the GHRH receptor (GHRHR) gene (GHRHR) are a frequent cause of isolated GH deficiency (IGHD). Although heterozygous carriers of these mutations appear normal, we hypothesized that heterozygosity for a GHRHR mutation might be associated with a subclinical phenotype. METHODS: We studied members of a large Brazilian kindred with IGHD (Itabaianinha cohort) caused by a homozygous null GHRHR mutation. We compared 76 adult subjects (age, 25-75 yr) heterozygous for the mutation (WT/MT) with 77 sex-matched controls from the same population who are homozygous for the wild-type GHRHR allele (WT/WT). RESULTS: We found no difference in adult height and sd score for serum IGF-I between the two groups. Body weight, body mass index, skin folds, waist and hip circumferences, and lean mass were all reduced in WT/MT subjects. Percentage fat mass and waist/hip ratio were similar in the two groups. Fasting insulin and homeostasis model assessment of insulin resistance were lower in WT/MT. The other biochemical parameters [total and fractionated cholesterol, triglycerides, lipoprotein (a), and C-reactive protein] were not different between the two groups. CONCLUSIONS: Heterozygosity for a null GHRHR mutation is not associated with reduction in adult stature or in serum IGF-I but is associated with changes in body composition and possibly an increase in insulin sensitivity. These effects do not seem to be modulated by changes in circulating IGF-I.

Once-daily administration of CJC-1295, a long-acting growth hormone-releasing hormone (GHRH) analog, normalizes growth in the GHRH knockout mouse.

Although the majority of children with isolated growth hormone (GH) deficiency have a good growth response to GH-releasing hormone (GHRH), the use of this therapeutic agent is limited by its very short half-life. Indeed, we have shown that, in mice with GHRH gene ablation (GHRH knockout; GHRHKO), even twice-daily injections of a GHRH analog are unable to normalize growth. CJC-1295 is a synthetic GHRH analog that selectively and covalently binds to endogenous albumin after injection, thereby extending its half-life and duration of action. We report the effects of CJC-1295 administration in GHRHKO animals. Three groups of 1-wk-old GHRHKO mice were treated for 5 wk with 2 microg of CJC-1295 at intervals of 24, 48, and 72 h. Placebo-treated GHRHKO mice and mice heterozygous for the GHRHKO allele served as controls. GHRHKO animals receiving daily doses of CJC-1295 exhibited normal body weight and length. Mice treated every 48 and 72 h reached higher body weight and length than placebo-treated animals, without full growth normalization. Femur and tibia length remained normal in animals treated every 24 and 48 h. Relative lean mass and subcutaneous fat mass were normal in all treated groups. CJC-1295 caused an increase in total pituitary RNA and GH mRNA, suggesting that proliferation of somatotroph cells had occurred, as confirmed by immunohistochemistry images. These findings demonstrate that treatment with once-daily administration of CJC-1295 is able to maintain normal body composition and growth in GHRHKO mice. The same dose is less effective when administered every 48 or 72 h.