The local expression and abundance of insulin-like growth factor (IGF) binding proteins in skeletal muscle are regulated by age and gender but not local IGF-I in vivo.

We wished to determine whether sustained IGF-I production in skeletal muscle increases local IGF binding protein (IGFBP) abundance, thereby mitigating the long-term stimulation of muscle growth by IGF-I. Muscle growth of transgenic mice that overexpress IGF-I in muscle (SIS2) and of wild-type (Wt) mice was compared. At 3, 5, 10, and 20 wk of age, hind-limb muscle weights and IGFBP-3, -4, -5, and -6 mRNA and protein abundances were quantified. Additional mice were injected with IGF-I or LR3-IGF-I, and phosphorylation of the type 1 IGF receptor (IGF-1R) was compared. Muscle mass was 20% greater in SIS2 compared with Wt mice by 10 wk of age (P < 0.01), and this difference was maintained to 20 wk. IGFBP mRNA and protein abundances were unaffected by genotype. IGFBP-4 and -5 protein abundances increased with age, whereas for IGFBP-3 and -6, there was a sexual dimorphic response (P < 0.01); after 5 wk of age, IGFBP-3 decreased in males but increased in females, whereas IGFBP-6 decreased in females and remained unchanged in males. These protein expression patterns resulted from differences at both the transcriptional and posttranscriptional levels. LR3-IGF-I stimulated IGF-1R phosphorylation to a greater extent than IGF-I at both 5 and 10 wk of age (P < 0.01), regardless of gender or genotype (P > 0.21). Thus, variations in local IGF-I levels do not appear to regulate muscle IGFBP expression. The age- and gender-specific differences in muscle IGFBP expression are not sufficient to alter the response of the muscle to the IGFs but may impact the IGF-independent effects of these IGFBPs.

Growth hormone releasing hormone plasmid supplementation, a potential treatment for cancer cachexia, does not increase tumor growth in nude mice.

Growth hormone releasing hormone (GHRH) is known to have multiple anabolic effects and immune-stimulatory effects. Previous studies suggest that treatment with anabolic hormones also has the potential to mitigate the deleterious effects of cancer cachexia in animals. We studied the effects of plasmid-mediated GHRH supplementation on tumor growth and the role of antitumor immune cells with two different human tumor cell lines, NCI-H358 human bronchioalveolar carcinoma and MDA-MB-468 human breast adenocarcinoma, subcutaneously implanted in nude mice. GHRH supplementation by delivery of human GHRH from a muscle-specific GHRH expression plasmid did not increase tumor progression in tumor-bearing nude mice. Male animals implanted with the NCI-H358 tumor cell line and treated with the GHRH-expressing plasmid exhibited a 40% decrease in the size of the tumors (P<.02), a 48% increase in white blood cells (P<.025) and a 300% increase in monocyte count (P<.0001), as well as an increase in the frequency of activated CD3+ and CD4+ cells in the tumors, compared to tumors of control animals. No adverse effects were observed in animals that received the GHRH-plasmid treatment. The present study shows that physiological stimulation of the GHRH-GH-IGF-I axis in mice with cancer does not promote tumor growth and may provide a viable treatment for cancer cachexia in humans.

Optimization of electroporation parameters for the intramuscular delivery of plasmids in pigs.

Increased transgene expression after plasmid transfer to the skeletal muscle is obtained with electroporation in many species, but optimum conditions are not well defined. Using a plasmid with a muscle-specific secreted embryonic alkaline phosphatase (SEAP) gene, we have optimized the electroporation conditions in a large mammal (pig). Parameters tested included electric field intensity, number of pulses, lag time between plasmid injection and electroporation, and plasmid delivery volume. Electric pulses, between 0.4 and 0.6 Amp constant current, applied 80 sec after the injection of 0.5 mg SEAP-expressing plasmid in a total volume of 2 mL produced the highest levels of expression. Further testing demonstrated that electroporation of a nondelineated injection site reduces the levels of SEAP expression. These results demonstrate that electroporation parameters such as amperage, lag time, and the number of pulses are able to regulate the levels of reporter gene expression in pigs.

Effects of plasmid-mediated growth hormone-releasing hormone supplementation on LL-2 adenocarcinoma in mice.

This study was designed to measure the effects of plasmid growth hormone-releasing hormone (GHRH) supplementation on LL-2 (Lewis lung adenocarcinoma) tumor-bearing immunocompetent mice. Male and female mice (n = 20/group/experiment) received 2.5 x 10(6) LL-2 cells in the left flank. One day later, we injected the mice intramuscularly with 20 micro g of a myogenic plasmid, pSP-hGHRH or pSP-betagal, as a control. Mean serum IGF-I was significantly higher in treated animals versus controls (P < 0.05). Male and female mice constitutively expressing GHRH exhibited a decline in tumor growth rate relative to controls (20% for males, P < 0.03, and 11% for females, P < 0.13). Histopathological analysis revealed that treated animals were less likely to develop lung metastases than controls (11%) and had no alternate-organ metastases. The number of metastases/lung was reduced by 57% in female mice with GHRH treatment (P < 0.006). When tumor size exceeded 8% of body weight, GHRH-treated mice showed normal urea, creatinine, and kidney volume, while controls displayed signs of renal insufficiency. This study provides evidence that with plasmid-mediated GHRH supplementation in tumor-bearing mice, tumor growth rate is not increased but is actually attenuated.

Maternal GHRH plasmid administration changes pituitary cell lineage and improves progeny growth of pigs.

Previous studies from our laboratory have demonstrated that administration of a myogenic plasmid that encodes a protease-resistant growth hormone-releasing hormone (HV-GHRH) to pregnant rat dams augmented long-term growth in first-generation progeny. In the present study, gilts were injected intra-muscularly at day 85 of gestation with 0, 0.1, 0.5, 1, or 5 mg of the HV-GHRH-expressing plasmid and were then electroporated. Piglets were weighed and bled periodically from birth to 100 kg. Piglets from gilts treated with 1 and 5 mg of HV-GHRH plasmid were larger at birth and weaning compared with controls. These two groups reached 100 kg 9 days earlier than the other groups. GHRH levels were increased at birth in piglets from treated gilts. IGF-I levels were significantly increased in the 5-mg group beginning at 21 days of age compared with controls. Pituitaries from the 5-mg group contained a significantly increased number of somatotrophs and lactotrophs from birth to 100 kg. This study confirms that enhanced maternal GHRH production results in intergenerational growth augmentation and that the magnitude of the response is dose dependent. The similarity of the response across species suggests that the effect is likely exerted as a fundamental component of gestational and developmental physiology.

Effects of plasmid-mediated growth hormone-releasing hormone in severely debilitated dogs with cancer.

Cachexia is a common manifestation of late stage malignancy and is characterized by anemia, anorexia, muscle wasting, loss of adipose tissue, and fatigue. Although cachexia is disabling and can diminish the life expectancy of cancer patients, there are still no effective therapies for this condition. We have examined the feasibility of using a myogenic plasmid to express growth hormone-releasing hormone (GHRH) in severely debilitated companion dogs with naturally occurring tumors. At a median of 16 days after intramuscular delivery of the plasmid, serum concentrations of insulin-like growth factor I (IGF-I), a measure of GHRH activity, were increased in 12 of 16 dogs (P < 0.01). These increases ranged from 21 to 120% (median, 49%) of the pretreatment values and were generally sustained or higher on the final evaluation. Anemia resolved posttreatment, as indicated by significant increases in mean red blood cell count, hematocrit, and hemoglobin concentrations, and there was also a significant rise in the percentage of circulating lymphocytes. Treated dogs maintained their weights over the 56-day study and did not show any adverse effects from the GHRH gene transfer. We conclude that intramuscular injection of a GHRH-expressing plasmid is both safe and capable of stimulating the release of growth hormone and IGF-I in large animals. The observed anabolic responses to a single dose of this therapy might be beneficial in patients with cancer-associated anemia and cachexia.

Nonhereditary enhancement of progeny growth.

The im electroporated injection of a protease-resistant GH-releasing hormone cDNA into rat dams at 16 d gestation resulted in enhanced long-term growth of the F(1) offspring. The offspring were significantly heavier by 2 wk of age, and the difference was sustained to 10 wk of age. Consistent with their augmented growth, the plasma IGF-I concentration of the F(1) progeny was increased significantly. The pituitary gland of the offspring was significantly heavier and contained an increased number of somatotrophs and PRL-secreting cells, which is indicative of modification of cell lineage differentiation. These unique findings demonstrate that enhanced GH-releasing hormone expression in pregnant dams can result in intergenerational growth promotion by altering development of the pituitary gland in the offspring.

Electrical enhancement of formulated plasmid delivery in animals.

Electroporation has been shown to significantly increase plasmid transfer to the skeletal muscle, but this procedure is also implicated in muscle damage. We are reporting a highly efficient in vivo transfer of a plasmid formulated with poly-(L-glutamate) (PLG) into murine, canine and porcine muscle fibers using electric pulses of low field intensity. In mice and pigs, the use of secreted embryonic alkaline phosphatase (SEAP) as the indicator gene caused increased PLG expression by 2-3 fold compared to naked plasmid; while delivery of a PLG-plasmid formulation to dogs showed a 10-fold increase in serum SEAP levels compared to plasmid alone. Muscle lesions were reduced by the protective PLG. Thus, PLG may constitute a useful adjuvant for increased expression and reduced muscle trauma to plasmid DNA delivered by electroporation.