GH-deficient Rats Research Articles

The role of prolactin and growth hormone in the regulation of casein gene expression and mammary cell survival: relationships to milk synthesis and secretion.

We have compared involution of the rat mammary gland, induced by litter removal, where milk accumulation occurs, with involution induced in the presence of the suckling young by combined PRL and GH deficiency. Both treatments induced involutionary processes involving apoptosis, as judged by DNA ladders and resulted in significant decreases in the DNA content of the gland. Surprisingly, the effects of hormone deprivation on protein output in milk were principally explained by the loss of secretory cells, as there were only modest decreases in casein messenger RNA (mRNA) expression and protein synthesis rates per U DNA in vitro. The association of casein mRNA with the polysome fraction was also unaffected by hormone deprivation, whereas involution induced by litter removal resulted in much greater decreases in steady state levels of casein mRNA and an increased association of the mRNAs with the monosome fraction. In PRL- and GH-deficient rats, PRL treatment could prevent all of these effects, GH was partially effective, whereas putative mediators of GH action, insulin-like growth factor I (IGF-I), IGF-II, and IGF-binding protein-3, were ineffective. This lack of effect of IGFs may be due to an inhibitory IGFBP, which we demonstrate to be present in increased amounts in the involuting mammary gland.

The effects of octreotide on GH receptor and IGF-I expression in the GH-deficient rat

Somatostatin has been suggested to influence the somatotrophic axis outside the central nervous system, in reducing GH-induced IGF-I mRNA and IGF-I generation. This study aimed to determine whether such effects were mediated via the GH receptor (GHR). GH-deficient dwarf rats aged 45-47 days (n = 8 per group) received twice daily subcutaneous injections of octreotide (1 mg/kg) (group O), saline (group S), octreotide (1 mg/kg) plus bovine GH (0.25 mg/kg) (group OG), or bovine GH (0.25 mg/kg) plus saline (group G) for 10 days. Octreotide-treated animals had less weight gain compared with saline-treated animals, but not when GH cotreated (group OG vs G). Octreotide had an overall effect on decreasing length gain (P < 0.01). Serum IGF-I (ng/ml) was reduced by octreotide (group O 171 +/- 11, group S 239 +/- 20, P < 0.01; group OG 283 +/- 30, group G 362 +/- 10, P < 0.001), as was serum insulin (P < 0.001). A significant decrease in hepatic and muscle IGF-I mRNA expression was found as expected, yet this was not associated with decreased hepatic GHR expression. Rather, an increase in hepatic 125I-bovine GH specific binding was observed (P < 0.001) and, in GH-cotreated animals (OG), hepatic GHR and GH binding protein (GHBP) mRNA expression were also increased by octreotide by approximately 40%. In muscle, octreotide was associated with an approximately 30% decrease in GHBP mRNA and no effect on GHR mRNA. This study suggests that the suppressive effects of octreotide on IGF-I metabolism, at least in liver, are not mediated via down-regulation of GHR expression, but more likely by direct effects on IGF-I expression.

The obese growth hormone (GH)-deficient dwarf rat: body fat responses to patterned delivery of GH and insulin-like growth factor-I.

We describe a new animal model of obesity and GH deficiency and report the effects on body fat of administering (GH) and insulin-like growth factor (IGF-I) in the model. Female GH-deficient dwarf rats fed a high-fat diet became obese and insulin-resistant compared with chow-fed controls. They were treated with recombinant human GH (rhGH 100-500 micrograms/day, s.c. for 14 days) by daily injection or minipump infusion with or without rhIGF-I (200 micrograms/day, sc infusion). Injections of rhGH increased body weight; infusions of rhGH caused weight loss. RhIGF-I by itself, or rhIGF-I plus GH injections had little effect, whereas rhGH infusions plus rhIGF-I caused a weight loss equivalent to the weight gained during the high-fat feeding and a decrease in fat pad weight. For some responses (serum IGF-1 and GHBP), the obese rats were GH resistant. Fat was lost from the internal fat pads when obese rats were returned to a chow diet, and injections of rhGH surprisingly attenuated this loss of fat. In obese dwarf rats, the lipolytic effects of rhGH are dose-regime dependent. By itself IGF-I is not insulin-like, but in the presence of GH it has antiinsulin actions causing a powerful net lipolysis. If GH plus IGF-I have similar effects in humans they may be useful for reducing body fat.

Chronic administration of a new potent agonist of growth hormone- releasing hormone induces compensatory linear growth in growth hormone-deficient rats: mechanism of action.

To assess the efficacy of a potent agonist analog of GH-releasing hormone (GH-RH), [Dat1,Gln8,Orn12,21,Abu15,Nle27,Asp28,A gm29]hGH-RH(1-29) (JI-38), we investigated the effects of its chronic administration on growth responses in monosodium glutamate (MSG)-lesioned and normal young rats. Body weight (BW), body length (BL), tibia length (TIL), and tail length (TAL) were monitored. Basal serum GH concentrations, GH responses to bolus injections of GH-RH, pituitary GH and serum IGF-I concentrations were measured by RIA. Pituitary GH-RH receptor concentration and binding affinity was also evaluated after the treatment. Neonatal treatment with MSG resulted, as expected, in blunted growth and a decrease in serum and pituitary GH concentration and serum IGF-I levels. A reduction in GH-RH receptor concentration, associated with increased binding affinity of the GH-RH receptor was also found. Chronic administration of GH-RH agonist JI-38 in doses of 2 micrograms at 12-hour intervals for 2 weeks markedly increased the GH responsiveness to GH-RH and stimulated growth, with MSG-treated animals achieving the growth rate of normal controls. Acceleration of growth was associated with stimulated GH synthesis and IGF-I secretion, although basal serum GH levels did not change. Pituitary GH-RH receptor concentration and binding affinity were not significantly modified by the treatment. Treatment of normal young growing rats with agonist JI-38 did not further increase the normal growth acceleration in these rats, but stimulated the GH synthesis and augmented the GH secretory responsiveness. The treatment of MSG-lesioned rats with GH-RH agonist was generally more effective in female than in male animals, and in some cases masked the sex differences in growth rate. Our findings provide the first evidence that the blunted growth rate of the MSG-lesioned rats is associated with a decreased pituitary GH-RH receptor concentration. Our work demonstrates that administration of GH-RH agonist JI-38 is able to restore the normal growth rate of the GH-deficient rats by stimulating GH synthesis and IGF-I secretion.

Upregulation of GH receptor and GH binding protein during pregnancy in the GH deficient rat

During pregnancy there are dramatic changes in the endocrine and metabolic status of the mother: growth hormone (GH) is an important regulator of growth and development. A proportion of GH is bound by specific GH binding proteins (GHBP) that closely resemble the GH receptor (GHR). In the rodent both GHBP and the GHR are considered to be GH dependent, and consequently during pregnancy the increase in serum GH is associated with an increase in GHBP. To examine whether an increase in maternal GH is obligatory for elevation of maternal GHBP or GHR during pregnancy, we used a unique GH-deficient (GHD) strain of rats, to avoid the methodological complications of hypophysectomy and assessed serum GH, GHBP and hepatic GHR binding during the course of pregnancy. In GH normal rats, serum GH concentrations increased twofold and GHBP levels increased threefold; there was no change in hepatic GHR binding. In GHD rats, serum GH concentrations were low and did not increase during pregnancy. Nonetheless, levels of both serum GHBP and hepatic GHR binding increased to that measured in normal rats. Thus, an increase in maternal GH concentration is not required for the gestational upregulation of maternal GHBP or hepatic GHR binding, suggesting that other hormones may be essential in modulating the GH axis during pregnancy.

Effects of continuous and repetitive administration of a potent analog of GH-RH(1-30)-NH2 on the GH release in rats treated with monosodium glutamate.

To assess the efficacy of a potent GH-releasing hormone (GH-RH) analog (D-Ala2,Nle27,Gaba30-GH-RH-(1-30)-amide) in the treatment of GH deficiency, we investigated the effects of chronic administration of this analog (A-495) on growth responses in monosodium glutamate (MSG)-lesioned rats. Basal serum GH concentrations, GH responses to bolus injections of GH-RH, as well as acceleration of body gain and linear growth were compared after long-term continuous and repetitive administration of A-495. The effects of continuous and repetitive administration of the analog on GH responses in vitro were also compared using the superfused pituitary cell system method. Treatment with MSG reduced the body weight and linear growth of the animals (-22% and -11%, respectively), the basal serum GH concentration (-66%), and the GH-RH-induced absolute GH responses (-61%) but did not alter the relative GH responses (to basal GH concentrations). Repetitive administration of 10 micrograms daily doses of A-495 at 24 h intervals for 2 weeks highly increased the GH responsiveness to GH-RH and induced catch-up growth, by which MSG-treated animals achieved the growth rate of normal controls. However, basal serum GH concentrations were only modestly enhanced. Continuous infusion of A-495 at the same daily dose resulted in slight increases in the GH-RH-induced GH rises, moderate acceleration of body gain, and no change in linear growth. Basal serum GH concentrations were not significantly influenced by this treatment. These results demonstrate that exogenous GH-RH pulses administered at lower frequency than the frequency of the physiological GH secretion are able to fully restore the normal growth rate of the GH deficient rats. The effectivity of the treatment is rather dependent on the magnitude of GH rises than the basal GH level. Although continuous administrations of the GH-RH is also have some effect on the body gain, repetitive administration is more effective at the same daily dose. Our results from in vitro experiments show that, in addition to the low magnitude of the GH-RH-stimulated GH rises, desensitization of the GH secretory response might also be accounted for the low effectivity of the continuously administered GH-RH. Present results demonstrate the therapeutic usefulness of our new GH-RH analog and are the first to evidence that GH-RH need not be administered as frequently as the appearance of the endogenous GH pulses to restore the normal growth of the GH deficient rats.

Insulin-like growth factor-1 and growth hormone (GH) have distinct and overlapping anabolic effects in GH-deficient rats

The anabolic activity of recombinant human growth hormone (rhGH) and insulin-like growth factor 1 (rhlGF-1) given either alone or together were studied in two models of GH deficiency, hypophysectomized and GH-deficient dwarf rats. A range of rhGH doses (0.08 to 50 mg/kg/day, seven daily sc injections) were given either alone or together with one dose of rhIGF-l (2.4 mg/kg/day, sc infusion). When given alone, or co-administered with rhlGF-1, rhGH produced dose dependent increases in weight gain, bone growth and organ weights. Weight gain in response to rhGH given with rhlGF-1 was comparable to that obtained by a 25-fold higher dose of rhGH given alone. In both animal models absolute weights of the kidneys, liver, spleen and thymus were increased by rhlGF-1 while kidney and liver weight were increased by rhGH. In the hypophysectomized rat, spleen and thymus weights were increased by rhGH but the relative potency of the combination was a 1000-fold that of rhGH alone. The effects of rhlGF-1 and rhGH were additive indicating that the effects of GH or IGF-1 can be greatly increased by their co-administration.

Growth hormone (GH) modulates insulin-like growth factor I (IGF-I) and type I IGF receptor mRNA levels in the ovary of prepubertal GH-deficient rats.

In order to explore the potential role of growth hormone (GH) in modulating insulin-like growth factor I (IGF-I) gene expression in the prepubertal rat ovary, female rats were rendered GH deficient by neonatal administration of monosodium glutamate (MSG). One group of rats received vehicle and served as the control. At 21 days of age, MSG-treated rats received either GH or vehicle for 2 weeks. On days 21, 24, 28 and 31 animals were weighed and subsets were sacrificed for liver RNA extraction. The remaining animals were sacrificed at day 35 when livers and ovaries were collected, and serum was obtained for GH determinations. The IGF-I mRNA levels were estimated by Northern blots and corroborated further by slot-blot analysis. The MSG-treated rats had lower body weights (p < 0.01) and GH levels (p < 0.05) than controls. Growth hormone replacement significantly accelerated the weight gain of MSG-treated rats. At day 24 and thereafter, three RNA IGF-I species (7.5, 1.8 and 0.8-1.2 kB) were seen in the liver. In the ovary, at age 35 days, two major IGF-I mRNA species (7.5 and 0.8-1.2 kb) were seen. The MSG treatment consistently reduced the levels of both IGF-I mRNA species in the ovary. Growth hormone administration partially restored their expression, both in the liver and in the ovary. In addition, ovarian type IIGF receptor mRNA levels were increased in the MSG-treated rats when compared to controls. This trend was reversed by GH replacement.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of deoxyribonucleic acid-protein interactions at a growth hormone-inducible nuclease hypersensitive site in the rat insulin-like growth factor-I gene.

Many of the growth-promoting effects of GH are mediated by insulin-like growth factor-I (IGF-I), a highly conserved, 70-residue basic peptide. Previous studies have demonstrated that GH rapidly stimulates IGF-I expression in vivo, and our laboratory has identified a GH-regulated alteration in chromatin configuration, manifested as a hormonally induced deoxyribonuclease-I (DNase-I)-hypersensitive site in the second IGF-I intron. In the current study, we have used in vivo DNase-I footprinting to map this hormonally responsive chromatin domain to an approximately 350-nucleotide region and have identified DNA-protein interactions within the hypersensitive site by in vitro gel mobility shift experiments and DNase-I footprinting studies. DNA-protein binding was localized to two adjacent segments of 32 and 48 nucleotides. In 1 of these regions, protein-DNA contacts were also detected in vivo on guanine residues by dimethylsulfate footprinting. DNA-binding activity was present in GH-deficient rats, but was not modified by hormone treatment. Our results define a rapid and reversible genomic alteration in response to GH in a GH-regulated gene and delineate a target within chromatin for GH action.

Characterization of deoxyribonucleic acid-protein interactions at a growth hormone-inducible nuclease hypersensitive site in the rat insulin-like growth factor-I gene

Many of the growth-promoting effects of GH are mediated by insulin-like growth factor-I (IGF-I), a highly conserved, 70-residue basic peptide. Previous studies have demonstrated that GH rapidly stimulates IGF-I expression in vivo, and our laboratory has identified a GH-regulated alteration in chromatin configuration, manifested as a hormonally induced deoxyribonuclease-I (DNase-I)-hypersensitive site in the second IGF-I intron. In the current study, we have used in vivo DNase-I footprinting to map this hormonally responsive chromatin domain to an approximately 350-nucleotide region and have identified DNA-protein interactions within the hypersensitive site by in vitro gel mobility shift experiments and DNase-I footprinting studies. DNA-protein binding was localized to two adjacent segments of 32 and 48 nucleotides. In 1 of these regions, protein-DNA contacts were also detected in vivo on guanine residues by dimethylsulfate footprinting. DNA-binding activity was present in GH-deficient rats, but was not modified by hormone treatment. Our results define a rapid and reversible genomic alteration in response to GH in a GH-regulated gene and delineate a target within chromatin for GH action.

Complex formation by human insulin-like growth factor-binding protein-3 and human acid-labile subunit in growth hormone-deficient rats.

Insulin-like growth factor-binding protein-3 (IGFBP-3), after first associating with IGF-I or IGF-II, is able to associate with the acid-labile subunit (ALS) and form a 140-kDa complex. To investigate the factors regulating ternary complex formation in vivo, human (h) IGFBP-3, hIGF-I, and hALS were administered in various combinations to GH-deficient (dw/dw) rats. hIGFBP-3 had a complex pattern of disappearance from the rat circulation, with an initial phase lasting minutes and a prolonged phase(s) lasting hours. If coinjected with hIGF-I, significantly more hIGFBP-3 was retained over 2 h. The molecular distribution of hIGFBP-3 was determined after size-separation chromatography. After an iv bolus of hIGFBP-3, 36.1 +/- 5.0% was in the 140-kilodalton complex at 5 min; this increased to 55.1 +/- 7.1% if hIGF-I was coinjected (P < 0.05). The 140-kDa complex disappeared slowly over hours, whereas 50- and 30-kDa forms of hIGFBP-3 cleared rapidly, with half-lives of minutes. To determine the importance of ALS in regulating the molecular distribution of hIGFBP-3, hALS was coinjected. Immunoreactive hALS disappeared slowly from the circulation and was shown to retain functional activity after 2 h in vivo. Coadministration of hALS did not influence the pattern of ternary complex formation, consistent with the presence of excess endogenous rat ALS. We conclude that ALS circulates in excess even in GH deficiency, is retained in the circulation for hours, and determines the stability of the 140-kDa complex, whereas IGF-I is a limiting factor in ternary complex formation by hIGFBP-3.

Administration of growth hormone (GH), but not insulin-like growth factor-I (IGF-I), by continuous infusion can induce the formation of the 150-kilodalton IGF-binding protein-3 complex in GH-deficient rats.

In the adult circulation, 70-90% of the serum insulin-like growth factors (IGFs) are carried by IGF-binding protein-3 (IGFBP-3), which exists as part of a 150-kilodalton (kDa) ternary complex including IGF and an acid-labile subunit (ALS). We have examined the hormonal regulation and molecular distribution of IGFBP-3 in the circulation of a uniquely GH-deficient (GHD) rat model. For 7 days, GHD rats were given GH by either twice daily injections (1 mg/kg) or continuous infusion (2.4 mg/kg.day) or IGF-I by continuous infusion (1.4 mg/kg.day). Each day, weight and feed and water intake were monitored, and on day 7, liver, kidney, spleen, heart, and lung were weighted, and sera were collected. Serum IGF-I was analyzed by immunoassay, and the molecular distribution of the IGFBPs was determined by neutral size-exclusion chromatography combined with Western ligand blot and Western immunoblot. The GHD rats were 40-60% lighter than their normal littermates, and all organs examined were proportionately smaller. Serum IGF-I and IGFBP-3 levels were less than 10% of those in normal rats. Incubation of serum from GHD rats with [125I]IGF-II showed that radiolabel was incorporated only into a 44-kDa IGFBP region that contained the smaller IGFBPs. IGFBP-3 eluted around 60 kDa. No 150-kDa IGFBP region was detected. The administration of GH or IGF-I to GHD rats resulted in significant increases in weight gained, although food and water intake remained unaltered. Weight gain was observed in all three treatments groups. Both GH treatment regimens significantly increased liver, spleen, and lung weight, whereas IGF-I therapy increased spleen, kidney, and heart. Administration of GH twice daily did not increase serum IGF-I or IGFBP-3 concentrations, and the molecular distribution of IGFBP-3 remained unchanged. In contrast, continuous infusion of GH resulted in 5-fold increases in serum IGF-I and increases in IGFBP-3 levels. Size-exclusion chromatography combined with Western ligand blot analysis revealed that radioligand was incorporated into 150- and 60-kDa regions, and that IGFBP-3 was detectable in both regions. Thus, GH infusion was able to induce formation of the 150-kDa ternary complex by increasing circulating levels of IGF-I, IGFBP-3, and presumably ALS. Administration of IGF-I also increased serum IGF-I and IGFBP-3 levels, although the increase in IGFBP-3 was only in the 60-kDa region of the chromatograph, suggesting that IGF-I can induce neither ALS nor formation of the 150-kDa complex.(ABSTRACT TRUNCATED AT 400 WORDS)

Administration of growth hormone (GH), but not insulin-like growth factor-I (IGF-I), by continuous infusion can induce the formation of the 150-kilodalton IGF-binding protein-3 complex in GH-deficient rats

Administration of growth hormone (GH), but not insulin-like growth factor-I (IGF-I), by continuous infusion can induce the formation of the 150-kilodalton IGF-binding protein-3 complex in GH-deficient rats

Multifactorial regulation of IGF‐I gene expression

Insulin-like growth factor-I (IGF-I) is a highly conserved 70-residue circulating peptide with diverse biological effects. In mammals IGF-I is an essential mediator of normal postnatal growth and its expression is influenced by hormonal, nutritional, tissue-specific, and developmental factors. Recent studies have demonstrated that the IGF-I gene is more complicated than might have been predicted from its simple protein sequence. In rats and in humans the single-copy six-exon gene is transcribed by adjacent promoters into nascent RNAs with different 5' leader sequences that undergo both alternative RNA splicing and differential polyadenylation to yield multiple mature transcripts. These observations suggest that trophic agents may modulate expression of IGF-I at any of several nodal points. In this report we review several of the mechanisms responsible for regulating production of IGF-I in the rat. During neonatal development IGF-I gene transcription is progressively activated, leading to a rise in both hepatic IGF-I mRNA and in serum IGF-I. The induction of IGF-I expression is limited to mRNAs directed by promoter 1, the more 5' of two rat IGF-I gene promoters, and precedes the ontogenic appearance of liver growth hormone (GH) receptors, indicating that mechanisms independent of GH activate IGF-I expression during early postnatal life. By contrast, in adult GH-deficient rats, a single intraperitoneal injection of GH causes a prompt rise in IGF-I gene transcription that is mediated equivalently by promoters 1 and 2.(ABSTRACT TRUNCATED AT 250 WORDS)

EFFECT OF TESTOSTERONE ON BONE MINERAL DENSITY (BMD) IN GROWTH HORMONE (GH) DEFICIENT RATS

The contribution of GH and sex steroids to attainment of peak bone mass are not clear and require longitudinal studies. We are examining the interactions between testosterone and GH on BMD using the GH deficient and GH sufficient rat models (Lewis strain). In the first part of study, forty male GH deficient rats were included with half the rats castrated at age 4 weeks and half remained as controls. All rats received identical diet and housing. BMD of total body, femur and lumbar spine (L1-L4) were measured from early adulthood at age 12 and 36 weeks, using Dual Energy X-ray Absorptiometry (DEXA, LUNAR) equipped with special software for small animals. This DEXA software was validated for small animals prior to the study. There was strong agreement between bone mineral content and ash weight for total body (r2=0.90) and femur (r2=0.83) in 17 rats with weight ranging from 146 to 212 grams. At age 12 weeks, there was no difference in total body, femur and L1-L4 BMD between two groups. However at 36 weeks, the castrated rats had significantly lower L1-L4 BMD compared to the controls (data not shown). From age 12 to 36 weeks, rats of both groups had significant increase in total body and femur BMD (left). The castrated group had no increase in L1-L4 BMD, which is significantly different from the control rats. We conclude that testosterone, even in the presence of GH deficiency, is critical for maintaining BMD of lumbar spine, which is predominantly trabecular bone. The impact of GH deficiency on BMD will be examined in the second part of study with GH deficient and sufficient models.

GROWTH HORMONE (GH) RECEPTOR GENE EXPRESSION IN THE HYPOTHALAMUS IS INCREASED DURING PUBERTY

The development of a pulsatile pattern of GH secretion occurs at puberty. Although the mechanisms responsible for the generation of GH pulses are unknown, a direct feedback effect of GH on the hypothalamus (via GH receptors) appears to play an important role. We tested the hypothesis that GH receptor gene expression changes over puberty by comparing GH receptor mRNA levels in the periventricular nucleus (PeN) of prepubertal (25 days) and adult (70 days) male rats. We performed in situ hybridization on coronal brain slices using a 35S-UTP-labeled cRNA probe and measured autoradiographic silver grains above GH receptor mRNA-containing cells. We observed that cellular levels of GH receptor mRNA (reported as grains/cell±SE) were significantly higher in adult compared to prepubertal animals (116±6 vs 92±4, respectively; p<0.05). The factors responsible for this pubertal increase in GH receptor message are unclear; however, mean plasma levels of GH increase during puberty and GH has been shown to increase GH receptor mRNA levels in other tissues. Therefore, we tested the hypothesis that GH regulates GH receptor gene expression in the PeN by comparing cellular levels of GH receptor mRNA among groups of control and GH-deficient rats (both dwarfs and GHRH antibody-treated). GH deficiency did not significantly alter the GH receptor mRNA levels when compared with control levels. Conclusion: An increase in GH receptor gene expression may be involved in the pubertal development of pulsatile GH secretion. This increase in GH receptor mRNA most likely occurs independent of increased mean plasma GH levels.

COMBINED TREATMENT WITH RECOMBINANT HUMAN INSULIN-LIKE GROWTH FACTOR-I (rhIGF-I) AND GROWTH HORMONE (rhGH) IN THE DWARF RAT

Daily injections of rhGH and infusions of rhIGF-1 have additive effects on body growth in GH-deficient rats. We now measure growth responses to a fixed daily dose of rhGH (240 μg/d, sc injection) given once or twice daily, with or without rhIGF-1 (120μg/d, sc infusion) for 8 d in female dw/dw rats (6-8 weeks old, 100 g). Rats were sacrificed on Day 8; organs weighed and serum and tibia taken. Data in the Table are means ± SD (n=5/group).For weight gain, cartilage and relative liver growth bid rhGH gave larger responses than daily rhGH. Spleen growth and serum IGF-I were optimal with rhIGF-1 alone. Combination treatment gave greater weight gain and higher serum IGF-I's but not greater cartilage, liver or spleen growth. We conclude that when rhGH is given in an optimal pattern at high doses additive whole body anabolic effects of rhGH and rhIGF-1 are observed.

Pregnancy in growth hormone-deficient rats: assessment of insulin-like growth factors (IGFs), IGF-binding proteins (IGFBPs) and IGFBP protease activity

Pregnancy in rodents is associated with important maternal metabolic changes. Early pregnancy is considered to be an anabolic phase of nutrient storing, while in late pregnancy, a catabolic phase develops to help meet the metabolic demands of the rapidly growing conceptus. Similarly, major changes also occur in the IGFs, IGF-binding proteins (IGFBPs) and GH axis. In the rat, maternal serum IGF-I levels increase from early to mid-pregnancy, after which IGF-I levels decline. Conversely, as IGF-I levels decline, pituitary-derived rat GH increases twofold. This coincides with a decrease in IGFBP-3 and the appearance of an IGFBP protease. However, the physiological role of these changes is unclear. The aim of our study was to examine the roles of GH, IGFs and IGFBPs during pregnancy in a unique isolated GH-deficient rat model, spontaneous dwarf rats. Pregnancy in GH-deficient dams resulted in a significantly reduced litter number, and maternal weight gain (day 21-day 1) was reduced by 28% when compared with dams with normal GH levels (GH-normal dams). In the sera of GH-normal dams, IGF-I levels increased 2.6-fold by day 4 of pregnancy and then progressively declined to below non-pregnant levels. Serum IGF-II levels were low and remained unchanged. Western-ligand blot analysis showed that IGFBP-3 was present in non-pregnancy and early pregnancy sera, but declined dramatically after day 12. This decline in IGFBP-3 coincided with the detection of an IGFBP protease. In contrast, in non-pregnancy sera from GH-deficient rats, serum IGF-I concentrations were 10% of the levels seen in GH-normal females, and neither IGF-I nor IGF-II concentrations changed with pregnancy. Furthermore, in GH-deficient dams, serum IGFBP-3 (as assessed by Western-ligand blot analysis) was low in non-pregnancy and early pregnancy sera, and became undetectable by day 12 of pregnancy. The low concentrations of IGFBP-3 in early pregnancy serum from GH-deficient rats coincided with IGFBP-proteolytic activity, and the decline in serum IGFBP-3 after day 16 was the result of increased protease activity. In conclusion, isolated GH deficiency results in: (1) reduced maternal weight gain and correspondingly smaller litter sizes; (2) low and unchanged maternal serum IGF-I levels; (3) low, but declining, serum IGFBP-3 levels; and (4) activity of IGFBP protease(s) detectable in early and late pregnancy, which may modulate the bioavailability and bioactivity of IGFs by regulating IGFBP-3.

Expression and Regulation of Insulin-Like Growth Factor-I in the Rat Incisor

Growth factors play an important role in the regulation of cell growth, division and differentiation. In this study the distribution and regulation of insulin-like growth factor-I (IGF-I) in the continuously erupting rat incisor was determined by immunohistochemistry. Results were evaluated both visually and with a computer-based image analysis system. The distribution and intensity of IGF-I immunoreactivity varied with developmental stage of the rat incisor. Strong IGF-I immunoreactivity was observed in differentiating odontoblasts and ameloblasts. The most intense immunoreactivity was observed in secretory ameloblasts, secretory odontoblasts and in maturation ameloblasts. Staining was weak or absent in post-secretory ameloblasts but persisted in post-secretory odontoblasts. Weak to moderate immunoreactivity was also seen in cells of the stratum intermedium and in the reduced enamel epithelium. Surrounding alveolar bone showed strong IGF-I immunoreactivity in osteoblasts and in the stratum basale and stratum spinosum of the adjacent labial gingival epithelium. In order to assess the role of GH in IGF-I expression, GH (65 micrograms/100 g bw) was administered for six days to dwarf GH deficient rats, producing a significant increase in body weight (P < 0.01). Measurements at different stages of odontogenesis showed that the staining intensity of secretory ameloblasts (P < 0.01) and maturation ameloblasts (P < 0.001) was significantly different between untreated and treated animals. These results indicate that IGF-I is present in cell populations of the enamel organ of the rat incisor found previously to exhibit growth hormone receptors, and that expression of IGF is GH dependent.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of intermittent growth hormone or insulin-like growth factor 1 administration in the neonatal dwarf rat.

To further evaluate the role of growth hormone (GH) in the neonatal period, the effects of GH or insulin-like growth factor 1 (IGF-1) administration were studied in the neonatal GH-deficient rat. Groups of pups from newborn litters were randomized to receive twice daily subcutaneous injections of recombinant bovine growth hormone (bGH, 5 micrograms), recombinant human IGF-1 (10 micrograms) or saline from the second to twelfth day of life. The effects on growth parameters, serum IGF-1 concentration, body composition and hepatic GH receptor binding were assessed. bGH-treated animals showed increases in body weight gain (p = 0.01), serum IGF-1 (p < 0.01), carcass nitrogen (p < 0.001) and carcass water (p < 0.001) compared to IGF-1 or saline-treated animals. No differences in these parameters were noted between IGF-1 and saline-treated groups. bGH-treated animals showed a significantly lower hepatic GH receptor binding (p < 0.01) compared to the other two groups. The demonstration of anabolic responses to GH administration in the neonatal period has implications for the possible role of GH in fetal and neonatal growth.