Dw Rats Research Articles

Asiaticoside-nitric oxide promoting diabetic wound healing through the miRNA-21-5p/TGF-β1/SMAD7/TIMP3 signaling pathway

Ethnopharmacological relevanceCentella asiatica (L.) Urban is an ethnobotanical herb. The main bioactive components of Centella asiatica are pentacyclic triterpenoid glycosides, namely asiaticoside and hydroxyasiaticoside. Asiaticoside possess a diverse array of pharmacological properties, such as wound-healing, anti-inflammatory, antioxidant, anti-allergic, antidepressant, anxiolytic, anti-fibrotic, antibacterial, anti-arthritic, anti-tumor, and immunomodulatory activities. Aim of the studyThe purpose of this investigation is to explore potential therapeutic interventions for the delayed healing of wounds in diabetic patients (DW) facilitated by Asiaticoside-Nitric Oxide. To clarify the key molecular mechanism of miRNA-21-5p in DW wound repair and to deepen the understanding of DW disease pathogenesis. Materials and methodsFirstly, miRNA microarray technology, bioinformatics, and RT-qPCR were used to analyze DW patients' and normal controls' skin tissue samples. Secondly, in order to investigate the role of miRNA-21-5p, a hyperglycemic model was established using HaCaT cells. Overexpressing as well as interfering HaCaT cell lines were constructed by lentiviral infection to further explore the proliferative and migratory effects of Asiaticoside-Nitric Oxide. The next step was to search for potential target genes of miRNA-21-5p and verify them with dual-luciferase reporter assay. Finally, the expression levels of target genes and proteins were detected through the utilization of RT-qPCR and Western blotting under the influence of Asiaticoside-Nitric Oxide. ResultsA library of miRNAs and target genes expressed explicitly in DW patients and rats was established. The study confirmed the upregulation of miRNA-21-5p in DW patients and identified its involvement in signaling pathways related to chronic ulcer wound repair. Overexpression of LV-miRNA-21-5p significantly promoted cell proliferation, while treatments of Asiaticoside-Low dose (AC-L) and Asiaticoside-Medium dose (AC-M) enhanced proliferation and migration, particularly when combined with nitroprusside (SNP). Further analysis revealed potential target genes of miRNA-21-5p, such as TGF-β1, SMAD7, and TIMP3. Their interaction with miRNA-21-5p was confirmed through dual luciferase assays. The study found that anti-DW drugs increased the expression of TGF-β1 and SMAD7 while inhibiting TIMP3 expression in a high-glucose environment. ConclusionsThe research concluded that miRNA-21-5p plays a crucial role in the delayed healing of diabetic wounds, and that the combination treatment of AC + SNP shows promise in promoting wound healing in DW rats. Target genes, including TGF-β1, SMAD7, and TIMP3, may contribute to the regulatory mechanisms involved in diabetic wound healing. These findings provide valuable insights for developing novel therapeutic approaches for DW.

Effect of zinc supplementation on immunotoxicity induced by subchronic oral exposure to glyphosate-based herbicide (GOBARA®) in Wistar rats

To evaluate the effect of zinc supplementation on immunotoxicity induced by subchronic oral exposure to glyphosate-based herbicide (GBH). Sixty adult male Wistar rats randomly divided equally into six groups were exposed to GBH by gavage daily for 16 weeks with or without zinc pretreatment. Group DW rats received distilled water (2 mL/kg), group Z rats received zinc (50 mg/kg), and group G1 and G2 rats received 187.5 and 375 mg/kg GBH, respectively. Group ZG1 and ZG2 rats were pretreated with 50 mg/kg zinc before exposure to 187.5 and 375 mg/kg GBH, respectively. Tumor necrosis factor alpha (TNF-α) and immunoglobulin (IgG, IgM, IgE) levels were measured by enzyme-linked immunosorbent assay. Spleen, submandibular lymph node, and thymus samples were processed for histopathology. Exposure to GBH (G1 and G2) significantly increased serum TNF-α concentrations and significantly decreased serum IgG and IgM concentrations compared with the control levels. Moderate-to-severe lymphocyte depletion occurred in the spleen, lymph nodes, and thymus in the GBH-exposed groups. Zinc supplementation mitigated the immunotoxic effects of GBH exposure. GBH exposure increased pro-inflammatory cytokine responses, decreased immunoglobulin production, and depleted lymphocytes in lymphoid organs in rats, but zinc supplementation mitigated this immunotoxicity.

Vasoprotective effects of lifespan‐extending peripubertal GH replacement in Lewis dwarf rats

In humans growth hormone deficiency (GHD) and low circulating levels of IGF-1 significantly increase the risk for cerebrovascular disease. Genetic GH/IGF-1-deficiency in Lewis dwarf rats (DW) significantly increases the incidence of late-life strokes, similar to the effects of GHD in elderly humans. Peripubertal treatment of DW rats with GH delays the occurrence of late-life stroke, which results in a significant extension of lifespan. The present study was designed to characterize the vascular effects of lifespan-extending peripubertal GH replacement in DW rats. We found</sup> peripubertal GHD in DW rats increases vascular oxidative stress, which is prevented by GH replacement. Peripubertal GHD did not alter vascular expression and activity of SOD and catalase. In contrast, cerebrovascular expression of Gpx-1 was significantly decreased in DW vessels and this effect was reversed by GH treatment. Peripubertal GHD significantly decreases expression of the Nrf2 target genes NQO1 and GCLC in the cerebral arteries, whereas it does not affect expression and activity of eNOS and vascular expression of IGF-1, IGF binding proteins and inflammatory markers. In conclusion, peripubertal GH/IGF-1 deficiency confers pro-oxidative cellular effects, which likely promote an adverse functional and structural phenotype in vasculature and results in accelerated vascular impairments later in life. (Funding: ADA, AFAR, NIH).

Dendritic stability in a model of adult-onset IGF-I deficiency

Objective A significant decrease in plasma levels of insulin-like growth factor-I (IGF-I) is one of the most robust hallmarks of aging and may contribute to functional changes associated with senescence. This study examined the role of IGF-I in the maintenance of adult dendritic morphology. Design We utilized a model of the aging-related decrease in plasma IGF-I to examine whether such a decrease, in itself, leads to dendritic changes in the cerebral cortex. The dw/ dw rat, originally of the Lewis strain, suffers from a spontaneous mutation in which growth hormone (GH) production is severely decreased. Since GH is responsible for the production of circulating IGF-I by the liver, these animals are deficient in plasma IGF-I. Homozygous dw/ dw rats were administered porcine GH to sustain IGF-I levels during development and then GH injections were stopped as adults in order to examine the effects of adult-onset GH and IGF-I deficiency. Animals sacrificed after two or eight weeks of GH and IGF-I deficiency were compared to age-matched dw/ dw animals that received GH both developmentally and throughout adulthood (GH/IGF-I replete). The dendritic arbors of pyramidal neurons in cingulate cortex were labeled by intracellular injection and reconstructed in three dimensions. Results Comparing GH/IGF-I replete and deficient dw/ dw rats, we found no differences in the apical or basal arbors of either layer two or layer five pyramidal neurons. Conclusions These findings indicate that a decrease in plasma levels of IGF-I is not sufficient in itself to produce dendritic changes like those seen in aging animals.

Effects of gender and functional overload on plantaris muscle morphology in the dwarf (HsdOla:dw-4) Lewis rat

To investigate relationships between pituitary function and gender on skeletal muscle growth and hypertrophy, fiber cross sectional area (CSA) and type were assessed in the plantaris muscle of normal and dwarf (Dw) male and female Lewis rats after 6 weeks of functional overload (FO). Serum growth hormone levels were 70–80% less in Dw rats of both genders and body mass was 62% greater in normal rats when compared to their Dw counterparts. Muscle weight was affected by gender, dwarfism, and FO as well as a significant gender∗Dw∗FO interaction. FO increased Type I, IIA, and IIX/B fiber CSA 120%, 102%, and 75%, respectively. Only type II fibers exhibited a reduction in CSA as a function of gender or dwarfism. Both type IIA and IIX/B fibers were affected by a significant gender∗Dw∗FO interaction. Our results suggest that the growth of type II fibers is sensitive to gender and pituitary function, while hypertrophy of type II muscle fibers is a function of the interaction between mechanical load, gender and pituitary function.

Decreased hexosamine biosynthesis in GH-deficient dwarf rat muscle. reversal with GH, but not IGF-I, therapy.

Enhanced glucose flux via the hexosamine biosynthesis pathway (HNSP) has been implicated in insulin resistance. We measured L-glutamine:D-fructose-6-phosphate amidotransferase activity (GFAT, a rate-limiting enzyme) and concentrations of UDP-N-acetyl hexosamines (UDP-HexNAc, major products of HNSP) in muscle and liver of growth hormone (GH)-deficient male dwarf (dw) rats. All parameters measured, except body weight, were similar in 5-wk-old control and dw rats. Muscle GFAT activity declined progressively with age in controls and dw rats but was consistently 30-60% lower in 8- to 14-wk-old dw rats vs. age-matched controls; UDP-HexNAc concentrations in muscle were concomitantly 30% lower in dw rats vs. controls (P < 0.01). Concentrations of UDP-hexoses, GDP-mannose, and UDP in muscle were similar in control and dw rats. Muscle HNSP activity was similarly diminished in fed and fasted dw rats. In liver, only a small difference in GFAT activity was evident between controls and dw rats, and no differences in UDP-HexNAc concentrations were observed. Treatment with recombinant human GH (rhGH) for 5 days restored UDP-HexNAc to control levels in dw muscles (P < 0.01) and partially restored GFAT activity. Insulin-like growth factor I treatment was ineffective. We conclude that GH participates in HNSP regulation in muscle.

Intrahypothalamic growth hormone feedback: from dwarfism to acromegaly in the rat.

Two different dwarf rat models with primary (dw/dw, DW) or secondary (transgenic growth retarded, WF/Tgr) GH deficiency and contrasting hypothalamic GH-releasing hormone (GHRH) and somatostatin (SRIH) expression were implanted sc with GC cells. These form encapsulated rat GH-secreting tumors that maintain high plasma rat GH levels for several weeks. In both strains, GC cell tumors stimulated growth and raised GHBP levels, without affecting pituitary GH content. In DW rats, GC cell implants increased SRIH expression in the periventricular nucleus (PeV), but not in the arcuate nucleus (ARC), whereas their high GHRH expression in ARC was decreased by GC cells. In contrast, GC cell implants in WF/Tgr rats had little effect on the already high SRIH expression in PeV or low GHRH expression in ARC, although they reduced SRIH expression in ARC. GC cell implants also reduced GH receptor expression in both ARC and PeV in the WF/Tgr dwarves. Thus, chronic GH overexposure stimulates rapid growth in both dwarf strains, but has differential hypothalamic effects in these models. This experimental approach now makes it possible to study the effects of pathophysiological concentrations of GH ranging from dwarfism to acromegaly in the same animal model.

Growth hormone (GH) status regulates GH receptor and GH binding protein mRNA in a tissue- and transcript-specific manner but has no effect on insulin-like growth factor-I receptor mRNA in the rat

The effect of growth hormone-deficiency (GHD) and treatment with recombinant bovine GH (bGH) or human IGF-I (hIGF-I) for 10 days on the expression of GH receptor (GHR). GH binding protein (GHBP) and of insulin-like growth factor-I receptor (IGF-IR) mRNA was examined using dw dw and normal Lewis rats. Hepatic GHR and GHBP mRNA expression was significantly lower in dw dw rats in comparison to Lewis rats ( P < 0.01) while specific 125I-bGH binding to hepatic microsomal membranes was significantly higher ( P < 0.01), suggesting a reduction in hepatic GHR turnover with GHD. Treatment with bGH reduced hepatic specific 125I-bGH binding in dw dw rats, but had no effect in Lewis rats. Treatment with hIGF-I increased hepatic specific 125I-bGH binding in Lewis rats. Hepatic GHR and GHBP mRNA expression was not changed by bGH or hIGF-I treatment, suggesting that differences in hepatic specific 125I-bGH binding may be due to posttranscriptional mechanisms. GHBP mRNA expression was higher in kidney, heart, and muscle of dw dw rats in comparison to Lewis rats ( P < 0.01), while GHR mRNA abundance was not changed. Treatment of dw dw rats with hIGF-I or bGH resulted in a coordinate reduction of GHR and GHBP mRNAs in kidney ( P < 0.01). IGF-IR mRNA was not detected in liver and despite reduced plasma IGF-I levels and IGF-I mRNA expression IGF-IR mRNA abundance was not changed in nonhepatic tissues by GHD. Our data suggest that changes in plasma IGF-I levels and local IGF-I mRNA do not influence IGF-IR mRNA expression, while GHR and GHBP mRNA expression in different rat tissues are regulated independently. The increased nonhepatic GHBP mRNA expression with GHD suggests that nonhepatic GHBP may have an important physiological function distinct from that of GHBP in liver or in plasma.

Pituitary growth hormone-releasing factor receptor expression in normal and dwarf rats.

Growth hormone-releasing factor (GRF) regulates GH release and somatotrope proliferation via a specific G-protein-coupled receptor. Little is known about the endocrine factors that regulate the expression of the GRF receptor (GRF-R) in the pituitary gland. We have developed a sensitive solution hybridization/RNAse protection assay for GRF-R mRNA in rat pituitary extracts. GRF-R transcripts were readily detectable in similar amounts in normal male and female rats, but were markedly reduced in extracts from age-matched growth hormone (GH)-deficient dwarf (dw) rats of either sex. The reduced GRF-R expression would appear to reflect somatotrope hypoplasia rather than GH deficiency per se since a similar reduction in GRF-R expression was seen in a transgenic model of dominant dwarfism, whereas GRF-R expression was significantly elevated in rats with GH deficiency induced by hypothyroidism. We were unable to demonstrate significant effects on GRF-R expression with infusions of human GH (hGH) or insulin-like growth factor 1, which stimulate growth in dw rats, but dexamethasone treatment induced a significant, time-related increase in GRF-R mRNA levels. We conclude that this assay can usefully quantify pituitary GRF-R expression in normal rats, and its reduction in two different strains of mutant dwarf rats with somatotrope hypoplasia.

In vivo evidence of a positive role for somatostatin to optimize pulsatile growth hormone secretion.

Despite convincing evidence that somatostatin (SRIF) and growth hormone (GH)-releasing factor (GRF) individually play crucial roles in GH regulation, the nature of the interplay between these two hypothalamic hormones is far from clear. In the present study, we used the long-acting SRIF analogue, octreotide, as a probe in both the normal and mutant dwarf (dw) rat 1) to further elucidate the temporal nature of the SRIF-GRF interaction in vivo and 2) to define possible mechanisms of action of SRIF in generating pulsatile GH secretion. Normal free-moving adult male rats pretreated with octreotide (25 and 50 micrograms iv) and subsequently challenged with GRF (1 micrograms iv) exhibited a markedly blunted GH response to exogenous GRF 1 h after treatment. In contrast, preexposure to octreotide for 3 h produced a two- to threefold augmentation in GH responsiveness to GRF. Compared with normal saline-pretreated controls, 3-h pretreatment with octreotide produced a 14- to 16-fold augmentation in the postinhibitory rebound release of GH after the coadministration of native SRIF-14 and GRF (P < 0.001). In dw rats, which show a selective reduction in GH synthesis and storage, 3-h preexposure to octreotide failed to significantly alter GRF-induced GH release. These results demonstrate that, in the normal male rat, a 3-h period of exposure to the SRIF analogue octreotide is sufficient to enhance GH responsiveness to GRF. Our findings suggest that this effect is due to a SRIF-mediated buildup of pituitary GH stores in a readily releasable poo.(ABSTRACT TRUNCATED AT 250 WORDS)

Growth hormone (GH) and insulin-like growth factor-I (IGF-I) treatment of the GH-deficient dwarf rat: differential effects on IGF-I transcription start site expression in hepatic and extrahepatic tissues and lack of effect on type I IGF receptor mRNA expression

The rat IGF-I gene consists of six exons, with exons 3 and 4 forming a ‘core’ mature IGF-I coding region to which alternate 5′ and 3′ regions are spliced. Transcription occurs from four dispersed start sites (ss) ≈ 382 (ss 1), ≈ 343 (ss 2), ≈ 245 (ss ≈ 30–40 (ss 4) basepairs (bp) from the 3′ end of exon 1, and from a region 50–70 bp from the 3′ end of exon 2. The expression of ss mRNAs displays tissue-specific and ontogenic regulation. Alternate splicing of exon 5 produces E-peptide coding domain variants (Ea and Eb mRNAs), with the Eb form found predominantly in the liver. The regulation of IGF-I mRNA expression by GH and IGF-I in the GH-deficient dwarf ( dw / dw) rat was investigated using antisense RNA probes in a solution hybridization RNase protection assay to detect leader exon and E domain variant mRNAs. GH treatment of dw / dw and normal Lewis rats increased the expression of all liver leader exon ss and E domain variants coordinately (1.6–1.9-fold increase, p < 0.01), although the increase observed in Eb transcripts was significantly higher in the dw / dw compared to the normal rat ( p < 0.05). In kidney, GH treatment significantly increased exon 1 ss 3 and ss 4 transcripts by approximately 40% ( p < 0.05). The expression of the other start sites was not affected by GH, suggesting that transcription factors may regulate start site usage independently. GH treatment was associated with a significant increase in IGF-I mRNA expression in skeletal muscle ( p < 0.05) but not cardiac muscle or spleen. IGF-I treatment was associated with minor (≈ 20%) but significant ( p < 0.05) reductions in IGF-I mRNA expression in the liver and kidney of dw / dw rats, suggesting that IGF-I can suppress IGF-I mRNA expression. IGF-I treatment did not affect IGF-I mRNA expression in cardiac and skeletal muscle of dw / dw rats. IGF-I receptor mRNA was detected in extrahepatic tissues only, and was not affected by either GH or IGF-I treatment. In summary, start site-specific regulation by GH was observed in kidney. GH increased IGF-I mRNA expression in muscle, kidney and liver, but had no effect in heart or spleen in the dw / dw rat. Our data suggest that systemic IGF-I can feedback on hepatic and renal IGF-I mRNA expression in the GH-deficient state.

Impaired growth hormone-releasing hormone signal transduction in the dwarf (dw) rat is independent of a generalized defect in the stimulatory G-protein, Gs alpha.

The homozygous dwarf (dw) rat pituitary is characterized by a 95% reduction in GH content as well as a 75-80% reduction in the number of somatotrophs. The nature of the mutation responsible for this phenotype is unknown. Previous investigations from our laboratory indicate that dw somatotrophs exhibit decreased sensitivity and a reduced GH secretory response to GH-releasing hormone in vitro, accompanied by a decreased generation of cAMP. We hypothesized that dw rats have a defect in the pathway linking the GH-releasing hormone receptor to adenylyl cyclase and focused on the expression and function of the stimulatory G-protein, Gs alpha. When corrected for differences in pituitary size and cell number, GH mRNA content was reduced by 78% compared to that in controls. However, there was no difference in Gs alpha mRNA content or size in dw pituitaries. Similarly, there was no difference in the content or size of mRNA for the pituitary transcription factor pit-1 in dw pituitaries. Immunoblot analysis of pituitary membrane proteins using a Gs alpha-specific antibody revealed no differences in size, quantity, or relative distribution of Gs alpha peptides between control and dw pituitaries. In addition, cholera toxin effectively ribosylated dw Gs alpha, and there were no differences in the size, quantity, or relative distribution of ribosylated membrane proteins between dw and control pituitaries. Finally, to examine for mutations in other regions of the Gs alpha-coding sequence, we cloned the full-length Gs alpha cDNA from dw rat pituitaries by polymerase chain reaction. The sequence of this clone was identical to that of normal rat Gs alpha cDNA. These results indicate that 1) 52-kilodalton Gs alpha appears to predominate in both normal and dw pituitary; 2) the content, function, and sequence of Gs alpha in adult dw rats are normal; and 3) a generalized Gs alpha regulatory or structural mutation as the cause of the observed phenotype can be excluded. The results also demonstrate that there is no decrease in pit-1 expression in the adult dw pituitary.

Growth hormone binding protein in the rat: effects of gonadal steroids.

In normal rats, females have higher circulating GH-binding protein (GHBP) levels than males, whereas in the GH-deficient dwarf (Dw) rat, there is no sexual dimorphism in plasma GHBP, suggesting that GH secretion may be involved in this difference. In order to study the relationship between gonadal steroids and GH on GHBP and GH receptor regulation, the levels of plasma GHBP, hepatic bovine GH, and human GH (hGH) binding as well as GHBP and GH receptor messenger RNA (mRNA) have now been studied in normal, Dw, hypophysectomized (Hx), or ovariectomized (Ovx) rats, subjected to different GH and gonadal steroid exposure. In normal male rats, estradiol (E2, 12.5-25 micrograms/day for 1 or 2 weeks) markedly increased plasma GHBP and hepatic hGH, and bGH binding. These effects of E2 were diminished in Dw rats, absent in Hx rats, but restored in Hx rats given exogenous hGH. Plasma GHBP rose in female rats given E2, and fell in females given the anti-estrogen tamoxifen. Ovx animals had lower plasma GHBP and hepatic GH binding which was reversed by E2, but not testosterone treatment. Continuous hGH infusions in Ovx rats restored hepatic GH binding, and increased plasma GHBP. In Dw males, hGH increased plasma GHBP and hepatic GH binding, whereas testosterone had no effect on GHBP or GH receptors and did not affect their up-regulation by hGH. Hepatic levels of GHBP-, and GH receptor mRNA transcripts showed the same trends in response to steroid or GH treatment, but the differences were rarely significant, except in Ovx animals which had higher GHBP mRNA transcripts after GH or E2 treatment. Thus E2 and GH increase both plasma GHBP and hepatic GH receptor binding. GH up-regulates GHBP in the absence of E2, whereas E2 treatment does not raise GHBP in the absence of GH. Whereas some of the effects of estrogen could be mediated via alterations in GH secretion, estrogen may also directly influence GHBP production at the liver, but only in the presence of GH.

Growth hormone receptor regulation in growth hormone-deficient dwarf rats

In the rat, many actions of GH depend upon the sexually dimorphic pattern of exposure to GH. Hepatic human GH (hGH) receptor binding differs between the sexes and is sensitive to GH deficiency, but this has mostly been studied in acutely hypophysectomized rats, which lack all pituitary hormones. We have used a strain of GH-deficient dwarf (Dw) rats to determine whether chronic GH deficiency alters the normal developmental pattern and sexually dimorphic expression of hepatic GH receptors. Adult female Dw rats had lower levels of 125I-labelled hGH binding (reflecting predominantly lactogenic receptors) than their normal counterparts whereas there was no difference between adult Dw and normal males; binding capacity increased from 25 days of age, becoming sexually dimorphic from 40 days to adulthood in both strains (% specific binding/mg protein: normal males 1.6 +/- 0.3, normal females 13.2 +/- 1.1, Dw males 2.1 +/- 0.4, Dw females 10.0 +/- 0.6). In contrast, hepatic 125I-labelled bovine GH (bGH) binding (somatogenic receptors) was much lower, and similar in both Dw and normal animals. A sex difference in 125I-labelled bGH binding was only seen in adult animals, and was considerably less marked in Dw rats compared with normal animals (normal males 1.3 +/- 0.1, normal females 2.5 +/- 0.2, Dw males 1.9 +/- 0.2, Dw females 2.4 +/- 0.2%/mg protein). Continuous hGH infusion stimulated growth in female Dw rats, and raised somatogenic and lactogenic GH binding (3.2 +/- 0.4 and 19.6 +/- 2.5%/mg protein) compared with sham-infused controls (2.4 +/- 0.2 and 7.9 +/- 0.6%/mg protein).(ABSTRACT TRUNCATED AT 250 WORDS)

Differential effects of central and peripheral administration of growth hormone (GH) and insulin-like growth factor on hypothalamic GH- releasing hormone and somatostatin gene expression in GH-deficient dwarf rats

The roles of GH and insulin-like growth factor-I (IGF-I) in the regulation of hypothalamic GH-releasing hormone (GRH) and somatostatin (SRIH) gene expression were investigated in the GH-deficient dwarf (dw) rat, in which endogenous feedback signals are lacking. Adult male and female dw rats were treated with GH or IGF-I by systemic (sc) administration or intracerebroventricular (icv) infusion, and hypothalamic GRH and SRIH mRNA were determined by Northern blotting and densitometric analysis. Systemic sc injection of rGH (75 micrograms every 12 h for 3 days) decreased GRH mRNA levels in both sexes. However, systemic sc injection of human IGF-I (150 micrograms every 12 h for 3 days) did not affect GRH mRNA levels in either sex despite significant stimulation of body weight gain. The use of a continuous sc infusion, which normalized serum IGF-I levels, and prolongation of the treatment period to 7 days also failed to change GRH mRNA levels. SRIH mRNA was unaffected by systemic administration of either GH or IGF-I. Continuous icv infusion of GH (1 microgram/h for 7 days) decreased GRH mRNA levels in both sexes, but did not alter SRIH mRNA levels. Continuous icv infusion of IGF-I (100 ng/h for 7 days) decreased GRH mRNA in both sexes. In contrast, SRIH mRNA levels were increased in both sexes. IGF-I decreased GRH mRNA levels at icv infusion rates of 100 and 300 ng/h and stimulated SRIH mRNA levels at infusion rates of 30 and 100 ng/h. Food intake was unaffected at these infusion rates. Changes in GRH and SRIH mRNA levels in response to systemic or central GH and IGF-I administration were similar in both sexes, except that the decrease in GRH mRNA levels produced by the icv infusion of IGF-I was greater in female than in male rats. The results provide evidence for a direct inhibitory feedback effect of GH in the central nervous system on the regulation of hypothalamic GRH gene expression that is independent of peripheral IGF-I. IGF-I feedback, in contrast, appears to originate with central nervous system-derived IGF-I and exhibits a stimulatory effect on SRIH mRNA as well as an inhibitory effect on GRH mRNA.

Differential effects of central and peripheral administration of growth hormone (GH) and insulin-like growth factor on hypothalamic GH-releasing hormone and somatostatin gene expression in GH-deficient dwarf rats.

The roles of GH and insulin-like growth factor-I (IGF-I) in the regulation of hypothalamic GH-releasing hormone (GRH) and somatostatin (SRIH) gene expression were investigated in the GH-deficient dwarf (dw) rat, in which endogenous feedback signals are lacking. Adult male and female dw rats were treated with GH or IGF-I by systemic (sc) administration or intracerebroventricular (icv) infusion, and hypothalamic GRH and SRIH mRNA were determined by Northern blotting and densitometric analysis. Systemic sc injection of rGH (75 micrograms every 12 h for 3 days) decreased GRH mRNA levels in both sexes. However, systemic sc injection of human IGF-I (150 micrograms every 12 h for 3 days) did not affect GRH mRNA levels in either sex despite significant stimulation of body weight gain. The use of a continuous sc infusion, which normalized serum IGF-I levels, and prolongation of the treatment period to 7 days also failed to change GRH mRNA levels. SRIH mRNA was unaffected by systemic administration of either GH or IGF-I. Continuous icv infusion of GH (1 microgram/h for 7 days) decreased GRH mRNA levels in both sexes, but did not alter SRIH mRNA levels. Continuous icv infusion of IGF-I (100 ng/h for 7 days) decreased GRH mRNA in both sexes. In contrast, SRIH mRNA levels were increased in both sexes. IGF-I decreased GRH mRNA levels at icv infusion rates of 100 and 300 ng/h and stimulated SRIH mRNA levels at infusion rates of 30 and 100 ng/h. Food intake was unaffected at these infusion rates. Changes in GRH and SRIH mRNA levels in response to systemic or central GH and IGF-I administration were similar in both sexes, except that the decrease in GRH mRNA levels produced by the icv infusion of IGF-I was greater in female than in male rats. The results provide evidence for a direct inhibitory feedback effect of GH in the central nervous system on the regulation of hypothalamic GRH gene expression that is independent of peripheral IGF-I. IGF-I feedback, in contrast, appears to originate with central nervous system-derived IGF-I and exhibits a stimulatory effect on SRIH mRNA as well as an inhibitory effect on GRH mRNA.

Impaired Generation of Cyclic Adenosine 3′,5′- Monophosphate in a Somatomammotroph Cell Line Derived from Dwarf (dw) Rat Anterior Pituitaries

The dwarf (dw) mutation in rats results in 40-50% growth retardation associated with a selective reduction in pituitary somatotroph number, GH content, and GH mRNA levels and a decreased GH secretory response to GH-releasing factor (GRF). Recent studies in freshly dispersed pituitary cells have provided evidence for a defect in adenylate cyclase-linked GRF signal transduction in dw somatotrophs. To further examine this defect in a more specific cell population, we developed a somatomammotroph cell line (DP) derived from anterior pituitaries of male dw rats. A similar cell line from normal rats (Po) was used as control. We studied acute GH (4-h release) and cAMP (30-min intracellular accumulation) responses to GH secretagogues known to interact with the adenylate cyclase system. Basal GH release in both cell lines was 80-130% of the cell content, thus limiting the capacity for further GH responses. GRF (10(-8) M) produced a doubling of cAMP levels in Po and DP cells (P less than 0.01), but inconsistent effects on GH release. (Bu)2cAMP (5 x 10(-3) M) increased GH secretion by 50-100% in both groups (P less than 0.01). Cholera toxin (10(-9) M) increased GH release by 50% in both Po and DP (P less than 0.01), but the cAMP response in DP cells was only half that in Po cells (P less than 0.01). Forskolin (10(-5) M), a direct stimulator of adenylate cyclase, doubled GH release in both groups (P less than 0.01). However, cAMP generation was impaired in DP, with a maximal response to forskolin less than one third that in Po (P less than 0.01). In somatotrophs, cAMP mediates not only GRF-stimulated GH release, but also GH synthesis and mitogenesis. The impairment in maximal cAMP generation in DP cells, while not affecting acute GH release, may underlie the defect in somatotroph cell number and GH content in the dw pituitary gland.

Evidence for a defect in growth hormone-releasing factor signal transduction in the dwarf (dw/dw) rat pituitary.

Dwarf (dw/dw) rats exhibit a 40% reduction in body growth, isolated GH deficiency (less than 5% of normal pituitary content), and a decreased number of pituitary somatotrophs (15-20% of normal). Since GH-releasing factor (GRF) stimulates GH synthesis and secretion and somatotroph proliferation, and its effects are probably mediated by cAMP, we have assessed GH secretion and cAMP production in dw rat pituitaries in response to various GH secretagogues. Dispersed pituitary cells from dw rats were less sensitive (2.5-fold) to stimulation of GH secretion by GRF and showed a 25% reduction in the maximal GH response even after normalization of their reduced GH content. Intracellular cAMP was elevated 63-fold over basal levels in normal cells after 4 h in response to maximal GRF stimulation, but only 1.9-fold in dw cells, and even larger differences between the groups were found at earlier time points. The GH responses of dw cells to exogenous cAMP, however, were indistinguishable from normal. Forskolin, a direct stimulator of adenylate cyclase, elicited comparable maximal GH and cAMP responses, but an increased ED50, in dw cells. Activation of GS alpha by cholera toxin showed an increased ED50 and reduced GH and cAMP responses in dw cells, and marked decreases in these responses were observed in response to prostaglandin E1. Phorbol ester stimulation resulted in a reduced maximal GH response in dw cells without a change in sensitivity. These results provide evidence for a defect in the GRF signal transduction pathway associated with a decreased ability of GS alpha to stimulate adenylate cyclase in dw rat somatotrophs that may be causally linked to their GH deficiency.

Growth hormone (GH) secretion in the dwarf rat: release, clearance and responsiveness to GH-releasing factor and somatostatin

The new mutant GH-deficient dwarf (Dw) rat was used to study the effects of GH-releasing factor (GRF) or somatostatin (SRIF) on GH release. In anaesthetized adult Dw female rats, i.v. injections of GRF (0.031-2.0 micrograms) elicited a dose-dependent release of GH. Although the peak plasma GH responses to maximal GRF doses were much lower in adult Dw rats compared with normal rats of this strain (AS), the responses largely reflected their relative pituitary GH contents (140 +/- 17 micrograms vs 2.9 +/- 0.4 micrograms, AS vs Dw (means +/- S.E.M.), P less than 0.001). Except at 20 days of age, normal AS rats were more sensitive to GRF than Dw rats despite their larger body weight. Peak GH responses to injection of 31.25 ng GRF increased nine-fold in normal rats between 20 and 40 days, whereas the GH responses to this GRF dose diminished in Dw rats over this age range, and their pituitary GH content was only 2-5% of that of age-matched AS rats. Treatment with human GH (200 micrograms/day for 7 days) stimulated growth in 40-day-old Dw rats and slightly increased the GH response to a low dose of GRF. Basal GH levels in adult Dw animals were sevenfold lower than in AS rats (2.4 +/- 0.3 vs 17.6 +/- 3.3 micrograms/l P less than 0.001) and were further suppressed by i.v. infusion of SRIF (25 micrograms/h). As in normal rats, a rebound GH secretion occurred in Dw rats after stopping SRIF, which was blocked by injection of anti-GRF serum.(ABSTRACT TRUNCATED AT 250 WORDS)