Enhanced dystrophic progression in mdx mice by exercise and beneficial effects of taurine and insulin-like growth factor-1

A preclinical screening for prompt-to-use drugs that are safer than steroids and beneficial in Duchenne muscular dystrophy was performed. Compounds able to reduce calcium-induced degeneration (taurine or creatine 10% in chow) or to stimulate regeneration [insulin-like growth factor-1 (IGF-1); 50 or 500 microg/kg s.c.] were administered for 4 to 8 weeks to mdx mice undergoing chronic exercise on a treadmill, a protocol to worsen dystrophy progression. alpha-Methyl-prednisolone (PDN; 1 mg/kg) was used as positive control. The effects were evaluated in vivo on forelimb strength and in vitro electrophysiologically on the macroscopic chloride conductance (gCl), an index of degeneration-regeneration events in mdx muscles, and on the mechanical threshold, a calcium-sensitive index of excitation-contraction coupling. The exercise produced a significant weakness and an impairment of gCl, by further decreasing the already low value of degenerating diaphragm (DIA) and fully hampering the increase of gCl typical of regenerating extensor digitorum longus (EDL) mdx muscle. The already negative voltage threshold for contraction of mdx EDL was also slightly worsened. Taurine > creatine > IGF-1 counteracted the exercise-induced weakness. The amelioration of gCl was drug- and muscle-specific: taurine was effective in EDL, but not in DIA muscle; IGF-1 and PDN were fully restorative in both muscles, whereas creatine was ineffective. An acute effect of IGF-1 on gCl was observed in vitro in untreated, but not in IGF-1-treated exercised mdx muscles. Taurine > PDN > IGF-1, but not creatine, significantly ameliorated the negative threshold voltage values of the EDL fibers. The results predict a potential benefit of taurine and IGF-1 for treating human dystrophy.     

Intravenous insulin-like growth factor-I receptor antisense treatment reduces angiotensin receptor expression and function in spontaneously hypertensive rats

The present study investigated the effects of a functional deficit in insulin-like growth factor-I signaling via chronic intravenous administration of insulin-like growth factor-I (IGF-I) receptor antisense in the conscious spontaneously hypertensive rat cardiovascular system. Insulin-like growth factor-I receptor (IGF-IR) antisense, but not full mismatch treatment, decreased IGF-IR expression in both conductance and resistance blood vessels. Aortic IGF-IR density was reduced by 67.4 +/- 6.0% in antisense-treated spontaneously hypertensive rat (SHR) compared with untreated animals, whereas mismatch treatment had no effect (analysis of variance, n = 3, P < 0.01). Aortic and tail artery angiotensin II type 1 receptor expression was significantly reduced by IGF-IR antisense treatment, whereas angiotensin II type 2 receptor expression was unaffected by administration of antisense and mismatch oligonucleotides. IGF-I receptor antisense treatment caused a significant decrease in pressor responses to angiotensin II in comparison with full-length mismatch treatment (E(max) was reduced to 65 +/- 7 mm Hg compared with 99 +/- 6 mm Hg, p < 0.05). Likewise, a reduction in pressor responses to noradrenaline was observed in hypertensive rats treated with IGF-IR antisense compared with full mismatch-treated rats (E(max) was reduced to 60 +/- 6 mm Hg compared with 108 +/- 5 mm Hg, p < 0.01). There was no clear antisense effect on resting blood pressure and no effect at on aortic medial thickness. These results suggest that although the proliferative and vasodilator effects of IGF-I are impaired in SHR, the effects on angiotensin receptor expression remain profound.     

Influence of circulating insulin-like growth factor-I compared with that of intrarenal insulin-like growth factor-I on proximal nephron receptor density in rats.

1. We examined the effects of dietary protein manipulations in partially nephrectomized (one and one-third nephrectomy) and normal rats to gain perspective on the relative importance of circulating versus intrarenal (collecting tubule) insulin-like growth factor-I in the control of proximal nephron receptor density. In addition, we studied the factors that influence liver insulin-like growth factor-I secretion in partially nephrectomized rats. 2. Dietary protein restriction (6% versus 40%) lowered circulating levels of insulin and insulin-like growth factor-I in both normal and partially nephrectomized rats up to 3 weeks after institution of the diets; however, growth hormone levels were little changed. Reduced renal mass stimulated intrarenal production of insulin-like growth factor-I regardless of the diet. 3. Scatchard analysis revealed that the density of insulin-like growth factor-I receptors on glomerular and proximal tubule basolateral membranes increased when circulating levels of insulin-like growth factor-I were diminished, despite raised levels of intrarenal insulin-like growth factor-I, in partially nephrectomized rats. 4. Circulating insulin-like growth factor-I, rather than the tissue level, plays the dominant role in the control of proximal nephron receptor density under physiological conditions. Insulin, but not growth hormone, may play a role in liver insulin-like growth factor-I secretion in partially nephrectomized rats during dietary manipulations.     

Recombinant human insulin-like growth factor-I accelerates recovery and reduces catabolism in rats with ischemic acute renal failure.

This study evaluated whether recombinant human insulin-like growth factor-I (rhIGF-I) enhances recovery of renal function and reduces catabolism in rats with ischemic acute renal failure (ARF). ARF and sham rats received subcutaneous injections of either rhIGF-I or vehicle three times daily starting 5 h after surgery. Serum creatinine and urea, which initially rose similarly in the ARF+vehicle and ARF+rhIGF-I rats, increased more slowly after commencing the rhIGF-I injections. 72 h after surgery, the ARF+rhIGF-I rats, in comparison with ARF+vehicle animals, showed significantly greater renal plasma flow and filtration fraction, a fivefold higher glomerular filtration rate, greater renal cortical IGF-I levels, increased proliferating cell nuclear antigen expression in proximal tubule nuclei and enhanced DNA synthesis in the renal cortex, corticomedullary junction, glomeruli, and tubules as demonstrated by [3H]thymidine incorporation and in corticomedullary junction tubules as determined by autoradiography. Estimated total nitrogen output (ETNO) was greater in ARF+vehicle than in ARF+rhIGF-I or sham rats throughout the study. ETNO in ARF+rhIGF-I rats returned to sham values by the second day after surgery. 72 h after surgery, protein degradation was increased and protein synthesis reduced in the epitrochlearis muscle of ARF+vehicle as compared with ARF+rhIGF-I or sham+vehicle rats. Thus, treatment with rhIGF-I starting 5 h after inducing ischemic ARF in rats increases recovery of renal function, enhances formation of new renal tubular cells, lowers protein degradation, and increases protein synthesis in skeletal muscle and reduces net catabolism.     

A novel, potent, and selective insulin-like growth factor-I receptor kinase inhibitor blocks insulin-like growth factor-I receptor signaling in vitro and inhibits insulin-like growth factor-I receptor dependent tumor growth in vivo

Insulin-like growth factor-I receptor (IGF-IR) and its ligands, IGF-I and IGF-II, are up-regulated in a variety of human cancers. In tumors, such as colorectal, non-small cell lung, ovarian, and pediatric cancers, which may drive their own growth and survival through autocrine IGF-II expression, the role of IGF-IR is especially critical. Here, we present a novel small-molecule IGF-IR kinase inhibitor, cis-3-[3-(4-methyl-piperazin-l-yl)-cyclobutyl]-1-(2-phenyl-quinolin-7-yl)-imidazo[1,5-a]pyrazin-8-ylamine (PQIP), which displayed a cellular IC(50) of 19 nmol/L for inhibition of ligand-dependent autophosphorylation of human IGF-IR with 14-fold cellular selectivity relative to the human insulin receptor. PQIP showed minimal activity against a panel of 32 other protein kinases. It also abolished the ligand-induced activation of downstream phosphorylated AKT and phosphorylated extracellular signal-regulated kinase 1/2 in both IGF-IR transfectant cells and a GEO human colorectal cancer cell line. Analysis of GEO cells revealed a significant level of both phosphorylated IGF-IR and IGF-II expression. Furthermore, inactivation of IGF-II in conditioned GEO culture medium by a neutralizing antibody diminished IGF-IR activation, indicating the presence of a functional IGF-II/IGF-IR autocrine loop in GEO cells. Once daily oral dosing of PQIP induced robust antitumor efficacy in GEO xenografts. The antitumor efficacy correlated with the degree and duration of inhibition of tumor IGF-IR phosphorylation in vivo by this compound. Moreover, when mice were treated for 3 days with a dose of PQIP that maximally inhibited tumor growth, only minor changes in blood glucose were observed. Thus, PQIP represents a potent and selective IGF-IR kinase inhibitor that is especially efficacious in an IGF-II-driven human tumor model.     

鞘脂激活蛋白原在mdx小鼠发病早期心肌的表达

目的 检测鞘脂激活蛋白原(prosaposin)mRNA和蛋白在mdx小鼠发病早期心肌内的表达情况.方法 取3～4周mdx鼠和C57BL/6J对照鼠心肌组织,4%多聚甲醛固定,石蜡切片,HE染色检测形态学改变,免疫组织化学染色检测prosaposin蛋白表达.新鲜取材做冰冻切片,原位杂交检测prosaposin mRNA表达情况并进行统计学分析.结果 mdx鼠心肌细胞在形态上无明显改变,原位杂交结果显示prosaposin mRNA在C57BL/6J对照鼠心肌主要以Ps+0形式存在,且在mdx鼠心肌组织表达明显减少(P<0.01),免疫组织化学结果亦提示prosaposin在mdx鼠心肌组织表达量减少.结论 在mdx小鼠发病早期,pmsaposin mRNA和蛋白在心肌表达量减少.     

Antisense-mediated reduction in insulin-like growth factor-I receptor expression suppresses the malignant phenotype of a human alveolar rhabdomyosarcoma.

The expression of the insulin-like growth factors (IGFs) and their receptors has been linked to cellular proliferation and tumorigenicity in a number of model systems. Since rhabdomyosarcoma cells express IGF-I receptors, an autocrine or paracrine loop involving this receptor and its ligands could be responsible in part for the growth characteristics of this tumor. To assess directly the role of the IGF-I receptor in rhabdomyosarcoma cell growth and tumorigenicity, a human alveolar rhabdomyosarcoma cell line with high IGF-I receptor expression was transfected with an amplifiable IGF-I receptor antisense expression vector. Four unique, transfected clones were analyzed and found to have reduced IGF-I receptor expression relative to the parental line. Integration of the antisense sequence was demonstrated by Southern blot analysis, and expression of antisense message in these clones was shown by S1 nuclease protection assay. Reduced IGF-I receptor surface expression in the transfectants was shown by decreased immunofluorescence with an IGF-I receptor monoclonal antibody and by decreased IGF-I binding as measured by Scatchard analysis. These clones had markedly reduced growth rates in vitro, impaired colony formation in soft agar, and failed to form tumors in immunodeficient mice when compared with vector-transfected clones. These results demonstrate that reduction of IGF-I receptor expression can inhibit both the in vitro and in vivo growth of a human rhabdomyosarcoma cell line and suggest a role for the IGF-I receptor in mediating neoplastic growth in this mesenchymally derived tumor.     

Increased expression of insulin/insulin-like growth factor-I hybrid receptors in skeletal muscle of noninsulin-dependent diabetes mellitus subjects.

Insulin receptors (IR) and IGF-I receptors (IGF-IR) have been shown to form hybrid receptors in tissues coexpressing both molecules. To date there is no information about the distribution of hybrids in tissues of normal or diabetic subjects. We developed a microwell-based immunoassay to quantitate hybrids in small human tissues samples. Microwells were coated with MA-20 anti-IR antibody or alpha-IGF-IR-PA antibody directed against the IGF-IR alpha-subunit, and incubated with skeletal muscle extracts of patients with noninsulin-dependent diabetes mellitus (NIDDM) and normal controls. Immobilized receptors were incubated with 125I-insulin or 125I-IGF-I in the presence or absence of the two unlabeled ligands. Hybrids were quantified as the fraction of 125I-IGF-I binding immunoadsorbed with MA-20 and expressed as percentage of total IGF-IR (type I+hybrids) immobilized with alpha-IGF-IR-PA. The immunoassay was validated using Western blotting analysis. Relative abundance of hybrids detected in NIDDM patients was higher than in controls. The percentage of hybrids was negatively correlated with IR number and in vivo insulin sensitivity measured by an insulin tolerance test, whereas the percentage was positively correlated with insulinemia. Insulin binding affinity was lower in NIDDM patients than in controls, and was correlated with the percentage of hybrids. Maximal IGF-I binding was significantly higher in muscle from NIDDM patients compared to controls and was positively correlated with the percentage of hybrid receptors whereas IGF-I binding affinity did not differ between the two groups. These results raise the possibility that alterations in expression of hybrid receptors may contribute to decreased insulin sensitivity, and to increased sensitivity to IGF-I. Because IGF-I has been proposed as a hypoglycemic agent in NIDDM, these results are relevant to the development of new approaches to the treatment of insulin resistance of NIDDM.     

Stabilizing insulin-like growth factor-I in poly(D,L-lactide-co-glycolide) microspheres.

This study aimed at developing a controlled drug delivery system for recombinant human insulin-like growth factor-I (IGF-I) for localized delivery in bone healing. IGF-I was microencapsulated into an end-group uncapped 14 kDa poly(D,L-lactide-co-glycolide) 50:50 (PLGA 50:50) by solvent extraction from a W(1)/O/W(2) dispersion. Prior to encapsulation, IGF-I was exposed to ultrasonication in a water/dichloromethane dispersion, and its stability tested in the presence and absence of various excipients in the W(1) phase. HPLC and RIA were used for the assessment of IGF-I stability. Microencapsulated IGF-I was tested again for its structural intactness and also for in vitro release from various formulations containing appropriate co-encapsulated excipients. A specific fat cell assay was used to determine the biological activity of released IGF-I. Moderate ultrasonic treatment of aqueous IGF-I/dichloromethane mixtures caused approx. 50% IGF-I degradation. However, IGF-I was fully protected when bovine serum albumin, succinylated gelatin or poly(ethyleneglycol) were added to the aqueous IGF-I. Co-encapsulation of these excipients protected efficiently the protein upon microencapsulation. IGF-I release from microsphere formulations was sustained for up to 13 days featuring a moderately pulsatile pattern, depending on the microsphere composition. Typically, the amounts of IGF-I released within the first 24 h (burst) and during the second release pulse were in the order of 20 and 40%, respectively, of the total dose. The biological activity of released IGF-I was confirmed at selected time-points by the fat cell assay. In conclusion, the developed microspheres proved to be suitable to release biologically intact IGF-I over up to 13 days, a time-period considered to be relevant to promote bone fracture healing.     

Interleukin 6 causes growth impairment in transgenic mice through a decrease in insulin-like growth factor-I. A model for stunted growth in children with chronic inflammation.

Stunted growth is a major complication of chronic inflammation and recurrent infections in children. Systemic juvenile rheumatoid arthritis is a chronic inflammatory disorder characterized by markedly elevated circulating levels of IL-6 and stunted growth. In this study we found that NSE/hIL-6 transgenic mouse lines expressing high levels of circulating IL-6 since early after birth presented a reduced growth rate that led to mice 50-70% the size of nontransgenic littermates. Administration of a monoclonal antibody to the murine IL-6 receptor partially reverted the growth defect. In NSE/hIL-6 transgenic mice, circulating IGF-I levels were significantly lower than those of nontransgenic littermates; on the contrary, the distribution of growth hormone pituitary cells, as well as circulating growth hormone levels, were normal. Treatment of nontransgenic mice of the same strain with IL-6 resulted in a significant decrease in IGF-I levels. Moreover, in patients with systemic juvenile rheumatoid arthritis, circulating IL-6 levels were negatively correlated with IGF-I levels. Our findings suggest that IL-6-mediated decrease in IGF-I production represents a major mechanism by which chronic inflammation affects growth.     

Adeno-associated virus vector-mediated minidystrophin gene therapy improves dystrophic muscle contractile function in mdx mice

Duchenne muscular dystrophy (DMD) is the most common disabling and lethal genetic muscle disorder, afflicting 1 of every 3500 males. Patients with DMD experience progressive muscle degeneration and weakness and succumb to respiratory or cardiac failure by their early twenties. No treatment is currently available for DMD. Mutations in the dystrophin gene result in lack of a functional dystrophin protein in striated muscle, which induces instability in the muscle cell membrane leading to persistent muscle injury after contraction. We have previously created novel minidystrophin genes and demonstrated that adeno-associated virus (AAV)-mediated intramuscular delivery of the minigenes effectively ameliorated mdx dystrophic histopathology and led to normal cell membrane integrity for more than 1 year. In this paper, we investigated whether AAV-minidystrophin could also improve mdx muscle contractile function. Two-month-old adult male mdx mice, with established muscular dystrophy, were given a single-dose injection of an AAV-minidystrophin vector in the tibialis anterior (TA) muscle of one leg, with the untreated contralateral leg used as a control. The treated TA muscle showed both (1) a significant increase in isometric force generation and (2) a significant increase in resistance to lengthening activation-induced muscle force decrements. We conclude that AAV-minidystrophin gene treatment is effective in improving mdx muscle contractile function.     

脉冲电磁场对去势大鼠骨组织胰岛素样生长因子-I表达的影响

目的观察脉冲电磁场(EM)对去势大鼠骨组织结构和胰岛素样生长因子-I(IGF-I)表达的影响,探讨EM在预防骨质疏松中的作用。方法3月龄SD大鼠随机抽签法分为正常对照组(Sham),去势组(OVX),雌激素治疗组(E2)及脉冲电磁场治疗组(EM),2个月后测定骨形态学参数,通过斑点杂交技术测定骨组织IGF-ImRNA的表达,通过免疫组织化学方法进行细胞定位。结果骨形态学测定显示E2组较OVX组骨小梁数目与厚度明显增加,髓腔变窄;EM组骨小梁数目与厚度接近或超过E2组。OVX组骨组织IGF-ImRNA的表达水平(27±6)OD较Sham组(78±6)OD显著下降P<0.01(t=1.823),EM组(69±7)OD与E2组(75±5)OD无显著性差异P>0.05(t=3.206),两组均接近Sham组水平,与OVX组比较显著性增高P<0.01(t=2.138,t=1.752)。结论EM显著抑制了雌激素缺乏导致的骨结构的衰退,改善骨结构特性,在一定程度上其作用优于雌激素替补治疗。EM可能是一种颇具潜力的预防OP的方法,其作用可能是通过IGF-I介导的。     

Myostatin propeptide gene delivery by adeno-associated virus serotype 8 vectors enhances muscle growth and ameliorates dystrophic phenotypes in mdx mice

Myostatin has been extensively documented as a negative regulator of muscle growth. Myostatin inhibition is therefore considered an attractive strategy for the treatment of muscle-wasting diseases such as muscular dystrophies. To investigate whether systemic gene delivery of myostatin propeptide (MRPO), a natural inhibitor of myostatin, could enhance body-wide skeletal muscle growth, we used adeno-associated virus serotype 8 (AAV8) vectors to deliver the MRPO gene into either normal mice or mdx mice, a murine model of Duchenne muscular dystrophy (DMD). In normal mice, a significant increase in skeletal muscle mass was observed after either an intraperitoneal injection of AAV-MPRO into neonates, or an intravenous injection of AAV-MPRO76AFc (a modified MPRO fused with IgG Fc) into adults. Enhanced muscle growth occurred because of myofiber hypertrophy, not hyperplasia. In mdx mice, a significant increase in skeletal muscle mass was also observed after AAV-MPRO76AFc injection. The treated mdx mice showed larger and more uniform myofibers, fewer infiltrating mononuclear cells, less fibrosis, and lower serum creatine kinase levels. In addition, a grip force test and an in vitro tetanic contractile force test showed improved muscle strength. A treadmill test, however, showed reduced endurance of the treated mdx mice compared with their untreated counterparts. Importantly, no cardiac hypertrophy was observed in either normal or mdx mice after myostatin inhibition by gene delivery. These results clearly demonstrate the efficacy of AAV8-mediated myostatin propeptide gene delivery in a rodent model of DMD, and warrant further investigation in large animal models and eventually in human patients.     

Myonuclear apoptosis in dystrophic mdx muscle occurs by perforin-mediated cytotoxicity.

Myonuclear apoptosis is an early event in the pathology of dystrophin-deficient muscular dystrophy in the mdx mouse. However, events that initiate apoptosis in muscular dystrophy are unknown, and whether elimination of apoptosis can ameliorate subsequent muscle wasting remains a major question. We have tested the hypothesis that cytotoxic T-lymphocytes initiate myonuclear apoptosis in dystrophic muscle, and examined whether perforin-mediated cytotoxicity plays a role in the pathophysiology of muscular dystrophy. Mdx mice showed muscle invasion by cytotoxic T cells and helper T cells at the onset of histologically detectable muscle fiber pathology. At this time, perforin-expressing cells were also present at elevated concentration. Mdx mice depleted of CD8(+) cells showed a significant reduction of apoptotic myonuclei concentration and a reduction in necrosis, judged by macrophage invasion of muscle fibers. Double-mutant mice, deficient in dystrophin and perforin, showed nearly complete absence of myonuclear apoptosis, and a significant reduction in the concentration of macrophages in the connective tissue surrounding muscle fibers. However, muscle fiber invasion by macrophages was not reduced significantly in double mutant mice. Thus, cytotoxic T-lymphocytes contribute significantly to apoptosis and necrosis in mdx dystrophy, and perforin-mediated killing is primarily responsible for myonuclear apoptosis.     

The measurement of insulin-like growth factor-I (IGF-I) concentration in random urine samples.

The aim of the present study was to assess a suitable expression of the urinary concentration of a protein/ peptide hormone such as insulin-like growth factor-I (IGF-I), measured in the urine of healthy individuals when the specimen collection is executed randomly. One hundred and twenty male subjects were divided by age into four groups, namely healthy sedentary young (SYA) and older (SOA) adults, older (OC) and young (YC) children. In a single urine specimen, randomly collected during the morning from each individual, total urinary IGF-I was measured by immunoradiometric method, and urinary creatinine (uCr) and total proteins (utPr) were measured by capillary electrophoresis and spectrophotometric methods, respectively. The urinary IGF-I concentrations were not significantly different in all groups investigated and they were (mean +/- SD): 82.7 +/- 82.8 ng/l, 103.5 +/- 83.3 ng/l, 80.4 +/- 64.4 ng/l in OC, SYA and SOA, respectively; only in the YC group there was a tendency to higher values (125.2 +/- 93.2 ng/l) compared with the other groups. utPr ranged from 26 to 40 mg/l and did not demonstrate significant differences between groups. The urinary IGF-I correlated with uCr and utPr, and statistical significance was observed in all measurements. The measurement of urinary IGF-I in random urine and its ratio to utPr is an innovative, useful way of investigation of urinary protein/peptide hormones.     

Protease-resistant form of insulin-like growth factor-binding protein 5 is an inhibitor of insulin-like growth factor-I actions on porcine smooth muscle cells in culture.

IGFs are pleiotrophic mitogens for porcine smooth muscle cells (pSMC) in culture. The effects of IGFs on cells are modulated by various insulin-like growth factor-binding proteins (IGFBP). IGFBP-5 is synthesized by pSMC and binds to the extracellular matrix. However, IGFBP-5 is also secreted into conditioned medium of cultured cells and is cleaved into fragments by a concomitantly produced protease. These fragments have reduced affinity for the IGFs and cleavage makes it difficult to assess the role of intact IGFBP-5. To study the consequence of accumulation of intact IGFBP-5 in medium, we determined the cleavage site in IGFBP-5 and prepared a protease resistant mutant. Amino acid sequencing of purified IGFBP-5 fragments suggested Arg138-Arg139 as the primary cleavage site. Arg138-Arg139-->Asn138-Asn139 mutations were introduced to create protease-resistant IGFBP-5, which has the same affinity for IGF-I as the native protein. This mutant IGFBP-5 remained intact even after 24 h of incubation and it inhibited several IGF-I actions when added to pSMC culture medium. The mutant IGFBP-5 (500 ng/ml) decreased IGF-I stimulated cellular DNA synthesis by 84%, protein synthesis by 77%, and it inhibited IGF-I stimulated migration of pSMC by 77%. It also inhibited IGF-I stimulation of IRS-1 phosphorylation. In contrast, the same amount of native IGFBP-5 did not inhibit IGF-I actions. The significance of inhibitory effects of the protease resistant IGFBP-5 was further demonstrated in pSMC transfected with mutant or native IGFBP-5 cDNAs. The mutant IGFBP-5 accumulated in culture medium of transfected cells, while native IGFBP-5 was degraded into fragments, PSMC overexpressing the mutant IGFBP-5 also responded poorly to IGF-I compared with mock transfected cells. IGF-I (5 ng/ml) increased [35S]methionine incorporation into control cells by 36% above the basal level, but it did not significantly change (4%) in pSMC cultures that were producing the mutant IGFBP-5. In conclusion, the accumulation of protease-resistant IGFBP-5 in the medium was inhibitory to IGF-I actions on pSMC. This suggests that proteolysis can prevent IGFBP-5 from acting as an inhibitor of IGF-I-stimulated effects and that it serves as an important mechanism for regulating cellular responsiveness to IGF-I.     

Decreased serum insulin-like growth factor-I associated with growth failure in newborn lambs with experimental cyanotic heart disease.

To determine whether chronic hypoxemia results in alterations in endocrine function that may contribute to growth failure, we measured growth hormone (GH), somatomedins (insulin-like growth factors I and II, IGF-I and IGF-2), hepatic growth hormone receptors, and circulating IGF-binding proteins IGFBP-3 and IGFBP-2 in 12 newborn lambs with surgically created pulmonic stenosis and atrial septal defect, and in 10 controls. During chronic hypoxemia (oxygen saturation of 60-74% for 2 wk), weight gain was 60% of control (hypoxemic, 135 +/- 20 vs. control, 216 +/- 26 g/d, P less than 0.02). IGF-I was decreased by 43% (hypoxemic 253.6 +/- 29.3 SE vs. control 448.0 +/- 75.5 ng/ml, P = 0.01), whereas GH was unchanged (19.9 +/- 5.1 vs. 11.9 +/- 3.0 ng/ml, NS). The increase in IGF-1 was associated with a decrease in IGFBP-3 (hypoxemic, 5.09 +/- 1.25 vs. control, 11.2 +/- 1.08 arbitrary absorbency units per mm (Au.mm), P less than 0.01), and increase in IGFBP-2 (0.47 +/- 0.03 vs. 0.19 +/- 0.13 Au.mm, P less than 0.05), but no significant downregulation of hepatic GH receptors (hypoxemic, 106.1 +/- 20.1 vs. control, 147.3 +/- 25.9 fmol/mg, NS). Thus, chronic hypoxemia in the newborn is associated with a decrease in IGF-I and IGFBP-3 in the face of normal GH. This suggests peripheral GH unresponsiveness, similar to protein-calorie malnutrition or GH receptor deficiency dwarfism, but mediated at a level distal to the hepatic GH receptor.     

Interactions between insulin-like growth factor-I (IGF-I) and the renin-angiotensin system in follicular growth and ovulation.

The interactions between insulin-like growth factor-I (IGF-I) and the renin-angiotensin system (RAS) in follicular growth and ovulation were studied with the use of an isolated perfused rabbit ovary preparation. Ovulation failed to occur in either control ovaries or the experimental ovaries perfused with IGF-I in a concentration of 1, 10, or 100 ng/ml in the absence of gonadotropin. Exposure to IGF-I stimulated the secretion rate of angiotensin II-like immunoreactivity (Ang II-IR) in perfused rabbit ovaries in a dose-dependent manner. The percent increase in follicle diameter in ovaries perfused with IGF-I for 12 h was significantly correlated with the secretion rate of Ang II-IR at 12 h after exposure to IGF-I. The addition of IGFBP-3 to the perfusate did not induce ovulation in the absence of gonadotropin, but exposure to IGFBP-3 inhibited hCG-induced ovulation in a dose-dependent manner. In addition, IGFBP-3 significantly reduced the ovarian secretion rate of Ang II-IR and prostaglandins stimulated by hCG administration. Intrafollicular plasminogen activator (PA) activity significantly increased within 4 h after exposure to 100 ng/ml of IGF-I, compared with that in control ovaries perfused with medium alone. The concomitant addition of IGFBP-3 to the perfusate significantly reduced the IGF-I-stimulated PA activity in the preovulatory follicles at 4, 6, and 8 h after exposure to IGF-I. However, IGFBP-3 alone affected neither the ovarian secretion rate of Ang II-IR nor intrafollicular PA activity. Exposure to streptokinase, an exogenous PA, in vitro stimulated both follicular growth and the intrafollicular Ang II-IR content. In conclusion, IGF-I enhances both ovarian Ang II production and follicular development by stimulating intrafollicular PA activity.