Insulin-like growth factor-II is produced by, signals to and is an important survival factor for the mature podocyte in man and mouse.

Podocytes are crucial for preventing the passage of albumin into the urine and, when lost, are associated with the development of albuminuria, renal failure and cardiovascular disease. Podocytes have limited capacity to regenerate, therefore pro-survival mechanisms are critically important. Insulin-like growth factor-II (IGF-II) is a potent survival and growth factor; however, its major function is thought to be in prenatal development, when circulating levels are high. IGF-II has only previously been reported to continue to be expressed in discrete regions of the brain into adulthood in rodents, with systemic levels being undetectable. Using conditionally immortalized human and ex vivo adult mouse cells of the glomerulus, we demonstrated the podocyte to be the major glomerular source and target of IGF-II; it signals to this cell via the IGF-I receptor via the PI3 kinase and MAPK pathways. Functionally, a reduction in IGF signalling causes podocyte cell death in vitro and glomerular disease in vivo in an aged IGF-II transgenic mouse that produces approximately 60% of IGF-II due to a lack of the P2 promoter of this gene. Collectively, this work reveals the fundamental importance of IGF-II in the mature podocyte for glomerular health across mammalian species.

Alteration of cardiovascular and neuronal function in M1 knockout mice.

We used gene targeting to generate mice lacking the M1 muscarinic acetylcholine receptor. These mice exhibit a decreased susceptibility to pilocarpine-induced seizures, loss of regulation of M-current potassium channel activity and of a specific calcium channel pathway in sympathetic neurons, a loss of the positive chronotropic and inotropic responses to the novel muscarinic agonist McN-A-343, and impaired learning in a hippocampal-dependent test of spatial memory.

Beta-adrenergic and muscarinic receptor mRNA accumulation in the sinoatrial node area of adult and senescent rat hearts.

The sinoatrial (SA) node is the cardiac pacemaker and changes in its adrenergic-muscarinic phenotype have been postulated as a determinant of age-associated modifications in heart rate variability. To address this question, right atria were microdissected, the SA node area was identified by acetylcholinesterase staining, and, using a RT-PCR method, the accumulation of mRNA molecules encoding beta1- and beta2-adrenergic (beta1- and beta2-AR) and muscarinic (M2-R) receptor was quantified to define the proportion between beta-AR and M2-R mRNAs within the sinoatrial area of adult (3 months) and senescent (24 months) individual rat hearts. In adult hearts, the highest M2-R/beta-AR mRNA ratio was observed within the sinoatrial area compared with adjacent atrial myocardium, while in the senescent hearts, no difference was observed between sinoatrial and adjacent areas. This change was specific of the sinoatrial area since adult and senescent whole atrial or ventricular myocardium did not differ in their M2-R/beta-AR mRNA ratio, and was associated with a fragmentation of acetylcholinesterase staining of the senescent SA node. Quantitative changes in the expression of genes encoding proteins involved in heart rate regulation specifically affect the sinoatrial area of the senescent heart.

beta-Adrenergic and muscarinic receptor expression are regulated in opposite ways during senescence in rat left ventricle.

The well-known attenuated sensitivity of senescent heart to isoproterenol is accompanied by a decreased beta1-adrenergic receptors (beta1-AR) density, a down regulation process which may involve several molecular modifications and whose understanding is incomplete. Data concerning the M2-R muscarinic receptors (M2-R) are more contradictory. Both the absolute and relative concentrations of beta1-AR and M2-R as well as the coupling protein G alpha s and G alpha(i2) mRNAs were determined by slot-blot analysis in the left ventricles (LVs) of 6-7 week and 22-month-old male Wistar rats. In addition, the beta-AR and M2-R densities were quantitated by radioactive ligand binding. (1) The M2-R mRNA concentration increases by 92+/-32% in senescent as compared to adult animals; by contrast, the density in M2-R remains unchanged, suggesting that the M2-R expression was not exclusively regulated at a pre-translational level. (2) The beta1-AR mRNA concentration was nearly halved (reduced by 46+/-9.5%) and paralleled the 51+/-5.6% diminution of the beta-AR density which resulted exclusively from the decrease of beta1-AR density without change in the beta2-AR concentration, suggesting a pre-translational regulation of the beta1-AR expression. (3) G alpha(i2) mRNA concentration was unchanged, while G alpha s mRNA concentration was reduced by 26+/-4.6% in senescent compared with adult LVs. To conclude, the different components of the adrenergic and muscarinic systems are differentially regulated during aging.

Heart rate and heart rate variability, a pharmacological target.

Heart rate varies with respiration, blood pressure, emotion, etc., and heart rate variability (HRV) is presently one of the best indices to predict fatal issues in cardiac failure and after myocardial infarction. HRV depends on various reflexes. In addition, parallel studies of HRV and the myocardial adrenergic and muscarinic transduction system in experimental models of cardiac hypertrophy (CH) have suggested that the myocardial phenotype at the sinus-node level may also play a role. A transgenic strain of mice with atrial overexpression of the beta 1-adrenergic receptors was generated with attenuated HRV, which demonstrates that the phenotype itself is a determinant of HRV. HRV is explored by noninvasive techniques, including simple determination of the standard error of the mean, time-domain analysis, and Fourier transformation. We recently developed a time and frequency domain method of analysis, the smoothed pseudo-Wigner-Ville transformation, which allows better exploration of nonstationarity. Nonlinear methods have also been applied due to the extreme complexity of the biological determinants, and have provided evidence of a chaotic attractor in certain conditions. It is proposed that in steady state a very simple process, which is not completely deterministic, could better explain intermit interval regulations than chaotic behavior. In contrast, under extreme circumstances the regulation proceeds using chaotic behavior. Arrhythmias and HRV can be quantitated in 16-month-old unanesthetized spontaneously hypertensive rats (SHR). Ventricular premature beats are more frequent in SHR than in age-matched controls; they disappear after converting enzyme inhibition (CEI) relative to the reduction of both cardiac hypertrophy and ventricular fibrosis. HRV is attenuated in SHR, as it is in compensatory CH in humans. When CH is prevented, HRV returns to normal. CEI is therefore antiarrhythmic. Another pharmacological application of this concept concerns the bradycardic agents that may improve HRV.

Molecular and cellular biology of the senescent hypertrophied and failing heart.

During aging, experimental studies have revealed various cellular changes, principal among which is myocyte hypertrophy, which compensates for the loss of myocytes and is associated with fibrosis. The expression of alpha-myosin heavy chain is replaced by that of the isogene beta-myosin, which leads to decreased myosin adenosine triphosphatase (ATPase) activity. In consequence, contraction is slower and more energetically economical. The Ca(2+)-ATPase of the sarcoplasmic reticulum and Na+/Ca2+ exchange activity are decreased, which probably explains the reduced velocity of relaxation. Membrane receptors are also modified, since the density of both the total beta-adrenergic and muscarinic receptors is decreased. The senescent heart is able to hypertrophy in response to overload and to adapt to the new requirements. Similar alterations are observed both in the senescent heart and in the overloaded heart, in clinical as well as in experimental studies; however, differences do exist, especially in terms of fibrosis and arrhythmias.

Phosphorylated forms of adrenocorticotropin and corticotropin-like intermediary lobe peptide in human tumors.

Many peptides contribute to the heterogeneity of immunoreactive adrenocorticotropin (ACTH) in man. The use of a radioimmunoassay (RIA) specifically directed against the C-terminal end of ACTH allowed us to study precisely the following four peptides: ACTH itself, corticotropin-like intermediary lobe peptide (CLIP) or ACTH (18-39) and their phosphorylated forms on Ser31. We have set up a high-performance liquid chromatography system that separates these four molecules in a single run, to establish their relative distributions in tumors responsible for Cushing's disease or for the ectopic ACTH syndrome, and to evaluate the possible interference of phospho-Ser31 on various RIA or immunoradiometric assay (IRMA) recognition systems for ACTH. In this system, alkaline phosphatase treatment shifted the retention time of the phosphorylated peptides to that of their non-phosphorylated counterparts. In three tumors responsible for the ectopic ACTH syndrome, CLIP peptides were predominant in two and phosphorylated molecules represented between 22% and 50% of immunoreactive materials. In five pituitary tumors responsible for Cushing's disease, ACTH peptides were predominant and the phosphorylated molecules varied between 35% and 75% in four of them. In the same tumor the ratios of phosphorylated to non-phosphorylated CLIP or ACTH were identical. The presence of phospho-Ser31 did not affect the recognition ability of two mid-ACTH and two C-terminal ACTH RIAs, nor of the ACTH IRMA (Allegro, Nichols).

Is the senescent heart overloaded and already failing?

Heart failure mainly occurs during the last decades of life, and it is important to know if the senescent heart is not an already failing heart. During aging, both contraction and relaxation of papillary muscle are impaired. Such an impairment is compensated in vivo and the cardiac output remains normal. In spite of a loss in myocytes, the heart weight/body weight ratio is unchanged, but the myocytes are bigger. Arrhythmias are permanent and are accompanied by a loss of the normal heart rate variability. Changes in specific mRNAs include: a shift in myosin heavy chain (MHC) isogene expression leading to an increased beta MHC content; decreased densities of Ca2+ ATPase of the sarcoplasmic reticulum, beta 1-adrenergic receptor, and muscarinic receptors; and attenuation of the Na+/Ca2+ exchange activity. Most of these changes, but not all, resemble those observed during cardiac overload and are accompanied by an increased duration of both the action potential and the intracellular calcium transient. However, the senescent heart is still able to further modify its phenotype in response to mechanical overload. The senescent heart is a diseased heart, and the origin of the "disease" is multifactorial and includes the general process of senescence, hormonal changes, and the myocardial consequences of senescence of the vessels.

Decreased accumulation of beta 1-adrenergic receptor, G alpha s and total myosin heavy chain messenger RNAs in the left ventricle of senescent rat heart.

The expression of genes coding for the beta 1-adrenergic receptor (beta 1-AR), the alpha subunit of Gs and total myosin heavy chain (MHC) was compared between left ventricles (LV's) from young (6-7 weeks old) and old (22 months old) rats. The mRNA levels were quantitated by Northern or Slot blots analyses using specific DNA probes. Ageing was found to be associated with a reduction in beta 1-AR (77%), G alpha s (33%) and, total MHC (51%) mRNA levels with no concomitant change in 18S RNA and poly(A+) mRNA levels. These results indicate that transcriptional and/or post-transcriptional mechanisms participate in the control of beta-adrenergic receptor density during ageing. As in the senescent LV, beta 1-AR mRNA level is reduced in the hypertrophied LV, whereas the level of G alpha s mRNA is reduced in the senescent but not in the hypertrophied LV. From our data we conclude (1) that a dual mechanism may operate during ageing, mechanical factors indirectly regulating beta 1-AR mRNA level, while changes in G alpha s mRNA level do not depend on hemodynamic load and (2) that the re-expression of beta-MHC mRNA does not compensate for the decreased accumulation of alpha-MHC mRNA which results in a large decrease in the level of total MHC mRNA in the senescent LV.

Expression of insulin-like growth factor binding protein-1 and -2 genes through the perinatal period in the rat.

Insulin-like growth factor binding proteins (IGFBPs) are essential mediators of the bioavailability and biological effects of the IGFs. Liver expression of the rat (r) IGFBP-1 and rIGFBP-2 genes has been characterized between day 16 in utero (16 diu) and 16 days postnatally (+16 dpn). Run-on experiments showed transcriptional activity of the rIGFBP-1 and rIGFBP-2 genes at birth (B) to be 25 and 5 times that at 16 diu, respectively. After B, transcriptional activity of the rIGFBP-1 gene remained high (140% B at +6 dpn), but that of the rIGFBP-2 gene dropped to 70% B by +6 dpn. Northern blot analysis done simultaneously showed rIGFBP-1 messenger RNA (mRNA) levels to increase approximately 50-fold between 16 diu and B, whereas rIGFBP-2 mRNA increased only 5- to 10-fold. rIGFBP-1 mRNA levels decreased after birth, reaching about 20% B by +6 dpn; rIGFBP-2 mRNA, however, remained stable (about 80% B) at least up to +6 dpn. Parallel Western ligand blot and immunoblot analyses of serum rIGFBPs revealed rIGFBP-1 and rIGFBP-2 concentrations to be increased 3- and 2-fold, respectively between 20 diu and B. Maximal expression of rIGFBP-1 was at +1 dpn (220% B), and of rIGFBP-2, at B. Both rIGFBPs then decreased, reaching about 5% B at adulthood. All these data indicate that increased transcriptional activity of the rIGFBP-1 and rIGFBP-2 genes at birth would determine the increased synthesis in the liver and circulating levels of these proteins. In addition, it would seem that post-transcriptional events (reduced half-life of the rIGFBP-1 messenger after birth, translation efficiency of the rIGFBP-2 messenger) modulate transcriptional regulation.

Serum insulin-like growth factors and insulin-like growth factor binding proteins in the human fetus. Relationships with growth in normal subjects and in subjects with intrauterine growth retardation.

IGF-I, IGF-II, and their binding proteins (BP) were studied in sera obtained by direct puncture of umbilical cords in utero between 20 and 37 wk of gestation in 103 normal fetuses and in 16 fetuses with intrauterine growth retardation, as well as in the cord blood of 37 normal newborns of 38- to 42-wk pregnancies. In normal fetuses, IGF-I levels were approximately 50 ng/mL and IGF-II levels approximately 350 ng/mL up to the 33rd wk of pregnancy. Thereafter, both increased to reach values two to three times higher at term. Correlations were found between fetal placental lactogen levels and those of IGF-I and IGF-II, which is consistent with the hypothesis that placental lactogen is involved in the regulation of IGF synthesis in the fetus. With weight (either measured at birth or deduced from echographical data) as index of fetal size, IGF-I levels were significantly (p less than 0.001) higher in fetuses with weights above the mean for gestational age than in fetuses with weights below the mean, whereas IGF-II levels were similar in the two groups. Similarly, IGF-I (but not IGF-II) levels in fetuses with intrauterine growth retardation were significantly lower than those in normal fetuses of the same age (p less than 0.01). These findings suggest that, during the latter months of intrauterine life, IGF-I (but not IGF-II) is involved in the control of fetal size. Total fetal BP concentrations were approximately 1/3 those of adults. The fetal electrophoretic profile obtained by Western-ligand blotting bore a strong resemblance to that of subjects with growth hormone deficiency.(ABSTRACT TRUNCATED AT 250 WORDS)

[Expression regulation of insulin-like growth factor binding proteins (IGFBP). Tissue-specific expression of IGFBP-1]. / Régulation de l'expression des protéines de liaison des "insulin-like growth factors" (IGFBP).

We have investigated the liver-specific expression of IGFBP-1 gene, an Insulin-like Growth Factor binding protein. Northern blot as well as transient transfection experiments, both carried out in differentiated (H4II, C2Rev7) and dedifferentiated (H5, C2) hépatoma cell lines, yielded clear cut data allowing the involvement of HNF1, a liver-specific trans acting factor, in basal IGFBP-1 liver-specific expression.

Molecular forms of serum insulin-like growth factor (IGF)-binding proteins in man: relationships with growth hormone and IGFs and physiological significance.

Insulin-like growth factor-I (IGF-I) and IGF-II are associated in the blood with specific binding proteins (BPs), forming complexes that elute in gel filtration with estimated mol wt around 40 and 150 kD. The latter appears to be under GH control. Five molecular forms of BP (41.5, 38.5, 34, 30, and 24 kD) have been identified by Western blotting using 125I-labeled IGF. All five forms are present in the smaller complexes, but only the 41.5- and 38.5-kD forms are found in the larger complexes. In this study immunoblotting showed that the 41.5- and 38.5-kD forms were recognized by antibodies directed against the GH-dependent BP purified from human plasma, and the 30-kD form was recognized by antibodies directed against the BP purified from amniotic fluid. The 34- and 24-kD forms proved to be immunologically unrelated to the other three. In sera with large quantities of the 41.5- and 38.5-kD forms, an additional band was often observed immediately ahead of the migration front of the 30 kD band. This was recognized by the anti-GH-dependent BP antibody and probably corresponds to a degradation product of the 41.5- and 38.5-kD BPs. Serum 41.5- and 38.5-kD BPs have been found to be elevated in acromegaly, where GH hypersecretion causes increased IGF-I levels, and diminished in cases of genetic or idiopathic GH deficiency and defects of the GH receptor (Laron's syndrome), where both IGF-I and IGF-II are decreased, as well as in Pygmy adults and children who have isolated IGF-I deficiency. In all of these conditions, the proportions of the 34- and 30-kD forms were inversely related to those of the 41.5- and 38.5-forms. Under treatment, the BP profiles tended to return to normal. In cases of GH deficiency caused by a tumor, the BP profiles resembled those of hypopituitary or normal serum, depending on whether IGF levels were diminished or normal. It, therefore, seems that BP synthesis is coordinated with IGF-I synthesis and may not be directly GH dependent. The results of neutral pH gel filtration analysis of hypopituitary (idiopathic and tumoral) and normal sera point to a relationship between the levels of circulating IGFs and those of the 150-kD IGF-BP complex whose binding units are the 41.5- and 38.5-kD BPs. It, therefore, seems that the 150-kD complex controls the bioavailability of IGF-I and IGF-II.(ABSTRACT TRUNCATED AT 400 WORDS)

Heterogeneity of insulin-like growth factor binding proteins and relationships between structure and affinity. 1. Circulating forms in man.

Circulating insulin-like growth factors (IGFs) are bound to specific, high-affinity binding proteins (BPs), and form complexes with relative molecular masses of about 150,000 ('large' complex) and 40,000 ('small' complex). The large complex appears to be under growth-hormone control and its proportions vary with those of the IGFs. Molecular heterogeneity among the binding proteins was revealed by polyacrylamide gel electrophoresis (SDS-PAGE) of serum in which they were cross-linked to 125I-labelled IGF I or II. Out of the six specific bands observed, of 150,000, 120,000, 49,000, 40,000 and 37,000 Mr, the last three appeared in both complexes, whereas the first three were visible only in the large complex. Some or all of the 49,000-37,000-Mr species may constitute the subunits of 150,000-Mr and/or 120,000-Mr IGF-BP complexes. With electrophoresis followed by transfer onto nitrocellulose and incubation with either 125I-labelled IGF I or II (western blot), the different binding proteins were identified per se. There were five molecular forms with Mr of 41,500, 38,500, 34,000, 30,000 and 24,000. In normal serum the 41,500 and 38,500-Mr forms were the major binding proteins. They appeared in both complexes, but were predominant in the large complex where they constitute the elementary binding units. These two proteins therefore bind to IGFs to form both 'monomeric' IGF-BP and 'oligomeric' (IGF-BP)n complexes. The 34,000, 30,000 and 24,000-Mr forms, by contrast, were visible only in the small complex. Different mechanisms appear to regulate the different binding proteins: in acromegalic serum the 41,500 and 38,500-Mr forms were augmented and the 34,000-Mr form diminished, whereas in hypopituitary serum the reverse was true and, in addition, the 30,000-Mr form was augmented. With chromatofocusing, the 34,000, 30,000 and 24,000-Mr forms eluted in three peaks between pH 6.0 and 4.0, whereas the 41,500 and 38,500-Mr forms eluted throughout the gradient, principally at pH 7.5 and 7.0. Competitive binding studies, done on binding proteins separated either by chromatofocusing or by SDS-PAGE and transfer onto nitrocellulose, revealed different affinities for the IGFs among the different molecular forms. The 41,500 and 24,000-Mr binding proteins preferentially bound IGF I and the 38,500, 34,000 and 30,000-Mr proteins preferentially bound IGF II. Our findings demonstrate the molecular heterogeneity of the binding proteins and the existence of a relationship between their structure and their affinities for the IGFs.(ABSTRACT TRUNCATED AT 400 WORDS)

Analysis of serum insulin-like growth factor binding proteins using western blotting: use of the method for titration of the binding proteins and competitive binding studies.

A nitrocellulose gel transfer technique has been adapted to study the insulin-like growth factor (IGF) binding proteins of human serum. Normal and hypopituitary sera were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by electroblotting to nitrocellulose or nylon membrane. Nonidet-P40 (3%) and Tween 20 (0.1%) were required for quenching and to allow detection of the IGF binding proteins by autoradiography after overlay with either 125I-labeled IGF I or IGF II. Several forms of IGF binding protein have been identified with molecular weights of 41,500, 38,500, 34,000, 30,000, and 24,000. Titration and competitive binding studies with IGF were performed on the transferred IGF binding proteins, indicating that binding proteins isolated by this technique can be characterized.

Somatomedin (insulin-like growth factors)-binding proteins. Molecular forms and regulation.

The insulin-like growth factor (IGF)-binding proteins present in the human serum and various biological media have been characterized by several methods: gel filtration, sucrose gradient sedimentation, polyacrylamide gel electrophoresis and chromatofocusing. Several forms have been identified with molecular weights of approximately 42,000, 39,000, 34,000, 30,000 and 24,000 daltons. Results of competitive binding studies suggest that the different forms of binding proteins have different affinities for IGF-I and IGF-II. The influence of various hormones and pathophysiological conditions on the biosynthesis of the binding proteins has been investigated.

Evidence for production by the liver of two IGF binding proteins with similar molecular weights but different affinities for IGF I and IGF II. Their relations with serum and cerebrospinal fluid IGF binding proteins.

Studies of the competitive binding of IGF I and IGF II and 125I-labelled IGF I and IGF II to the specific binding proteins (BPs) produced by the liver in culture suggest that two BPs exist, one with a selective affinity for IGF I and the other with a selective affinity for IGF II. The ratio of the former BP to the latter appeared to be higher in the culture medium of young rat livers and of fetal human livers than that in the culture medium of adult human livers. The two BPs form complexes with their IGFs with the same apparent molecular weight of approximately 40K. With gel filtration of adult human serum both types of BP eluted with the greater than 100K and the 40-70K molecular weight material. The BP with the selective affinity for IGF II predominated in the 40-70K material as well as in the cerebrospinal fluid of adult human subjects.