beta-cell-specific deletion of the Igf1 receptor leads to hyperinsulinemia and glucose intolerance but does not alter beta-cell mass.

Regulation of glucose homeostasis by insulin depends on the maintenance of normal beta-cell mass and function. Insulin-like growth factor 1 (Igf1) has been implicated in islet development and differentiated function, but the factors controlling this process are poorly understood. Pancreatic islets produce Igf1 and Igf2, which bind to specific receptors on beta-cells. Igf1 has been shown to influence beta-cell apoptosis, and both Igf1 and Igf2 increase islet growth; Igf2 does so in a manner additive with fibroblast growth factor 2 (ref. 10). When mice deficient for the Igf1 receptor (Igf1r(+/-)) are bred with mice lacking insulin receptor substrate 2 (Irs2(-/-)), the resulting compound knockout mice show a reduction in mass of beta-cells similar to that observed in pancreas of Igf1r(-/-) mice (ref. 11), suggesting a role for Igf1r in growth of beta-cells. It is possible, however, that the effects in these mice occur secondary to changes in vascular endothelium or in the pancreatic ductal cells, or because of a decrease in the effects of other hormones implicated in islet growth. To directly define the role of Igf1, we have created a mouse with a beta-cell-specific knockout of Igf1r (betaIgf1r(-/-)). These mice show normal growth and development of beta-cells, but have reduced expression of Slc2a2 (also known as Glut2) and Gck (encoding glucokinase) in beta-cells, which results in defective glucose-stimulated insulin secretion and impaired glucose tolerance. Thus, Igf1r is not crucial for islet beta-cell development, but participates in control of differentiated function.

Inactivation of the IGF-I receptor gene in primary Sertoli cells highlights the autocrine effects of IGF-I.

IGF-I regulates pituitary and gonadal functions, and is pivotal for sexual development and fertility in mammalian species. To better understand the function of autocrine IGF-I in Sertoli cell physiology, we established a system for Cre-mediated conditional inactivation of the IGF-I receptor (IGF-IR) in cultured Sertoli cells. We show here that loss of IGF-IR decreased the number of viable Sertoli cells as a consequence of diminished Sertoli cell proliferation and increased Sertoli cell death. Furthermore, the lack of IGF-IR altered the morphology of cultured Sertoli cells and decreased lactate and transferrin secretions. Collectively, our data indicate that autocrine IGF-I contributes significantly to Sertoli cell homeostasis. The described in vitro system for loss-of-function analysis of the IGF-IR can be readily transposed to study the role of other intratesticular growth factors involved in spermatogenesis.

Cre-mediated germline mosaicism: a method allowing rapid generation of several alleles of a target gene.

Conditional gene targeting uses the insertion of expression cassettes for the selection of targeted embryonic stem cells. The presence of these cassettes in the final targeted chromosomal locus may affect the normal expression of the targeted gene and produce interesting knock down phenotypes. We show here that the selection cassette may then be selectively removed in vivo, using three appropriately positioned loxP sites in the targeted gene and the transgenic mouse EIIaCre. This strategy was applied to two different target genes and we demonstrated that it is reliable and reproducible. First, we generated double transgenic EIIaCre/loxP mice (F1) that showed variable degrees of mosaicism for partially CRE-recombined floxed alleles. Efficiency of EIIaCre at creating mosaicism was dependent on the target gene and on parental transmission of the transgene. The segregation of partially recombined alleles and EIIaCre transgene was obtained in the next generation using mosaic F1 males. Mosaic females were unsuitable for this purpose because they systematically generated complete excisions during oogenesis. Our strategy is applicable to other approaches based on three loxP sites. As this procedure allows generation of knock down (presence of neo), knockout (total exision of the loxP-flanked sequences) and floxed substrains (excision of the selection cassette) from a single, targeted germline mutation and in a single experiment, its use may become more widespread in conditional mutagenesis.

A targeted partial invalidation of the insulin-like growth factor I receptor gene in mice causes a postnatal growth deficit.

The insulin-like growth factor (IGF) system is a major regulator of somatic growth in vertebrates. Both ligands (IGF-I and IGF-II) signal via the same IGF receptor (IGF-IR). Classical IGF-IR invalidation is lethal at birth, so that conditional models are needed to study the postnatal role of this receptor. To establish a genetically inducible invalidation of IGF-IR, we targeted the IGF-IR gene using a construct that introduced a neomycin resistance cassette into intron 2, leaving the rest of the gene intact. This neomycin resistance cassette interfered with the processing of the primary transcript, resulting in there being 12% fewer IGF-binding sites at the cell surface in heterozygous mice and 41% fewer in homozygous mice. Hetero- and homozygous offspring grew more slowly than their wild-type littermates. This difference was noticeable from 4 weeks after birth and was significant from 5 weeks after birth in males. In females, the effect on postnatal growth of insertion of the neo cassette was not significant. In males, IGF-I levels increased moderately (+26%) but significantly, indicating effective feedback regulation of the IGF system. IGF-binding protein-4 (IGFBP-4) levels, estimated by Western ligand blotting, were low in homozygotes (-38%), whereas IGFBP-1, -2, and -3 levels were unaffected. In females, IGF-I and IGFBP-1, -2, -3, and -4 levels did not differ significantly among heterozygous, homozygous, and wild-type animals. We investigated the molecular mechanism involved and characterized two RNA-splicing events that could account for the decrease in IGF-IR. The phenotype of these mice developed exclusively postnatally, and body proportions were maintained. IGF-IRneo mice constitute a new model for human postnatal growth deficiency.

Experimental IGF-I receptor deficiency generates a sexually dimorphic pattern of organ-specific growth deficits in mice, affecting fat tissue in particular.

Reduced IGF type I receptor levels diminish postnatal growth rate and adult body weight in mice. Here, we studied the impact of experimental IGF receptor deficiency on tissue-specific growth by Cre-lox-mediated dosage of a floxed IGF-IR gene. We generated mice with a wide spectrum of receptor deficiency (5-82%), and separated them into two groups with either strong (> or =50%) IGF-IR deficiency (XS mice) or moderate deficiency (<50%, M mice). The growth of XS mice was significantly retarded from 3 wk after birth onward, with respect to M littermates. This effect was twice as strong in males as in females. Growth deficits persisted throughout adult life, and at 10-12 months, most organs and tissues showed specific weight defects. Skin, bone and connective tissue, muscle, spleen, heart, lung, and brain were the most severely affected organs in the XS males. With the exception of muscle and spleen, the same tissues were also significantly reduced in size in females, although to a lesser extent. The most severe growth defect, however, concerned adipose tissue. Fat pad size in XS males was only 29% (females, 44%) of M mice. The estimated number of adipocytes in XS male fat pads was only 21% that of M males (XS female, 27%). Lipid content per cell was significantly higher in XS adipocytes, whereas plasma glucose and insulin levels were low in XS males. Thus, IGF type I receptor deficiency produced mice with disproportionate postnatal organ growth, and these effects depended strongly on sex. A marked reduction in IGF-IR levels resulted in a major defect in adipose tissue.

IGF type 1 receptor ligand binding characteristics are altered in a subgroup of children with intrauterine growth retardation.

The IGFs, IGF-I and IGF-II, regulate fetal growth by activating IGF type 1 receptors (IGF-IR). We aimed to quantify the binding of IGF-I to its cognate receptors in intrauterine growth-retarded children (IUGR). We measured the affinity of the erythrocyte IGF-IR and the number of IGF-IR receptors in 17 children with retarded growth (mean height, -2.7 SD), normal levels of GH, and a history of idiopathic intrauterine growth retardation (height at birth, -10 to -2 SD; mean, -3.1 SD). These children had reduced receptor affinity (Kd = 0.47 nM; P < 0.01) and more receptors per cell [binding capacity (Bmax) = 11.7 binding sites/cell; P < 0.05)] compared with control children (Kd = 0.32 nM; Bmax = 7.8 binding sites/cell). Moreover, the distributions of Kd and Bmax suggested that there were two groups of IUGR children. Group 1 included subjects with normal receptor binding function (Kd = 0.36 nM; Bmax = 8.2 sites/cell) and normal levels of circulating IGF-I. Group 2 comprised children with low receptor affinity (Kd = 0.56 nM) and increased receptor number (Bmax = 14.7 sites/cell). This group showed significantly decreased IGF-I levels (-2.1 SD; P < 0.01). We investigated these IGF-IR binding parameters in two additional groups of growth-retarded children (Turner syndrome and patients with chronic renal failure), in whom the IGF-I axis was not believed to be the primary cause, and found that Kd and Bmax were normal or nearly normal. We also measured IGF-IR binding parameters in 4 Seckel syndrome patients with IUGR and severely retarded growth (mean height, -7.9 SD). Their receptor affinity was reduced, but not statistically different, from that in controls, and their receptor number was normal, whereas IGF-I levels were elevated. Our results suggest heterogeneous alterations in IGF-IR binding function in IUGR patients.

Genotyping of Cre-lox mice and detection of tissue-specific recombination by multiplex PCR.

Conditional gene targeting, based on Cre-lox or other systems, requires frequent genotyping of transgenic mouse populations and monitoring of tissue-specific Cre recombinatory efficiency. This is currently achieved by Southern analysis from tail- and tissue-derived DNA. Multiplex PCR amplification of the floxed (flanked by loxP sites) genomic region, combined with the PCR detection of the Cre transgene, simplifies this task. Here, we show that complete genotyping of a floxed locus is possible with three appropriately placed primers and that this triplex PCR can be performed simultaneously with a universal PCR assay for the detection of Cre transgenes. Using this approach, we also determined the ratios of recombined versus non-recombined floxed genomic segments in genomic DNA samples. This allowed us to estimate the efficiency of in vivo conditional inactivation from biopsy material and tissue samples that were too small for Southern analysis. As many new conditional knockouts are spatiotemporally restricted, such assays will become increasingly useful. The proposed PCR strategy is flexible and may be adapted to the structural specificities of any target gene.

Expression of insulin-like growth factor-I (IGF-I) and IGF-II in the avian brain: relationship of in situ hybridization patterns with IGF type 1 receptor expression.

Insulin-like growth factors (IGFs) are expressed in defined spatiotemporal patterns during the development of the mammalian central nervous system (CNS). Since IGF expression in avian species is less well documented, we studied here the expression of IGF-I and IGF-II during chicken CNS development, using in situ hybridization and reverse transcriptase-PCR, and compared the results with the expression of the IGF type 1 receptor (IGF-1R). IGF-II expression started early in embryonic life, shortly after the onset of IGF-1R expression. During organogenesis, IGF-II was strongly expressed in kidney, liver and gut primordia, in contrast with IGF-1R mRNA, which is highly enriched in proliferating neuroepithelia. During the second half of embryonic development, IGF-I and IGF-II had distinct expression patterns, suggesting specific roles for each ligand during brain maturation. IGF-II mRNA was found in numerous brainstem nuclei and in the optic tectum, whereas IGF-I mRNA was found predominantly in telencephalic regions. Both ligands were expressed in the cerebellum, but each by different cell layers. Some brain regions (olfactory bulb and olivo-cerebellar system) did not exhibit the postnatal downregulation typical of extrahepatic IGF-I expression, but continued to express IGF-I into adulthood. Purkinje cells expressed IGF-II in the embryo, but switched to IGF-I expression in the adult. The conservation of embryonic and postnatal IGF expression patterns in the CNS between avians and mammals suggests that the involvement of the IGF system in neurogenesis and differentiation, and possibly in neural plasticity and learning, may have arisen early during tetrapode/vertebrate evolution.

The avian IGF type 1 receptor: cDNA analysis and in situ hybridization reveal conserved sequence elements and expression patterns relevant for the development of the nervous system.

Insulin-like growth factor type 1 receptor (IGF-1R) is a tyrosine kinase with a key role in development. The primary structure of IGF-1R is known for mammalian species, but not for birds. The avian embryo, however, provides an ideal system for the experimental study of neurogenesis. We therefore cloned the complete coding sequence of the chicken IGF-1R from a cDNA library and analyzed its embryonic expression by Northern blot and in situ hybridization. The deduced chicken IGF-1R precursor of 1363 amino acids was 85% identical to human IGF-1R and did not show deletions or insertions in critical positions, when compared to its mammalian homologues. Notably, all cysteine residues in the extracellular domains, and 15 of the 17 N-linked glycosylation sites found in human IGF-1R were also present in the chicken receptor. An 11 kb transcript was abundant in developing nervous tissues, kidney, pancreas and the gastrointestinal tract. The early in situ expression patterns in 20-somite embryos revealed high levels of IGF-1R mRNA in the neuroepithelia, notochord and somites. At embryonic day 4 (E4), high concentrations of IGF-1R transcripts were found again primarily in the neuroepithelia and, to a lesser degree, in the sensory ganglia and diverse mesenchymal derivatives. During the second half of embryonic development, IGF-1R expression in the CNS was particularly abundant in telencephalic regions, including the olfactory bulb, hippocampus, striatum and piriform cortex, and also in the optic tectum and cerebellum. By the use of cDNA cloning and in situ hybridization this study reveals conserved amino acid sequence elements between birds and mammals, and developmental expression patterns that are compatible with an important role of this receptor in growth, differentiation and maturation of the avian CNS.

Tropoelastin gene expression in the developing vascular system of the chicken: an in situ hybridization study.

Temporal and spatial patterns in the accumulation of Tropoelastin (TE) mRNA during development of the chick embryo were established by in situ hybridization. Radiolabeled oligonucleotide probes of high specific activity were hybridized to serial sections of the cardiovascular system from embryonic day 3.5 (ED 3.5) to ED 19. Tropoelastin mRNA was observed as early as ED 3.5 in the dorsal part of the arterial trunk. During septation varying levels of TE mRNA were seen in the pulmonary trunk, the aorta and the aorticopulmonary septum. Thereafter TE mRNA levels increased up to ED 12, and the appearance of message was distributed distally in the walls of developing arteries. From ED 4.5 on, we found a decreasing proximo-distal gradient of the hybridization signal along the trunks and later along the main arteries (longitudinal gradient), and a radial gradient through the arterial vessel wall with the highest levels of TE mRNA in the outer layers of the media. Both gradients persisted in all major arterial vessels except in the proximal systemic and pulmonary trunks, where the original radial gradient was inverted or locally bimodal during the second half of development. The valvular region of aortic and pulmonary trunks showed particularly striking patterns of TE mRNA distribution, notably a prominent label on the endothelial cell layer on aortic and pulmonary valves. Outside the cardiovascular system, TE mRNA was mainly present in prochondral or perichondral cells in trachea and growing skeleton, and in the gap of growing joints. In kidney or nephric primordia, TE mRNA was only detectable in the wall of renal arteries. A hybridization signal was observed on mesenchyme of pulmonary septae at ED 16. Our results suggest a complex regulation of elastin gene expression during development, particularly within the proximal regions of the large arterial vessels.

Body surface area as a parameter of age decline.

We determined the age loss of body surface area (DuBois) in a cross-sectional and longitudinal combined study of 209 men and women aged between 40 and 96 from a rural region in the northwest of Spain. We established a secular growth trend of 1.01 +/- 0.30 mm per year, observable in this region since 1880, in 424 men joining the army between 1850 and 1970. Even after consideration of this growth trend, the loss of body surface area was shown to be strongly associated with age in adults of both sexes, with up to 2.5% loss per year in the oldest participants. Similarly the loss of body height becomes an individually dramatic process, in the sense that proportions of the body are no longer preserved. The secular growth trend in this region started long before the improvement of living conditions, commonly cited as causally involved in acceleration processes.

Decelerated growth and longevity in men.

Severe dietary restriction delays the physical development of rodents and leads to adult animals of reduced body size but significantly increased life expectancy. We tried to find a similar relationship in human populations using demographical and statistical methods. We show for the total Spanish male population that the mean adult body height reliably reflects the regional living and nutritional conditions. This relation does not only hold for todays socioeconomic data but can also be reproduced using data on family income in the mid 19th century. We calculated the mean height of young men liable to the military service around 1860 and determined their longevity retrospectively using posterior census data. This was done separately for all the Spanish provinces. The linear regression between both parameters manifests a statistically highly significant relationship: the smaller the mean height at age 18 in a province, the higher the chance for people living there to reach high chronological ages. Migrational movements, selection, mortality due to epidemics or unreliability of the population censuses can be largely ruled out as explications for the described correlation. Furthermore, we determined the secular growth trend in Spain for the last 130 years. From 1860 to 1920 the mean height increased by 2.7 cm, from 1920 to 1987 by another 9.8 cm. Since 1950 the trend is above 2 cm per decade country-wide. From 1974 onwards it amounts to 2.89 +/- 0.17. Such high values were worldwide looked upon as isolated cases found only in geographically and socially isolated population subgroups during very limited periods.

[Two strains of genetically hypertensive rats, LH and SRH, have distinct profiles of postnatal expression of tropoelastin and type III collagen in the aortic wall]. / Deux souches de rats génétiquement hypertendues, LH et SHR, se différencient par leur profil postnatal d'expression de tropoélastine et de collagène de type III dans la paroi aortique.

Experimental pharmacology and studies on hypertension frequently use genetically hypertensive animal models like the SHR or the Lyon hypertensive rat LH. In order to further characterize these two models we measured the expression levels of three major extracellular matrix components in the aortic wall, tropoelastin (TE) and type I and type III collagen, during postnatal development. The type I collagen expression decreases progressively during the first twelve weeks of postnatal development without significant differences between SHR and LH, or their normotensive controls, WKY or LN respectively. No differences were detected either for the expression levels of TE and type III collagen between the hypertensive strains and their respective controls. However, direct comparison of the two hypertensive strains SHR and LH, revealed a specific, strong increase of TE and type III expression for the LH at 5 and 12 weeks (p < 0.001 and 0.005 respectively). The evolution of the ratios of expression levels between the two collagens (type III/type I) on one side and of TE and collagen type I (TE/type I) on the other side were similar for the hypertensive strains and their respective controls, but diverged significantly for LH and SHR animals (up to p < 0.001 depending on the age group). Both indicators, III/I and TE/I, are considerably higher in LH compared to SHR from 5 weeks of postnatal development onwards. Our results indicate that the genes for TE and type I and III collagen are regulated during postnatal development of LH and SHR. It is however not possible at this point to establish a link between mRNA levels and hypertension in these animals. Nevertheless, the ratios III/I and TE/I seem to be good phenotypic markers for the characterisation of LH and SHR strains.

The role of insulin-like signalling in the regulation of ageing.

Inactivation of insulin-like growth factor I (IGF-I) signalling pathways have been shown to extend lifespans in various lower species, including the nematode Caenorhabditis elegans. In order to investigate this relationship in a mammalian species, a series of experiments were carried out with a mouse model heterozygous for a mutation in the IGF-I receptor gene. These heterozygous mice only had slight post-natal growth retardation, but had a lifespan 26% longer than normal. Their fertility and dietary intake were unaffected. The mechanism for increased lifespan in these mutant mice appears to be enhanced resistance to oxidative stress: heterozygous mice had a greater survival rate subsequent to severe oxidative stress generated in vivo than wild-type mice, and cells from heterozygous animals had a better resistance to hydrogen peroxide in vitro than cells from wild-type animals. Resistance to oxidative stress in these mutant animals could be caused by decreased phosphorylation of molecules downstream of the IGF-I receptor in the IGF-I signalling pathway, one of which is thought to be p66shc. Whether this link between reduced IGF-I signalling and longevity is conserved in other mammalian species, including humans, is presently not known. If it was, it could have implications for growth hormone therapy, which increases serum IGF-I levels.

[Objective assessment of biological aging in the human: results]. / Zur Objektivierung des biologischen Alters des Menschen: Ergebnisse.

From a population sample of 139 healthy persons, six specially selected aging parameters were tested for their significance to vitality, by means of four mathematical models referring to the concept of vitality. As reference values, tm, is the age expressed in years when maximal vitality occurs, and the aging constant is beta, also known as an aging factor. Our results for tm extended from 16.6 to 39.6 years, depending on the respective mathematical model applied. We find a similar characteristic for the beta-values. A comparison with results in other publications on the subject, obtained by processing a considerably larger number of variables than we did, underlined the utility of our parameters. This is also shown by the quality of results we obtained by regression analysis, by the adjustment to the functions of the vitality concept and by the simplicity of the required laboratory methods of analysis.

Selective expression of insulin-like growth factor II in the songbird brain.

Neuronal replacement occurs in the forebrain of juvenile and adult songbirds. To address the molecular processes that govern this replacement, we cloned the zebra finch insulin-like growth factor II (IGF-II) cDNA, a factor known to regulate neuronal development and survival in other systems, and examined its expression pattern by in situ hybridization and immunocytochemistry in juvenile and adult songbird brains. The highest levels of IGF-II mRNA expression occurred in three nuclei of the song system: in the high vocal center (HVC), in the medial magnocellular nucleus of the neostriatum (mMAN), which projects to HVC, and to a lesser extent in the robust nucleus of the archistriatum (RA), which receives projections from HVC. IGF-II mRNA expression was developmentally regulated in zebra finches. In canary HVC, monthly changes in IGF-II mRNA expression covaried with previously reported monthly differences in neuron incorporation. Combining retrograde tracers with in situ hybridization and immunocytochemistry, we determined that the HVC neurons that project to area X synthesize the IGF-II mRNA, whereas the adjacent RA-projecting neurons accumulate the IGF-II peptide. Our findings raise the possibility that within HVC IGF-II acts as a paracrine signal between nonreplaceable area X-projecting neurons and replaceable RA-projecting neurons, a mode of action that is compatible with the involvement of IGF-II with the replacement of neurons. Additional roles for IGF-II expression in songbird brain are likely, because expression also occurs in some brain areas outside the song system, among them the cerebellar Purkinje cells in which neurogenesis is not known to occur.

Quantitation of tropoelastin mRNA and assessment of alternative splicing in human skin fibroblasts by reverse transcriptase-polymerase chain reaction.

We have developed a reverse transcriptase-polymerase chain reaction (RT-PCR) assay for the quantitative measurement of levels of tropoelastin mRNA in total RNA preparations from skin fibroblasts. This method facilitates the reproducible detection of low abundance tropoelastin mRNA in the range of 10-1000 copies per cell. The procedure is based on a competitive RT-PCR assay where a tropoelastin cDNA-derived internal RNA standard is cotranscribed and coamplified together with the sample derived-endogenous target mRNA. In addition, RT-PCR of several domains of tropoelastin mRNA, followed by DNA sequence analysis of asymmetric PCR products, revealed a previously unknown pattern of alternate exon usage at the 3' end of the tropoelastin gene in human skin fibroblasts.