A human iPSC model of Ligase IV deficiency reveals an important role for NHEJ-mediated-DSB repair in the survival and genomic stability of induced pluripotent stem cells and emerging haematopoietic progenitors.

DNA double strand breaks (DSBs) are the most common form of DNA damage and are repaired by non-homologous-end-joining (NHEJ) or homologous recombination (HR). Several protein components function in NHEJ, and of these, DNA Ligase IV is essential for performing the final 'end-joining' step. Mutations in DNA Ligase IV result in LIG4 syndrome, which is characterised by growth defects, microcephaly, reduced number of blood cells, increased predisposition to leukaemia and variable degrees of immunodeficiency. In this manuscript, we report the creation of a human induced pluripotent stem cell (iPSC) model of LIG4 deficiency, which accurately replicates the DSB repair phenotype of LIG4 patients. Our findings demonstrate that impairment of NHEJ-mediated-DSB repair in human iPSC results in accumulation of DSBs and enhanced apoptosis, thus providing new insights into likely mechanisms used by pluripotent stem cells to maintain their genomic integrity. Defects in NHEJ-mediated-DSB repair also led to a significant decrease in reprogramming efficiency of human cells and accumulation of chromosomal abnormalities, suggesting a key role for NHEJ in somatic cell reprogramming and providing insights for future cell based therapies for applications of LIG4-iPSCs. Although haematopoietic specification of LIG4-iPSC is not affected per se, the emerging haematopoietic progenitors show a high accumulation of DSBs and enhanced apoptosis, resulting in reduced numbers of mature haematopoietic cells. Together our findings provide new insights into the role of NHEJ-mediated-DSB repair in the survival and differentiation of progenitor cells, which likely underlies the developmental abnormalities observed in many DNA damage disorders. In addition, our findings are important for understanding how genomic instability arises in pluripotent stem cells and for defining appropriate culture conditions that restrict DNA damage and result in ex vivo expansion of stem cells with intact genomes.

Randomized multicenter clinical trial of myofascial physical therapy in women with interstitial cystitis/painful bladder syndrome and pelvic floor tenderness.

PURPOSE: We determined the efficacy and safety of pelvic floor myofascial physical therapy compared to global therapeutic massage in women with newly symptomatic interstitial cystitis/painful bladder syndrome. MATERIALS AND METHODS: A randomized controlled trial of 10 scheduled treatments of myofascial physical therapy vs global therapeutic massage was performed at 11 clinical centers in North America. We recruited women with interstitial cystitis/painful bladder syndrome with demonstrable pelvic floor tenderness on physical examination and a limitation of no more than 3 years' symptom duration. The primary outcome was the proportion of responders defined as moderately improved or markedly improved in overall symptoms compared to baseline on a 7-point global response assessment scale. Secondary outcomes included ratings for pain, urgency and frequency, the O'Leary-Sant IC Symptom and Problem Index, and reports of adverse events. We compared response rates between treatment arms using the exact conditional version of the Mantel-Haenszel test to control for clustering by clinical center. For secondary efficacy outcomes cross-sectional descriptive statistics and changes from baseline were calculated. RESULTS: A total of 81 women randomized to the 2 treatment groups had similar symptoms at baseline. The global response assessment response rate was 26% in the global therapeutic massage group and 59% in the myofascial physical therapy group (p=0.0012). Pain, urgency and frequency ratings, and O'Leary-Sant IC Symptom and Problem Index decreased in both groups during followup, and were not significantly different between the groups. Pain was the most common adverse event, occurring at similar rates in both groups. No serious adverse events were reported. CONCLUSIONS: A significantly higher proportion of women with interstitial cystitis/painful bladder syndrome responded to treatment with myofascial physical therapy than to global therapeutic massage. Myofascial physical therapy may be a beneficial therapy in women with this syndrome.

Genetic manipulation of IRS proteins: animal modelsfor understanding the molecular basis of diabetes

The identifi cation of insulin receptor substrate (IRS) proteins in the 1990srepresents a key phase of diabetes research as it has enabled ourpresent understanding of the molecular basis of insulin and insulin-likegrowth factor (IGF) action. The generation of mice with targeted deletionsof the four major IRS proteins has revealed invaluable information aboutthe biological functions of these signaling molecules and has providednovel insights into the role of defective insulin signaling in the developmentof diabetes and metabolic diseases. Irs1-defi ciency in mice causesreduced body size, beta cell hyperplasia, and increased life-span. Disruptionof Irs2 has demonstrated that this branch of the insulin/IGF signalingcascade has an important role in peripheral insulin action and pancreaticbeta-cell growth and function. Global disruption of IRS2 signalingin mice causes diabetes due to failed beta cell compensation in the presenceof peripheral insulin resistance. Gene targeting of Irs3 or Irs4 didnot produce remarkable phenotypes suggesting that either they play veryspecifi c roles in limited tissues or that their absence may be compensatedfor by other signaling mechanisms. A complete understanding ofthe cellular events mediated by IRS1 and IRS2 will reveal new strategiesto prevent or cure diabetes and other metabolic diseases(AU)

Expression of the mu-opioid receptor in the anterior pituitary gland is influenced by age and sex.

To analyze whether opioids are able to modulate endocrine regulation by acting directly on rat pituitary cells, an immunohistochemical study of micro-opioid receptor expression in these cells was performed, with attention given to the analysis of potential age- and sex-related variations in receptor expression patterns. In both sexes, the micro-opioid receptor was detected in the pituitary pars distalis. However, significant age-related differences were observed. Both in male and female rats, the percentage of micro-opioid receptor-expressing cells decreased significantly from postnatal week one through the 24 months of our study. Interestingly, pituitary cells containing the micro-opioid receptor were significantly more numerous in male than in female, with exception of the pre-pubertal phase and old rats. According to two-way analysis of variance, the gender-related differences in micro-receptor expression were independent of age-related variations. Thus, without excluding hypothalamic actions, our results suggest that opioids may exert their endocrine function by acting directly on pituitary cells.

Dopaminergic modulation of nNOS expression in the pituitary gland of male rat.

Nitric oxide is an unconventional transmitter since it is not transported and released by exocytosis. In the pituitary gland, nitric oxide is locally synthesised by gonadotroph and folliculo-stellate cells. Dopamine, the principal central inhibitory signal in prolactin release, may exert its inhibitory effects by stimulation of nitric oxide production. However, the effects of dopaminergic modulation on nitric oxide-producing pituitary cells have not been analysed. Therefore, we examined the effects of intraventricular administration of the dopamine antagonist haloperidol (40 microg) on the pituitary expression of neuronal nitric oxide synthase (nNOS) in male adult rats. In untreated and control animals, nNOS-positive cells were very similar. Two types of nNOS-positive cells appeared in the pars distalis: round or polygonal cells and stellate cells. Although some isolated cells were found, the nNOS-positive cells commonly appeared grouped in clusters close to blood vessels. nNOS immunoreactivity appeared as a uniform staining throughout the cytoplasm, including cell prolongations. The number and size of nNOS-expressing cells in the pituitary gland decreased significantly after treatment with haloperidol (p<0.01). To evaluate the potential direct effect of dopamine on pituitary cells, pituitary monolayer cultures were treated with dopamine during a time-course of 12 h. Our in vitro studies revealed that dopamine increases the percentage of nNOS-positive cells and augments cellular area (p<0.05). These results demonstrate that: (1) treatment of rats in vivo with a dopamine antagonist significantly decreases expression of nNOS in the pituitary and (2) in vitro dopamine exerts a direct effect on pituitary cultures by increasing nNOS-positive cells. Thus, these findings suggest that dopamine may function as a physiological stimulator of nNOS expression in the rat pituitary gland.

Increases in the size, number and proliferation rate of VIP-immunoreactive pituitary cells induced in vitro by TRH are associated with increases in VIP release / Los aumentos del tamaño, número y proliferación de las células hipofisarias inmunorreactivas a VIP, inducidos in vitro por TRH, están asociados con incrementos de la liberación de VIP

Pituitary VIP levels and pituitary VIP-expressing cell numbers are increased in hypothyroidism, but the regulating role of TRH as regards morphological changes remains obscure. In order to determine whether TRH is involved in regulating the maintenance of pituitary VIP-expressing cells and its cellular proliferation a double immunocytochemical study for VIP and proliferating cell nuclear antigen (PCNA) was carried out in pituitary monolayer cultures treated with TRH. TRH induced a significant in vitro increase in the numerical density (p<0.01) and percentage (p<0.001) of VIP-expressing cells. These changes were accompanied by increases in VIP release (p<0.01) and in the size (p<0.01) of VIP-expressing cells. Our results suggest that TRH could regulate the maintenance of the percentage of pituitary VIP-expressing cells, their activity and their proliferation in a similar way to the way in which it regulates the release of VIP (AU)

Los niveles hipofisarios de VIP y el número de células que expresan VIP están aumentados en el hipotiroidismo, pero el papel regulador de TRH respecto a los cambios morfológicos permanece inaclarado. Con el fin de determinar si la TRH está implicada en la regulación del mantenimiento de las células hipofisarias que expresan VIP y su proliferación celular, se llevó a cabo un estudio inmunocitoquímico doble para VIP y el antígeno nuclear de proliferación celular (PCNA) en cultivos hipofisarios monocapa tratados con TRH. TRH indujo un aumento significativo in vitro de la densidad numérica (p<0.01) y el porcentaje (p<0.001) de las células que expresan VIP. Estos cambios estuvieron acompañados por aumentos de la liberación de VIP (p<0.01) y del tamaño (p<0.01) de las células VIP-positivas. Nuestros resultados sugieren que la TRH podría regular el mantenimiento del porcentaje, actividad y proliferación de las células que expresan VIP, de forma similar al modo en que regula la liberación de VIP (AU)

A pilot clinical trial of oral pentosan polysulfate and oral hydroxyzine in patients with interstitial cystitis.

PURPOSE: This pilot study was designed to evaluate the feasibility of a multicenter, randomized, clinical trial in interstitial cystitis (IC). Secondary objectives were to evaluate the safety and efficacy of oral pentosan polysulfate sodium (PPS), hydroxyzine, and the combination to consider their use in a larger randomized clinical trial. MATERIALS AND METHODS: A 2 x 2 factorial study design was used to evaluate PPS and hydroxyzine. Participants met the National Institutes of Health-National Institute for Diabetes and Digestive and Kidney Diseases criteria for IC and reported at least moderate pain and frequency for a minimum of 6 months before study entry. The primary end point was a patient reported global response assessment. Secondary end points included validated symptom indexes and patient reports of pain, urgency and frequency. The target sample size was 136 participants recruited during 10 months. RESULTS: A total of 121 (89% of goal) participants were randomized over 18 months and 79% provided complete followup data. The response rate for hydroxyzine was 31% for those treated and 20% for those not treated (p = 0.26). A nonsignificant trend was seen in the PPS treatment groups (34%) as compared to no PPS (18%, p = 0.064). There were no treatment differences for any of the secondary end points. Adverse events were mostly minor and similar to those in previous reports. CONCLUSIONS: The low global response rates for PPS and hydroxyzine suggest that neither provided benefit for the majority of patients with IC. This trial demonstrated the feasibility of conducting a multicenter randomized clinical trial in IC using uniform procedures and outcomes. However, slow recruitment underscored the difficulties of evaluating commonly available IC drugs.

Postnatal differentiation of the immunohistochemical expression of aromatase P450 in the rat pituitary gland.

At our laboratory, we have recently demonstrated the immunohistochemical expression of aromatase P450 in the pituitary glands of adult rats; this expression was seen to be sex-dependent. In order to determine whether the changes in the expression of the enzyme are related to changes in the gonadal sphere and whether the expression of the enzyme is related to the postnatal differentiation of hypophyseal cytology, in the present work we performed an immunohistochemical study in the rat pituitary gland from birth to old age. The immunohistochemical reaction to aromatase was evident and very generalized at 7 days after birth, with no large differences between the male and female animals. At 14 days the immunohistochemical reaction was decreased in the females, with no changes in the males. At 17 days, aromatase immunoreactivity in the pituitary glands of female rats was very weak whereas the males showed large numbers of reactive cells. These observations were further pronounced at 21 days and 2 months of life. At 24 months, the immunoreactivity found in the pituitary glands of the male rats had almost completely disappeared. Our results show that a postnatal differentiation in the immunohistochemical expression of aromatase occurs; this is tightly linked to sexual activity and is lost in old age. This suggests that hypophyseal aromatase would be related to the mechanisms of action of gonadal steroids on hypophyseal differentiation and secretion.

Immunocytochemical and morphometric evidence of the existence of different cellular populations reactive to r-GHRH in the rat pituitary gland / Evidencia inmunocitoquímica y morfométrica de la existencia de diferentes poblaciones celulares reactivas a r-GHRH en la hipófisis de la rata

In order to characterize the pituitary cells reactive for r-GRF in the adult rat, an immunocytochemicaI and morphometric study was made of the cells immunopositive for r-GHRH and those immunopositive for GH and for GHRH using a double labelling method. The existence of two populations of r-GRF-immunoreactive cells was observed; one of these was only stained with specific serum against r-GRF and the other was immunopositively stained for GH and for r-GHRH. Morphometrically, in both sexes the GH-immunoreactive cells were seen to have a cellular size that was significantly larger (p<0.01) than the r-GHRH-immunoreactive cells. The cells that were only stained for r-GHRH were significantly smaller than those immunoreactive for GH and r-GHRH. Our findings suggest the existence of at least two cellular populations in the pituitary gland of the adult rat able to react with anti-r-GHRH serum (AU)

Con el fin de caracterizar las células hipofisarias reactivas a r-GRF en la rata adulta, se realizó un estudio inmunocitoquímico y morfométrico de las células inmunopositivas a r-GHRH y de aquellas inmunopositivas a GH y a GHRH usando un método de doble marcaje. Se observó la existencia de dos poblaciones de células inmunorreactivas a r-GRF; una de ellas solamente fue teñida con suero específico anti r-GRF y la otra fue teñida inmunopositivamente para GH y para r-GHRH. Morfométricamente, las células inmunorreactivas a GH presentaron un tamaño celular significativamente mayor (p<0.01) que las células inmunorreactivas a r-GHRH. Las células que solamente se tiñeron con el suero anti r-GHRH fueron significativamente más pequeñas que aquellas inmunorreactivas a GH y r-GHRH. Nuestros resultados sugieren la existencia de al menos dos poblaciones celulares en la hipófisis de la rata adulta capaces de reaccionar con el suero anti r-GHRH (AU)

Las rutas de señales de la insulina: mecanismos de integración de la homeostasis energética y la reproducción / Insulin signaling pathways: mechanisms integrating energy homeostasis and reproduction

La reproducción en las hembras de mamíferos es extremadamente sensible a la disponibilidad de combustibles metabólicos. Esta relación entre la homeostasis energética y la reproducción ha sido reconocida durante décadas. Así, la restricción dietética intensa, los estados catabólicos y el exceso del gasto energético deterioran la fertilidad humana. Del mismo modo, la obesidad resultante de una mayor ingestión que la requerida por el cuerpo está asociada con situaciones de infertilidad como en el síndrome de ovarios policísticos. Por ello, la reproducción, la ingestión de alimento y la utilización de combustibles constituyen las respuestas homeostáticas reguladas por señales metabólicas. Así, la capacidad reproductiva en organismos inferiores como el gusano y la mosca está también influida por la disponibilidad de alimento y las reservas de energía. Sin embargo, el modo preciso de la regulación de la actividad reproductiva por la nutrición y la energía metabólica continúa siendo una cuestión sin resolver de la biología moderna. Se postula que la insulina es la principal hormona reguladora del metabolismo de hidratos de carbono en el organismo y junto con las rutas de señales dependientes de ella, desempeña un papel primordial en la homeostasis de combustibles metabólicos. Verificaciones recientes procedentes de modelos animales demuestran que la acción de la insulina también regula la ingestión de comida, el peso corporal y la capacidad de reproducción, implicando a las rutas de señalización de la insulina como la conexión mecanística entre el metabolismo y la regulación neuroendocrina de la fertilidad. La deleción de IRS-2, uno de los principales sustratos del receptor de insulina, produce en el ratón defectos tanto metabólicos como reproductivos. Las ratonas deficientes en IRS-2 demuestran una moderada intolerancia a glucosa, resistencia a insulina, hiperfagia y una discreta obesidad. Curiosamente, estos animales son infértiles debido a defectos en el ovario y/o en el eje hipotálamo-pituitario-ovárico. Aquí se revisan las perspectivas históricas del control metabólico de la reproducción haciendo un especial énfasis en las contribuciones de la señalización de la insulina. Además, nuestra revisión se centra en las observaciones recientes del modelo knockout de IRS-2, que nos ha proporcionado una evidencia directa de que la fertilidad requiere la integración de señales metabólicas y reproductivas. Basándonos en el papel de las rutas de señalización de la insulina en la regulación de la fertilidad, el metabolismo y la longevidad en C. elegans y Drosophila, nuestra hipótesis de trabajo es que las rutas de señales mediadas por IRS-2 representan un mecanismo conservado evolutivamente que comunica la homeostasis energética con la fisiología de la reproducción (AU)

Vitellogenin gene transcription: a relative quantitative exposure indicator of environmental estrogens.

We report the development of a quantifiable exposure indicator for measuring the presence of environmental estrogens in aquatic systems. Synthetic oligonucleotides, designed specifically for the vitellogenin gene (Vg) transcription product, were used in a reverse transcription-polymerase chain reaction (RT-PCR) protocol. This extremely sensitive and rapid method was able to detect vitellogenin gene transcription in male common carp (Cyprinus carpio) injected with 17beta-estradiol. Sequence analysis of the induced mRNA product confirmed a vitellogenin gene transcript with homology to rainbow trout and fathead minnow vitellogenin cDNA sequences. Relative levels of vitellogenin gene induction among individuals were quantified by incorporating 18S ribosomal RNA universal primers and Competimers in a PCR multiplex reaction with primers for vitellogenin. This method is more sensitive than current protocols, such as mortality, visible signs of stress, or other techniques that look for unscheduled gene expression, because it measures the appearance of primary transcripts at the nanogram level. In addition, this procedure does not sacrifice accuracy or reliability, even though the exposure to estrogen is within 1 d. This research will support the construction of regional stressor profiles, thereby providing a method for comparative environmental exposure assessment. It may also provide an in vivo screening method for potential endocrine-disrupting compounds.

Extinction, working memory, and line bisection in spatial neglect.

The authors studied four patients with spatial neglect, using a task in which lines contain an off-centered bisection mark and a task in which the right and left segments of these bisected lines are presented independently and sequentially. In the prebisected line task, subjects reported the position of the bisection. In the segments task, subjects compared the length of the segments. Accuracy was greater with the sequential presentation of line segments, suggesting that an extinction-like phenomenon plays a role in line bisection bias.

IRS proteins and beta-cell function.

Insulin receptor substrate (IRS) proteins mediate a variety of the metabolic and growth-promoting actions of insulin and IGF-1. After phosphorylation by activated receptors, these intracellular signaling molecules recruit various downstream effector pathways including phosphatidylinositol 3-kinase and Grb2. Ablation of the IRS-2 gene produces a diabetic phenotype; mice lacking IRS-2 display peripheral insulin resistance and beta-cell dysfunction characterized by a 50% reduction in beta-cell mass. In contrast, deletion of IRS-1 retards somatic growth and enhances beta-cell mass. IRS1-/- mice are 50% smaller than controls but have a twofold increase in pancreatic beta-cell mass. Thus, observations from these recently developed animal models implicate the IRS signaling systems in the response of classical insulin target tissues, and they suggest a critical role for these proteins in the regulation of beta-cell function. In humans, type 2 diabetes generally occurs when insulin-secretory reserves fail to compensate for peripheral insulin resistance. Study and identification of the signals downstream of IRS proteins in beta-cells may provide unique insights into the compensatory mechanisms by which these cells respond to insulin resistance. Therefore, the intent of this review is to summarize recent observations regarding the regulation of beta-cell function by members of the IRS protein family.

IRS-2 pathways integrate female reproduction and energy homeostasis.

Severe dietary restriction, catabolic states and even short-term caloric deprivation impair fertility in mammals. Likewise, obesity is associated with infertile conditions such as polycystic ovary syndrome. The reproductive status of lower organisms such as Caenorhabditis elegans is also modulated by availability of nutrients. Thus, fertility requires the integration of reproductive and metabolic signals. Here we show that deletion of insulin receptor substrate-2 (IRS-2), a component of the insulin/insulin-like growth factor-1 signalling cascade, causes female infertility. Mice lacking IRS-2 have small, anovulatory ovaries with reduced numbers of follicles. Plasma concentrations of luteinizing hormone, prolactin and sex steroids are low in these animals. Pituitaries are decreased in size and contain reduced numbers of gonadotrophs. Females lacking IRS-2 have increased food intake and obesity, despite elevated levels of leptin. Our findings indicate that insulin, together with leptin and other neuropeptides, may modulate hypothalamic control of appetite and reproductive endocrinology. Coupled with findings on the role of insulin-signalling pathways in the regulation of fertility, metabolism and longevity in C. elegans and Drosophila, we have identified an evolutionarily conserved mechanism in mammals that regulates both reproduction and energy homeostasis.

Role of brain insulin receptor in control of body weight and reproduction.

Insulin receptors (IRs) and insulin signaling proteins are widely distributed throughout the central nervous system (CNS). To study the physiological role of insulin signaling in the brain, we created mice with a neuron-specific disruption of the IR gene (NIRKO mice). Inactivation of the IR had no impact on brain development or neuronal survival. However, female NIRKO mice showed increased food intake, and both male and female mice developed diet-sensitive obesity with increases in body fat and plasma leptin levels, mild insulin resistance, elevated plasma insulin levels, and hypertriglyceridemia. NIRKO mice also exhibited impaired spermatogenesis and ovarian follicle maturation because of hypothalamic dysregulation of luteinizing hormone. Thus, IR signaling in the CNS plays an important role in regulation of energy disposal, fuel metabolism, and reproduction.

Skin cleaning with kerosene facilitates passage of carcinogens to the lungs of animals treated with used gasoline engine oil.

Solvents such as kerosene or gasoline may be used by workers to clean their skin following contact with oily materials. This practice is not recommended, as it is well known that the solvent will defat the skin. Many also suspect that solvent washing may increase exposure by carrying materials through the skin; however, there is little documentation of this. Auto mechanics may be exposed to used gasoline engine oil (UGEO), an animal carcinogen which forms carcinogen-DNA adducts in skin and lung following topical application. This study was designed to determine if cleaning with kerosene following exposure to UGEO altered absorption of carcinogens from this material. UGEO or new oil (NO) was applied to the shaved skins of groups of HSD-ICR mice for five days. At 1 or 8 hours after application, the treated skins were cleaned with either kerosene or a commercial cleaner, or were not cleaned. Animals were sacrificed 24 hours after the last application, skins and lungs harvested, and DNA analyzed for carcinogen-DNA adducts by 32P-postlabeling. Five applications of UGEO significantly increased carcinogen-DNA adduct levels in both lungs and skin compared to animals treated with NO. DNA adduct levels in the skin were reduced significantly in groups washed with kerosene or commercial cleaner. Washing at one as opposed to eight hours after UGEO application resulted in lower adduct levels regardless of cleaner. DNA adduct levels in the lung were reduced when the commercial cleaner was used, again in a time-related fashion. However, cleaning with kerosene resulted in mean carcinogen-DNA adduct levels in the lung which were significantly higher than even the positive controls, regardless of cleaning time. This is the first demonstration that kerosene cleaning facilitates passage of carcinogens through the skin, resulting in higher levels of genetic damage in a critical internal organ.

Gab-1-mediated IGF-1 signaling in IRS-1-deficient 3T3 fibroblasts.

The insulin receptor substrate (IRS) family of proteins mediate a variety of intracellular signaling events by serving as signaling platforms downstream of several receptor tyrosine kinases including the insulin and insulin-like growth factor-1 (IGF-1) receptors. Recently, several new members of this family have been identified including IRS-3, IRS-4, and growth factor receptor-binding protein 2-associated binder-1 (Gab-1). 3T3 cell lines derived from IRS-1-deficient embryos exhibit a 70-80% reduction in IGF-1-stimulated S-phase entry and a parallel decrease in the induction of the immediate-early genes c-fos and egr-1 but unaltered activation of the mitogen-activated protein kinases extracellular signal-regulated kinase-1 and extracellular signal-regulated kinase-2. Reconstitution of IRS-1 expression in IRS-1-deficient fibroblasts by retroviral mediated gene transduction is capable of restoring these defects. Overexpression of Gab-1 in IRS-1-deficient fibroblasts also results in the restoration of egr-1 induction to levels similar to those achieved by IRS-1 reconstitution and markedly increases IGF-1-stimulated S-phase progression. Gab-1 is capable of regulating these biological end points despite the absence of IGF-1 stimulated tyrosine phosphorylation. These data provide evidence that Gab-1 may serve as a unique signaling intermediate in insulin/IGF-1 signaling for induction of early gene expression and stimulation of mitogenesis without direct tyrosine phosphorylation.

Tissue-specific insulin resistance in mice with mutations in the insulin receptor, IRS-1, and IRS-2.

Type 2 diabetes is characterized by abnormalities of insulin action in muscle, adipose tissue, and liver and by altered beta-cell function. To analyze the role of the insulin signaling pathway in these processes, we have generated mice with combined heterozygous null mutations in insulin receptor (ir), insulin receptor substrate (irs-1), and/or irs-2. Diabetes developed in 40% of ir/irs-1/irs-2(+/-), 20% of ir/irs-1(+/-), 17% of ir/irs-2(+/-), and 5% of ir(+/-) mice. Although combined heterozygosity for ir/irs-1(+/-) and ir/irs-2(+/-) results in a similar number of diabetic mice, there are significant differences in the underlying metabolic abnormalities. ir/irs-1(+/-) mice develop severe insulin resistance in skeletal muscle and liver, with compensatory beta-cell hyperplasia. In contrast, ir/irs-2(+/-) mice develop severe insulin resistance in liver, mild insulin resistance in skeletal muscle, and modest beta-cell hyperplasia. Triple heterozygotes develop severe insulin resistance in skeletal muscle and liver and marked beta-cell hyperplasia. These data indicate tissue-specific differences in the roles of IRSs to mediate insulin action, with irs-1 playing a prominent role in skeletal muscle and irs-2 in liver. They also provide a practical demonstration of the polygenic and genetically heterogeneous interactions underlying the inheritance of type 2 diabetes.

Irs-2 coordinates Igf-1 receptor-mediated beta-cell development and peripheral insulin signalling.

Insulin receptor substrates (Irs proteins) mediate the pleiotropic effects of insulin and Igf-1 (insulin-like growth factor-1), including regulation of glucose homeostasis and cell growth and survival. We intercrossed mice heterozygous for two null alleles (Irs1+/- and Irs2+/-) and investigated growth and glucose metabolism in mice with viable genotypes. Our experiments revealed that Irs-1 and Irs-2 are critical for embryonic and post-natal growth, with Irs-1 having the predominant role. By contrast, both Irs-1 and Irs-2 function in peripheral carbohydrate metabolism, but Irs-2 has the major role in beta-cell development and compensation for peripheral insulin resistance. To establish a role for the Igf-1 receptor in beta-cells, we intercrossed mice heterozygous for null alleles of Igf1r and Irs2. Our results reveal that Igf-1 receptors promote beta-cell development and survival through the Irs-2 signalling pathway. Thus, Irs-2 integrates the effects of insulin in peripheral target tissues with Igf-1 in pancreatic beta-cells to maintain glucose homeostasis.

IRS pleckstrin homology domains bind to acidic motifs in proteins.

Using a yeast two-hybrid system, we identified several proteins that interact with the PH domains in IRS-1 and IRS-2, including Lon protease, myeloblast protein, and nucleolin. Although the roles of these molecules in insulin action are not yet known, each protein contained an acidic motif that interacted with the PH domain of IRS-2. However, only the acidic motif in nucleolin bound to IRS-1, suggesting that the PH domain in IRS-1 and IRS-2 are not identical. Moreover, synthetic peptides based on the acidic motif in Lon protease and myeloblast protein inhibited the binding of nucleolin to the PH domain of IRS-2 but not to the PH domain of IRS-1, confirming the selectivity of these PH domains. The ability to bind acidic motifs may be a specific function of the PH domain in IRS proteins, because the PH domains in betaARK, phospholipase Cgamma, or spectrin did not bind nucleolin. In 32D cells, nucleolin bound to both IRS-1 and IRS-2, and expression of the acidic motif of nucleolin inhibited insulin-stimulated tyrosine phosphorylation of IRS-1 and IRS-2. These results suggest that the binding of acidic motifs to the PH domain of IRS-1 and IRS-2 disrupts coupling to the activated insulin receptor. Our results are consistent with the hypothesis that the PH domain in the IRS proteins may ordinarily bind acidic peptide motifs in membrane proteins or other acidic membrane elements that couple IRS proteins to activated membrane receptors.