A High-Throughput In Situ Method for Estimation of Hepatocyte Nuclear Ploidy in Mice.

When the liver is injured, hepatocyte numbers decrease, while cell size, nuclear size and ploidy increase. The expansion of non-parenchymal cells such as cholangiocytes, myofibroblasts, progenitors and inflammatory cells also indicate chronic liver damage, tissue remodeling and disease progression. In this protocol, we describe a simple high-throughput approach for calculating changes in the cellular composition of the liver that are associated with injury, chronic disease and cancer. We show how information extracted from two-dimensional (2D) tissue sections can be used to quantify and calibrate hepatocyte nuclear ploidy within a sample and enable the user to locate specific ploidy subsets within the liver in situ. Our method requires access to fixed/frozen liver material, basic immunocytochemistry reagents and any standard high-content imaging platform. It serves as a powerful alternative to standard flow cytometry techniques, which require disruption of freshly collected tissue, loss of spatial information and potential disaggregation bias.

Genetic inactivation of the LIGHT (TNFSF14) cytokine in mice restores glucose homeostasis and diminishes hepatic steatosis.

AIMS/HYPOTHESIS: Non-alcoholic fatty liver disease (NAFLD) is frequently associated with type 2 diabetes mellitus. Progression of NAFLD is mediated, among other things, by activation of inflammatory pathways. In the present study, the role of the proinflammatory cytokine LIGHT (TNFSF14) was explored in NAFLD and type 2 diabetes mellitus in mice deficient for the cytokine. METHODS: Light-deficient (Light-/-) mice and WT controls were fed a regular chow diet (RCD) or a high-fat high-cholesterol diet (HFHCD) for 16 weeks. The expression of LIGHT and its receptors, herpes virus entry mediator (HVEM) and lymphotoxin ß receptor (LTßR), was investigated in both dietary regimens. Glucose tolerance, insulin sensitivity, non-alcoholic fatty liver (NAFL), systemic and tissue inflammation, and metabolic gene expression were explored in Light-/- and WT mice fed an RCD and an HFHCD. The effect of Light deficiency was also evaluated in hepatic tissue and in inflammation in HFHCD-fed Irs2+/- mice with impaired insulin signalling. RESULTS: Light deficiency did not have an effect on metabolism, in NAFL or in tissue and systemic inflammation, in RCD-fed WT mice. HVEM and LTßR were markedly increased in livers of HFHCD-fed WT mice compared with RCD-fed WT controls. In WT mice under HFHCD, Light deficiency improved glucose tolerance and insulin sensitivity. Non-alcoholic fatty liver disease activity (NAS) score, hepatic CD3+ T lymphocytes and F4/80+ macrophages were decreased in HFHCD-fed Light-/- mice compared with HFHCD-fed WT controls. Consistent with a potential role of adipose tissue in hepatic homeostasis, Light-/- mice exhibited augmented anti-inflammatory F4/80+CD206+ adipose tissue macrophages and reduced proinflammatory F4/80+CD11c+ adipose tissue macrophages. Moreover, adipose tissue explants from Light-/- mice showed diminished secretion of monocyte chemoattractant protein 1 (MCP1), TNF-α and IL-17 cytokines. Circulating Light-/- leucocytes consistently displayed augmented levels of the patrolling Ly6Clow monocytes, decreased Th9 T cell subset and diminished plasma TNF-α and IL-6 levels. Similarly, Light deficiency in Irs2+/- mice, which display impaired insulin signalling, also reduced NAFL as well as systemic and adipose tissue inflammation. Analysis of hepatic gene expression in Light-/- mouse livers showed reduced levels of Zbtb16, the transcription factor essential for natural killer T (NKT) cell function, and two genes related to NAFLD and fibrosis, Klf6 and Tlr4. CONCLUSIONS/INTERPRETATION: These results indicate that Light deficiency in HFHCD improves hepatic glucose tolerance, and reduces hepatic inflammation and NAFL. This is accompanied by decreased systemic inflammation and adipose tissue cytokine secretion and by changes in the expression of key genes such as Klf6 and Tlr4 involved in NAFLD. These results suggest that therapies to block LIGHT-dependent signalling might be useful to restore hepatic homeostasis and to restrain NAFLD.

Insulin resistance disrupts epithelial repair and niche-progenitor Fgf signaling during chronic liver injury.

Insulin provides important information to tissues about feeding behavior and energy status. Defective insulin signaling is associated with ageing, tissue dysfunction, and impaired wound healing. In the liver, insulin resistance leads to chronic damage and fibrosis, but it is unclear how tissue-repair mechanisms integrate insulin signals to coordinate an appropriate injury response or how they are affected by insulin resistance. In this study, we demonstrate that insulin resistance impairs local cellular crosstalk between the fibrotic stroma and bipotent adult liver progenitor cells (LPCs), whose paracrine interactions promote epithelial repair and tissue remodeling. Using insulin-resistant mice deficient for insulin receptor substrate 2 (Irs2), we highlight dramatic impairment of proregenerative fibroblast growth factor 7 (Fgf7) signaling between stromal niche cells and LPCs during chronic injury. We provide a detailed account of the role played by IRS2 in promoting Fgf7 ligand and receptor (Fgfr2-IIIb) expression by the two cell compartments, and we describe an insulin/IRS2-dependent feed-forward loop capable of sustaining hepatic re-epithelialization by driving FGFR2-IIIb expression. Finally, we shed light on the regulation of IRS2 and FGF7 within the fibrotic stroma and show-using a human coculture system-that IRS2 silencing shifts the equilibrium away from paracrine epithelial repair in favor of fibrogenesis. Hence, we offer a compelling insight into the contribution of insulin resistance to the pathogenesis of chronic liver disease and propose IRS2 as a positive regulator of communication between cell types and the transition between phases of stromal to epithelial repair.

Targeting neuroblastoma stem cells with retinoic acid and proteasome inhibitor.

BACKGROUND: Neuroblastma cell lines contain a side-population of cells which express stemness markers. These stem-like cells may represent the potential underlying mechanism for resistance to conventional therapy and recurrence of neuroblastoma in patients. METHODOLOGY/PRINCIPAL FINDINGS: To develop novel strategies for targeting the side-population of neurobastomas, we analyzed the effects of 13-cis-retinoic acid (RA) combined with the proteasome inhibitor MG132. The short-term action of the treatment was compared with effects after a 5-day recovery period during which both chemicals were withdrawn. RA induced growth arrest and differentiation of SH-SY5Y and SK-N-BE(2) neuroblastoma cell lines. Inhibition of the proteasome caused apoptosis in both cell lines, thus, revealing the critical role of this pathway in the regulated degradation of proteins involved in neuroblastoma proliferation and survival. The combination of RA with MG132 induced apoptosis in a dose-dependent manner, in addition to promoting G2/M arrest in treated cultures. Interestingly, expression of stem cell markers such as Nestin, Sox2, and Oct4 were reduced after the recovery period of combined treatment as compared with untreated cells or treated cells with either compound alone. Consistent with this, neurosphere formation was significantly impaired by the combined treatment of RA and MG132. CONCLUSIONS: Given that stem-like cells are associated with resistant to conventional therapy and are thought to be responsible for relapse, our results suggest that dual therapy of RA and proteasome inhibitor might be beneficial for targeting the side-population of cells associated residual disease in high-risk neuroblastoma.

Loss of protein tyrosine phosphatase 1B increases IGF-I receptor tyrosine phosphorylation but does not rescue retinal defects in IRS2-deficient mice.

PURPOSE: Mice with deletion of insulin receptor substrate (IRS) 2 develop type 2 diabetes and photoreceptor degeneration. Loss of protein tyrosine phosphatase 1B (PTP1B) in diabetic IRS2(-/-) mice restores insulin sensitivity and normalizes glucose homeostasis. Since insulin-like growth factor (IGF)-IR promotes survival of photoreceptors and is a substrate of PTP1B, we investigated IGF-IR-mediated survival signaling and visual function in PTP1B(-/-) and double mutant IRS2(-/-)/PTP1B(-/-) mice. METHODS: IGF-IR-mediated Akt signaling was evaluated in IGF-I-stimulated retinal explants. Histologic and electroretinogram analysis was performed in wild-type (WT), IRS2(-/-), PTP1B(-/-), and the double mutant IRS2(-/-)/PTP1B(-/-) mice. RESULTS: IGF-I stimulated the tyrosine phosphorylation of its receptor and Akt activation in retinal explants of WT mice. In PTP1B(-/-) retinal explants, these responses were enhanced. Conversely, in retinas from IRS2(-/-) mice, expression of PTP1B was increased, coincident with decreased IGF-I-mediated Akt serine 473 phosphorylation. PTP1B deletion in IRS2(-/-) mice also enhanced IGF-IR tyrosine phosphorylation but, unexpectedly, did not rescue Akt activation in response to IGF-I. One potential explanation is that PTEN was increased in retinas of IRS2(-/-) and IRS2(-/-)/PTP1B(-/-) mice. Histologic evaluation revealed alterations in various structures of the retina in IRS2(-/-) and IRS2(-/-)/PTP1B(-/-) mice, specifically in the outer nuclear layer (ONL) and retinal outer segments (ROS). Electroretinogram (ERG) analysis confirmed that PTP1B deficiency did not restore visual function in IRS2(-/-) mice. CONCLUSIONS: Although loss of PTP1B enhances tyrosine phosphorylation of the IGF-IR in retinal explants of IRS2(-/-) mice, Akt activation remains defective owing to elevated PTEN levels and, thus, structural and functional visual defects persist in this model.

Increased dosage of Ink4/Arf protects against glucose intolerance and insulin resistance associated with aging.

Recent genome-wide association studies have linked type-2 diabetes mellitus to a genomic region in chromosome 9p21 near the Ink4/Arf locus, which encodes tumor suppressors that are up-regulated in a variety of mammalian organs during aging. However, it is unclear whether the susceptibility to type-2 diabetes is associated with altered expression of the Ink4/Arf locus. In the present study, we investigated the role of Ink4/Arf in age-dependent alterations of insulin and glucose homeostasis using Super-Ink4/Arf mice which bear an extra copy of the entire Ink4/Arf locus. We find that, in contrast to age-matched wild-type controls, Super-Ink4/Arf mice do not develop glucose intolerance with aging. Insulin tolerance tests demonstrated increased insulin sensitivity in Super-Ink4/Arf compared with wild-type mice, which was accompanied by higher activation of the insulin receptor substrate (IRS)-PI3K-AKT pathway in liver, skeletal muscle and heart. Glucose uptake studies in Super-Ink4/Arf mice showed a tendency toward increased (18)F-fluorodeoxyglucose uptake in skeletal muscle compared with wild-type mice (P = 0.079). Furthermore, a positive correlation between glucose uptake and baseline glucose levels was observed in Super-Ink4/Arf mice (P < 0.008) but not in wild-type mice. Our studies reveal a protective role of the Ink4/Arf locus against the development of age-dependent insulin resistance and glucose intolerance.

IRS2 signalling is required for the development of a subset of sensory spinal neurons.

Insulin and insulin-like growth factor-I play important roles in the development and maintenance of neurons and glial cells of the nervous system. Both factors activate tyrosine kinase receptors, which signal through adapter proteins of the insulin receptor substrate (IRS) family. Although insulin and insulin-like growth factor-I receptors are expressed in dorsal root ganglia (DRG), the function of IRS-mediated signalling in these structures has not been studied. Here we address the role of IRS2-mediated signalling in murine DRG. Studies in cultured DRG neurons from different embryonic stages indicated that a subset of nerve growth factor-responsive neurons is also dependent on insulin for survival at very early time points. Consistent with this, increased apoptosis during gangliogenesis resulted in a partial loss of trkA-positive neurons in DRG of Irs2 mutant embryos. Analyses in adult Irs2(-/-) mice revealed that unmyelinated fibre afferents, which express calcitonin gene-related peptide/substance P and isolectin B4, as well as some myelinated afferents to the skin were affected by the mutation. The diminished innervation of glabrous skin in adult Irs2(-/-) mice correlated with longer paw withdrawal latencies in the hot-plate assay. Collectively, these findings indicate that IRS2 signalling is required for the proper development of spinal sensory neurons involved in the perception of pain.

Differential sensitivity to adrenergic stimulation underlies the sexual dimorphism in the development of diabetes caused by Irs-2 deficiency.

The diabetic phenotype caused by the deletion of insulin receptor substrate-2 (Irs-2) in mice displays a sexual dimorphism. Whereas the majority of male Irs-2(-/-) mice are overtly diabetic by 12 weeks of age, female Irs-2(-/-) animals develop mild obesity and progress less rapidly to diabetes. Here we investigated ß-cell function and lipolysis as potential explanations for the gender-related differences in this model. Glucose-stimulated insulin secretion was enhanced in islets from male null mice as compared to male WT whereas this response in female Irs-2(-/-) islets was identical to that of female controls. The ability of α(2)-adrenoceptor (α(2)-AR) agonists to inhibit insulin secretion was attenuated in male Irs2 null mice. Consistent with this, the expression of the α(2A)-AR was reduced in male Irs-2(-/-) islets. The response of male Irs-2(-/-) islets to forskolin was enhanced, owing to increased production of cAMP. Basal lipolysis was increased in male Irs-2(-/-) but decreased in female Irs-2(-/-) mice, concordant with the observation that adipose tissue is sparse in males whereas female Irs2 null mice are mildly obese. Adipocytes from both male and female Irs-2(-/-) were resistant to the anti-lipolytic effects of insulin but female Irs-2(-/-) fat cells were additionally resistant to the catabolic effects of beta-adrenergic agonists. This catecholamine resistance was associated with impaired generation of cAMP. Consequently, targets of cAMP-dependent protein kinase (PKA) which mediate lipolysis were not phosphorylated in adipose tissue of female Irs-2(-/-) mice. Our findings suggest that IRS-2 deficiency in mice alters the expression and/or sensitivity of components of adrenergic signaling.

Hypoxia promotes efficient differentiation of human embryonic stem cells to functional endothelium.

Early development of mammalian embryos occurs in an environment of relative hypoxia. Nevertheless, human embryonic stem cells (hESC), which are derived from the inner cell mass of blastocyst, are routinely cultured under the same atmospheric conditions (21% O(2)) as somatic cells. We hypothesized that O(2) levels modulate gene expression and differentiation potential of hESC, and thus, we performed gene profiling of hESC maintained under normoxic or hypoxic (1% or 5% O(2)) conditions. Our analysis revealed that hypoxia downregulates expression of pluripotency markers in hESC but increases significantly the expression of genes associated with angio- and vasculogenesis including vascular endothelial growth factor and angiopoitein-like proteins. Consequently, we were able to efficiently differentiate hESC to functional endothelial cells (EC) by varying O(2) levels; after 24 hours at 5% O(2), more than 50% of cells were CD34+. Transplantation of resulting endothelial-like cells improved both systolic function and fractional shortening in a rodent model of myocardial infarction. Moreover, analysis of the infarcted zone revealed that transplanted EC reduced the area of fibrous scar tissue by 50%. Thus, use of hypoxic conditions to specify the endothelial lineage suggests a novel strategy for cellular therapies aimed at repair of damaged vasculature in pathologies such as cerebral ischemia and myocardial infarction.

Inhibition of PTP1B restores IRS1-mediated hepatic insulin signaling in IRS2-deficient mice.

OBJECTIVE: Mice with complete deletion of insulin receptor substrate 2 (IRS2) develop hyperglycemia, impaired hepatic insulin signaling, and elevated gluconeogenesis, whereas mice deficient for protein tyrosine phosphatase (PTP)1B display an opposing hepatic phenotype characterized by increased sensitivity to insulin. To define the relationship between these two signaling pathways in the regulation of liver metabolism, we used genetic and pharmacological approaches to study the effects of inhibiting PTP1B on hepatic insulin signaling and expression of gluconeogenic enzymes in IRS2(-/-) mice. RESEARCH DESIGN AND METHODS: We analyzed glucose homeostasis and insulin signaling in liver and isolated hepatocytes from IRS2(-/-) and IRS2(-/-)/PTP1B(-/-) mice. Additionally, hepatic insulin signaling was assessed in control and IRS2(-/-) mice treated with resveratrol, an antioxidant present in red wine. RESULTS: In livers of hyperglycemic IRS2(-/-) mice, the expression levels of PTP1B and its association with the insulin receptor (IR) were increased. The absence of PTP1B in the double-mutant mice restored hepatic IRS1-mediated phosphatidylinositol (PI) 3-kinase/Akt/Foxo1 signaling. Moreover, resveratrol treatment of hyperglycemic IRS2(-/-) mice decreased hepatic PTP1B mRNA and inhibited PTP1B activity, thereby restoring IRS1-mediated PI 3-kinase/Akt/Foxo1 signaling and peripheral insulin sensitivity. CONCLUSIONS: By regulating the phosphorylation state of IR, PTB1B determines sensitivity to insulin in liver and exerts a unique role in the interplay between IRS1 and IRS2 in the modulation of hepatic insulin action.

Murine models to investigate the influence of diabetic metabolism on the development of atherosclerosis and restenosis.

Atherosclerosis and related forms of cardiovascular disease (CVD) are associated with several genetic and environmental risk factors, including hypercholesterolemia, diabetes mellitus (DM), hypertension, obesity and smoking. Human DM is a multi-system disorder that results from progressive failure of insulin production and insulin resistance. Most diabetic patients die from complications of atherosclerosis and CVD, and DM is also associated with increased risk of restenosis post-angioplasty. Furthermore, the incidence of DM, particularly type 2-DM, is expected to increase significantly during the next decades owing to the unhealthy effects of modern life-style habits (e.g., obesity and lack of physical exercise). Thus, it is of utmost importance to develop novel preventive and therapeutic strategies to reduce the social and health-care burden of CVD and DM. Although a number of physiological alterations thought to promote atherosclerosis have been identified in diabetic patients, the precise molecular mechanisms that link DM and atherosclerosis are largely unknown. Thus, the aim of this review is to discuss current murine models of combined DM and atherosclerosis and to explore how these experimental systems are being utilized to gain mechanistic insights into diabetes-induced neointimal lesion development, as well as their potential use in evaluating the efficacy of new therapies. Our discussion includes models generated by streptozotocin treatment and those resulting from naturally occurring or targeted mutations in the mouse.

In vitro immunoblockade of VIP inhibits the proliferation of pituitary prolactin cells.

VIP is a peptide synthesised in the pituitary gland and is involved in the stimulation of prolactin secretion. However, to date it has not been determined whether VIP is able to regulate the proliferation of pituitary prolactin-producing cells, like other factors involved in the regulation of prolactin such as estradiol or dopamine. The aim of the present study was to address whether VIP is involved in regulating the proliferation of pituitary prolactin-secreting cells. Thus, we performed an in vitro study on monolayer cultures of rat pituitary cells, neutralising the possible paracrine effect of VIP by immunoblockade of the peptide and later determining the degree of proliferation of prolactin-secreting cells. The effects of immunoblockade were validated by determining the levels of VIP in the culture media, which were decreased (P < 0.01), and modifications in the patterns of the immunohistochemical reaction to prolactin-positive cells. Immunoblockade of VIP decreased the proliferation of pituitary prolactin-positive cells at all antibody concentrations analysed, mainly between 3 and 12 h (P < 0.01). Moreover, immunoblockade decreased the sizes of the cellular and nuclear areas, except at 1 h, at which point it only decreased the nuclear area of prolactin-positive cells. The results obtained suggest that-in the same way as it regulates the secretion of the hormone-VIP could be involved in regulating the proliferation of prolactin cells, like estradiol or dopamine.

Insulin receptor substrate 2 is essential for maturation and survival of photoreceptor cells.

Insulin receptor substrates (Irs-proteins) integrate signals from the insulin and insulin-like growth factor-1 (IGF1) receptors with other processes to control cellular growth, function, and survival. Here, we show that Irs2 promoted the maturation and survival of photoreceptors in the murine retina immediately after birth. Irs2 was mainly localized to the outer plexiform layer as well as to photoreceptor inner segments. It was also seen in ganglion cells and inner plexiform layer but in smaller amounts. Compared with control littermates, Irs2 knock-out mice lose 10% of their photoreceptors 1 week after birth and up to 50% by 2 weeks of age as a result of increased apoptosis. The surviving photoreceptor cells developed short organized segments, which displayed proportionally diminished but otherwise normal electrical function. However, IGF1-stimulated Akt phosphorylation was barely detected, and cleaved/activated caspase-3 was significantly elevated in isolated retinas of Irs2-/- mice. When diabetes was prevented, which allowed the Irs2-/- mice to survive for 2 years, most photoreceptor cells were lost by 16 months of age. Because apoptosis is the final common pathway in photoreceptor degeneration, pharmacological strategies that increase Irs2 expression or function in photoreceptor cells could be a general treatment for blinding diseases such as retinitis pigmentosa.

IRS-2 mediates the antiapoptotic effect of insulin in neonatal hepatocytes.

To assess the role of insulin action and inaction in the liver, immortalized hepatocyte cell lines have been generated from insulin receptor substrate (IRS)-2(-/-) and wild-type mice. Using this model, we have recently demonstrated that the lack of IRS-2 in neonatal hepatocytes resulted in insulin resistance. In the current study, we show that immortalized neonatal hepatocytes undergo apoptosis on serum withdrawal, with caspase-3 activation and DNA laddering occurring earlier in the absence of IRS-2. Insulin rescued wild-type hepatocytes from serum withdrawal-induced caspase-3 activation and DNA fragmentation in a dose-dependent manner, but it failed to rescue hepatocytes lacking IRS-2. In IRS-2(-/-) cells, insulin failed to phosphorylate Bad. Furthermore, in these cells, insulin was unable to translocate Foxo1 from the nucleus to the cytosol. Adenoviral infection of wild-type cells with constitutively active Foxo1 (ADA) induced caspase-8 and caspase-3 activities, proapoptotic gene expression, DNA laddering and apoptosis. Dominant negative Foxo1 regulated the whole pathway in an opposite manner. Prolonged insulin treatment (24 hours) increased expression of antiapoptotic genes (Bcl-xL), downregulated proapoptotic genes (Bim and nuclear Foxo1), and decreased caspase-3 activity in wild-type hepatocytes but not in IRS-2(-/-) cells. Infection of IRS-2(-/-) hepatocytes with adenovirus encoding IRS-2 reconstituted phosphatidylinositol 3-kinase (PI 3-kinase)/Akt/Foxo1 signaling, restored pro- and antiapoptotic gene expression, and decreased caspase-3 activity in response to insulin, thereby blocking apoptosis. In conclusion, IRS-2 signaling is specifically required through PIP3 generation to mediate the survival effects of insulin. Epidermal growth factor, via PIP3/Akt/Foxo1 phosphorylation, was able to rescue IRS-2(-/-) hepatocytes from serum withdrawal-induced apoptosis, modulating pro- and anti-apoptotic gene expression and downregulating caspase-3 activity.

Molecular mechanisms of insulin resistance in IRS-2-deficient hepatocytes.

To assess the role of insulin receptor (IR) substrate (IRS)-2 in insulin action and resistance in the liver, immortalized neonatal hepatocyte cell lines have been generated from IRS-2(-/-), IRS-2(+/-), and wild-type mice. These cells maintained the expression of the differentiated liver markers albumin and carbamoyl phosphate synthetase, as well as bear a high number of IRs. The lack of IRS-2 did not result in enhanced IRS-1 tyrosine phosphorylation or IRS-1-associated phosphatidylinositol (PI) 3-kinase activity on insulin stimulation. Total insulin-induced PI 3-kinase activity was decreased by 50% in IRS-2(-/-) hepatocytes, but the translocation of PI-3,4,5-trisphosphate to the plasma membrane in these cells was almost completely abolished. Downstream PI 3-kinase, activation of Akt, glycogen synthase kinase (GSK)-3 (alpha and beta isoforms), Foxo1, and atypical protein kinase C were blunted in insulin-stimulated IRS-2(-/-) cells. Reconstitution of IRS-2(-/-) hepatocytes with adenoviral IRS-2 restored activation of these pathways, demonstrating that IRS-2 is essential for functional insulin signaling in hepatocytes. Insulin induced a marked glycogen synthase activity in wild-type and heterozygous primary hepatocytes; interestingly, this response was absent in IRS-2(-/-) cells but was rescued by infection with adenoviral IRS-2. Regarding gluconeogenesis, the induction of phosphoenolpyruvate carboxykinase and glucose 6-phosphatase by dibutyryl cAMP and dexamethasone was observed in primary hepatocytes of all genotypes. However, insulin was not able to suppress gluconeogenic gene expression in primary hepatocytes lacking IRS-2, but when IRS-2 signaling was reconstituted, these cells recovered this response to insulin. Suppression of gluconeogenic gene expression in IRS-2-deficient primary hepatocytes was also restored by infection with dominant negative Delta 256Foxo1.

Ras-GRF1 signaling is required for normal beta-cell development and glucose homeostasis.

Development of diabetes generally reflects an inadequate mass of insulin-producing beta-cells. beta-cell proliferation and differentiation are regulated by a variety of growth factors and hormones, including insulin-like growth factor I (IGF-I). GRF1 is a Ras-guanine nucleotide exchange factor known previously for its restricted expression in brain and its role in learning and memory. Here we demonstrate that GRF1 is also expressed in pancreatic islets. Interestingly, our GRF1-deficient mice exhibit reduced body weight, hypoinsulinemia and glucose intolerance owing to a reduction of beta-cells. Whereas insulin resistance is not detected in peripheral tissues, GRF1 knockout mice are leaner due to increased lipid catabolism. The reduction in circulating insulin does not reflect defective glucose sensing or insulin production but results from impaired beta-cell proliferation and reduced neogenesis. IGF-I treatment of isolated islets from GRF1 knockouts fails to activate critical downstream signals such as Akt and Erk. The observed phenotype is similar to manifestations of preclinical type 2 diabetes. Thus, our observations demonstrate a novel and specific role for Ras-GRF1 pathways in the development and maintenance of normal beta-cell number and function.

Variations in the cellular proliferation of prolactin cells from late pregnancy to lactation in rats.

Lactation is a physiological process associated with hyperactivity of hypophyseal prolactin-producing cells. It is known that the percentage of these cells is increased during lactation, although there are discrepancies in the reports regarding the mechanisms responsible for increasing the number of prolactin cells. In order to analyse whether this increase is a result of previous proliferation, variations in the proliferation rate of prolactin-positive cells were determined from late pregnancy to lactation in adult female rats by means of observation of the immunohistochemical expression of PCNA as a marker of cellular proliferation. During late pregnancy, a very significant increase in the percentage of proliferating prolactin cells was observed in comparison to non-pregnant females in the proestrus phase (p < 0.01). Although the percentage of prolactin-positive cells after one week of lactation was higher than in non-lactating or in pregnant females (p < 0.01), the proliferation rate was lower than in the other groups studied. In sum, our results suggest that late pregnancy constitutes a preliminary proliferative phase preparatory to the ensuing lactation phase and that endocrine changes in late pregnancy involve the cellular proliferation of hypophyseal prolactin cells in order to prepare the gland for later demands and to prevent proliferative changes from occurring during lactation.