Hepatocyte growth factor overexpression in the islet of transgenic mice increases beta cell proliferation, enhances islet mass, and induces mild hypoglycemia

Hepatocyte growth factor (HGF) is produced in pancreatic mesenchyme-derived cells and in islet cells. In vitro, HGF increases the insulin content and proliferation of islets. To study the role of HGF in the islet in vivo, we have developed three lines of transgenic mice overexpressing mHGF using the rat insulin II promoter (RIP). Each RIP-HGF transgenic line displays clear expression of HGF mRNA and protein in the islet. RIP-mHGF mice are relatively hypoglycemic in post-prandial and fasting states compared with their normal littermates. They display inappropriate insulin production, striking overexpression of insulin mRNA in the islet, and a 2-fold increase in the insulin content in islet extracts. Importantly, beta cell replication rates in vivo are two to three times higher in RIP-HGF mice. This increase in proliferation results in a 2-3-fold increase in islet mass. Moreover, the islet number per pancreatic area was also increased by approximately 50%. Finally, RIP-mHGF mice show a dramatically attenuated response to the diabetogenic effects of streptozotocin. We conclude that the overexpression of HGF in the islet increases beta cell proliferation, islet number, beta cell mass, and total insulin production in vivo. These combined effects result in mild hypoglycemia and resistance to the diabetogenic effects of streptozotocin.     

Targeted expression of placental lactogen in the beta cells of transgenic mice results in beta cell proliferation, islet mass augmentation, and hypoglycemia

The factors that regulate pancreatic beta cell proliferation are not well defined. In order to explore the role of murine placental lactogen (PL)-I (mPL-I) in islet mass regulation in vivo, we developed transgenic mice in which mPL-I is targeted to the beta cell using the rat insulin II promoter. Rat insulin II-mPL-I mice displayed both fasting and postprandial hypoglycemia (71 and 105 mg/dl, respectively) as compared with normal mice (92 and 129 mg/dl; p < 0.00005 for both). Plasma insulin concentrations were inappropriately elevated, and insulin content in the pancreas was increased 2-fold. Glucose-stimulated insulin secretion by perifused islets was indistinguishable from controls at 7.5, 15, and 20 mm glucose. Beta cell proliferation rates were twice normal (p = 0. 0005). This hyperplasia, together with a 20% increase in beta cell size, resulted in a 2-fold increase in islet mass (p = 0.0005) and a 1.45-fold increase in islet number (p = 0.0012). In mice, murine PL-I is a potent islet mitogen, is capable of increasing islet mass, and is associated with hypoglycemia over the long term. It can be targeted to the beta cell using standard gene targeting techniques. Potential exists for beta cell engineering using this strategy.     

Intrasplenic xenotransplantation of human fetal pancreatic islet cell cultures in rats with experimental diabetes mellitus

Islet cell cultures obtained from the pancreas of human embryos were transplanted to the spleen pulp of rats with alloxan diabetes mellitus. During 1-2 weeks after transplantation, 6 of the 8 recipients manifested a decrease in glycemia to normal or almost normal. The antidiabetic effect of xenotransplantation of islet cell cultures was well preserved throughout the entire observation period (up to 4 months). Two recipient rats with stable normoglycemia were subjected to splenectomy. One week after operation the animals manifested the recurrent grave diabetic status. Histological study of the removed spleen has shown the red pulp to contain the accumulations of implanted islet cells.     

Insulin resistance (HOMA) in relation to plasma cortisol,IGF-I and IGFBP-3. A study in normal short-statured and GH-deficient children

OBJECTIVE: To investigate the possible contribution of plasma cortisol and growth hormone (GH) as reflected by insulin-like growth factor-I (IGF-I)/insulin-like growth factor-binding protein-3 (IGFBP-3) on insulin action in short-statured children. METHODS: In this study, insulin resistance (HOMA) was determined in 34 normal short-statured (age 9.4 +/- 3.5 years) and in 19 GH-deficient children (age 10.4 +/- 2.2 years). HOMA was examined in relation to fasting plasma cortisol, IGF-I, IGFBP-3 and in addition to birthweight and body mass index (BMI). RESULTS: Birthweight was not correlated to insulin resistance. In GH-deficient children, BMI was significantly augmented and was associated with HOMA (p < 0.02). In both groups of patients, fasting plasma cortisol was related to HOMA (normal: r = 0.295, p < 0.05, GH-deficient: r = 0.495, p < 0.02). Only in normal short-statured children IGF-I (r = 0.338, p < 0.03) and IGFBP-3 (r = 0.493, p < 0.002) were associated with insulin resistance. CONCLUSION: The results indicated that at a young age cortisol contributed to insulin resistance in short-statured children. In normal short-statured children HOMA was associated with IGF-I and IGFBP-3. Possibly GH, a known cause of insulin resistance, contributed to HOMA as IGF-I and IGFBP-3 do not mediate insulin resistance but reflect growth hormone secretion. The results in GH-deficient children supported this conclusion as in the absence of GH insulin resistance was not associated with IGF-I/IGFBP-3.     

Beta cell function, peripheral sensitivity to insulin and islet cell autoimmunity in cystic fibrosis patients with normal glucose tolerance

Beta cell function, peripheral sensitivity to insulin and specific pancreatic autoimmunity were studied in 30 youngsters with cystic fibrosis (CF) accurately selected in order to fulfill the criteria for normal glucose tolerance. With respect to weight-matched controls, patients with CF exhibited a significantly lower glucose tolerance and a globally preserved, although delayed, insulin response to oral glucose tolerance test, while first-phase insulin secretion after i.v. glucose was blunted. Peripheral sensitivity to insulin, assessed in vivo by both the euglycemic clamp technique and the number of insulin receptors, directly measured in circulating monocytes, was superimposable in patients and controls. Serum islet-cell antibodies were not found in any of the patients. In conclusion, disorders of beta cell function may be observed in CF patients even when glucose tolerance is within the normal range. Such abnormalities are not associated with changes in peripheral sensitivity to insulin and do not seem to depend on specific autoimmune events.     

Selective deletion of Pten in pancreatic beta cells leads to increased islet mass and resistance to STZ-induced diabetes

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a lipid phosphatase. PTEN inhibits the action of phosphatidylinositol-3-kinase and reduces the levels of phosphatidylinositol triphosphate, a crucial second messenger for cell proliferation and survival, as well as insulin signaling. In this study, we deleted Pten specifically in the insulin producing beta cells during murine pancreatic development. Pten deletion leads to increased cell proliferation and decreased cell death, without significant alteration of beta-cell differentiation. Consequently, the mutant pancreas generates more and larger islets, with a significant increase in total beta-cell mass. PTEN loss also protects animals from developing streptozotocin-induced diabetes. Our data demonstrate that PTEN loss in beta cells is not tumorigenic but beneficial. This suggests that modulating the PTEN-controlled signaling pathway is a potential approach for beta-cell protection and regeneration therapies.     

IFN-gamma induces islet cell MHC antigens and enhances autoimmune, streptozotocin-induced diabetes in the mouse

To explore the role of the lymphokine, IFN-gamma, in the development of autoimmune-mediated insulin-dependent diabetes, we examined the effects of systemically administered IFN-gamma on the clinical features and pancreatic immunohistology of CBA mice made diabetic with multiple low doses of the pancreatic islet beta-cell toxin, streptozotocin. Mice given streptozotocin and IFN-gamma were significantly more hyperglycemic than those given streptozotocin alone and had significantly decreased body weight. Mice given IFN-gamma alone did not differ in glycemia or weight from vehicle-injected mice. On day 11, Ia proteins were detected on islet cells from mice given streptozotocin and their expression was potentiated by IFN-gamma; they could not be detected on islet cells from mice given IFN-gamma alone or vehicle. H-2K protein expression was increased on islet cells from mice given streptozotocin and was potentiated by IFN-gamma. IFN-gamma alone also increased H-2K protein expression on islet cells compared with vehicle-treated mice. These findings show that IFN-gamma enhances the severity of diabetes in mice given multiple-low doses of streptozotocin, in association with enhanced expression of Ia and H-2K proteins on islet cells. They indicate an important role for IFN-gamma in amplifying the autoimmune process leading to beta-cell destruction in diabetes. The ability of IFN-gamma to worsen autoimmune disease has implications for its use in man.     

Production of immunoreactive insulin-like growth factor I and response to exogenous IGF-I in small cell lung cancer cell lines

Small cell lung cancer (SCLC) cell lines were examined for the presence of insulin-like growth factor I-related protein (IGF-I) in cell pellets and culture media. IGF-I immunoreactivity was detected in 11/14 pellets, ranging from 12 to 76 mIU/mg soluble protein. The IGF-I levels in the cell pellets showed a correlation to the corresponding culture media. IGF-I binding sites were found in all tested cell lines. The maximum binding (Bmax) ranged from 131 to 1230 fmol/mg protein and the dissociation constant (KD) from 0.89 to 5.21 nM. The incorporation of [3H]thymidine in the presence of recombinant human IGF-I resulted in a clearly increased DNA synthesis in two of seven cell lines. Thus, IGF-I may be an important growth factor in SCLC.     

Cyclin D1 overexpression induces epidermal growth factor-independent resistance to apoptosis linked to BCL-2 in human A431 carcinoma

Overexpression of EGF receptors and constitutive cyclin D1 expression are frequently associated with human squamous carcinomas. We have now investigated whether these parameters influence susceptibility to okadaic acid induced cell death in EGF-receptor overexpressing mutant p53 A431 human carcinoma. Exposure of these cells to 20 nM okadaic acid induced apoptosis-associated caspase 3 activation, DNA fragmentation, cleavage of Poly ADP-Ribose Polymerase (PARP), p53-independent expression of pro-apoptotic bax, and loss of proliferation-promoting cyclin D1. All these alterations were antagonized by concurrent addition of exogenous EGF. Ectopic overexpression of the cyclin D1 gene in A431 carcinoma conferred resistance to 20 nM okadaic acid irrespective of exogenous EGF, associated with a parallel induction of anti-apoptotic bcl-2. Treatment with a subtoxic concentration of a bispecific bcl-2/bcl xL antisense oligonucleotide cooperated with okadaic acid to down-regulate bcl-2 and sensitize cyclin D1-overexpressing cells to okadaic acid. Although EGF protects EGF-R proficient epithelial cells from diverse apoptotic stimuli through Mcl-1, this is the first report demonstrating that cyclin D1 overexpression provides an EGF independent protection from okadaic acid-induced cell death through induction of bcl-2. We also show that this anti-apoptotic effect of cyclin D1 overexpression, can be partly antagonized with antisense strategies that down-regulate anti-apoptotic bcl-2 family members.     

Nerve growth factor prevents both neuroretinal programmed cell death and capillary pathology in experimental diabetes.

BACKGROUND: Chronic diabetes causes structural changes in the retinal capillaries of nearly all patients with a disease duration of more than 15 years. Acellular occluded vessels cause hypoxia, which stimulates sight-threatening abnormal angiogenesis in 50% of all type I diabetic patients. The mechanism by which diabetes produces acellular retinal capillaries is unknown. MATERIALS AND METHODS: In this study, evidence of programmed cell death (PCD) was sought in the retinas of early diabetic rats, and the effect of nerve growth factor (NGF) on PCD and capillary morphology was evaluated. RESULTS: Diabetes induced PCD primarily in retinal ganglion cells (RGC) and Muller cells. This was associated with a transdifferentiation of Muller cells into an injury-associated glial fibrillary acidic protein (GFAP)-expressing phenotype, and an up-regulation of the low-affinity NGF receptor p75NGFR on both RGC and Muller cells. NGF treatment of diabetic rats prevented both early PCD in RGC and Muller cells, and the development of pericyte loss and acellular occluded capillaries. CONCLUSIONS: These data provide new insight into the mechanism of diabetic retinal vascular damage, and suggest that NGF or other neurotrophic factors may have potential as therapeutic agents for the prevention of human diabetic retinopathy.     

Insulin-like growth factor I (IGF-I) and insulin binding to erythrocytes of normal prepubertal children and adults.

Erythrocyte insulin-like growth factor I (IGF-I) and insulin receptors were characterized in 10 normal prepubertal children (5 girls and 5 boys) aged 4-11 yrs and 10 normal adults (4 women and 6 men) aged 32-47 yrs. erythrocytes were purified from 5 ml of blood by Ficoll-Paque gradient centrifugation. Reticulocytes count in the erythrocyte suspensions were lower than 1%. Insulin and IGF-I binding assays were performed simultaneously. Maximal percent binding of [125I] labelled IGF-I was significantly higher in prepubertal children than in adults (8.7 +/- 0.7% versus 6.2 +/- 0.5% at a concentration of 5 x 10(9) erythrocytes/ml). Scatchard analysis revealed the high affinity constant was better in prepubertal children (Ka = 4.6 +/- 1.3 nM-1 versus 1.8 +/- 0.2 nM-1), whereas the binding capacity was similar (5.8 +/- 1.1 versus 7.7 +/- 0.8 high affinity binding sites/cell). In both groups, unlabelled IGF-I inhibited tracer-binding half maximally at about 1 nM. Insulin was 100-fold less potent. In adults, specific binding of [125I] labelled IGF-I was higher in women (7.6 +/- 0.7%) than in men (5.3 +/- 0.4%). No significant difference was observed in maximal specific binding of [125I] labelled insulin between prepubertal children (8.2 +/- 0.5%) and adults (7.2 +/- 0.7%). In both groups, competition by unlabelled insulin for [125I] labelled insulin binding gave 50% displacement for approximately 0.25 nM and IGF-I was about 80-fold less potent. Both IGF-I and insulin binding parameters were not significantly correlated with plasma hormone levels. In prepubertal children, the high-affinity IGF-I receptors number decreased with increasing high-affinity insulin receptors number.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chronic alcohol consumption,type 2 diabetes mellitus,insulin-like growth factor-I (IGF-I), and growth hormone (GH) in ethanol-treated diabetic rats

Aims

Alcohol has deleterious influences on glucose metabolism which may contribute to the development of type 2 diabetes mellitus (T2DM). Insulin-like growth factor I (IGF-I) and growth hormone (GH), which interact with insulin to modulate metabolic control, have been shown to be related to impaired glucose tolerance. This study was conducted to assess the possibility that altered circulating IGF-I and GH levels contribute to the exacerbation of T2DM by alcohol use in type 2 diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats and non-diabetic Long-Evans Tokushima Otsuka (LETO) rats.

Main method

OLETF rats were pair-fed a Lieber-DeCarli Regular Ethanol diet and LETO rats were pair-fed a control diet for 6 weeks. At 6 weeks, an Intraperitoneal Glucose Tolerance Test (IP-GTT) was performed and IGF-I and GH levels were evaluated.

Key findings

Prior to an IP-GTT, OLETF-Ethanol (O-E) group had significantly a decrease in the mean glucose levels compared to OLETF-Control (O-C) group. At 120 min post IP-GTT, the O-E group had significantly an increase in the mean glucose levels compared to O-C group. The serum IGF-I levels were significantly lower and the serum GH levels were significantly higher in the O-E group than in L-C group.

Significance

These results suggest that IGF-I and GH are prominent in defining the risk and development of T2DM, and may be adversely affected by heavy alcohol use, possibly mediating its diabetogenic effects. Thus, the overall glucose intolerance in the setting of alcoholism may be attributable to inappropriate alteration of IGF-I and GH levels.     

NIM1 overexpression in Arabidopsis potentiates plant disease resistance and results in enhanced effectiveness of fungicides

The NIM1 (for noninducible immunity, also known as NPR1) gene is required for the biological and chemical activation of systemic acquired resistance (SAR) in Arabidopsis. Overexpression of NIM1 in wild-type plants (hereafter referred to as NIM1 plants or lines) results in varying degrees of resistance to different pathogens. Experiments were performed to address the basis of the enhanced disease resistance responses seen in the NIM1 plants. The increased resistance observed in the NIM1 lines correlated with increased NIM1 protein levels and rapid induction of PR1 gene expression, a marker for SAR induction in Arabidopsis, following pathogen inoculation. Levels of salicylic acid (SA), an endogenous signaling molecule required for SAR induction, were not significantly increased compared with wild-type plants. SA was required for the enhanced resistance in NIM1 plants, however, suggesting that the effect of NIM1 overexpression is that plants are more responsive to SA or a SA-dependent signal. This hypothesis is supported by the heightened responsiveness that NIM1 lines exhibited to the SAR-inducing compound benzo(1,2,3)-thiadiazole-7-car-bothioic acid S-methyl ester. Furthermore, the increased efficacy of three fungicides was observed in the NIM1 plants, suggesting that a combination of transgenic and chemical approaches may lead to effective and durable disease-control strategies.     

Immune islet killing mechanisms associated with insulin-dependent diabetes: in vitro expression of cellular and antibody-mediated islet cell cytotoxicity in humans

In previous work we have shown that some bacteria can bind to human lymphocytes and can be used to identify lymphocyte subpopulations in conventionally stained blood smears. These bacteria are of different species or genera, which makes it difficult to study the binding mechanism. Also, the main marker for B cells, Brucella melitensis, is of very small size and highly pathogenic. Here we show that B cells as well as some of the T cell subpopulations can be identified by different mutants obtained from a strain of an Escherichia coli. Two procedures were used to generate mutants. First, E. coli-YS57 (pro-his-trp-) was mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine and the binding to mouse spleen cells was used as a selective pressure. Second, phage-resistant mutants of E. coli-YS57 were obtained and tested for the ability to bind to lymphocytes. Out of 10 strains selected by the former procedure, 5 bound to a significant number of human lymphocytes. All four phage-resistant mutants bound to human lymphocytes. Out of the total of nine mutants that bound to lymphocytes, six bound consistently, i.e., they bound to similar percentages of peripheral blood lymphocytes from different normal donors. One phage-resistant mutant, E. coli USC-106, bound only to B cells. The subpopulations of lymphocytes identified by the mutants were essentially the same as those identified by different species or genera of bacteria. We concluded that E. coli mutants can be obtained that identify human lymphocyte subpopulations and that one of these mutants recognizes B cells; these mutants may be used to study the nature of the receptors for bacteria on lymphocytes, which appear to have a lectin-like nature.