Probucol attenuated hyperglycemia in multiple low-dose streptozotocin-induced diabetic mice.

The present studies were undertaken to examine the effects of probucol on the protection against pancreatic beta-cell damage by multiple low-dose streptozotocin (STZ: 40 mg/kg, ip). The degree of hyperglycemia at 7, 14 and 17 days after STZ injection was attenuated by probucol. Serum immunoreactive insulin (IRI) levels were increased in the rats fed probucol containing diet at Day 14 and 17. Serum IRI levels after intraperitoneal injection of 2.0 g/kg glucose was reduced in STZ mice and the reduction of serum IRI levels was attenuated in the rats fed probucol, accompanied with a significant reduction of the degree of hyperglycemia after bolus of glucose. Probucol attenuated the reduction of pancreatic IRI content by STZ. The percentage of Thy 1.2-positive splenocytes was increased by STZ and probucol reduced the percentage of Thy 1.2-positive splenocytes, although there were no differences in the populations of splenocytes, positive with Lyt 2 or L3/T4. These data suggest that probucol has a protective action against pancreatic insulitis by multiple low-dose STZ administration.     

Zinc effects on hyperglycemia and hypoleptinemia in streptozotocin-induced diabetic mice.

Zinc has an antihyperglycemic effect. Zinc can also influence the production of leptin, a satiety factor that reduces appetite and blood sugar level. In this study, we investigated the effect of zinc supplementation on food intake and circulating leptin and glucose concentrations in streptozotocin-induced diabetic mice. Male diabetic mice received zinc supplementation (20 ppm) from drinking water for two weeks. The results showed that zinc treatment did not affect body weight gain, body fat content or food intake in these diabetic mice. However, zinc supplementation markedly ameliorated the hyperglycemia of diabetic mice. After zinc treatment, serum leptin concentrations tended to increase in the diabetic mice. This study suggests that zinc is a mediator of leptin production.     

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.     

Comparison of nerve growth factor mRNA expression in cardiac and skeletal muscle in streptozotocin-induced diabetic mice

To address the role of nerve growth factor (NGF) in diabetes mellitus (DM)-induced cardiac autonomic neuropathy, we quantitated and compared the expression of NGF mRNA in the cardiac and the skeletal muscle in experimental DM mice with the RT-PCR-HPLC method, which we have developed previously, using a NGF deletion mutant RNA as an internal standard. DM was induced in ICR mice via intraperitoneal injection of streptozotocin. RT-PCR was performed using total RNA extracted from left ventricle and soleus muscle, and the levels of NGF mRNA were quantitated by HPLC analysis. NGF mRNA content of the cardiac muscle was 17-fold higher than the skeletal muscles in control mice. NGF mRNA content of the cardiac muscle in diabetic mice at 6 weeks was 4.0-fold higher than that in the control mice, while that of the skeletal muscle in diabetic mice was not different from the controls. These results indicated that the DM-induced increase in NGF mRNA content was higher in cardiac muscle than skeletal muscle, and that NGF might play an important role in cardiac autonomic neuropathy.     

Hepatocyte growth factor increases mitochondrial mass in glioblastoma cells

Hepatocyte growth factor/scatter factor (HGF) is a multifunctional growth factor that is linked to the initiation and/or progression of numerous malignancies. HGF also alters cancer cell responses to DNA damaging cytotoxic agents. Many cell responses to Met activation require alterations in metabolic activity but how the metabolic machinery responds to Met activation remains poorly defined. Treating human glioblastoma cells with HGF followed by the topoisomerase inhibitor camptothecin was found to increase the activity per cell of the mitochondrial respiratory chain enzyme succinate-tetrazolium reductase (>80% increase, p < 0.05) and the tricarboxylic acid cycle enzyme succinate dehydrogenase (>25% increase, p < 0.05). Treatment with either HGF or camptothecin alone had no effect on enzyme activity. The mitochondrial enzymatic response to HGF was dose- and time-dependent with the maximum increase occurring in cells pre-treated with 30 ng/ml HGF for 48h prior to camptothecin exposure. This enzymatic response was associated with a concurrent increase in mitochondrial mass of comparable magnitude (approximately 56%, p < 0.05) as measured by fluorescent mitochondrial staining and flow cytometry. The mitochondrial mass response to HGF was prevented by the MAP-kinase pathway inhibitor PD98059 and was unaffected by the phosphatidylinositol 3-kinase inhibitors LY294002 and wortmannin. These findings suggest that HGF influences cell responses to chemotherapeutic stress, in part, by altering mitochondrial functions through a MAP-kinase dependent increase in mitochondrial mass.     

Hepatocyte growth factor significantly suppresses collagen-induced arthritis in mice

Hepatocyte growth factor (HGF) plays an important role in angiogenesis, cell proliferation, antifibrosis, and antiapoptosis. Moreover, recent studies have highlighted the immunosuppressive effect of HGF in animal models of allogenic heart transplantation and autoimmune myocarditis and in studies in vitro as well. We also reported that HGF significantly suppresses dendritic cell function, thus down-regulating Ag-induced Th1-type and Th2-type immune responses in allergic airway inflammation. However, the immunosuppressive effect of HGF in many other situations has not been fully clarified. In the present study, using a mouse model of collagen-induced arthritis (CIA) and experiments in vitro, we examined the effect of HGF on autoimmune arthritis and then elucidated the mechanisms of action of HGF. To achieve sufficient delivery of HGF, we used biodegradable gelatin hydrogels as a carrier. HGF suppressed Ag-induced T cell priming by regulating the functions of dendritic cells in the Ag-sensitization phase with down-regulation of IL-10. In contrast, under continuous Ag stimulation HGF induced IL-10-producing immunocytes both in vivo and in vitro. Moreover, HGF potently inhibited the development of CIA with enhancing the Th2-type immune response. We also confirmed that HGF significantly suppressed the production of IL-17 by immunocytes. These results indicate that HGF suppresses the development of CIA through different ways at different phases. They also suggest that HGF could be an attractive tool for treating patients with rheumatoid arthritis.     

Beneficial effects of Phellodendri Cortex extract on hyperglycemia and diabetic nephropathy in streptozotocin-induced diabetic rats

This study investigated the effect of Phellodendri Cortex extract on hyperglycemia and diabetic nephropathy in streptozotocin-induced diabetic rats. Male Sprague-Dawley rats were divided into normal control (NC), diabetic control (DC), and diabetic treatment with Phellodendri Cortex extract (DP). Over a 4-week experimental period, Phellodendri Cortex extract was administered orally at 379 mg/kg BW/day. The final fasting serum glucose level, urine total protein level, and relative left kidney weight in the DP group were significantly lower than the DC group. Renal XO and SOD activities in the DP group were significantly lower than the DC group and renal CAT activity in the DP group was significantly higher than the DC group. Tubular epithelial change was reduced in the DP group compared to the DC group. These results indicated that Phellodendri Cortex can reduce glucose level and prevent or retard the development of diabetic nephropathy in streptozotocin-induced diabetic rats.     

Nerve growth factor increases in pancreatic beta cells after streptozotocin-induced damage in rats

We investigated short-term in vivo and in vitro effects of streptozotocin (STZ) on pancreatic beta cells. Male Wistar rats were treated with 75 mg/kg STZ, and, after 4 hrs blood glucose and insulin were measured and islet cells were isolated, cultured for 16 hrs, and challenged with 5.6 and 15.6 mM glucose. Treated rats showed hyperglycemia (approximately 14 mM) and a 70% decrease in serum insulin levels as compared with controls. Although insulin secretion by isolated beta cells from STZ-treated rats was reduced by more than 80%, in both glucose concentrations, nerve growth factor (NGF) secretion by the same cells increased 10-fold. Moreover, NGF messenger RNA (mRNA) expression increased by 30% as compared with controls. Similar results were obtained in an in vitro model of islet cells, in which cells were exposed directly to STZ for 1, 2, and 4 hrs and then challenged for 3 hrs with the same glucose concentrations. Our data strongly suggest that an early increase in NGF production and secretion by beta cells could be an endogenous protective response to maintain cell survival and that diabetes mellitus may occur when this mechanism is surpassed.     

Hepatocyte growth factor is a potent angiogenic factor which stimulates endothelial cell motility and growth

beta-Nerve growth factor (NGF) is expressed in spermatogenic cells and has testosterone-downregulated low-affinity receptors on Sertoli cells suggesting a paracrine role in the regulation of spermatogenesis. An analysis of the stage-specific expression of NGF and its low affinity receptor during the cycle of the seminiferous epithelium in the rat revealed NGF mRNA and protein at all stages of the cycle. Tyrosine kinase receptor (trk) mRNA encoding an essential component of the high-affinity NGF receptor was also present at all stages. In contrast, expression of low affinity NGF receptor mRNA was only found in stages VIIcd and VIII of the cycle, the sites of onset of meiosis. The low-affinity NGF receptor protein was present in the plasma membrane of the apical Sertoli cell processes as well as in the basal plasma membrane of these cells at stages VIIcd to XI. NGF was shown to stimulate in vitro DNA synthesis of seminiferous tubule segments with preleptotene spermatocytes at the onset of meiosis while other segments remained nonresponsive. We conclude that NGF is a meiotic growth factor that acts through Sertoli cells.     

Functional alterations in pancreatic beta cells as a factor in virus-induced hyperglycemia in mice

Alterations in the functional capacity of pancreatic beta cells appear to contribute to coxsackievirus B4-induced, long-term hyperglycemia in mice. Mice infected with prototype B4 or its diabetogenic E2 variant were monitored for abnormalities in sugar metabolism (by the glucose tolerance test), for total protein and insulin synthesis in intact beta cells, for alterations in beta cell proteins, and for virus replication. The infected mice were hypoglycemic at 72 h postinfection and hyperglycemic at 6 weeks. At 8 weeks postinfection, few of the prototype- but most of the E2-infected mice remained hyperglycemic. Total protein and synthesis of immunoprecipitable insulin decreased during early infection. At 8 weeks postinfection, insulin synthesis in the prototype-infected mice increased almost to the level of control mice. Although insulin synthesis increased likewise in the E2-infected mice, it remained well below the control level. Two-dimensional gel electrophoresis revealed the disappearance of many cellular proteins in beta cells from E2-infected mice but of very few in cells from prototype-infected mice at 72 h postinfection. Many of the disappearing proteins reappeared gradually in the E2-infected group. Infectious virus was recovered from the infected beta cells only at 72 h postinfection. Functional impairment in these cells appears to be a factor in virus-induced hyperglycemia.     

Loss of autophagy diminishes pancreatic beta cell mass and function with resultant hyperglycemia

Autophagy is a cellular degradation-recycling system for aggregated proteins and damaged organelles. Although dysregulated autophagy is implicated in various diseases including neurodegeneration, its role in pancreatic beta cells and glucose homeostasis has not been described. We produced mice with beta cell-specific deletion of Atg7 (autophagy-related 7). Atg7 mutant mice showed impaired glucose tolerance and decreased serum insulin level. beta cell mass and pancreatic insulin content were reduced because of increased apoptosis and decreased proliferation of beta cells. Physiological studies showed reduced basal and glucose-stimulated insulin secretion and impaired glucose-induced cytosolic Ca2+ transients in autophagy-deficient beta cells. Morphologic analysis revealed accumulation of ubiquitinated protein aggregates colocalized with p62, which was accompanied by mitochondrial swelling, endoplasmic reticulum distension, and vacuolar changes in beta cells. These results suggest that autophagy is necessary to maintain structure, mass and function of pancreatic beta cells, and its impairment causes insulin deficiency and hyperglycemia because of abnormal turnover and function of cellular organelles.     

Biotin amelioration of nephrotoxicity in streptozotocin-induced diabetic mice

The current study was carried out to investigate the protective role of biotin in kidney injury and oxidative stress in diabetic mice type 1. Male Swiss albino mice were randomly divided into 3 groups. Control group received saline. Diabetes type 1 was induced in second and third groups by intraperitoneal injection of streptozotocin as a single dose (150 mg/kg). Second group remained as the untreated diabetic group and the third group received 15 mg/kg daily oral dose of biotin for 12 successive days. Biochemical results showed significant elevation in blood glucose and urea levels in both diabetic groups. Also, there is an increase in glomerular areas and decrease in glomerular cellularity in both diabetic groups. Histopathological results showed severe alterations in the untreated diabetic group represented by distorted glomeruli, inflammatory cells, and giant macrophages. In addition, there was an intense immune-reaction response toward acrolein indicator of oxidative damage. Upon biotin administration of diabetic mice, the above mentioned histopathological changes were reduced and also acroline reaction of oxidative damage was diminished. Our findings prove that biotin has a protective role against streptozotocin-induced oxidative damage in kidneys of laboratory mice.     

Hepatocyte growth factor induces epithelial cell motility through transactivation of the epidermal growth factor receptor

Hepatocyte growth factor (HGF) is a potent inducer of motility in epithelial cells. Since we have previously found that activation of the epidermal growth factor receptor (EGFR) is an absolute prerequisite for induction of motility of corneal epithelial cells after wounding, we investigated whether induction of motility in response to HGF is also dependent on activation of the EGFR. We now report that HGF induces transactivation of the EGFR in an immortalized line of corneal epithelial cells, in human skin keratinocytes, and in Madin-Darby canine kidney cells. EGFR activation is unconditionally required for induction of motility in corneal epithelial cells, and for induction of a fully motile phenotype in Madin-Darby canine kidney cells. Activation of the EGFR occurs through amphiregulin and heparin-binding epidermal growth factor-like growth factor. Early after HGF stimulation, blocking EGFR activation does not inhibit extracellular-signal regulated kinase 1/2 (ERK1/2) activation by HGF, but the converse is seen after approximately 1 h, indicating the existence of EGFR-dependent and -independent routes of ERK1/2 activation. In summary, HGF induces transactivation of the EGFR in epithelial cells, and this is a prerequisite for induction of full motility.     

Petalonia improves glucose homeostasis in streptozotocin-induced diabetic mice

The anti-diabetic potential of Petalonia binghamiae extract (PBE) was evaluated in vivo. Dietary administration of PBE to streptozotocin (STZ)-induced diabetic mice significantly lowered blood glucose levels and improved glucose tolerance. The mode of action by which PBE attenuated diabetes was investigated in vitro using 3T3-L1 cells. PBE treatment stimulated 3T3-L1 adipocyte differentiation as evidenced by increased triglyceride accumulation. At the molecular level, peroxisome proliferator-activated receptor γ (PPARγ) and terminal marker protein aP2, as well as the mRNA of GLUT4 were up-regulated by PBE. In mature adipocytes, PBE significantly stimulated the uptake of glucose and the expression of insulin receptor substrate-1 (IRS-1). Furthermore, PBE increased PPARγ luciferase reporter gene activity in COS-1 cells. Taken together, these results suggest that the in vivo anti-diabetic effect of PBE is mediated by both insulin-like and insulin-sensitizing actions in adipocytes.     

Leptin downregulates ghrelin levels in streptozotocin-induced diabetic mice

Ghrelin, an orexigenic peptide produced in the stomach, is increased in streptozotocin (STZ)-induced diabetic (DM) mice. This study clarifies the regulation of ghrelin levels by leptin in STZ-DM mice. STZ-DM mice had higher plasma ghrelin concentrations and greater ghrelin mRNA expression than control mice. Changes in ghrelin levels were dose dependently attenuated by the subcutaneous injection of leptin (0-27 nmol x kg(-1) x day(-1) over 7 days). Leptin treatment also partially reversed the hyperphagia and hyperglycemia observed in STZ-DM mice, but not the hypoinsulinemia, and there was a decrease in plasma ghrelin concentrations and ghrelin mRNA levels compared with STZ-LEP pair-fed mice. These results indicate that leptin treatment partially reverses elevated plasma ghrelin levels in STZ-DM mice independent of food intake and insulin, and suggest that hypoleptinemia in STZ-DM mice upregulates ghrelin.     

Contribution of neural cell death to depressive phenotypes of streptozotocin-induced diabetic mice

Major depression disorder (MDD) or depression is highly prevalent in individuals with diabetes, and the depressive symptoms are more severe and less responsive to antidepressant therapies in these patients. The underlying mechanism is little understood. We hypothesized that the pathophysiology of comorbid depression was more complex than that proposed for MDD and that neural cell death played a role in the disease severity. To test this hypothesis, we generated streptozotocin (STZ)-induced diabetic mice. These mice had blood glucose levels threefold above controls and exhibited depressive phenotypes as judged by a battery of behavioral tests, thus confirming the comorbidity in mice. Immunohistological studies showed markedly increased TUNEL-positive cells in the frontal cortex and hippocampus of the comorbid mice, indicating apoptosis. This finding was supported by increased caspase-3 and decreased Bcl-2 proteins in these brain regions. In addition, the serum brain-derived neurotrophic factor (BDNF) level of comorbid mice was reduced compared with controls, further supporting the neurodegenerative change. Mechanistic analyses showed an increased expression of mitochondrial fission genes fission protein 1 (Fis1) and dynamin-related protein 1 (Drp1), and a decreased expression of mitochondrial fusion genes mitofusin 1 (Mfn1), mitofusin 2 (Mfn2) and optical atrophy 1 (Opa1). Representative assessment of the proteins Drp1 and Mfn2 mirrored the mRNA changes. The data demonstrated that neural cell death was associated with the depressive phenotype of comorbid mice and that a fission-dominant expression of genes and proteins mediating mitochondrial dynamics played a role in the hyperglycemia-induced cell death. The study provides new insight into the disease mechanism and could aid the development of novel therapeutics aimed at providing neuroprotection by modulating mitochondrial dynamics to treat comorbid depression with diabetes.KEY WORDS: Depression, Diabetes, Comorbidity, Apoptosis, Neurodegeneration, Mitochondria     

Hepatocyte growth factor affects satellite cell activation and differentiation in regenerating skeletal muscle

Hepatocyte growth factor (HGF) is the onlyknown growth factor that activates quiescent satellite cells inskeletal muscle. We hypothesized that local delivery of HGF may enhanceregeneration after trauma by increasing the number of myoblastsavailable for restoring normal tissue architecture. Injection of HGFinto muscle at the time of injury increases myoblast number but doesnot enhance tissue repair as determined using quantitative histologicalanalyses. Rather, depending on the dose and the timing of HGFadministration relative to the injury, regeneration can be inhibited.The greatest inhibitory effect is observed when HGF is administered onthe day of injury and continued for 3 days, corresponding to the time when satellite cell activation, proliferation, and earlydifferentiation normally occur. To establish a mechanism for thisinhibition, we show that HGF can act directly on primary muscle cellsto block differentiation. These results demonstrate that1) exogenous HGF synergizes withfactors in damaged muscle to increase myoblast number,2) regeneration is not regulatedsolely by myoblast number, and 3)HGF inhibits muscle differentiation both in vitro and in vivo.

     

Genetic engineering of a suboptimal islet graft with A20 preserves beta cell mass and function

Transplantation of an excessive number of islets of Langerhans (two to four pancreata per recipient) into patients with type I diabetes is required to restore euglycemia. Hypoxia, nutrient deprivation, local inflammation, and the beta cell inflammatory response (up-regulation of NF-kappaB-dependent genes such as inos) result in beta cell destruction in the early post-transplantation period. Genetic engineering of islets with anti-inflammatory and antiapoptotic genes may prevent beta cell loss and primary nonfunction. We have shown in vitro that A20 inhibits NF-kappaB activation in islets and protects from cytokine- and death receptor-mediated apoptosis. In vivo, protection of newly transplanted islets would reduce the number of islets required for successful transplantation. Transplantation of 500 B6/AF(1) mouse islets into syngeneic, diabetic recipients resulted in a cure rate of 100% within 5 days. Transplantation of 250 islets resulted in a cure rate of only 20%. Transplantation of 250 islets overexpressing A20 resulted in a cure rate of 75% with a mean time to cure of 5.2 days, comparable to that achieved with 500 islets. A20-expressing islets preserve functional beta cell mass and are protected from cell death. These data demonstrate that A20 is an ideal cytoprotective gene therapy candidate for islet transplantation.     

The reversal of hyperglycemia after transplantation of mouse embryonic stem cells induced into early hepatocyte-like cells in streptozotocin-induced diabetic mice

Cellular replacement therapy is a potential therapeutic strategy for diabetes. In this study, we investigated the effect of transplantation of induced mouse embryonic stem cells (mESCs) into endoderm and early hepatocyte-like cells in streptozotocin (STZ)-diabetic mice. After embryoid body (EB) formation from mESC, the EBs were cultured in the presence of dexamethasone (DEX) and insulin for 4 days then was added acidic fibroblast growth factor (aFGF), hepatocyte growth factor (HGF) and oncostatin M (OSM) for 10 days, respectively. Blood glucose levels, intraperitoneal glucose tolerance (IGT) test and islet histology were assessed. The result revealed that transplantation of induced mESCs into early hepatocyte-like cells could repair pancreatic islets of control group. Blood glucose levels and intraperitoneal glucose tolerance test were significantly improved in test group compared to control group. Furthermore, there was significant increase in the number of islets in test group compared to control group. The findings declare that induced mESCs into endoderm and early hepatocyte-like cells, are appropriate candidate for regenerative therapy of pancreatic islets in type I diabetes.