Continuous stimulation of human glucagon-like peptide-1 (7-36) amide in a mouse model (NOD) delays onset of autoimmune type 1 diabetes.

AIMS/HYPOTHESIS: We examined the effect of glucagon-like peptide-1 (GLP-1) on the development of diabetes and islet morphology in NOD mice by administering GLP-1 to prediabetic mice. METHODS: Eight-week-old female NOD mice were infused subcutaneously with human GLP-1 via a mini-osmotic pump for 4 or 8 weeks. In mice treated with GLP-1 for 4 weeks, blood glucose levels and body weight were measured. An intraperitoneal glucose tolerance test (IPGTT) and evaluation of insulitis score were also performed. Beta cell area, proliferation, apoptosis, neogenesis from ducts and subcellular localisation of forkhead box O1 (FOXO1) were examined by histomorphometrical, BrdU-labelling, TUNEL, insulin/cytokeratin and FOXO1/insulin double-immunostaining methods, respectively. RESULTS: Mice treated with human GLP-1 for 4 weeks had lower blood glucose levels until 2 weeks after completion of treatment, showing improved IPGTT data and insulitis score. This effect continued even after cessation of the treatment. In addition to the increase of beta cell neogenesis, BrdU labelling index was elevated (0.24 vs 0.13%, p < 0.001), while apoptosis was suppressed by 54.2% (p < 0.001) in beta cells. Beta cell area was increased in parallel with the translocation of FOXO1 from the nucleus to the cytoplasm. The onset of diabetes was delayed in mice treated with GLP-1 for 4 weeks, while mice treated with GLP-1 for 8 weeks did not develop diabetes by age 21 weeks compared with a 60% diabetes incidence in control mice at this age. CONCLUSIONS/INTERPRETATION: Continuous infusion of human GLP-1 to prediabetic NOD mice not only induces beta cell proliferation and neogenesis, but also suppresses beta cell apoptosis and delays the onset of type 1 diabetes.

Production of adiponectin, an anti-inflammatory protein, in mesenteric adipose tissue in Crohn's disease.

BACKGROUND AND AIMS: A characteristic feature of Crohn's disease (CD) is mesenteric adipose tissue hypertrophy. Mesenteric adipocytes or specific proteins secreted by them may play a role in the pathogenesis of CD. We recently identified adiponectin as an adipocyte specific protein with anti-inflammatory properties. Here we report on expression of adiponectin in mesenteric adipose tissue of CD patients. METHODS AND RESULTS: Mesenteric adipose tissue specimens were obtained from patients with CD (n = 22), ulcerative colitis (UC) (n = 8) and, for controls, colon carcinoma patients (n = 28) who underwent intestinal resection. Adiponectin concentrations were determined by enzyme linked immunosorbent assay, and adiponectin mRNA levels were determined by real time quantitative reverse transcription-polymerase chain reaction. Tissue concentrations and release of adiponectin were significantly increased in hypertrophied mesenteric adipose tissue of CD patients compared with normal mesenteric adipose tissue of CD patients (p = 0.002, p = 0.040, respectively), UC patients (p = 0.002, p = 0.003), and controls (p<0.0001, p<0.0001). Adiponectin mRNA levels were significantly higher in hypertrophied mesenteric adipose tissue of CD patients than in paired normal mesenteric adipose tissue from the same subjects (p = 0.024). Adiponectin concentrations in hypertrophied mesenteric adipose tissue of CD patients with an internal fistula were significantly lower than those of CD patients without an internal fistula (p = 0.003). CONCLUSIONS: Our results suggest that adipocytes in hypertrophied mesenteric adipose tissue produce and secrete significant amounts of adiponectin, which could be involved in the regulation of intestinal inflammation associated with CD.

High titres of IgA antibodies to enterovirus in fulminant type-1 diabetes.

AIMS/HYPOTHESIS: We have recently proposed that fulminant type-1 diabetes is a novel subtype of type-1 diabetes with abrupt onset of insulin-deficient hyperglycaemia without islet-related autoantibodies. The pathogenesis is still unknown, but flu-like symptoms are frequently observed before the onset of disease of this subtype. Enterovirus infection is a candidate environmental factor causing type-1 diabetes. The aim of this study was to determine whether enterovirus infection contributes to the development of fulminant type-1 diabetes. METHODS: We investigated 19 patients with recent-onset fulminant type-1 diabetes, 18 patients with recent-onset typical type-1A diabetes, and 19 healthy controls. IgM, IgG, and IgA subclasses of antibodies to enterovirus were determined by ELISA. RESULTS: IgA antibody titres to enterovirus were significantly higher in fulminant type-1 diabetes than in typical type-1A diabetes (p=0.033) and controls (p=0.0003). IgM antibodies to enterovirus were not detected in any subject. IgG titres were lower in autoimmune diabetes than fulminant type and controls (p=0.014 and 0.019, respectively). CONCLUSIONS/INTERPRETATION: High titres of enterovirus IgA antibodies in serum suggest recurrent enterovirus infection in fulminant type-1 diabetic patients, indicating higher susceptibility to enteroviral infections among them. Such infections might have pathogenetic importance in the triggering of fulminant type-1 diabetes.

Plasma adiponectin levels in women with anorexia nervosa.

Adiponectin is a plasma protein exclusively secreted by adipose tissue, which plays a role in modulating lipid and glucose metabolism. The plasma adiponectin concentration shows an inverse correlation with the body mass index in normal and obese individuals, but it has not been investigated in subjects with an extremely low body weight and undernutrition such as anorexia nervosa patients. We investigated plasma adiponectin levels in 21 females with anorexia nervosa. Nineteen healthy females served as the lean control group. The subjects with anorexia nervosa had a significantly lower weight and showed a tendency towards higher adiponectin levels than the control group. No correlation between adiponectin and BMI was found in patients with anorexia nervosa, while a linear negative correlation was seen in lean controls. The patient who showed the lowest adiponectin level reached a life-threatening state and required intravenous feeding in hospital. In association with improved nutrition and weight gain, the adiponectin level increased gradually until the body mass index was about 16 and then decreased subsequently as would be expected in lean normal subjects. These observations suggest that adipose tissue secretes less adiponectin and the adiponectin levels do not show an inverse correlation simply with body mass index in some subjects with severe undernutrition.

Pancreatic biopsy as a procedure for detecting in situ autoimmune phenomena in type 1 diabetes: close correlation between serological markers and histological evidence of cellular autoimmunity.

To better understand the pathogenesis of type 1 diabetes, we have developed pancreatic biopsy under laparoscope for recent-onset type 1 diabetic patients. The patients included 29 acute-onset type 1 diabetic patients, 5 latent-onset type 1 diabetic patients, and 1 type 2 diabetic patient. Their median age was 28 years, and the duration of diabetes at the time of biopsy was approximately 3 months. In 31 of 35 patients, we could obtain the pancreas tissue by punching. No serious complications, such as heavy bleeding, peritonitis, or pancreatitis, have been experienced. Pneumoderma was observed in two patients, and abdominal dull pain had continued for 2 days in two patients. However, special treatment was not necessary for these complications. T-cell-predominant infiltration to islets (insulitis) and hyperexpression of major histocompatibility complex class I antigens on islet cells were the two major findings and were observed in 17 of 29 recent-onset type 1 diabetic patients. These findings could be regarded as evidence of immune attack against beta-cells, and their presence was closely correlated with the presence of either anti-GAD or anti-IA-2 antibodies (P = 0.02). In conclusion, pancreatic biopsy under laparoscope is a safe procedure without serious complications, according to our findings, for detecting in situ autoimmune phenomenon in recent-onset type 1 diabetic patients.

Ectopic expression of alpha1,6 fucosyltransferase in mice causes steatosis in the liver and kidney accompanied by a modification of lysosomal acid lipase.

The alpha1,6 fucosyltransferase (alpha1,6 FucT) catalyzes the transfer of a fucose from GDP-fucose to the innermost GlcNAc residue of N-linked glycans via an alpha1,6 linkage. alpha1,6 FucT was overexpressed in transgenic mice under the control of a combined cytomegalovirus and chicken beta-actin promoter. Histologically numerous small vacuoles, in which lipid droplets had accumulated, were observed in hepatocytes and proximal renal tubular cells. Electron microscopic studies showed that the lipid droplets were membrane-bound and apparently localized within the lysosomes. Cholesterol esters and triglycerides were significantly increased in liver and kidney of the transgenic mice. Liver lysosomal acid lipase (LAL) activity was significantly lower in the transgenic mice compared to the wild mice, whereas LAL protein level, which was detected immunochemically, was increased, indicating that the specific activity of LAL was much lower in the transgenic mice. In all of the transgenic and nontransgenic mice examined, the activity of liver LAL was negatively correlated with the level of alpha1,6 FucT activity. As evidenced by lectin and immunoblot analysis, LAL was found to be more fucosylated in the transgenic mice, suggesting that the aberrant fucosylation of LAL causes an accumulation of inactive LAL in the lysosomes. Such an accumulation of inactive LAL could be a likely cause for a steatosis in the lysosomes of the liver and kidney in the case of the alpha1,6 FucT transgenic mice.

Ectodomain shedding of epidermal growth factor receptor ligands is required for keratinocyte migration in cutaneous wound healing.

Keratinocyte proliferation and migration are essential to cutaneous wound healing and are, in part, mediated in an autocrine fashion by epidermal growth factor receptor (EGFR)-ligand interactions. EGFR ligands are initially synthesized as membrane-anchored forms, but can be processed and shed as soluble forms. We provide evidence here that wound stimuli induce keratinocyte shedding of EGFR ligands in vitro, particularly the ligand heparin-binding EGF-like growth factor (HB-EGF). The resulting soluble ligands stimulated transient activation of EGFR. OSU8-1, an inhibitor of EGFR ligand shedding, abrogated the wound-induced activation of EGFR and caused suppression of keratinocyte migration in vitro. Soluble EGFR-immunoglobulin G-Fcgamma fusion protein, which is able to neutralize all EGFR ligands, also suppressed keratinocyte migration in vitro. The application of OSU8-1 to wound sites in mice greatly retarded reepithelialization as the result of a failure in keratinocyte migration, but this effect could be overcome if recombinant soluble HB-EGF was added along with OSU8-1. These findings indicate that the shedding of EGFR ligands represents a critical event in keratinocyte migration, and suggest their possible use as an effective clinical treatment in the early phases of wound healing.

Integrin-associated protein/CD47 regulates motile activity in human B-cell lines through CDC42.

Cell migration requires a dynamic interaction between the cell, its substrate, and the cytoskeleton-associated motile apparatus. Integrin-associated protein (IAP)/CD47 is a 50-kd cell surface protein that is physically associated with beta3 integrins and that modulates the functions of beta3 integrins in various cells. However, in B-lymphocytes that express beta1 integrins but few beta3 integrins, the roles of IAP/CD47 remain to be determined. Cross-linking of IAP/CD47 by the immobilized anti-IAP/CD47 monoclonal antibody (mAb) B6H12, but not 2D3, produced signals to promote polarization with lamellipodia, a characteristic morphology during leukocyte migration, in pre-B and mature B-cell lines (BALL, Nalm6, ONHL-1, Daudi), but not in myeloma cell lines (RPMI8226, OPM-2). In the presence of the immobilized fibronectin (FN), soluble B6H12 could increase the rate of the polarization and activate migratory activity of BALL cells to FN in a transwell filter assay. Furthermore, the dominant-negative form of CDC42 completely blocked B6H12-induced morphologic and functional changes without inhibiting phorbol 12-myristate 13-acetate-induced spreading on FN in BALL cells, whereas the dominant-negative form of Rac1 inhibited all these changes. These findings demonstrate that in B-lymphocytes, IAP/CD47 may transduce the signals to activate the migratory activity, in which CDC42 may be specifically involved, and that IAP/CD47 shows synergistic effect with alpha4beta1 on B-cell migration. These findings would provide new insight into the role of IAP/CD47 on B-cell function.

Stable overexpression of the glucose-6-phosphatase catalytic subunit attenuates glucose sensitivity of insulin secretion from a mouse pancreatic beta-cell line.

Glucose-6-phosphatase (G-6-Pase) hydrolyzes glucose-6-phosphate to glucose, reciprocal with the so-called glucose sensor, glucokinase, in pancreatic beta cells. To study the role of G-6-Pase in glucose-stimulated insulin secretion from beta cells, we have introduced rat G-6-Pase catalytic subunit cDNA and have established permanent clones with 3-, 7- and 24-fold G-6-Pase activity of the mouse beta-cell line, MIN6. In these clones, glucose usage and ATP production in the presence of 5.5 or 25 mM glucose were reduced, and glucose-stimulated insulin secretion was decreased in proportion to the increased G-6-Pase activity. In addition, insulin secretory capacity in response to d-fructose and pyruvate was unchanged; however, 25 mM glucose-stimulated insulin secretion and intracellular calcium response were completely inhibited. In the clone with 24-fold G-6-Pase activity, changes in intracellular NAD(P)H autofluorescence in response to 25 mM glucose were reduced, but the changes with 20 mM fructose and 20 mM pyruvate were not altered. Stable overexpression of G-6-Pase in beta cells resulted in attenuation of the overall glucose-stimulated metabolic responses corresponding to the degree of overexpression. This particular experimental manipulation shows that the possibility exists of modulating glucose-stimulated insulin release by thoroughly altering glucose cycling at the glucokinase/G-6-Pase step.

Mutation/polymorphism scanning of glucose-6-phosphatase gene promoter in noninsulin-dependent diabetes mellitus patients.

Glucose-6-phosphatase (G6Pase) catalyzes the rate-limiting step of gluconeogenesis, and hepatic G6Pase activity is increased in diabetes. We have cloned and analyzed the human G6Pase gene promoter region and identified putative regulatory sequences for insulin, cAMP, glucocorticoid, and hepatocyte nuclear factors. The promoter region of the G6Pase gene was analyzed in 154 noninsulin-dependent diabetes mellitus patients and 90 control subjects by PCR-single strand conformation polymorphism and direct sequencing methods. Polymorphisms were not found in any subjects. The results suggested that in noninsulin-dependent diabetic patients, the major cause of the hepatic glucose overproduction was not attributed to dysregulation of the G6Pase gene due to mutation/polymorphism of its promoter region.

Demonstration of two different processes of beta-cell regeneration in a new diabetic mouse model induced by selective perfusion of alloxan.

To clarify the regeneration process of pancreatic beta-cells, we established a new mouse model of diabetes induced by selective perfusion of alloxan after clamping the superior mesenteric artery. In this model, diabetes could be induced by the destruction of beta-cells in alloxan-perfused segments, while beta-cells in nonperfused segments were spared. Intraperitoneal glucose tolerance tests showed glucose intolerance, which gradually ameliorated and was completely normalized in 1 year with a concomitant increase of insulin content in the pancreas. Histological examination showed neo-islet formation in the alloxan-perfused segment and the proliferation of spared beta-cells in the nonperfused segment. In the alloxan-perfused segment, despite a marked reduction of islets in size and number at an early stage, both the number of islets, including islet-like cell clusters (ICCs), and the relative islet area significantly increased at a later stage. Increased single beta-cells and ICCs were located in close contact with duct cell lining, suggesting that they differentiated from duct cells and that such extra-islet precursor cells may be important for beta-cell regeneration in beta-cell-depleted segment. In addition to beta-cells, some nonhormone cells in ICCs were positive for nuclear insulin promoter factor 1, which indicated that most, if not all, nonhormone cells positive for this factor were beta-cell precursors. In the nonperfused segment, the islet area increased significantly, and the highest 5-bromo-2-deoxyuridine-labeling index in beta-cells was observed at day 5, while the number of islets did not increase significantly. This indicated that the regeneration of islet endocrine cells occurs mostly through the proliferation of preexisting intra-islet beta-cells in the nonperfused segment. In conclusion, the regeneration process of beta-cells varied by circumstance. Our mouse model is useful for studying the mechanism of regeneration, since differentiation and proliferation could be analyzed separately in one pancreas.

Early morphologic changes of atherosclerosis induced by ventromedial hypothalamic lesion in the spontaneously diabetic Goto-Kakizaki rat.

It is generally thought that typical atherosclerotic lesions do not develop in the rodent. The Goto-Kakizaki (GK) rat is a nonobese strain in which a spontaneous type of non-insulin-dependent diabetes mellitus develops without apparent macroangiopathy. In our previous study, making ventromedial hypothalamic (VMH) lesions in GK rats induced hyperphagia and a further deterioration in glucose metabolism. In the current study, male GK rats in which VMH lesions were made were examined for vascular changes, with special reference to atherosclerotic lesions. Marked hyperglycemia in GK rats with VMH lesions (hereafter referred to as VMH lesion rats) was revealed over an observation period (plasma glucose levels 16 weeks after the operation: VMH lesion GK rats, 19.3 +/- 2.0 mmol/L, vs sham-operated GK rats, 10.1 +/- 1.3 mmol/L; p < 0.0001). Light microscopic observation of the descending aorta in VMH lesion GK rats 16 weeks after the surgery revealed that the intimal thickening and the number of infiltrating cells into the intima were significantly increased as compared with sham-operated GK rats (17531 +/- 3747 microm2 vs 3072 +/- 1192 microm2, p < 0.0001; 15.6 +/- 3.1 per one transverse section vs 6.8 +/- 2.5 per one transverse section, p < 0.0005). Electron microscopic observations demonstrated an increased number of microvilli and lysosomes in endothelial cells, infiltration of macrophages and lymphocytes into the intima, and migration of medial smooth muscle cells into the intima that are considered to be early events in atherosclerosis. These morphologic changes could be induced by a deterioration in glucose metabolism. This rat may thus be useful for studying the process of the initiation of atherosclerosis in diabetes mellitus.

Over-expression of bcl-xL gene in human gastric adenomas and carcinomas.

The present study was designed to clarify whether bcl-xL is involved in the development of carcinoma in the stomach. Levels of bcl-xL and bcl-2 mRNA were determined by a reverse-transcription/polymerase-chain reaction in endoscopic gastric biopsy specimens from 10 control subjects, 11 patients with adenomas and 14 patients with carcinomas. In 6 of 11 adenomas, 5 of 8 early carcinomas and 3 of 6 advanced carcinomas, the bcl-xL gene was over-expressed. In carcinomas, over-expression of the bcl-xL gene was observed in 6 of 9 intestinal-type carcinomas and 2 of 5 diffuse-type carcinomas. No correlation was observed between bcl-xL and bcl-2 gene expression. In cases in which the bcl-xL gene was over-expressed, an apparent increase in the protein level of Bcl-xL was observed by immunoblot analysis and intense Bcl-x immunoreactivity was detected immunohistochemically within the tumor cells. In conclusion, we showed that bcl-xL is over-expressed in gastric carcinomas at both the RNA and protein levels, suggesting that over-expression of bcl-xL may play a role in gastric carcinogenesis.

Suppression of human pancreatic cancer growth in BALB/c nude mice by manumycin, a farnesyl:protein transferase inhibitor.

Activating mutations of Ki-ras have been detected in most human pancreatic adenocarcinomas. Since Ras protein requires farnesylation to function, we investigated the effects of manumycin, a potent farnesyl:protein transferase inhibitor, on the growth in nude mice of a human pancreatic cancer cell line, MIA PaCa-2, with a point mutation in the Ki-ras gene. Tumor-bearing mice received intraperitoneal injection of 1 or 5mg/kg manumycin daily for 5 days, or 2 mg/kg manumycin daily for 2 weeks. Growth of inoculated tumors was significantly inhibited by the treatment. The treatment significantly (P<0.05) lowered the numbers of bromodeoxyuridine-incorporating tumor cells. Manumycin did not have apparent hepatotoxicity in vivo. Farnesyl:protein transferase inhibitors could offer a new approach for cancer chemotherapy.