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Hiroyuki Konya,1 Yuzo Yano,1 Satoshi Matsutani,1 Taku Tsunoda,2 Takashi Ikawa,2 Yoshiki Kusunoki,2 Toshihiro Matsuo,2 Masayuki Miuchi,2 Tomoyuki Katsuno,3 Tomoya Hamaguchi,4 Jun-ichiro Miyagawa,2 Mitsuyoshi Namba21Department of Internal Medicine, Ashiya Municipal Hospital, Ashiya, 2Division of Diabetes, Endocrinology and Metabolism, Department of Internal Medicine, 3Division of Innovative Diabetes Treatment, Hyogo College of Medicine, Nishinomiya, 4Division of Diabetes, Department of Internal Medicine, Itami City Hospital, Itami, Hyogo, JapanAbstract: Saxagliptin is a selective and potent dipeptidyl peptidase (DPP)-4 inhibitor, approved as an adjunct to diet and exercise to improve glycemic control in type 2 diabetes mellitus (T2DM) in the USA on July 2009, and had been launched globally in over 86 countries by September 2013. In patients with T2DM, once-daily administration of saxagliptin before breakfast achieves sustained inhibition of plasma DPP-4 activity and reduction of postprandial hyperglycemia, including after dinner, associated with an increase in plasma glucagon-like peptide-1 levels. This paper reviews the safety and efficacy of saxagliptin in Japanese patients with T2DM. The clinical development study in Japan supported its usefulness for the disease. Saxagliptin 1, 2.5, and 5 mg led to significant improvements in glycated hemoglobin (HbA1c), and was generally well tolerated. Treatment with saxagliptin 5 mg induced a sustained reduction in HbA1c over 52 weeks. Long-term combination therapy with saxagliptin and other oral hypoglycemic agents also provided sustained glycemic control and was well tolerated for up to 52 weeks. Saxagliptin as add-on to sulfonylureas or glinides has a tendency to increase hypoglycemia, but not with other oral antidiabetic agents, such as α-glucosidase inhibitors, metformin, or thiazolidinediones. The results of clinical trials have confirmed the long-term efficacy and safety of saxagliptin monotherapy as well as its use as add-on combination therapy, and support its usefulness as a therapeutic agent for T2DM. Saxagliptin has less concern for hypoglycemia and weight gain, which often becomes problematic in routine care of T2DM. Meta-analysis of clinical trials in the USA showed no evidence of increased risk of cardiovascular events associated with saxagliptin, suggesting the superior of saxagliptin in terms of safety. Recently, investigators in the SAVOR-TIMI (Saxagliptin Assessment of Vascular Outcomes Recorded in Patients with Diabetes Mellitus-Thrombolysis in Myocardial Infarction) 53 study suggested that DPP-4 inhibition with saxagliptin did not increase or decrease the rate of ischemic events, although the rate of hospitalization for heart failure was increased. Although saxagliptin improves glycemic control, other approaches are necessary to reduce cardiovascular risk in patients with diabetes. Saxagliptin is applicable for various pathological conditions, and is considered to be clinically significant as a new therapeutic option for Japanese patients with T2DM.Keywords: dipeptidyl peptidase-4, incretin hormones, saxagliptin, type 2 diabetes mellitus, Japan, efficacy, safety, patient acceptability

Hiroyuki Konya,1 Tomoyuki Katsuno,2 Taku Tsunoda,1 Yuzo Yano,1 Mai Kamitani,1 Masayuki Miuchi,2 Tomoya Hamaguchi,3 Jun-Ichiro Miyagawa,2 Mitsuyoshi Namba2 1Department of Internal Medicine, Ashiya Municipal Hospital, Ashiya, Hyogo, Japan; 2Division of Diabetes, Endocrinology and Metabolism, Department of Internal Medicine, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan; 3Division of Innovative Diabetes Treatment, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan Abstract: Patients with diabetes mellitus are at increased risk from cardiovascular-related morbidity and mortality as compared with healthy individuals. An association between the postprandial metabolic state and atherogenesis has been observed in patients with diabetes mellitus. In the Study to Prevent Non-Insulin-Dependent Diabetes Mellitus (STOP-NIDDM), treatment with an α-glucosidase inhibitor (α-GI) in patients with impaired glucose tolerance not only reduced the rate of conversion from impaired glucose tolerance to type 2 diabetes mellitus (T2DM), but was also associated with a reduction in the risk of cardiovascular events. These results suggested the importance of treating postprandial hyperglycemia in the early stages of T2DM. Glinides are rapid and short-acting insulin secretagogues that bind to the sulfonylurea receptors on pancreatic β-cells to facilitate rapid insulin secretion, restore postprandial early insulin secretion, and reduce the postprandial glucose spike. Moreover, α-GIs reduce postprandial hyperglycemia and insulin secretion by delaying the digestion of carbohydrates and polysaccharides in the small intestine. Then, both glinides and α-GI have beneficial effects for treating patients with T2DM and impaired glucose tolerance. Considering the ameliorating effects of these drugs on postprandial metabolic disorders, combinations of glinides and α-GI might constitute a promising therapeutic strategy for managing patients with T2DM, and also appear to be suitable for Japanese people, who consume more carbohydrates, such as polished rice, than Caucasians. It has recently been reported that combined use of mitiglinide and voglibose reduces postprandial insulin secretion and blunts diurnal glycemic changes in T2DM patients. This therapy can thus be regarded as being suitable for achieving strict postprandial glycemic control. In this report, we outline the effects of this combination therapy on postprandial plasma glucose and assess its safety. Keywords: mitiglinide, voglibose, combination therapy, type 2 diabetes mellitus, postprandial hyperglycemia

Background and Objective: Pancreatic fat is a form of ectopic fat. Lipid droplets (LDs) are also observed in β cells; however, the pathophysiological significance, especially for β cell function, has not been elucidated. Our aim was to assess LD accumulation in β cells in various stages of glucose intolerance and to clarify its relationship with clinical and histological parameters., Methods: We examined 42 Japanese patients who underwent pancreatectomy. The BODIPY493/503-positive (BODIPY-positive) area in β cells was measured in pancreatic sections from 32 patients. The insulin granule numbers were counted in an additional 10 patients using electron microscopy., Results: The BODIPY-positive area in β cells in preexisting type 2 diabetes patients was higher than that in normal glucose tolerance patients (p = 0.031). The BODIPY-positive area in β cells was positively correlated with age (r = 0.45, p = 0.0097), HbA1c (r = 0.38, p = 0.0302), fasting plasma glucose (r = 0.37, p = 0.045), and homeostasis model assessment insulin resistance (r = 0.41, p = 0.049) and negatively correlated with an increase in the C-peptide immunoreactivity level by the glucagon test (r = -0.59, p = 0.018). The ratio of mature insulin granule number to total insulin granule number was reduced in the patients with rich LD accumulation in β cells (p = 0.039)., Conclusions: Type 2 diabetes patients had high LD accumulation in β cells, which was associated with insulin resistance, hyperglycemia, aging and β cell dysfunction involving decreased mature insulin granules., Competing Interests: J-IM is employed by Keiseikai Medical Corporation, Osaka, Japan. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Horii, Kozawa, Fujita, Kawata, Ozawa, Ishibashi, Yoneda, Nammo, Miyagawa, Eguchi and Shimomura.)

Introduction: This trial was conducted to assess the long-term safety, efficacy, and benefit of early add-on of linagliptin to insulin in patients with type 2 diabetes mellitus (T2DM)., Methods: This trial enrolled 246 subjects. The subjects were randomized to the linagliptin group or the control group and were observed for 156 weeks. After week 16, subjects in the control group were also allowed to add linagliptin to evaluate the benefit of early add-on of linagliptin to insulin. The primary end point was a change in HbA1c from baseline to week 16. Secondary end points included fasting plasma glucose, daily insulin dose, and frequency of adverse events., Results: HbA1c and fasting plasma glucose levels significantly decreased from baseline to week 16 in the linagliptin group compared with the control group. The significant improvement in HbA1c continued until week 52. The daily insulin dose significantly decreased in the linagliptin group compared with the control group. The frequency of hypoglycemia and adverse events was comparable in both groups., Conclusions: Add-on of linagliptin to insulin was tolerated, improved glycemic control, and reduced the daily insulin dose. This study demonstrates the long-term safety, efficacy and benefit of early add-on of linagliptin to insulin in Japanese T2DM patients.

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.

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.

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.

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.

CD47 modulates a variety of cell functions such as adhesion, spreading, and migration. Using a fusion protein consisting of the extracellular region of Src homology 2 domain bearing protein tyrosine phosphatase substrate-1 (SHPS-1) and the Fc portion of human Ig (SHPS-1-Ig) we investigated the effects of SHPS-1 as a ligand for CD47 on B lymphocytes. Although SHPS-1-Ig binding to human B cell lines was solely mediated via CD47, their binding capacity for soluble and immobilized SHPS-1-Ig varied among cell lines irrespective of the similar expression levels of CD47, suggesting that distinctive affinity/avidity states exist during B cell maturation. Nalm6 cell line and tonsilar B lymphocytes adhered to immobilized SHPS-1-Ig and showed polarization-like morphology. These effects of SHPS-1-Ig were blocked by anti-CD47 mAbs (B6H12 and SE5A5). Wortmannin, a phosphatidylinositol-3 kinase inhibitor, but not pertussis toxin significantly inhibited the polarization induced by the immobilized SHPS-1-Ig. Thus, SHPS-1 acts as an adhesive substrate via CD47 in human B lymphocyte. Immunohistochemical analyses indicated that SHPS-1 is expressed on high endothelial venule as well as macrophages in human tonsils. HUVECs also express SHPS-1 in the absence of any stimuli, and the adhesion of tonsilar B lymphocytes to nonactivated HUVECs was significantly inhibited by SE5A5, indicating that SHPS-1/CD47 interaction is involved in the adhesion. Our findings suggest that SHPS-1/CD47 interaction may contribute to the recruitment of B lymphocytes via endothelial cells under steady state conditions.

Background/aims: Recent work has shown that the signal transducer and activator of transcription (STAT) 3 activation is important for the initiation of the proliferative response following partial hepatectomy (PH). However, the issue of where STAT3 is activated and how it is regulated is unclear. The aims of this study were to identify STAT3-activated cells and to clarify the expression of suppressor of cytokine signal (SOCS), a negative feedback molecule of STAT3, after PH., Methods: STAT3-activated cells and SOCS1-positive cells were identified by immunohistochemistry after a two-thirds PH in rats. SOCS mRNA was examined by Northern analysis., Results: STAT3 was activated in hepatocytes from those localized in the periportal zones of hepatic lobules after PH. STAT3 activation was also detected in Kupffer cells and sinusoidal endothelial cells prior to its detection in hepatocytes. After STAT3 activation, SOCS1 protein in response to PH was detected immunohistochemically in regenerating liver. SOCS1 and SOCS3 mRNA were induced in regenerating liver after PH., Conclusions: STAT3 signaling can occur in Kupffer cells and sinusoidal endothelial cells prior to in hepatocytes from those localized in the periportal zones. SOCS1 as well as SOCS3 may regulate STAT3 signaling negatively after PH.

Long-term exposure of the pancreatic beta cells to free fatty acid (FFA) reportedly inhibits glucose-stimulated insulin secretion. We here studied the impact of FFA on glucose and lipid metabolism in pancreatic beta cells with special reference to insulin secretion. Pancreatic beta-cell line MIN6 was exposed to various concentrations of palmitate for 3 days. Glucose-stimulated insulin secretion and insulin content were decreased corresponding to the concentration of the palmitate exposed. Glycolytic flux and ATP synthesis was unchanged, but pyruvate-stimulated change in NAD(P)H concentration was decreased. Pyruvate carboxylase was decreased at the protein level, which was restored by the removal of palmitate or the inhibition of beta-oxidation. Intracellular content of triglyceride and FFA were elevated, beta-oxidation was increased, and de novo lipogenesis from glucose was decreased. NADPH content and citrate output into the medium, which reflected pyruvate malate shuttle flux, were decreased, but malic enzyme activity was unaffected. The malic enzyme inhibitor alone inhibited insulin response to glucose. In conclusion, long-term exposure of FFA to beta cells inhibits glucose-stimulated insulin secretion via the decreased NADPH contents due to the inhibition of pyruvate carboxylase and malate pyruvate shuttle flux.

Mutations in the transcription factor hepatocyte nuclear factor-1alpha (HNF-1alpha; gene symbol TCF1) cause maturity-onset diabetes of the young type 3 (MODY3), a form of diabetes mellitus characterized by autosomal dominant inheritance, early onset, and pancreatic beta-cell dysfunction. Recent genetic studies, however, also found mutations in patients diagnosed with idiopathic (non-autoimmune based) type 1 diabetes. We identified a novel frameshift mutation (142delG) in the TCF1 gene in a family with a strong family history of type 1 diabetes and examined the functional properties of the mutant HNF 1alpha. The expression of the mutant protein was not detected in COS-7 cells by Western blot analysis after transfection of the mutant cDNA. This is the first case of an unstable mutant HNF-1alpha protein. Reporter gene analysis indicated that the mutant HNF-1alpha had no transactivation activity in HeLa and MIN6 cells. Haploinsufficiency for HNF-1alpha may lead to severe forms of diabetes like type 1 diabetes., (Copyright 2001 Wiley-Liss, Inc.)

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.

N-Acetylglucosaminyltransferase III (GnT-III) is a key enzyme that inhibits the extension of N-glycans by introducing a bisecting N-acetylglucosamine residue. In this study we investigated the effect of GnT-III on epidermal growth factor (EGF) signaling in HeLaS3 cells. Although the binding of EGF to the epidermal growth factor receptor (EGFR) was decreased in GnT-III transfectants to a level of about 60% of control cells, the EGF-induced activation of extracellular signal-regulated kinase (ERK) in GnT-III transfectants was enhanced to approximately 1.4-fold that of the control cells. A binding analysis revealed that only low affinity binding of EGF was decreased in the GnT-III transfectants, whereas high affinity binding, which is considered to be responsible for the downstream signaling, was not altered. EGF-induced autophosphorylation and dimerization of the EGFR in the GnT-III transfectants were the same levels as found in the controls. The internalization rate of EGFR was, however, enhanced in the GnT-III transfectants as judged by the uptake of (125)I-EGF and Oregon Green-labeled EGF. When the EGFR internalization was delayed by dansylcadaverine, the up-regulation of ERK phosphorylation in GnT-III transfectants was completely suppressed to the same level as control cells. These results suggest that GnT-III overexpression in HeLaS3 cells resulted in an enhancement of EGF-induced ERK phosphorylation at least in part by the up-regulation of the endocytosis of EGFR.

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.

During the late phase of megakaryopoiesis, megakaryocytes undergo polyploidization, which is characterized by DNA duplication without concomitant cell division. However, it remains unknown by which mechanisms this process occurs. AIM-1 and STK15 belong to the Aurora/increase-in-ploidy (Ipl)1 serine/threonine kinase family and play key roles in mitosis. In a human interleukin-3-dependent cell line, F-36P, the expressions of AIM-1 and STK15 mRNA were specifically observed at G2/M phase of the cell cycle during proliferation. In contrast, the expressions of AIM-1 and STK15 were continuously repressed during megakaryocytic polyploidization of human erythro/megakaryocytic cell lines (F-36P, K562, and CMK) treated with thrombopoietin, activated ras (H-ras(G12V)), or phorbol ester. Furthermore, their expressions were suppressed during thrombopoietin-induced polyploidization of normal human megakaryocytes. Activation of AIM-1 by the induced expression of AIM-1(wild-type) canceled TPA-induced polyploidization of K562 cells significantly, whereas that of STK15 did not. Moreover, suppression of AIM-1 by the induced expression of AIM-1 (K/R, dominant-negative type) led to polyploidization in 25% of K562 cells, whereas STK15(K/R) showed no effect. Also, the induced expression of AIM-1(K/R) in CMK cells provoked polyploidization up to 32N. These results suggested that downregulation of AIM-1 at M phase may be involved in abortive mitosis and polyploid formation of megakaryocytes.

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.

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.

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.

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.

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.

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.

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.

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.