Durability of protective effect of dulaglutide on pancreatic ß-cells in diabetic mice: GLP-1 receptor expression is not reduced despite long-term dulaglutide exposure.

AIMS: It is well-known that chronic exposure to large amounts of ligand leads to downregulation of its receptor. It is not known, however, whether a GLP-1R agonist downregulates its receptor. For this reason, our study examined whether GLP-1R expression is reduced after long-term exposure to dulaglutide (Dula) in non-diabetic and diabetic mice. METHODS: Seven-week-old male db/db and db/m mice were given either Dula (0.6mg/kg×2/week) or a control vehicle (CTL) for 17 weeks. Various metabolic parameters, such as glucose-stimulated insulin secretion (GSIS), insulin and TG content in islets, were evaluated after the intervention. ß-cell-related gene expression was also analyzed by real-time RT-PCR. RESULTS: In db/m mice, GLP-1R expression in ß-cells did not decrease, not even after long-term administration of Dula, compared with control mice, while GLP-1R expression in 24-week-old db/db mice treated with Dula was augmented, rather than downregulated, compared with 24-week-old CTL db/db mice. This was probably due to improved glycaemic control. In db/db mice treated with Dula, food intake and blood glucose levels were significantly decreased up to 24 weeks of age compared with CTL db/db mice, and their expression levels of various ß-cell-related genes, insulin content and GSIS were also enhanced. In contrast, oxidative and endoplasmic reticulum stress, inflammation, fibrosis and apoptosis were suppressed with Dula treatment. CONCLUSION: Dula exerts beneficial effects on glycaemic control and has long-lasting protective effects on pancreatic ß-cells. GLP-1R expression levels were not reduced at all in non-diabetic as well as diabetic mice despite long-term dulaglutide exposure.

Transforming growth factor ß1 T868C gene polymorphism is associated with cerebral infarction in Japanese patients with type 2 diabetes.

It is likely that the C allele of the polymorphism at position 29 of the translated sequence of transforming growth factor (TGF)-ß1 gene, which codes a pleiotropic cytokine expressed in a variety of cells, is a susceptibility allele for cerebral infarction in Japanese type 2 diabetic patients.

Aldose reductase C-106T gene polymorphism is associated with diabetic retinopathy in Japanese patients with type 2 diabetes.

It is likely that the C allele of the polymorphism at position -106 in the promoter of aldose reductase gene, which codes a rate-limiting enzyme of the polyol pathway, is a susceptibility allele for diabetic retinopathy in Japanese type 2 diabetic patients.

Combined effect of oxidative stress-related gene polymorphisms on atherosclerosis.

It is well known that oxidative stress plays critical roles in the pathogenesis of atherosclerosis. In this study, we enrolled 1746 type 2 diabetic subjects, determined 4 common genetic variants related to oxidative stress (glutamate-cysteine ligase modifier subunit (GCLM) C-588T, myeloperoxidase G-463A, human paraoxonase 1 Gln192Arg and NAD(P)H oxidase p22phox C242T polymorphisms), and measured carotid intima-media thickness (IMT) as a surrogate marker for atherosclerosis. GCLM C-588T polymorphism was associated with average IMT (AveIMT) (r=0.090, p=0.0008), but the association between the other 3 polymorphisms and AveIMT did not reach the statistical significance. However, AveIMT was significantly greater as the total number of 4 concomitant "pro-oxidant alleles" in each subject was increased (r=0.108, p<0.0001). Furthermore, the number of "pro-oxidant alleles" was a risk factor for a high AveIMT independently of conventional risk factors (p=0.0003). In conclusion, accumulation of oxidative stress-associated alleles was associated with carotid atherosclerosis in type 2 diabetic patients.

Regeneration of beta cells in the native pancreata after syngeneic and allogeneic pancreas transplantations in spontaneously type 2 diabetic Torii rats.

BACKGROUND: We previously demonstrated that syngeneic pancreas transplantation has a potential to reverse diabetes even in a rat model of type 2 diabetes mellitus, namely Spontaneously Diabetic Torii (SDT; RT1a). The onset of diabetes was significantly delayed in the pancreas transplant recipients. We speculated that perfect diabetic control achieved by pancreas transplantation showed a beneficial effect on the native pancreata & recipients. MATERIALS AND METHODS: Twenty-five-week-old diabetic SDT rats were divided into 3 groups: untreated controls and syngeneic and allogeneic transplant recipients. We transplanted pancreaticoduodenal grafts from nondiabetic 10-week-old SDT rats and from 10-week-old allogeneic Dark Agouti (DA; RT1a) rats using daily administration of FK506. RESULTS: Untreated SDT rats showed disappearance of pancreatic and duodenal homeobox-1 (PDX-1) expression in the pancreas and a marked decrease in beta-cell mass. Among syngeneic and allogeneic pancreas transplant recipients, islet-like cell clusters were found in the native pancreata. The beta-cell mass at 40 weeks of age was significantly increased in the native pancreata of recipients compared with age-matched controls. Interestingly, we observed the reexpression of PDX-1 in the nuclei of islet-like cell clusters. CONCLUSIONS: Our results indicated the benefits of avoiding glucose toxicity by pancreas transplantation which induced PDX-1 expression in the native pancreata of type 2 diabetic recipients, resulting in regeneration of beta cells in the native pancreata.

Association of soluble CD40 ligand with carotid atherosclerosis in Japanese type 1 diabetic patients.

AIMS/HYPOTHESIS: It has recently been shown that the soluble form of CD40 ligand (sCD40L) interacts with CD40 on vascular cells, leading to a variety of proinflammatory responses, and that serum sCD40L levels can be a predictive marker of cardiovascular events. The aim of this study was to estimate sCD40L levels in type 1 diabetic patients to examine a possible association with carotid atherosclerosis. SUBJECTS AND METHODS: Human sCD40L levels in serum and intima-media thickness (IMT) of carotid artery were examined in 80 Japanese type 1 diabetic patients (27 men and 53 women, age 22.8+/-3.4 years (mean+/-SD), duration of diabetes 13.2+/-6.1 years) and 20 healthy age-matched non-diabetic individuals. RESULTS: Serum sCD40L levels were significantly (p=0.0185) higher in subjects with type 1 diabetes (2.10+/-1.33 ng/ml) compared with non-diabetic subjects (1.35+/-0.88 ng/ml). The greatest IMT (Max-IMT) and averaged IMT (Mean-IMT) were also significantly greater in patients with type 1 diabetes than in control subjects (0.73+/-0.14 vs 0.64+/-0.07 mm, p=0.0041, 0.63+/-0.09 vs 0.57+/-0.06 mm, p=0.0066, respectively). Levels of sCD40L were statistically significantly associated with Max-IMT (r=0.383, p<0.001) and Mean-IMT (r=0.275, p=0.0058). Furthermore, stepwise multivariate regression analyses demonstrated that sCD40L is a determinant of both Max- and Mean-IMT, independently of conventional risk factors. CONCLUSIONS/INTERPRETATION: It is suggested that increased levels of serum sCD40L are associated with accelerated atherosclerotic change observed in young patients with type 1 diabetes.

Increased stress protein ORP150 autoantibody production in Type 1 diabetic patients.

AIMS: Various genetic and environmental stresses interfere with protein folding in the endoplasmic reticulum (ER), which leads to the induction of ER stress. It has recently been reported that ER stress is involved in the development of diabetes in diabetic animal models. The aim of this study is to estimate ER stress levels in Type 1 diabetic patients. METHODS: We recruited Type 1 diabetic patients undergoing periodic follow-up examinations (n = 91) and healthy non-diabetic individuals (n = 37), and measured their serum anti-oxygen-related protein (ORP)150 autoantibody levels. RESULTS: Anti-ORP150 autoantibody levels in Type 1 diabetic patients were significantly higher compared with those in healthy non-diabetic subjects. Furthermore, the serum autoantibody levels in Type 1 diabetic patients correlated with HbA(1c) (F > 3.0, P = 0.079), indicating that hyperglycaemia itself induces ER stress in diabetes. CONCLUSIONS: Anti-ORP150 autoantibody levels in Type 1 diabetic patients are higher compared with non-diabetic subjects, suggesting that ER stress is increased in Type 1 diabetes.

Metformin or gliclazide, rather than glibenclamide, attenuate progression of carotid intima-media thickness in subjects with type 2 diabetes.

AIM/HYPOTHESIS: Metformin is a well-known oral hypoglycaemic agent and has been commonly used, in combination with sulphonylurea, to treat type 2 diabetes. However, the advantageous effect of metformin plus sulphonylurea on diabetic macroangiopathy has yet to be clarified. To evaluate whether sulphonylurea or sulphonylurea plus metformin prevent diabetic macroangiopathy, we examined the progression of carotid artery intima-media thickness (IMT) as a surrogate end point. METHODS: Subjects with type 2 diabetes were divided into three groups, receiving the following treatments: (i) glibenclamide (n=59); (ii) gliclazide (n=30); and (iii) glibenclamide + metformin (n=29). Maximum IMT and average IMT (the greatest value among 6 average values of each 3 points including greatest thickness) were measured at the beginning and end of the observation period. RESULTS: For the follow-up period of 3 years, the annual change in average IMT of the glibenclamide plus metformin group (0.003+/-0.048 mm) was smaller than that of the glibenclamide group (0.064+/-0.045 mm) and gliclazide group (0.032+/-0.036 mm) (p<0.0001 and p=0.043 respectively). In the gliclazide group, average IMT increased during the follow-up period, but annual change in average IMT was significantly smaller than that of the glibenclamide group (p=0.005). Glibenclamide + metformin or gliclazide also attenuated the progression of maximum IMT, compared with that of glibenclamide (0.041+/-0.105, 0.044+/-0.106, 0.114+/-0.131 mm/year respectively, p=0.029 and p=0.035 respectively). Multivariable regression analysis implied that administration of metformin or gliclazide significantly and independently (p<0.05) reduces the progression of average IMT, compared with glibenclamide monotherapy. CONCLUSIONS/INTERPRETATION: These data indicate that metformin or gliclazide, rather than glibenclamide, have a potent anti-atherogenic effect in type 2 diabetes.

Adenovirus-mediated gene transfer of glutamine: fructose-6-phosphate amidotransferase antagonizes the effects of interleukin-1beta on rat chondrocytes.

OBJECTIVE: To determine whether overexpression of glutamine: fructose-6-phosphate amidotransferase (GFAT) in synoviocytes will antagonize the response to interleukin-1beta (IL-1beta) of chondrocytes and synovial fibroblasts in co-culture. METHODS: Synovial fibroblasts from the rat were transduced by an adenovirus carrying the cDNA for GFAT and then co-cultured with rat chondrocytes encapsulated in alginate beads. Following challenge with 1, 5, or 10 ng/ml of IL-1beta for 24 h, proteoglycan synthesis by the chondrocytes was determined by incorporation of Na2(35)SO4. Production of nitric oxide (NO) and prostaglandin E2 (PGE2) were monitored by assay of conditioned medium from the co-culture. RESULTS: IL-1beta treatment of untransduced-synoviocyte/chondrocyte co-cultures resulted in markedly decreased proteoglycan synthesis by the chondrocytes, and increased NO and PGE2 levels in the culture medium. In contrast, adenovirus-mediated transfer of GFAT in synoviocytes prevented both the decrease in chondrocyte proteoglycan synthesis and increases in NO and PGE2 provoked by IL-1beta. CONCLUSIONS: Our study suggests that in a synoviocyte/chondrocyte co-culture system, overexpression of GFAT by synoviocytes significantly inhibits subsequent stimulation by IL-1beta in vitro. Since GFAT is the rate limiting enzyme in the synthesis of intracellular glucosamine and its derivatives, these results may open new possibilities for osteoarthritis treatment.

Ectopically expressed PDX-1 in liver initiates endocrine and exocrine pancreas differentiation but causes dysmorphogenesis.

To date, the potency of pancreatic and duodenal homeobox gene 1 (PDX-1) in inducing differentiation into insulin-producing cells has been demonstrated in some cells and tissues. In order to carry out efficient screening of somatic tissues and cells that can transdifferentiate into beta-cell-like cells in response to PDX-1, we generated CAG-CAT-PDX1 transgenic mice carrying a transgene cassette composed of the chicken beta-actin gene (CAG) promoter and a floxed stuffer DNA sequence (CAT) linked to PDX-1 cDNA. When the mice were crossed with Alb-Cre mice, which express the Cre recombinase driven by the rat albumin gene promoter, PDX-1 was expressed in more than 50% of hepatocytes and cholangiocytes. The PDX-1 (+) livers expressed a variety of endocrine hormone genes such as insulin, glucagon, somatostatin, and pancreatic polypeptide. In addition, they expressed exocrine genes such as elastase-1 and chymotrypsinogen 1B. However, the mice exhibited marked jaundice due to conjugated hyperbilirubinemia, and the liver tissue displayed abnormal lobe structures and multiple cystic lesions. Thus, the in vivo ectopic expression of PDX-1 in albumin-producing cells was able to initiate but not complete the differentiation of liver cells into pancreatic cells. The conditional PDX-1 transgenic mouse system developed in this study appeared to be useful for efficient screening of PDX-1 responsive somatic tissues and cells.

[Usefulness of CT-guided RFA for hepatocellular carcinoma].

US-guided puncture is the simplest and most popular method in the RFA treatment for HCC. However, depending on the location of tumors, it is often difficult to detect them by US. We report here the utility of CT-guided RFA for the treatment of HCC. We performed CT-guided RFA for 27 nodules in 21 patients with HCC from July 1999 to June, 2001. We used the LeVeen Needle Electrode made by Boston Company and the Cool-tip type electrode made by Radionics Company. We judged the effects of the treatment by dynamic CT within 7 days after RFA. We were able to accomplish the treatment for all patients with the exception of one case who developed severe pain during RFA. We experienced transient increases of AST/ALT in a few cases, subcutaneous emphysema in one case, pleural effusion and ascites in two cases, but conservative treatments were effective for all cases. US-guided puncture was especially useful for the treatment of the tumors localized below the diaphragm that were hardly detectable by US.