Anti-hypertensive azelnidipine preserves insulin signaling and glucose uptake against oxidative stress in 3T3-L1 adipocytes.

It is known that reactive oxygen species (ROS) are involved in the development of insulin resistance as well as pancreatic ß-cell dysfunction both of which are often observed in type 2 diabetes. In this study, we evaluated the effects of azelnidipine, a calcium channel blocker, on ROS-mediated insulin resistance in adipocytes. When 3T3-L1 adipocytes were exposed to ROS, insulin-mediated glucose uptake was suppressed, but such phenomena were not observed in the presence of azelnidipine. Phosphorylation of insulin receptor and phosphorylation of Akt were suppressed by ROS, which was mitigated by azelnidipine treatment. Activation of the JNK pathway induced by ROS was also reduced by azelnidipine. Various inflammatory cytokine levels were increased by ROS, which was also suppressed by azelnidipine treatment. In contrast, adiponectin mRNA and secreted adiponectin levels were reduced by ROS, which was refilled by azelnidipine treatment. In conclusion, azelnidipine preserves insulin signaling and glucose uptake against oxidative stress in 3T3-L1 adipocytes.

Combination of DPP-4 inhibitor and PPARγ agonist exerts protective effects on pancreatic ß-cells in diabetic db/db mice through the augmentation of IRS-2 expression.

We investigated the effects of long- and short-term treatment with pioglitazone (Pio) and/or alogliptin (Alo) on ß-cells in diabetic db/db mice. Six-week-old male db/db mice received Pio (25 mg/kg, oral) and/or Alo (30 mg/kg, oral) for 4 weeks and for 2 days. Blood glucose levels were decreased after 4-week intervention, but not after 2-day intervention. Pio increased adiponectin levels, and Alo decreased glucagon levels and increased active GlP-1 levels. Insulin sensitivity was restored by Pio. After 4-week treatment, ß-cell mass was increased (over 2-fold increase) and expression levels of various ß-cell-related factors were restored. Expression levels of IRS-2 and various downstream factors were up-regulated by Pio and Alo after 2-day and 4-week intervention. In addition, mRNA and protein levels of IRS-2 and various downstream factors were up-regulated in MIN6 cells after 24-h exposure to Pio and exendin-4. These results suggest that Pio and Alo additively up-regulate IRS-2 expression independently of the alteration of glycemic control. Taken together, combination of Pio and Alo exerts protective effects on ß-cells in diabetic db/db mice, at least in part, through the augmentation of IRS-2 expression.

Dietary restriction preserves the mass and function of pancreatic ß cells via cell kinetic regulation and suppression of oxidative/ER stress in diabetic mice.

To assess the molecular mechanisms by which dietary restriction preserves the ß-cell mass and function in diabetic db/db mice. Male db/db mice were divided into two groups with or without diet restriction. Daily food intake of db/db mice was adjusted to that of the control db/m mice, which was determined in advance. A dietary restriction was implemented for 6 weeks from 6 weeks of age. Islet morphology, ß-cell function and gene expression profiles specific for pancreatic islet cells were compared. Food intake in db/m mice was 50% of that in db/db mice. Impaired glucose tolerance and insulin sensitivity were significantly ameliorated in db/db mice with dietary restriction. The pancreatic ß-cell mass was greater in mice with dietary restriction than that in mice without intervention. The dietary restriction significantly increased cyclin D gene expression and down-regulated CAD gene expression at 12 weeks compared with untreated db/db mice. Antiapoptotic bcl-2 gene expression was significantly increased, whereas genes related to oxidative stress, ER stress and inflammatory processes, such as NADPH oxidase, CHOP10 and TNF, were markedly down-regulated in mice with dietary restriction. Dietary restriction preserved the pancreatic ß-cell function and ß-cell mass in diabetic db/db mice, suggesting that alimentary therapy prevented ß-cell loss by suppressing cellular apoptosis and antioxidative stress in the pancreatic ß cells.

Protective effects of pioglitazone and/or liraglutide on pancreatic ß-cells in db/db mice: Comparison of their effects between in an early and advanced stage of diabetes.

The aim was to compare the protective effects of pioglitazone (PIO) and/or liraglutide (LIRA) on ß-cells with the progression of diabetes. Male db/db mice were treated with PIO and/or LIRA for 2 weeks in an early and advanced stage. In an early stage insulin biosynthesis and secretion were markedly increased by PIO and LIRA which was not observed in an advanced stage. In concomitant with such phenomena, expression levels of various ß-cell-related factors were up-regulated by PIO and LIRA only in an early stage. Furthermore, ß-cell mass was also increased by the treatment only in an early stage. Although there was no difference in apoptosis ratio between the two stages, ß-cell proliferation was augmented by the treatment only in an early stage. In conclusion, protective effects of pioglitazone and/or liraglutide on ß-cells were more powerful in an early stage of diabetes compared to an advanced stage.

Self-inducible secretion of glucagon-like peptide-1 (GLP-1) that allows MIN6 cells to maintain insulin secretion and insure cell survival.

Based on the hypothesis that MIN6 cells could produce glucagon-like peptide-1 (GLP-1) to maintain cell survival, we analyzed the effects of GLP-1 receptor agonist, exendin-4 (Ex4), and antagonist, exendin-(9-39) (Ex9) on cell function and cell differentiation. MIN6 cells expressed proglucagon mRNAs and produced GLP-1, which was accelerated by Ex4 and suppressed by Ex9. Moreover, Ex4 further enhanced glucose-stimulated GLP-1 secretion, suggesting autocrine loop-contributed amplification of the GLP-1 signal. Ex4 up-regulated cell differentiation- and cell function-related CREBBP, Pdx-1, Pax6, proglucagon, and PC1/3 gene expressions. The confocal laser scanning images revealed that GLP-1 positive cells were dominant in the early stage of cells, but positive for insulin were more prominent in the mature stage of cells. Ex4 accelerated cell viability, while Ex9 and anti-GLP-1 receptor antibody enhanced cell apoptosis. MIN6 cells possess a mechanism of GLP-1 signal amplification in an autocrine fashion, by which the cells maintained insulin production and cell survival.

Molecular mechanism by which pioglitazone preserves pancreatic beta-cells in obese diabetic mice: evidence for acute and chronic actions as a PPARgamma agonist.

Pioglitazone preserves pancreatic beta-cell morphology and function in diabetic animal models. In this study, we investigated the molecular mechanisms by which pioglitazone protects beta-cells in diabetic db/db mice. In addition to the morphological analysis of the islets, gene expression profiles of the pancreatic islet were analyzed using laser capture microdissection and were compared with real-time RT-PCR of db/db and nondiabetic m/m mice treated with or without pioglitazone for 2 wk or 2 days. Pioglitazone treatment (2 wk) ameliorated dysmetabolism, increased islet insulin content, restored glucose-stimulated insulin secretion, and preserved beta-cell mass in db/db mice but had no significant effects in m/m mice. Pioglitazone upregulated genes that promote cell differentiation/proliferation in diabetic and nondiabetic mice. In db/db mice, pioglitazone downregulated the apoptosis-promoting caspase-activated DNase gene and upregulated anti-apoptosis-related genes. The above-mentioned effects of pioglitazone treatment were also observed after 2 days of treatment. By contrast, the oxidative stress-promoting NADPH oxidase gene was downregulated, and antioxidative stress-related genes were upregulated, in db/db mice treated with pioglitazone for 2 wk, rather than 2 days. Morphometric results for proliferative cell number antigen and 4-hydroxy-2-noneal modified protein were consistent with the results of gene expression analysis. The present results strongly suggest that pioglitazone preserves beta-cell mass in diabetic mice mostly by two ways; directly, by acceleration of cell differentiation/proliferation and suppression of apoptosis (acute effect); and indirectly, by deceleration of oxidative stress because of amelioration of the underlying metabolic disorder (chronic effect).

Gallbladder edema in type 1 diabetic patient due to delayed-type insulin allergy.

A 29-year-old woman was diagnosed as having type 1 diabetes mellitus and received insulin aspart and NPH insulin (NovolinN). On day 22, she had leg edema and right abdominal pain. The serum hepatobiliary enzyme levels were markedly elevated. Computed tomography revealed gallbladder edema. After an injection of human regular insulin and NPH insulin (HumacartN), the elevated liver enzyme levels were no longer observed. Challenge testing demonstrated that protamine was the cause of her allergy. Furthermore, tests revealed increased VEGF levels. This is an extremely rare case with a delayed-type protamine allergy caused by NovolinN resulting in gallbladder edema.

Molecular analysis of db gene-related pancreatic beta cell dysfunction; evidence for a compensatory mechanism inhibiting development of diabetes in the db gene heterozygote.

The db gene homozygous, but not heterozygous, mice develop diabetes with severe beta-cell damage. We investigated the molecular mechanism underlying db gene-associated pancreatic beta-cell dysfunction. Islet morphology, beta-cell function, and gene expression profiles specific for pancreatic islet cells were compared among db gene homozygous(db/db), heterozygous (db/m) and unrelated m/m mice. The beta-cell ratio decreased in db/db mice with age, but not in non-diabetic db/m and m/m mice. The islet insulin content was lower, but the triglyceride content was higher in db/db than other mice. The islet cell specific gene expression profiles analyzed by laser capture microdissection method and morphological findings suggested an augmentation of beta-cell apoptosis, oxidative stress and ER stress in db/db mice. Interestingly, insulin I and II, anti-apoptotic bcl-2, and proliferation promoting ERK-1 gene expressions were significantly upregulated in db/m mice. An impaired glucose tolerance was shown in m/m mice fed a high fat diet, but not in db/m mice, in which a higher insulin response and increased beta-cell mass were observed. Expressions of insulin I and II, bcl-2, and ERK-1 gene were increased in db/m mice, but not in m/m fed a high fat diet. The present results strongly suggest that the db gene heterozygote, but not homozygote, acquires a compensatory mechanism suppressing beta-cell apoptosis and augmenting the capacity of beta-cell function.

Analysis of waist circumference in Japanese subjects with type 2 diabetes mellitus: lack of propriety to define the current criteria of metabolic syndrome.

A necessary condition of advocated criteria to determine the metabolic syndrome (MetS) in Japan is waist circumference (WC), which varies among races. In this study, we measured WC and visceral fat area (VFA) in subjects with type 2 diabetes (T2DM) and assessed the propriety of new criteria of MetS in Japan. Four hundred and nineteen patients (M/F: 258/161, age: 60.4+/-0.7 years, BMI: 24.4+/-0.2 kg/m(2)) who received abdominal CT examination were analyzed, and 178 (M/F: 111/67) subjects sufficed the criteria of MetS. Average VFA was significantly larger in subjects with MetS (162+/-3 cm(2) versus 82+/-3 cm(2), p<0.01). The WC and VFA were correlated significantly in both male (r=0.78, p<0.001) and female (r=0.82, p<0.001), and corresponding VFA at 85 cm of WC in male and at 90 cm in female were 125 cm(2) and 120 cm(2). Incidence of cardio- and cerebro-vascular diseases (CVD) was not different between subjects with and without MetS. The present cross-sectional study strongly suggests that the recommended WC is not suitable to define the current criteria of MetS (VFA, > or =100 cm(2)) and its criteria is not appropriate to segregate a risk of CVD in Japanese T2DM subjects. Further prospective analysis should be required to validate the criteria and clinical significance of MetS in T2DM.