Linagliptin provides effective, well-tolerated add-on therapy to pre-existing oral antidiabetic therapy over 1 year in Japanese patients with type 2 diabetes.

AIMS: To evaluate the long-term safety and efficacy of linagliptin as add-on therapy to one approved oral antidiabetic drug (OAD) in Japanese patients with type 2 diabetes mellitus and insufficient glycaemic control. METHODS: This 52-week, multicentre, open-label, parallel-group study evaluated once-daily linagliptin 5 mg as add-on therapy to one OAD [biguanide, glinide, glitazone, sulphonylurea (SU) or α-glucosidase inhibitors (A-GI)] in 618 patients. After a 2-week run-in, patients on SU or A-GI were randomized to either linagliptin (once daily, 5 mg) or metformin (twice or thrice daily, up to 2250 mg/day) as add-on therapy. Patients receiving the other OADs received linagliptin add-on therapy (non-randomized). RESULTS: Adverse events were mostly mild or moderate, and rates were similar across all groups. Hypoglycaemic events were rare, except in the SU group. Overall, 26 (5.8%) hypoglycaemic events were reported in patients receiving linagliptin (non-randomized). Hypoglycaemic events were similar for linagliptin and metformin added to A-GI (1/61 vs. 2/61, respectively) or SU (17/124 vs. 10/63, respectively). Significant reductions in glycated haemoglobin (HbA1c) levels (between -0.7 and -0.9%) occurred throughout the study period for the background therapy groups that received linagliptin (baseline HbA1c 7.9-8.1%). The decline in HbA1c levels was indistinguishable between linagliptin and metformin groups when administered as add-on therapy to A-GI or SU. CONCLUSIONS: Once-daily linagliptin showed safety and tolerability over 1 year and provided effective add-on therapy leading to significant HbA1c reductions, similar to metformin, over 52 weeks in Japanese patients.

Long-term safety of linagliptin monotherapy in Japanese patients with type 2 diabetes.

AIMS: In a phase III study conducted among Japanese patients with type 2 diabetes mellitus (T2DM), linagliptin 5 and 10 mg showed clinically meaningful improvements in glycaemic parameters after 12 and 26 weeks compared with placebo and voglibose, respectively. This extension study assessed long-term tolerability of linagliptin over 52 weeks. METHODS: Japanese patients with T2DM who completed either phase of a 12-week/26-week study comparing linagliptin monotherapy with placebo or voglibose were eligible to enrol. In the extension study, the comparator groups switched to linagliptin 5 or 10 mg, while the linagliptin groups maintained dosage. RESULTS: In all, 540 patients received at least one dose of linagliptin 5 or 10 mg and 494 completed the extension. Long-term treatment with linagliptin was well tolerated; adverse events (AEs) of special interest and serious AEs occurred in small percentages of patients. Drug-related AEs occurred in 10.2 and 10.6% of patients in the linagliptin 5- and 10-mg groups, respectively, and discontinuations due to drug-related AEs occurred in 1.1 and 0.7%, respectively. Only one (0.4%) patient in each dose group experienced investigator-defined hypoglycaemia during the treatment period (both events were non-severe). Body weight was not clinically altered in either group. The glycated haemoglobin A1c profiles over time were similar with linagliptin 5 and 10 mg. CONCLUSIONS: These findings provide evidence for the safety and tolerability of oral linagliptin at either 5 or 10 mg for up to 52 weeks for the treatment of Japanese patients with T2DM, without clinically relevant increase in the risk of hypoglycaemia or weight gain.

Linagliptin monotherapy provides superior glycaemic control versus placebo or voglibose with comparable safety in Japanese patients with type 2 diabetes: a randomized, placebo and active comparator-controlled, double-blind study.

AIMS: To evaluate the efficacy and safety of linagliptin 5 and 10 mg vs. placebo and voglibose in Japanese patients with type 2 diabetes mellitus (T2DM). METHODS: This study enrolled patients with inadequately controlled T2DM who were previously treated with one or two oral antidiabetics or were drug naÏve. After a 2 to 4-week washout and placebo run-in, 561 patients were randomized (2 : 2 : 2 : 1) to double-blind treatment with linagliptin 5 or 10 mg qd, voglibose 0.2 mg tid or placebo. The primary endpoint was the change from baseline in haemoglobin A1c (HbA1c) with linagliptin vs. placebo after 12 weeks and vs. voglibose after 26 weeks. RESULTS: Baseline characteristics were well balanced across treatment groups (overall mean HbA1c was 8.01%). The adjusted mean (95% confidence interval) treatment differences at week 12 were -0.87% (-1.04, -0.70; p < 0.0001) and -0.88% (-1.05, -0.71; p < 0.0001) for linagliptin 5 and 10 mg vs. placebo and at week 26 were -0.32% (-0.49, -0.15; p = 0.0003) and -0.39% (-0.56, -0.21; p < 0.0001) for linagliptin 5 and 10 mg vs. voglibose. At week 12, mean HbA1c was 7.58, 7.48 and 8.34% in patients receiving linagliptin 5 mg, linagliptin 10 mg and placebo, respectively. At week 26, mean HbA1c was 7.63% with linagliptin 5 mg, 7.50% with linagliptin 10 mg and 7.91% with voglibose. Drug-related adverse event rates were comparable across treatment groups over 12 weeks (9.4% linagliptin 5 mg, 8.8% linagliptin 10 mg and 10.0% placebo) and 26 weeks (11.3% linagliptin 5 mg, 10.6% linagliptin 10 mg and 18.5% voglibose). There were no documented cases of hypoglycaemia. CONCLUSIONS: Linagliptin showed superior glucose-lowering efficacy and comparable safety and tolerability to both placebo and voglibose in Japanese patients with T2DM.

Effects of Arterial Stiffness on Cerebral WM Integrity in Older Adults: A Neurite Orientation Dispersion and Density Imaging and Magnetization Transfer Saturation Imaging Study.

BACKGROUND AND PURPOSE: Arterial stiffness is reported to be able to cause axonal demyelination or degeneration. The present study aimed to use advanced MR imaging techniques to examine the effect of arterial stiffness on the WM microstructure among older adults. MATERIALS AND METHODS: Arterial stiffness was measured using the cardio-ankle vascular elasticity index (CAVI). The high-CAVI (mean CAVI ≥ 9 points) and the low-CAVI groups (mean CAVI < 9 points) were created. The neuronal fiber integrity of the WM was evaluated by neurite orientation dispersion and density imaging and magnetization transfer saturation imaging. Tract-Based Spatial Statistics and the tracts-of-interest analysis were performed. Specific WM regions (corpus callosum, internal capsule, anterior thalamic radiation, corona radiata, superior longitudinal fasciculus, forceps minor, and inferior fronto-occipital fasciculus) were selected in the tracts-of-interest analysis. RESULTS: In Tract-Based Spatial Statistics, the high-CAVI group showed a significantly lower myelin volume fraction value in the broad WM and significantly higher radial diffusivity and isotropic volume fraction values in the corpus callosum, forceps minor, inferior fronto-occipital fasciculus, internal capsule, corona radiata, and anterior thalamic radiation than the low-CAVI group. In tracts-of-interest analysis using multivariate linear regression, significant associations were found between the mean CAVI and radial diffusivity in the anterior thalamic radiation and the corona radiata; isotropic volume fraction in the anterior thalamic radiation and the corona radiata; and myelin volume fraction in the superior longitudinal fasciculus (P < .05). Additionally, partial correlation coefficients were observed for the significant associations of executive function with radial diffusivity and myelin volume fraction (P < .05). CONCLUSIONS: Arterial stiffness could be associated with demyelination rather than axonal degeneration.

An Investigation of Water Diffusivity Changes along the Perivascular Space in Elderly Subjects with Hypertension.

BACKGROUND AND PURPOSE: Hypertension may be related to alterations of the glymphatic system, a waste metabolite drainage system in the brain. We aimed to investigate analysis along the perivascular space index changes in elderly subjects with hypertension. MATERIALS AND METHODS: Diffusion-weighted images were acquired from 126 subjects, including 63 subjects with hypertension (25 men and 38 women; mean age, 72.45 years) and 63 age- and sex-matched controls (25 men and 38 women; mean age, 72.16 years). We calculated the analysis along the perivascular space index as a ratio of the mean of x-axis diffusivities in the projection and association areas to the mean of y-axis diffusivity in the projection area and z-axis diffusivity in the association area. The left, right, and mean analysis along the perivascular space indices of both hemispheres were compared between the hypertension and control groups using a Mann-Whitney U test. The Spearman correlation coefficient was used to assess the correlation between the left, right, and mean ALPS indices and blood pressure and pulse pressure. RESULTS: The left (P = .011) and mean (P = .024) analysis along the perivascular space indices of the hypertension group were significantly lower than that of the control group. The left, right, and mean analysis along the perivascular space indices of all subjects were significantly negatively correlated with blood pressure values (r = -0.200 to -0.278, P = .002-0.046) and pulse pressure values (r = -0.221 to -0.245, P = .006-0.013). CONCLUSIONS: Our results are consistent with a model in which hypertension causes glymphatic dysfunction.

Genetic variants at CDC123/CAMK1D and SPRY2 are associated with susceptibility to type 2 diabetes in the Japanese population.

AIMS/HYPOTHESIS: Recently, rs10906115 in CDC123/CAMK1D, rs1359790 near SPRY2, rs1436955 in C2CD4A/C2CD4B and rs10751301 in ODZ4 were identified as genetic risk variants for type 2 diabetes by a genome-wide association study in a Chinese population. The aim of the present study was to ascertain the role of these four variants in conferring susceptibility to type 2 diabetes in the Japanese population. METHODS: We genotyped 11,530 Japanese individuals (8,552 type 2 diabetes cases, 2,978 controls) for the above single nucleotide polymorphisms (SNPs) and used logistic regression analysis to determine whether they were associated with type 2 diabetes. RESULTS: In accordance with the findings in a Chinese population, rs10906115 A, rs1359790 C and rs1436955 G were found to be risk alleles. Both rs10906115 and rs1359790 were significantly associated with susceptibility to type 2 diabetes in our study (rs10906115 OR 1.15, 95% CI 1.08, 1.22; p = 6.10 × 10(-6); rs1359790 OR 1.14, 95% CI 1.06, 1.21; p = 2.24 × 10(-4)). Adjustment for age, sex and BMI had no significant effects on the association between these variants and the disease. We did not observe any significant associations between the SNPs and any metabolic traits, e.g. BMI, fasting plasma glucose (determined for 1,332 controls), HOMA of beta cell function (900 controls) and HOMA of insulin resistance (900 controls; p > 0.05). CONCLUSIONS/INTERPRETATION: The SNPs rs10906115 A and rs1359790 C are significantly associated with susceptibility to type 2 diabetes in the Japanese population, confirming that these alleles are common susceptibility variants for type 2 diabetes in East Asian populations.

Repetitive hypoglycaemia increases serum adrenaline and induces monocyte adhesion to the endothelium in rat thoracic aorta.

AIMS/HYPOTHESIS: Severe hypoglycaemia associated with diabetes management is a potential risk for cardiovascular diseases. However, the effect and mechanism of hypoglycaemia on the progression of atherosclerosis remains largely unknown. As a first step towards elucidating the above, we investigated the effect of hypoglycaemia on monocyte-endothelial interaction. METHODS: Insulin was injected intraperitoneally once every 3 days for 5 weeks in Goto-Kakizaki rats, a non-obese rat model of type 2 diabetes. We counted the number of monocytes adherent to the endothelium of thoracic aorta as an index of early atherosclerogenesis. Cultured HUVEC were used to investigate the mechanism of action. RESULTS: Insulin treatment increased the number of monocytes adherent to the vascular endothelium. This increase was abrogated by injection of glucose with insulin. Amosulalol, an α-1 and ß-adrenoreceptor antagonist, suppressed monocyte adhesion to endothelium and levels of adhesion molecules (intercellular adhesion molecule-1 and vascular cell adhesion molecule-1) in the endothelial surface, which had been enhanced by insulin-induced hypoglycaemia. In HUVEC, adrenaline (epinephrine) significantly increased nuclear translocation of nuclear factor-κB (NF-κB) p65 and levels of adhesion molecules, effects that were abrogated following addition of SQ22536, a specific adenyl cyclase inhibitor. CONCLUSIONS/INTERPRETATION: Our data indicate that repetitive hypoglycaemia induced by insulin enhanced monocyte adhesion to endothelial cells in Goto-Kakizaki rat aorta through enhanced adrenaline activity and that the latter stimulated intracellular cAMP, leading to nuclear translocation of NF-κB with subsequent production of adhesion molecules in endothelial cells.

Impact of whole body irradiation and vascular endothelial growth factor-A on increased beta cell mass after bone marrow transplantation in a mouse model of diabetes induced by streptozotocin.

AIMS/HYPOTHESIS: Recent studies have shown that bone marrow transplantation reduces hyperglycaemia in a mouse model of diabetes induced by streptozotocin. However, the essential factors for the improvement of hyperglycaemia by bone marrow transplantation have not been fully elucidated. The aim of this study was to search for such factors. METHODS: We investigated the effect of irradiation to whole body, to abdomen alone or to whole body excluding abdomen, followed by infusion or no infusion of bone marrow cells. We also investigated the effect of bone marrow transplantation on beta cell-specific vascular endothelial growth factor-A gene (Vegfa) knockout mice. RESULTS: Bone marrow transplantation improved streptozotocin-induced hyperglycaemia and partially restored islet mass. This change was associated with increased islet vascularisation. Among the other methods investigated, low-dose irradiation of the whole body without infusion of bone marrow cells also improved blood glucose level. In streptozotocin-treated beta cell-specific Vegfa knockout mice, which exhibit impaired islet vascularisation, bone marrow transplantation neither improved hyperglycaemia, relative beta cell mass nor islet vascularisation. CONCLUSION/INTERPRETATION: Our results indicate that whole body irradiation is essential and sufficient for restoration of beta cell mass after streptozotocin treatment independent of infusion of bone marrow cells. Vascular endothelial growth factor-A produced in beta cells is also essential for this phenomenon.

Effects of metformin on peripheral insulin sensitivity and intracellular lipid contents in muscle and liver of overweight Japanese subjects.

AIM: Recent studies suggest that insulin resistance is associated with increased intrahepatic lipid (IHL) and intramyocellular lipid (IMCL) contents. While metformin improves insulin resistance mainly in liver, its effects on IHL and IMCL have not been clarified yet. The aim of this study was to investigate the effects of low-dose metformin (750 mg/day) on peripheral insulin sensitivity, IHL and IMCL. METHODS: Before and 3 months after low-dose metformin therapy, eight overweight/obese Japanese subjects [body mass index (BMI) >25 kg/m2] were studied with blood sampling, measurement of IHL and IMCL by 1H magnetic resonance spectroscopy and glucose infusion rate (GIR) during euglycaemic-hyperinsulinaemic clamp as an index of peripheral insulin sensitivity. RESULTS: A 3-month low-dose metformin therapy did not alter body weight, total body fat, fat distribution or physical activity level but increased GIR by 31% (from 6.24 +/- 0.86 to 7.82 +/- 0.82 mg/kg/min, p < 0.01). Although metformin treatment did not alter IMCL (from 4.1 +/- 1.0 to 4.2 +/- 0.9, not significant), it decreased IHL by 21% (from 15.9 +/- 2.8 to 11.8 +/- 2.2%, p < 0.05). CONCLUSIONS: A 3-month low-dose metformin treatment improved peripheral insulin sensitivity and reduced IHL, without significantly changing BMI, adiposity or IMCL.

Suppression of transcription factor PDX-1/IPF1/STF-1/IDX-1 causes no decrease in insulin mRNA in MIN6 cells.

The insulin gene transcription factor PDX-1/IPF1/STF-1/ IDX-1 plays a key role in directing beta cell-specific gene expressions. Recently, impairment of PDX-1 expression or activity has been observed in beta cell-derived HIT cells cultured under high glucose concentrations, and this has been suggested as a possible cause of the decrease in insulin gene transcription. To investigate the pathophysiological significance of PDX-1 as a determinant of the rate of insulin gene transcription, we suppressed its expression in beta cell-derived MIN6 cells using an antisense oligodeoxynucleotide (ODN) and searched for possible changes in the beta cell-specific gene expression. Treatment of MIN6 cells with an 18-mer phosphorothioate ODN complementary to a sequence starting at the translation initiation codon of PDX-1 caused a potent, concentration-dependent reduction in PDX-1 expression; addition of 2 microM antisense ODN could reduce PDX-1 expression to 14+/-4% of the control. There was also a decrease in its DNA binding to the insulin gene A element. Despite such suppression of PDX-1, Northern blot analysis revealed no decrease in the amount of insulin mRNA in the MIN6 cells. Similarly, no changes were detected in the transcription of the glucokinase or islet amyloid polypeptide gene, for which PDX-1 was shown to function as a transcription factor. Thus, our findings dispute the physiological significance of PDX-1 in determining the rate of insulin gene transcription. This means that other components constituting the transcription-controlling machinery need to be evaluated in order to understand the molecular basis of impaired insulin biosynthesis such as that observed due to glucose toxicity.

Glycation-dependent, reactive oxygen species-mediated suppression of the insulin gene promoter activity in HIT cells.

Prolonged poor glycemic control in non-insulin-dependent diabetes mellitus patients often leads to a decline in insulin secretion from pancreatic beta cells, accompanied by a decrease in the insulin content of the cells. As a step toward elucidating the pathophysiological background of the so-called glucose toxicity to pancreatic beta cells, we induced glycation in HIT-T15 cells using a sugar with strong deoxidizing activity, D-ribose, and examined the effects on insulin gene transcription. The results of reporter gene analyses revealed that the insulin gene promoter is more sensitive to glycation than the control beta-actin gene promoter; approximately 50 and 80% of the insulin gene promoter activity was lost when the cells were kept for 3 d in the presence of 40 and 60 mM D-ribose, respectively. In agreement with this, decrease in the insulin mRNA and insulin content was observed in the glycation-induced cells. Also, gel mobility shift analyses using specific antiserum revealed decrease in the DNA-binding activity of an insulin gene transcription factor, PDX-1/IPF1/STF-1. These effects of D-ribose seemed almost irreversible but could be prevented by addition of 1 mM aminoguanidine or 10 mM N-acetylcysteine, thus suggesting that glycation and reactive oxygen species, generated through the glycation reaction, serve as mediators of the phenomena. These observations suggest that protein glycation in pancreatic beta cells, which occurs in vivo under chronic hyperglycemia, suppresses insulin gene transcription and thus can explain part of the beta cell glucose toxicity.

Identification of a portable repression domain and an E1A-responsive activation domain in Pax4: a possible role of Pax4 as a transcriptional repressor in the pancreas.

Pax4 is a paired-domain (PD)-containing transcription factor which plays a crucial role in pancreatic beta/delta-cell development. In this study, we characterized the DNA-binding and transactivation properties of mouse Pax4. Repetitive rounds of PCR-based selection led to identification of the optimal DNA-binding sequences for the PD of Pax4. In agreement with the conservation of the optimal binding sequences among the Pax family transcription factors, Pax4 could bind to the potential binding sites for Pax6, another member of the Pax family also involved in endocrine pancreas development. The overexpression of Pax4 in HIT-T15 cells dose dependently inhibited the basal transcriptional activity as well as Pax6-induced activity. Detailed domain mapping analyses using GAL4-Pax4 chimeras revealed that the C-terminal region of Pax4 contains both activation and repression domains. The activation domain was active in the embryonic kidney-derived 293/293T cells and embryonal carcinoma-derived F9 cells, containing adenoviral E1A protein or E1A-like activity, respectively but was inactive or very weakly active in other cells including those of pancreatic beta- and alpha-cell origin. Indeed, the exogenous overexpression of type 13S E1A in heterologous cell types could convert the activation domain to an active one. On the other hand, the repression domain was active regardless of the cell type. When the repression domain was linked to the transactivation domain of a heterologous transcription factor, PDX-1, it could completely abolish the transactivation potential of PDX-1. These observations suggest a primary role of Pax4 as a transcriptional repressor whose function may involve the competitive inhibition of Pax6 function. The identification of the E1A-responsive transactivation domain, however, indicates that the function of Pax4 is subject to posttranslational regulation, providing further support for the complexity of mechanisms that regulate pancreas development.

Monocyte and Basophil Counts as Predictors of Neutrophil Count Recovery in Patients with Thiamazole-Induced Agranulocytosis.

Purpose: Thiamazole (MMI) is frequently used for the treatment of Graves' disease, but it occasionally induces agranulocytosis at the beginning of the treatment. To date, the predictive factors of recovery from MMI-induced agranulocytosis remain unclear. The primary aim of this study was to evaluate the predictive factor of the recovery time from MMI-induced agranulocytosis. Method: This was a retrospective cohort study performed in a university hospital and a thyroid hospital. We included 27 Japanese patients with Graves' disease with MMI-induced agranulocytosis diagnosed during follow-up. All patients were administrated recombinant human granulocyte colony-stimulating factor daily until they had a neutrophil count>1 000/µL, which was defined as recovery. The predictive factors associated with recovery time were estimated using multivariable regression analysis. Results: At the onset of agranulocytosis, the median administration period of MMI was 33 days, the average white blood cell count was 1 896/µL, and the median neutrophil count was 22/µL. The median recovery time was 4 days. Stepwise multivariate regression analysis identified the monocyte and basophil counts to be significant predictors of MMI-induced agranulocytosis. Conclusion: Patients with agranulocytosis and decreased monocyte and basophil counts at onset may recover late and require careful treatment.

The human glucokinase gene beta-cell-type promoter: an essential role of insulin promoter factor 1/PDX-1 in its activation in HIT-T15 cells.

The glycolytic enzyme glucokinase plays a primary role in the glucose-responsive secretion of insulin, and defects of this enzyme can cause NIDDM. As a step toward understanding the molecular basis of glucokinase (GK) gene regulation, we assessed the structure and regulation of the human GK gene beta-cell-type promoter. The results of reporter gene analyses using HIT-T15 cells revealed that the gene promoter was comprised of multiple cis-acting elements, including two primarily important cis-motifs: a palindrome structure, hPal-1, and the insulin gene cis-motif A element-like hUPE3. While both elements were bound specifically by nuclear proteins, it was the homeodomain-containing transcription factor insulin promoter factor 1 (IPF1)/STF-1/PDX-1 that bound to the hUPE3 site: IPF1, when expressed in CHO-K1 cells, became bound to the hUPE3 site and activated transcription. An anti-IPF1 antiserum used in gel-mobility shift analysis supershifted the DNA protein complex formed with the hUPE3 probe and nuclear extracts from HIT-T15 cells, thus supporting the involvement of IPF1 in GK gene activation in HIT-T15 cells. In contrast to the insulin gene, however, neither the synergistic effect of the Pan1 expression on the IPF1-induced promoter activation nor the glucose responsiveness of the activity was observed for the GK gene promoter. These results revealed some conservative but unique features for the transcriptional regulation of the beta-cell-specific genes in humans. Being implicated in insulin and GK gene regulations as a common transcription factor, IPF1/STF-1/PDX-1 is likely to play an essential role in maintaining normal beta-cell functions.

PDX-1 induces insulin and glucokinase gene expressions in alphaTC1 clone 6 cells in the presence of betacellulin.

The pancreatic beta- and alpha-cells are developmentally related to each other but reveal diverse gene expression patterns. Among the two important transcription factors for insulin gene expression, IEF1 is present both in alpha- and beta-cells, but PDX-1/IPF1/STF-1/IDX-1, a homeodomain-containing transcription factor, is present in beta-cells but not in alpha-cells. To elucidate the function of PDX-1 in the expression of beta-cell-specific genes, we established stable alphaTC1 clone 6 (alphaTC1.6)-derived transfectants expressing PDX-1 and examined the changes in the gene expression patterns in them. The exogenous expression of PDX-1 in alphaTC1.6 cells alone could induce islet amyloid polypeptide (IAPP) mRNA expression in the cells but not the expression of insulin, glucokinase, or GLUT2 gene. However, when betacellulin was added to the medium, the PDX-1-expressing alphaTC1.6 cells, but not the control alphaTC1.6 cells, came to express insulin and glucokinase mRNAs. This did not occur with other growth factors such as epidermal growth factor, transforming growth factor alpha, and insulin-like growth factor I. GLUT2 mRNA remained undetectable in the PDX-1--expressing alphaTC1.6 cells. These observations demonstrate the potency of PDX-1 for the expression of the insulin, glucokinase, and IAPP genes and suggest that certain regulatory factors, which can partially be modified by betacellulin, also contribute to the beta-cell specificity of gene expression.

Regulation of the pancreatic pro-endocrine gene neurogenin3.

Neurogenin3 (ngn3), a basic helix-loop-helix (bHLH) transcription factor, functions as a pro-endocrine factor in the developing pancreas: by itself, it is sufficient to force undifferentiated pancreatic epithelial cells to become islet cells. Because ngn3 expression determines which precursor cells will differentiate into islet cells, the signals that regulate ngn3 expression control islet cell formation. To investigate the factors that control ngn3 gene expression, we mapped the human and mouse ngn3 promoters and delineated transcriptionally active sequences within the human promoter. Surprisingly, the human ngn3 promoter drives transcription in all cell lines tested, including fibroblast cell lines. In contrast, in transgenic animals the promoter drives expression specifically in regions of ngn3 expression in the developing pancreas and gut; and the addition of distal sequences greatly enhances transgene expression. Within the distal enhancer, binding sites for several pancreatic transcription factors, including hepatocyte nuclear factor (HNF)-1 and HNF-3, form a tight cluster. HES1, an inhibitory bHLH factor activated by Notch signaling, binds to the proximal promoter and specifically blocks promoter activity. Together with previous genetic data, these results suggest a model in which the ngn3 gene is activated by the coordinated activities of several pancreatic transcription factors and inhibited by Notch signaling through HES1.

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.

Dobutamine stress echocardiography predicts reversible dysfunction and quantitates the extent of irreversibly damaged myocardium after reperfusion of anterior myocardial infarction.

OBJECTIVES: This study was designed to evaluate dobutamine stress echocardiography in identifying reversible dysfunction and assessing the extent of irreversibly damaged myocardium early in acute myocardial infarction. BACKGROUND: Several experimental and clinical studies have suggested that dobutamine enhances contractile function of stunned or hibernating, or both, myocardium. It is important for clinical strategy to predict the magnitude of improvement in myocardial function early in acute myocardial infarction. METHODS: We studied 21 patients with a reperfused first anterior myocardial infarction. Two-dimensional echocardiography was performed before and during dobutamine infusion (10 micrograms/kg body weight per min) at a mean of 3 days after the infarction. Follow-up echocardiography was performed at a mean of 25 days later. To assess segmental wall motion, we divided the left ventricle into 17 segments and assigned a wall motion abnormality score: 3 = dyskinesia or akinesia; 0 = normal. Improvement in wall motion was indicated by a decrease of at least one grade in segmental score. For quantitative assessment, the ratio of endocardial length showing dyskinesia or akinesia to a left ventricular endocardial length (akinetic length ratio) was determined in the apical long-axis view at each stage. RESULTS: Sensitivity and specificity of dobutamine infusion in detecting improvement in wall motion at follow-up echocardiography were 83% (55 of 66 segments) and 86% (43 of 50 segments), respectively. Excellent correlation was found (r = 0.93, p < 0.001; absolute difference [mean +/- SD] 0.03 +/- 0.05) between the akinetic length ratios measured during dobutamine infusion and in the late convalescent stage. CONCLUSIONS: In the early stage of acute myocardial infarction, low dose dobutamine stress echocardiography provides a useful method for predicting reversible dysfunction with excellent sensitivity and specificity and can also be used to quantitate the extent of irreversibly damaged myocardium.

Ubiquitous, but variable, expression of two alternatively spliced mRNAs encoding mouse homologues of transcription factors E47 and E12.

Two basic helix-loop-helix (bHLH) transcription factors, E47 and E12, are involved in cell-specific gene expression as part of dimeric complexes which interact with the cis-acting motif E-box. Although both generated from a single gene (E2A) by means of alternative splicing, the structural difference in these bHLH regions between the two suggests that the two bHLH proteins may differ in some of their functions. As a step toward elucidating the individual implications of E47 and E12, we investigated the mRNA expression ratios of their homologues (A1 and kA1, respectively) in mouse tissues and cell lines. Both the A1 and kA1 mRNAs were ubiquitously expressed in all tissues examined. However, their ratios varied: e.g., skeletal muscle, 2.2 +/- 0.3 (mean +/- SE); spleen, 2.0 +/- 0.2; pancreatic islet cells, 1.2 +/- 0.2. The A1/kA1 ratios in the cell lines investigated were similar to those of their original tissues. In conclusion, the ubiquity in mRNA expression observed for both the E47 and E12 homologues in mouse provides support for their involvement in a broad range of transcriptional regulation. The variation in the A1/kA1 expression ratios, on the other hand, supports the idea that A1 (E47) and kA1 (E12) each have some unique roles in the functions of these E2A gene-encoded bHLH proteins.

Identification of amino-acid polymorphism within the leucine zipper motif of mouse transcription factor A1.

Mouse transcription factor A1 (A1) is a mouse homologue of human E47, a ubiquitously expressed DNA-binding protein which contains a basic region, helix-loop-helix (HLH) and leucine zipper (LZ) motifs [Walker et al., Nucleic Acids Res. 18 (1990) 1159-1166]. Analyses of the nucleotide (nt) sequences of A1 cDNAs isolated from various mouse strains revealed amino acid (aa) polymorphism in the highly conserved region within the LZ motif. Interestingly, the location and pattern of aa deletions are identical to those previously described for the aa polymorphism within the human counterpart, E47 (E12) [Kamps et al., Cell 60 (1990) 547-555].