Yoshitaka Kajimoto

Possible novel therapy for diabetes with cell-permeable JNK-inhibitory peptide

The JNK pathway is known to be activated in several tissues in the diabetic state, and is possibly involved in the development of insulin resistance and suppression of insulin biosynthesis. Here we show a potential new therapy for diabetes using cell-permeable JNK-inhibitory peptide. Intraperitoneal administration of the peptide led to its transduction into various tissues in vivo, and this treatment markedly improved insulin resistance and ameliorated glucose tolerance in diabetic mice. These data indicate that the JNK pathway is critically involved in diabetes and that the cell-permeable JNK-inhibitory peptide may have promise as a new therapeutic agent for diabetes.

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.

The Human Glucokinase Gene β-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 β-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 β-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 β-cell functions.

Role of Oxidative Stress in Pancreatic β‐Cell Dysfunction

Abstract: Oxidative stress is produced under diabetic conditions and is likely involved in progression of pancreatic β‐cell dysfunction found in diabetes. Possibly caused by low levels of antioxidant enzyme expressions, pancreatic β‐cells are vulnerable to oxidative stress. When β‐cell‐derived HIT‐T15 cells or isolated rat islets were exposed to oxidative stress, insulin gene expression was markedly decreased. To investigate the significance of oxidative stress in the progression of pancreatic β‐cell dysfunction in type 2 diabetes, we evaluated the effects of antioxidants in diabetic C57BL/KsJ‐db/db mice. According to an intraperitoneal glucose tolerance test, the treatment with antioxidants retained glucose‐stimulated insulin secretion and moderately decreased blood glucose levels. Histological analyses of the pancreata revealed that the β‐cell mass was significantly larger in the mice treated with the antioxidants, and the antioxidant treatment suppressed apoptosis in β‐cells without changing the rate of β‐cell proliferation. The antioxidant treatment also preserved the amounts of insulin content and insulin mRNA, making the extent of insulin degranulation less evident. As possible mechanism underlying the phenomena, expression of pancreatic and duodenal homeobox factor‐1 (also known as IDX‐1/STF‐1/IPF1), an important transcription factor for the insulin gene, was more clearly visible in the nuclei of islet cells after the antioxidant treatment. Under diabetic conditions, JNK is activated by oxidative stress and involved in the suppression of insulin gene expression. This JNK effect appears to be mediated in part by nucleocytoplasmic translocation of PDX‐1, which is also downstream of JNK activation. Taken together, oxidative stress and consequent activation of the JNK pathway are involved in progression of β‐cell dysfunction found in diabetes. Antioxidants may serve as a novel mechanism‐based therapy for type 2 diabetes.

Atherosclerosis in carotid artery of young IDDM patients monitored by ultrasound high-resolution B-mode imaging

Ultrasound high-resolution B-mode imaging was used to assess the carotid arteries in 105 patients with insulin-dependent diabetes mellitus (IDDM), 4–25 years of age, with duration of diabetes ranging from 0.5–17 years, 529 patients with non-insulin-dependent diabetes (NIDDM), 31–86 years of age, with duration of diabetes ranging from 0.5–49 years, and 104 nondiabetic healthy subjects, 7–76 years of age, to determine the intimal plus medial thickness (IMT) of the arterial wall. The IMT values for IDDM patients 10–19 years of age (0.525 ± 0.123 mm, n = 68) or 20–25 years of age (0.696 ± 0.124 mm, n = 14) were significantly > those in age-matched nondiabetic subjects (0.444 ± 0.057 mm, n = 12, P = 0.01169; 0.538 ± 0.098 mm, n = 34, P < 0.00006). NIDDM patients showed IMT values equivalent to those in normal adults ≥ 20 years of age. Multiple regression analysis showed that IMT in IDDM patients was positively related to the duration of diabetes (P = 0.00061) as well as to age (P = 0.00046). No other possible risk factors, such as serum total cholesterol level, serum high-density lipoprotein (HDL)-cholesterol level, serum low-density lipoprotein-cholesterol level, serum triglycerides, serum lipoprotein(a) level, or systolic or diastolic blood pressure, have shown significant correlations with IMT in IDDM patients. However, non-HDL-cholesterol, smoking, and systolic hypertension were independently responsible for increases in IMT values of NIDDM patients as well as age and duration of diabetes. The partial regression coefficient of IMT for duration of diabetes (0.00978 mm/year) consistent with that of IMT for age (0.00848–0.00909 mm/year) in nondiabetic and diabetic subjects indicated that diabetes led to carotid atherosclerosis that was twice as advanced in children and adolescents with IDDM. Ultrasound high-resolution B-mode imaging revealed atherosclerosis in the carotid arteries of young IDDM patients on in vivo examination. Diabetes as well as aging can advance atherosclerosis in the carotid arteries of young diabetic patients. The IMT of carotid arteries, which can be measured noninvasively and frequently, may be an important indicator of atherosclerosis, even in young IDDM patients.

Effect of Troglitazene on Microalbuminuria in Patients With Incipient Diabetic Nephropathy

OBJECTIVE Although some studies have suggested a direct action of troglitazone on vascular cells, its effects on diabetic vascular diseases have not been reported. We therefore investigated the effect of troglitazone on microalbuminuria in patients with incipient diabetic nephropathy. RESEARCH DESIGN AND METHODS A total of 30 patients with type 2 diabetes associated with microalbuminuria (urinary albumin-to-creatinine ratio [ACR] [milligrams per gram creatinine] ranging from 30 to 300 mg/g creatinine) were studied. They were randomly divided into two groups: patients treated with metformin (500 mg/day, n = 13) or with troglitazone (400 mg/day, n = 17) for 12 weeks. ACR, lipid profile, blood pressure, glycated hemoglobin, and plasma glucose during meal-load tests were measured every 4 weeks. RESULTS Anthropometric indices (BMI and percent fat), lipid profile, and blood pressure did not change with either treatment. Fasting and postmeal glucose levels decreased similarly in the two groups. Decrements in glycated hemoglobin were greater in the metformin group at 4 and 8 weeks after the initiation of treatment (P < 0.05). Troglitazone reduced ACR (median [25–75th percentiles]) from 70 (49–195) to 40 (31–90) mg/g creatinine at 4 weeks (P = 0.021) and maintained these reduced levels throughout the treatment period (8 weeks: 35 [26–68], P = 0.007; 12 weeks: 43 [26–103], P = 0.047). Metformin did not change ACR throughout the 12 weeks. CONCLUSIONS Troglitazone ameliorated microalbuminuria in diabetic nephropathy. Furthermore, our findings suggest that troglitazone has some effects on vascular cells other than lowering plasma glucose levels. Troglitazone might be useful for diabetic angiopathy, including nephropathy and coronary artery disease.

PDX-1 Induces Insulin and Glucokinase Gene Expressions in αTC1 Clone 6 Cells in the Presence of Betacellulin

The pancreatic β- and α-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 α- and β-cells, but PDX-1/IPF1/STF-1/IDX-1, a homeodomain-containing transcription factor, is present in β-cells but not in α-cells. To elucidate the function of PDX-1 in the expression of β-cell-specific genes, we established stable αTC1 clone 6 (αTC1.6)-derived transfectants expressing PDX-1 and examined the changes in the gene expression patterns in them. The exogenous expression of PDX-1 in αTC1.6 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 βcellulin was added to the medium, the PDX-1-expressing αTC1.6 cells, but not the control αTC1.6 cells, came to express insulin and glucokinase mRNAs. This did not occur with other growth factors such as epidermal growth factor, transforming growth factor α, and insulin-like growth factor I. GLUT2 mRNA remained undetectable in the PDX-1–expressing αTC1.6 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 βcellulin, also contribute to the β-cell specificity of gene expression.

Antihypertensive effect of casein hydrolysate in a placebo-controlled study in subjects with high-normal blood pressure and mild hypertension

We describe a clinical trial to study the efficacy of a casein hydrolysate, prepared using an Aspergillus oryzae protease, containing the major angiotensin-I-converting enzyme inhibitory peptides Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP) in a single-blind, placebo-controlled study. A total of 131 volunteers with high-normal blood pressure and mild hypertension were randomly divided into four groups (n 32 or 33 in each group). Each volunteer was given two tablets containing four different dosages of VPP and IPP (VPP+IPP: 0, 1.8, 2.5 and 3.6 mg), daily for 6 weeks. A significant decrease in systolic blood pressure was observed at 6 weeks in the active group receiving 1.8 mg (P<0.01) VPP and IPP; in the active groups receiving either 2.5 mg or 3.6 mg, systolic blood pressure was decreased at both 3 weeks (P<0.05 and P<0.05) and 6 weeks (P<0.001 and P<0.0001) compared with systolic blood pressure measured before treatment. Changes in the systolic blood pressure after 6 weeks of treatment in the four groups were --1.7, --6.3, --6.7 and --10.1 mmHg, and these effects were dose dependent. In addition, a significant difference in systolic blood pressure between the placebo group and the VPP and IPP group receiving 3.6 mg was observed (P<0.001) by two-way ANOVA. The antihypertensive effect was greater in mildly hypertensive subjects (n 20 or 21 in each group) than in any of the other subjects. No significant change of diastolic blood pressure was observed for all the test groups, and no differences in diastolic blood pressure in the test sample groups compared with the placebo group were observed during the test period.

Asymptomatic hyperglycaemia is associated with increased intimal plus medial thickness of the carotid artery.

SummaryAtherosclerotic changes have not been demonstrated directly in asymptomatic hyperglycaemic non-diabetic subjects, although high mortality due to coronary heart disease has been reported. We measured arterial wall thickness non-invasively, in order to directly demonstrate atherosclerosis of the carotid arteries of hyperglycaemic non-diabetic subjects and to evaluate its risk factors.The thicknesses of the intimal plus medial complex (IMT) of the carotid arteries of 112 asymptomatic hyperglycaemic non-diabetic subjects (aged 22–81, 95 males and 17 females) were compared with those of 55 healthy male subjects and 211 non-insulin-dependent NIDDM male diabetic patients. The subjects were subgrouped into impaired glucose-tolerant (IGT) subjects who had a 2-h glycaemic level of more than 7.8 mmol/l, and non-IGT subjects whose 2-h glycaemic levels were within 6.7–7.7 mmol/l.Non-IGT and IGT subjects showed significantly greater IMTs than age-matched healthy males and showed no significant differences compared to age-matched NIDDM patients. Multivariate analysis demonstrated that the risk factors for IMT of non-IGT and IGT subjects were age and systolic blood pressure. According to data on the accumulation of atherogenic risks (hypertension, dyslipidaemia, and smoking), IMT increased linearly in non-IGT and IGT subjects. However, non-IGT and IGT subjects without hyperlipidaemia, hypertension, or smoking risk still had significantly greater IMT than age-matched normal males (1.019±0.063 vs 0.770±0.111 mm, p<0.05). Prevalence of ECG-indicated coronary heart disease was significantly higher in hyperglycaemic non-diabetic subjects and NIDDM with increased carotid arterial wall thickness (IMT ≥1.1 mm) than in those without increased thickness (IMT<1.1 mm). Asymptomatic hyperglycaemic non-diabetic subjects have increased thickness of their carotid arteries compared to age-matched male NIDDM patients. As one of several independent risk factors, mild hyperglycaemia advances atherosclerosis, which leads to coronary heart disease.

Probucol preserves pancreatic β-cell function through reduction of oxidative stress in type 2 diabetes

Oxidative stress is induced under diabetic conditions and causes various forms of tissue damage in patients with diabetes. Recently, pancreatic beta-cells have emerged as a putative target of oxidative stress-induced tissue damage and this seems to explain in part the progressive deterioration of beta-cell function in type 2 diabetes. As a step toward clinical trial of antioxidant for type 2 diabetes, we investigated the possible anti-diabetic effects of probucol, an antioxidant widely used as an anti-hyperlipidemic agent, on preservation of beta-cell function in diabetic C57BL/KsJ-db/db mice. Probucol-containing diet was given to mice from 6 to 16 weeks of age. Immunostaining for oxidative stress markers such as 4-hydroxy-2-nonenal (HNE)-modified proteins and heme oxygenase-1 revealed that probucol treatment decreased reactive oxygen species (ROS) in pancreatic islets of diabetic animals. Oxidative stress is known to enhance apoptosis of beta-cells and to suppress insulin biosynthesis, but probucol treatment led to preservation of beta-cell mass and the insulin content. According to intraperitoneal glucose tolerance tests, the probucol treatment preserved glucose-stimulated insulin secretion and improved glucose tolerance at 10 and 16 weeks: insulin, 280+/-82 vs. 914+/-238 pmol/l (120 min, at 16 weeks; P<0.05); glucose, 44.6+/-2.4 vs. 35.2+/-2.6 mmol/l (120 min, at 16 weeks; P<0.05). Thus, our present observations demonstrate the potential usefulness of probucol for treatment of type 2 diabetes.