Determinants of Glycated Hemoglobin in Subjects With Impaired Glucose Tolerance: Subanalysis of the Japan Diabetes Prevention Program.

BACKGROUND: Limited evidence is available about the relationship of lifestyle factors with glycated hemoglobin (HbA1c) in subjects with impaired glucose tolerance. The aim of study was to identify such determinant factors of HbA1c in subjects with impaired glucose tolerance. METHODS: This cross-sectional study included 121 men and 124 women with impaired glucose tolerance, who were diagnosed based on a 75-g oral glucose tolerance test. Demographic and biochemical parameters, including the body mass index (BMI), fasting plasma glucose (FPG), 2-h post-load glucose (2-h PG), and HbA1c, were measured. The pancreatic ß-cell function and insulin resistance were assessed using homeostasis model assessment (HOMA-ß). Dietary intake was assessed by a food frequency questionnaire. RESULTS: The levels of FPG, 2-h PG, and carbohydrate intake were correlated with the HbA1c level in men, while the FPG and 2-h PG levels were correlated with the HbA1c level in women. In multiple regression analyses, BMI, FPG, 2-h PG, and white rice intake were associated with HbA1c levels in men, while BMI, FPG, HOMA-ß, and bread intake were associated with HbA1c levels in women. CONCLUSIONS: The present findings suggest that a substantial portion of HbA1c may be composed of not only glycemic but also several lifestyle factors in men with impaired glucose tolerance. These factors can be taken into consideration as modifiable determinants in assessing the HbA1c level for the diagnosis and therapeutic monitoring of the disease course.

Effects of lifestyle intervention on weight and metabolic parameters in patients with impaired glucose tolerance related to beta-3 adrenergic receptor gene polymorphism Trp64Arg(C/T): Results from the Japan Diabetes Prevention Program.

The beta-3 adrenergic receptor (ADRB3), primarily expressed in adipose tissue, is involved in the regulation of energy metabolism. The present study hypothesized that ADRB3 (Trp64Arg, rs4994) polymorphisms modulate the effects of lifestyle intervention on weight and metabolic parameters in patients with impaired glucose tolerance. Data were analyzed from 112 patients with impaired glucose tolerance in the Japan Diabetes Prevention Program, a lifestyle intervention trial, randomized to either an intensive lifestyle intervention group or usual care group. Changes in weight and metabolic parameters were measured after the 6-month intervention. The ADRB3 polymorphisms were determined using the polymerase chain reaction restriction fragment length polymorphism method. Non-carriers showed a greater weight reduction compared with the carriers in both the lifestyle intervention group and usual care group, and a greater increase of high-density lipoprotein cholesterol levels than the carriers only in the lifestyle intervention group. ADRB3 polymorphisms could influence the effects of lifestyle interventions on weight and lipid parameters in impaired glucose tolerance patients.

Effect of baseline HbA1c level on the development of diabetes by lifestyle intervention in primary healthcare settings: insights from subanalysis of the Japan Diabetes Prevention Program.

OBJECTIVES: To determine the effects of a lifestyle intervention on the development of type 2 diabetes mellitus (T2DM) among participants with impaired glucose tolerance (IGT), in particular in the subgroup with baseline glycated hemoglobin (HbA1c) levels ≥5.7%, in primary healthcare settings. DESIGN: Randomized controlled trial. SETTING: 32 healthcare centers in Japan. PARTICIPANTS: Participants with IGT, aged 30-60 years, were randomly assigned to either an intensive lifestyle intervention group (ILG) or a usual care group (UCG). INTERVENTIONS: During the initial 6 months, participants in the ILG received four group sessions on healthy lifestyles by public health providers. An individual session was further conducted biannually during the 3 years. Participants in the UCG received usual care such as one group session on healthy lifestyles. OUTCOME MEASURES: The primary endpoint was the development of T2DM based on an oral glucose tolerance test. RESULTS: The mean follow-up period was 2.3 years. The annual incidence of T2DM were 2.7 and 5.1/100 person-years of follow-up in the ILG (n=145) and UCG (n=149), respectively. The cumulative incidence of T2DM was significantly lower in the ILG than in the UCG among participants with HbA1c levels ≥5.7% (log-rank=3.52, p=0.06; Breslow=4.05, p=0.04; Tarone-Ware=3.79, p=0.05), while this was not found among participants with HbA1c levels <5.7%. CONCLUSIONS: Intensive lifestyle intervention in primary healthcare setting is effective in preventing the development of T2DM in IGT participants with HbA1c levels ≥5.7%, relative to those with HbA1c levels <5.7%. TRIAL REGISTRATION NUMBER: UMIN000003136.

Potent lipolytic activity of lactoferrin in mature adipocytes.

Lactoferrin (LF) is a multifunctional glycoprotein found in mammalian milk. We have shown in a previous clinical study that enteric-coated bovine LF tablets decreased visceral fat accumulation. To address the underlying mechanism, we conducted in vitro studies and revealed the anti-adipogenic action of LF in pre-adipocytes. The aim of this study was to assess whether LF could increase the lipolytic activity in mature adipocytes. Pre-adipocytes were prepared from rat mesenteric fat and differentiated into mature adipocytes for assays of lipolysis. The addition of LF significantly increased the glycerol concentration in the medium in a dose-dependent manner, whereas pepsin-degraded LF did not. A DNA microarray analysis demonstrated that LF decreased the expression of perilipin and affected the cAMP pathway. These findings are supported by the results of quantitative RT-PCR of perilipin and assays of cAMP. These data collectively indicate that visceral fat reduction by LF may result from the promotion of lipolysis and the additional anti-adipogenic activity of LF.

Proinsulin C-peptide activates α-enolase: implications for C-peptide--cell membrane interaction.

Proinsulin C-peptide shows beneficial effects on microvascular complications of Type 1 diabetes. However, the possible occurrence of membrane C-peptide receptor(s) has not been elucidated. The aim of this study was to identify and characterize membrane proteins to which C-peptide binds. The enzyme α-enolase was co-immunoprecipitated with C-peptide after chemical cross-linking to HL-60 cell surface proteins and identified by mass spectrometry. Recombinant α-enolase activity was modulated by C-peptide, with a significant decrease in K(m) for 2-phosphoglycerate without affecting V(max). The enzyme modulation by C-peptide was abolished when C-terminal basic lysine residue (K434) of the enzyme was replaced by neutral alanine or acidic glutamate, but not with basic arginine. The enzyme modulation by C-peptide was reproduced with the C-peptide fragments containing glutamate corresponding to position 27 (E27) of the full-length C-peptide. Addition of a lysine analogue to the assay and A31 cell culture abrogated the enzyme modulation and MAP kinase activation by C-peptide, respectively. The results indicate that C-peptide has the capacity to activate α-enolase through a specific interaction between E27 of the peptide and K434 of the enzyme. Since α-enolase plays a role as a cell surface receptor for plasminogen, it may conceivably also serve as a receptor for C-peptide in vivo.

Prevention of type 2 diabetes in a primary healthcare setting: three-year results of lifestyle intervention in Japanese subjects with impaired glucose tolerance.

BACKGROUND: A randomized control trial was performed to test whether a lifestyle intervention program, carried out in a primary healthcare setting using existing resources, can reduce the incidence of type 2 diabetes in Japanese with impaired glucose tolerance (IGT). The results of 3 years' intervention are summarized. METHODS: Through health checkups in communities and workplaces, 304 middle-aged IGT subjects with a mean body mass index (BMI) of 24.5 kg/m2 were recruited and randomized to the intervention group or control group. The lifestyle intervention was carried out for 3 years by public health nurses using the curriculum and educational materials provided by the study group. RESULTS: After 1 year, the intervention had significantly improved body weight (-1.5 ± 0.7 vs. -0.7 ± 2.5 kg in the control; p = 0.023) and daily non-exercise leisure time energy expenditure (25 ± 113 vs. -3 ± 98 kcal; p = 0.045). Insulin sensitivity assessed by the Matsuda index was improved by the intervention during the 3 years. The 3-year cumulative incidence tended to be lower in the intervention group (14.8% vs.8.2%, log-rank test: p = 0.097). In a sub-analysis for the subjects with a BMI > 22.5 kg/m2, a significant reduction in the cumulative incidence was found (p = 0.027). CONCLUSIONS: The present lifestyle intervention program using existing healthcare resources is beneficial in preventing diabetes in Japanese with IGT. This has important implications for primary healthcare-based diabetes prevention. TRIAL REGISTRATION NUMBER: UMIN000003136.

Effects of pepsin and trypsin on the anti-adipogenic action of lactoferrin against pre-adipocytes derived from rat mesenteric fat.

Lactoferrin (LF) is a multifunctional glycoprotein in mammalian milk. In a previous report, we showed that enteric-coated bovine LF tablets can decrease visceral fat accumulation, hypothesising that the enteric coating is critical to the functional peptides reaching the visceral fat tissue and exerting their anti-adipogenic activity. The aim of the present study was to assess whether ingested LF can retain its anti-adipogenic activity. We therefore investigated the effects of LF and LF treated with digestive enzymes (the stomach enzyme pepsin and the small intestine enzyme trypsin) on lipid accumulation in pre-adipocytes derived from the mesenteric fat tissue of male Sprague-Dawley rats. Lipid accumulation in pre-adipocytes was significantly reduced by LF in a dose-dependent manner and was associated with reduction in gene expression of CCAAT/enhancer binding protein delta, CCAAT/enhancer binding protein alpha and PPARγ as revealed by DNA microarray analysis. Trypsin-treated LF continued to show anti-adipogenic action, whereas pepsin-treated LF abrogated the activity. When an LF solution (1000 mg bovine LF) was administered by gastric intubation to Sprague-Dawley rats, immunoreactive LF determined by ELISA could be detected in mesenteric fat tissue at a concentration of 14·4 µg/g fat after 15 min. The overall results point to the importance of enteric coating for action of LF as a visceral fat-reducing agent when administered in oral form.

Potent anti-obesity effect of enteric-coated lactoferrin: decrease in visceral fat accumulation in Japanese men and women with abdominal obesity after 8-week administration of enteric-coated lactoferrin tablets.

Lactoferrin (LF), a multifunctional glycoprotein in mammalian milk, is reported to exert a modulatory effect on lipid metabolism. The aim of the present study was to elucidate whether enteric-coated LF (eLF) might improve visceral fat-type obesity, an underlying cause of the metabolic syndrome. Using a double-blind, placebo-controlled design, Japanese men and women (n 26; aged 22-60 years) with abdominal obesity (BMI>25 kg/m2, and visceral fat area (VFA)>100 cm2) consumed eLF (300 mg/d as bovine LF) or placebo tablets for 8 weeks. Measurement of the total fat area, VFA and subcutaneous fat area from computed tomography images revealed a significant reduction in VFA ( - 14.6 cm2) in the eLF group, as compared with the placebo controls ( - 1.8 cm2; P = 0.009 by ANCOVA). Decreases in body weight, BMI and hip circumference in the eLF group ( - 1.5 kg, - 0.6 kg/m2, - 2.6 cm) were also found to be significantly greater than with the placebo (+1.0 kg, +0.3 kg/m2, - 0.2 cm; P = 0.032, 0.013, 0.041, respectively). There was also a tendency for a reduction in waist circumference in the eLF group ( - 4.4 cm) as compared with the placebo group ( - 0.9 cm; P = 0.073). No adverse effects of the eLF treatment were found with regard to blood lipid or biochemical parameters. From these results, eLF appears to be a promising agent for the control of visceral fat accumulation.

Lack of association of the Trp64Arg polymorphism of beta3-adrenergic receptor gene with energy expenditure in response to caffeine among young healthy women.

A close relationship between coffee intake and certain metabolic disorders is known. Caffeine, one of coffee components, can increase energy expenditure (EE), but there are considerable individual differences in the caffeine effects on EE, and the causes have not been fully established in humans. The Arg allele in the beta(3)-adrenergic receptor gene (beta(3)-AR), a marker for obesity-related traits, may be a contributor to individual variations in EE. This study investigated the effect of the Arg allele of beta(3)-AR on caffeine-induced increases in EE. In 44 healthy young women (21 +/- 1 years), physical characteristics, blood pressure, biochemical profiles and dietary nutritional intake were measured. A caffeine-loading test was conducted at a dosage of 4 mg per body weight (kg). EE was measured using an indirect open-circuit calorimeter for a 10-min period before, and at 30 min and 60 min after the caffeine-loading test. The beta(3)-AR Trp64Arg polymorphism was detected with a PCR-restriction fragment length polymorphism method. The frequency of the Arg allele was 24%. The distribution of the Trp/Trp, Trp/Arg, and Arg/Arg genotypes was 58%, 36%, and 6%, respectively. At the baseline, subjects with the Arg/Arg genotype had a significantly lower EE level than those with the Trp/Trp or Trp/Arg genotype. After the caffeine-loading test, there were caffeine-induced increases in EE in all genotypes, but there were no differences in the levels of increase among the genotypes. These findings suggest that the genotypes of beta(3)-AR Trp64Arg polymorphism might be not associated with caffeine-induced increases in EE levels.

Proinsulin C-peptide abrogates type-1 diabetes-induced increase of renal endothelial nitric oxide synthase in rats.

BACKGROUND: Proinsulin C-peptide shows ameliorative effects on diabetic complications, possibly through the production of nitric oxide (NO). On the contrary, increased local availability of NO and expression of endothelial NO synthase (eNOS) in the renal endothelium are shown to be involved in the progression of diabetic nephropathy. The aim of this study was to elucidate the effect of C-peptide and insulin as a reference on the eNOS expression in the early phase of type 1 diabetic rat kidney. METHODS: Type 1 diabetes in rats was produced by streptozotocin injection and some of the rats were treated with either C-peptide or insulin by the aid of an osmotic pump for 1 week. Conventional biochemical and histological analyses were performed on tissue samples. RESULTS: The diabetic rats showed hyperglycemia with over 90% reduction of endogenous insulin and C-peptide. Replacement with C-peptide or insulin resulted in recovery of weight lost, but only insulin infusion lowered plasma-glucose concentration. The eNOS protein was localized in glomeruli and endothelial cells of arterioles, and its amounts in the kidneys, but not in the lungs, of diabetic rats was increased. Replacement with C-peptide or insulin-abrogated diabetes-induced increase of renal eNOS protein. CONCLUSION: The results indicate that C-peptide suppresses diabetes-induced abnormal renal eNOS expression, by which C-peptide may exert beneficial effects on diabetic nephropathy.

Up-regulation of muscle uncoupling protein 3 gene expression by calcium channel blocker, benidipine hydrochloride in rats.

To examine whether benidipine hydrochloride, one of the calcium channel blockers, up-regulate uncoupling protein 3 (UCP3) expression in two skeletal muscles (gastrocnemius and soleus) in rats. Wistar rats were treated orally with benidipine hydrochloride at 4 mg/kg for 7 days. Blood pressure was measured after 4 days. At the end of experiments, the rats were weighed, and brown adipose tissue (BAT) and skeletal muscles (gastrocnemius and soleus muscles) were removed. The mRNA levels of uncoupling protein 1 (UCP1) and UCP3 were measured using the real-time quantitative reverse transcription-polymerase chain reaction method. Benidipine reduced body weight and also had a hypotensive effect. In rats treated with benidipine, UCP1 mRNA levels were significantly increased 1.4-fold in BAT, and UCP3 mRNA levels in BAT and gastrocnemius muscle were significantly increased 1.7 and 3.0-fold, respectively, compared with the control rats. There was no difference in UCP3 mRNA levels in soleus muscle between the two groups. We concluded that benidipine up-regulates not only UCP1 gene expression in BAT but also UCP3 gene expression in BAT and gastrocnemius muscle, which may contribute to thermogenesis in rats.

Relationship between polymorphisms 804C/A and 252A/G of lymphotoxin-alpha gene and -308G/A of tumor necrosis factor alpha gene and diabetic retinopathy in Japanese patients with type 2 diabetes mellitus.

To clarify whether polymorphisms of the lymphotoxin-alpha (LTA) gene and tumor necrosis factor alpha (TNF-alpha) gene were related to diabetic retinopathy (DR), we performed a case-control study in 251 Japanese patients with type 2 diabetes mellitus participating in a multicenter research protocol. Genetic analyses were performed by using a fluorescent allele-specific DNA primer assay system. Diabetic retinopathy was diagnosed in a masked manner by an independent ophthalmologist using fundus photographs and was classified as nondiabetic retinopathy (NDR), nonproliferative retinopathy (NPDR), and proliferative retinopathy (PDR). The results showed that the genotype frequencies of 804C/A in exon 3 and 252A/G in intron 1 of the LTA gene were not significantly different among patients with NDR, NPDR, and PDR. A allelic frequency of the TNF-alpha gene (-302A/G in promoter) was also identical among NDR, NPDR, and PDR groups. Multivariate logistic regression analyses showed that significant associations with DR were glycosylated hemoglobin level and diabetes duration, but not polymorphisms of the LTA gene or TNF-alpha gene. In conclusion, the present study showed no association between polymorphisms 804C/A and 252A/G of the LTA gene and -302A/G of the TNF-alpha gene and DR in Japanese type 2 diabetic patients.

Angiotensinogen gene polymorphism (Met235Thr) influences visceral obesity and insulin resistance in obese Japanese women.

To investigate the relationship between angiotensinogen (AGT) Met235Thr polymorphism (M235T) and human obesity, because AGT is regarded as one of the cytokines produced from adipocytes and serum AGT concentrations are reported to be positively correlated with body mass index. One hundred and twenty obese Japanese women (age, 58.8+/-9.4 years; body mass index, 32.2+/-4.9 kg/m(2)) were enrolled. Angiotensinogen genotypes were determined with a fluorescent allele-specific DNA primer assay system. Subjects were divided into M/M, M/T, and T/T groups. Control subjects comprised 146 healthy age-matched women. Clinical characteristics and the effects of diet and exercise therapy for 6 months were compared among the 3 genotypes. The genotype frequencies of AGT M235T polymorphism were in accordance with the Hardy-Weinberg equation (obese: M/M, 6.7%; M/T, 27.5%; T/T, 65.8%; control: M/M, 6.8%; M/T, 21.2%; T/T, 71.9%). The frequency of the T allele did not differ between obese and control subjects (0.80 vs 0.83). As the number of obese women with M/M genotype was only 8, comparisons of the characteristics and outcomes of weight reduction therapy were performed only between subjects with M/T genotype and T/T genotype. In the T/T group, % body fat and waist circumference at baseline were significantly greater than in the M/T group (36.3%+/-4.8% vs 33.8%+/-4.7%, P=.0105; 107.9+/-10.9 vs 102.6+/-7.9 cm, P=.0428, respectively). Before the weight reduction therapy, significantly higher insulin and higher homeostasis model assessment (HOMA-R) were demonstrated in the T/T group than in the M/T group (9.1+/-5.5 microU/mL vs 5.9+/-4.4 microU/mL, P=.0056; 2.3+/-1.4 vs 1.6+/-1.3, P=.0252, respectively). Both systolic and diastolic blood pressure at baseline in the T/T group tended to be higher than those in the M/T group, but the differences were not significant. No genotype-dependent difference in energy expenditure or outcome of weight reduction therapy was observed with respect to AGT M235T polymorphism. After the diet and exercise therapy, the blood pressure in the T/T group tended to be higher than that in the M/T group, but the difference was not significant. We demonstrated that the T/T genotype of the AGT M235T gene polymorphism was positively related to visceral obesity and hyperinsulinemia in obese Japanese women. Blood pressure did not show genotype-specific differences before or after the treatment. Further studies of the association between obesity and this gene polymorphism should contribute to understanding and treating obesity-related diseases.

In vivo brain microdialysis studies on the striatal dopamine and serotonin release in zitter mutant rats.

In the present study, using in vivo brain microdialysis, we investigated the basal extracellular dopamine (DA) and serotonin (5-HT) release in the caudal striatum (cSTR) of young (4-6 months old) and aged (10-12 months old) zitter mutant rats. The basal extracellular levels of DA release in both young and aged zitter rats were significantly lower than that of age-matched Sprague-Dawley (SD) rats, whereas only aged zitter rats showed a significant difference in the basal 5-HT release. Dopaminergic neurons were more vulnerable than serotonergic neurons in the cSTR of zitter mutant rats during aging. Perfusion of 60 mM potassium (K+) enhanced the extracellular levels of cSTR DA in the young zitter rats and the extracellular levels of both DA and 5-HT in the cSTR of the aged zitter rats. The firing rate of K+-stimulated monoamine release in the cSTR was significantly higher in the zitter rats than in the age-matched SD rats. These findings suggest that there are innate quantitative differences in the releasable pool and the availability of monoamines in the cSTR of zitter mutant rats.

Relation between polymorphisms G1704T and G82S of rage gene and diabetic retinopathy in Japanese type 2 diabetic patients.

OBJECTIVE: To clarify whether polymorphisms G1704T and G82S of the rage gene were related to diabetic retinopathy, we performed a case-control study in Japanese type 2 diabetic patients. PATIENTS AND METHODS: Two hundred and sixty-eight patients with type 2 diabetes were examined for polymorphisms G1704T and G82S of the RAGE gene. The genotypes of G1704T and G82S of the RAGE gene were determined with a fluorescent allele-specific DNA primer assay system. Diabetic retinopathy (DR) was diagnosed in a masked manner by independent ophthalmologists using fundus photographs and was classified as non-diabetic retinopathy (NDR), non-proliferative retinopathy (NPDR), and proliferative retinopathy (PDR). RESULTS: The T allele frequency of G1704T and S allele frequency of G82S in patients with DR did not significantly differ from those without retinopathy. There were no differences among the genotypes of G1704T and G82S of the RAGE gene regarding age, duration of diabetes, BMI, HbA(1c), blood pressure, and lipids levels. CONCLUSION: These data suggest that polymorphisms G1704T and G82S of the RAGE gene are not related to DR in Japanese type 2 diabetic patients.

Association study of G1704T and G82S polymorphisms of RAGE gene for microalbuminuria in Japanese type 2 diabetic patients.

To clarify whether polymorphisms G1704T and G82S of the RAGE gene were related to microalbuminuria, we performed a case-control study in Japanese type 2 diabetic patients. Polymorphisms G1704T and G82S of the RAGE gene were examined with genomic DNA obtained from 116 type 2 diabetic patients with microalbuminuria (urinary albumin/creatinine ratio between 30 and 300 mg/g of creatinine) (microalbuminuria group), and 232 patients with normoalbuminuria (urinary albumin/creatinine ratio <30 mg/g of creatinine) (normoalbuminuria group). The genotype distribution and T allele frequency of G1704T (9.9%) and S allele frequency of G82S (14.2%) in the microalbuminuria group did not significantly differ from those (T allele frequency, 8.4%; S allele frequency, 12.3%) in the normoalbuminuria group. There were no differences among the genotypes of G1704T and G82S of the RAGE gene regarding age, duration of diabetes, body mass index, glycosylated hemoglobin (HbA1c), blood pressure, and serum lipid levels. These data suggest that G1704T and G82S polymorphisms of the RAGE gene are not related to microalbuminuria in Japanese type 2 diabetic patients.