Metformin, but not pioglitazone, decreases postchallenge plasma ghrelin levels in type 2 diabetic patients: a possible role in weight stability?

AIM: Effects of metformin and pioglitazone on body weight are clearly different. Recently, the role of ghrelin, an orexigenic peptide derived from stomach, has been appreciated. Plasma ghrelin levels display a preprandial peak and postprandial suppression, suggesting its physiological role. We hypothesized that metformin or pioglitazone may modulate circulating ghrelin levels and this modulation may be related to differential effects on body weight with these agents. METHODS: Thirty-five Japanese type 2 diabetic patients [21 men and 14 women, age 62 +/- 2 years, body mass index (BMI) 26.6 +/- 0.5 kg/m(2) and haemoglobin A1c (HbA1c) 8.2 +/- 0.1%, mean +/- s.e.] were randomly assigned to groups for the addition of metformin or pioglitazone. At baseline and 4 months later, a 75-g oral glucose tolerance test (OGTT) was performed to measure plasma ghrelin levels. RESULTS: In 33 subjects who completed the study, the overall decrease in HbA1c ( approximately 1%) was comparable between the two groups. As expected, BMI increased in the pioglitazone group but not in the metformin group. After the treatment, plasma ghrelin levels at each point of OGTT remained unchanged in the pioglitazone group. In the metformin group, fasting ghrelin levels were unaltered, whereas the absolute levels at 30, 60 and 120 min decreased significantly. The area under the curve for the 2-h ghrelin profile also decreased significantly. CONCLUSIONS: Metformin, but not pioglitazone, decreased plasma ghrelin levels after the glucose load. This decrease may in part account for weight stability in type 2 diabetic patients treated with metformin.

Long-range correlated glucose fluctuations in diabetes.

OBJECTIVES: Our objective is to investigate diabetes-related alteration of glucose control in diurnal fluctuations in normal daily life by detrended fluctuation analysis (DFA). METHODS: The fluctuations of glucose of 12 non-diabetic subjects and 15 diabetic patients were measured using a continuous glucose monitoring system (CGMS) over a period of one day. The glucose data was calculated by the DFA method, which is capable of revealing the presence of long-range correlations in time series with inherent non-stationarity. RESULTS: Compared with the non-diabetic subjects, the mean glucose level and the standard deviation are significantly higher in the diabetic group. The DFA exponent alpha is calculated, and glucose time series are searched for the presence of negatively (0.5 < alpha < 1.5) or positively (1.5 < alpha) correlated fluctuations. A crossover phenomenon, i.e. a change in the level of correlations, is observed in the non-diabetic subjects at about two hours; the net effects of glucose flux/reflux causing temporal changes in glucose concentration are negatively correlated in a "long-range" (> two hours) regime. However, for diabetic patients, the DFA exponent alpha = 1.65 +/- 0.30, and in the same regime positively correlated fluctuations are observed, suggesting that the net effects of the flux and reflux persist for many hours. CONCLUSIONS: Such long-range positive correlation in glucose homeostasis may reflect pathogenic mechanisms of diabetes, i.e., the lack of the tight control in blood glucose regulation. Using modern time series analysis methods such as DFA, continuous evaluation of glucose dynamics could promote better diagnoses and prognoses of diabetes and a better understanding of the fundamental mechanism of glucose dysregulation in diabetes.

Effects of statins on the adipocyte maturation and expression of glucose transporter 4 (SLC2A4): implications in glycaemic control.

AIMS/HYPOTHESIS: Hyperlipidaemia often occurs in patients with type 2 diabetes mellitus. Though HMG-CoA reductase inhibitors (statins) are widely used for controlling hypercholesterolemia, atorvastatin has also been reported to have an adverse effect on glucose metabolism. Based on these findings, the aim of this study was to investigate the effects of statins on adipocytes, which play pivotal roles in glucose metabolism. METHODS: In 3T3-L1 cells, effects of statins on adipocyte maturation were determined morphologically. Protein and mRNA levels of SLC2A4 and adipocyte marker proteins were determined by immunoblotting and RT-PCR, respectively. Type 2 diabetic NSY mice were treated with atorvastatin for 15 weeks, followed by glucose and insulin tolerance tests and examination of SLC2A4 expression in white adipose tissue (WAT). Seventy-eight Japanese subjects with type 2 diabetes and hypercholesterolaemia were treated with atorvastatin (10 mg/day), and its effects on lipid and glycaemic profiles were measured 12 weeks after treatment initiation. RESULTS: Treatment with atorvastatin inhibited adipocyte maturation, SLC2A4 and C/EBPalpha expressions and insulin action in 3T3-L1 cells. Atorvastatin also attenuated SLC2A4 and C/EBPalpha expressions in differentiated 3T3-L1 adipocytes. These effects were reversed by L. mevalonate or geranylgeranyl pyrophosphate. In NSY mice, atorvastatin accelerated glucose intolerance as a result of insulin resistance and decreased SLC2A4 expression in WAT. In addition to improving hyperlipidaemia, atorvastatin treatment significantly increased HbA(1c) but not fasting glucose levels in diabetic patients, and this effect was greater in the non-obese subgroup. CONCLUSIONS/INTERPRETATION: These results demonstrate that atorvastatin attenuates adipocyte maturation and SLC2A4 expression by inhibiting isoprenoid biosynthesis, and impairs glucose tolerance. These actions of atorvastatin could potentially affect the control of type 2 diabetes.

Urinary excretion of the aquaporin-2 water channel exaggerated in pathological states of impaired water excretion.

OBJECTIVE: The present study was undertaken to determine whether the hydro-osmotic action of arginine vasopressin (AVP) is exaggerated in pathological states of impaired water excretion by measuring urinary excretion of the aquaporin-2 (AQP-2) water channel. PATIENTS AND MEASUREMENTS: Eighteen hyponatraemic patients with impaired water excretion and 12 control subjects were studied during an acute oral water load (20 ml/kg body weight). RESULTS: In the patient group plasma AVP levels were 1.6 pmol/l, relatively high compared to plasma osmolality of 279.8 mmol/kg. Urinary excretion of AQP-2 under ad libitum water drinking was 41.1 fmol/micromol creatinine in the patient group, a value significantly greater than that of 21.7 fmol/micromol creatinine in the control subjects. The acute water load verified the impairment in water excretion in the patient group, as the excretion of the water load was only 28.2% (control, 77.3%, P < 0.001) and the minimum urinary osmolality was as high as 437.3 mmol/kg (control, 122.9 mmol/kg, P < 0.001). Also, the minimum urinary excretion of AQP-2 was significantly greater in the patient group than that in the control. There was a positive correlation between plasma AVP levels and urinary excretion of AQP-2 in the control subjects (r = 0.56, P < 0.01). In contrast, the urinary excretion of AQP-2 was exaggerated compared to the respective plasma AVP levels in the patient group, and thus the positive correlation disappeared. CONCLUSION: These results indicate that hydroosmotic action of AVP is exaggerated more than that expected from plasma AVP levels in pathological states of impaired water excretion, with non-suppressible, but normal, arginine vasopressin levels in spite of the hypo-osmotic condition.

Chronic hypernatremia derived from hypothalamic dysfunction: impaired secretion of arginine vasopressin and enhanced renal water handling.

We analyzed the disorder of water metabolism in a 32 year-old female with chronic hypernatremia. She had meningitis at 4 years, and ventriculo-peritoneal shunt operation at 13 years because of normal pressure hydrocephalus. At 14 years hypernatremia of 166 mmol/l was initially found and thereafter hypernatremia ranging from 150 to 166 mmol/l has been persisted for the last 18 years. Physical and laboratory findings did not show dehydration. Urine volume was 750-1700 ml per day and urinary osmolality (Uosm) 446-984 mmol/kg, suggesting no urinary concentrating defect. Plasma arginine vasopressin (AVP) levels ranged from 0.4 to 1.2 pmol/l despite hyperosmolality of 298 through 343 mmol/kg under ad libitum water drinking. There was no correlation between plasma osmolality (Posm) and plasma AVP levels, but Uosm had a positive correlation with Posm (r=0.545, P < 0.05). Hypertonic saline (500 NaCl) infusion after a water load increased Uosm from 377 to 679 mmol/kg, and plasma AVP from 0.2 to 1.3 pmol/l. There was a positive correlation between Posm and plasma AVP levels in the hypertonic saline test (r=0.612, P<0.05). In contrast, an acute water load (20 ml/kg BW) verified the presence of impaired water excretion, as the percent excretion of the water load was only 8.5% and the minimal Uosm was as high as 710 mmol/kg. Urinary excretion of aquaporin-2 remained low in concert with plasma AVP levels. No abnormality in pituitary-adrenocortical function was found. These results indicate that marked hypernatremia is derived from partial central diabetes insipidus and elevated threshold of thirst, and that enhanced renal water handling may contribute to maintenance of body water in the present subject.

Clinical implication of serial leptin measurement in subjects with type 2 diabetes mellitus.

Plasma leptin concentration is closely associated with body fat in humans, with energy restriction inducing a greater decrease in plasma leptin than in body fat. Since adequate energy restriction is mandatory in diet therapy of diabetes mellitus especially in obese subjects, the present study was undertaken to evaluate the clinical implication of serial leptin measurement in the management of diabetic patients. Fifty-four consecutive subjects with type 2 diabetes, who were subjected to adjusted energy restriction during hospitalization, were enrolled in the study. During their hospitalization period (24+/-4 days), plasma leptin concentrations decreased from 6.9+/-0.7 to 5.7+/-0.6 microg/l (P<0.0001) in the overall subjects, and the %change in plasma leptin (-13.9%) was greater than the %changes in body mass index (BMI) and percent body fat (-1.7% and -4.7%, respectively). The %change in plasma leptin was positively correlated with the %changes in BMI and plasma C-peptide (r=0.526, P<0.0001 and r=0.446, P<0.002, respectively) and negatively with a %change in plasma ketone bodies (r=-0.516, P<0.005). Multiple regression analysis revealed that the %changes in BMI and plasma C-peptide were independent determinants of the %change in plasma leptin. In addition, 38 subjects were followed up after discharge. Three months after discharge, plasma leptin concentrations significantly increased by 25.6%, which was again much greater than the %change in BMI (+0.9%). In 28 subjects who showed increase in plasma leptin levels after discharge, BMI was also increased. In contrast, the remaining 10 subjects without the increase in plasma leptin kept their BMI unchanged. Throughout the observation period, the changes in plasma leptin were prominent in the subjects with BMI greater than 25 kg/m2. In conclusion, plasma leptin concentrations showed greater changes than the alterations in anthropometric indexes during the observation period. Serial leptin measurement may be useful to estimate adherence to energy restriction especially in obese subjects with type 2 diabetes.

Close association of urinary excretion of aquaporin-2 with appropriate and inappropriate arginine vasopressin-dependent antidiuresis in hyponatremia in elderly subjects.

The present study was undertaken to determine whether urinary excretion of aquaporin-2 (AQP-2) participates in the involvement of arginine vasopressin (AVP) in hyponatremia less than 130 mmol/L in 33 elderly subjects (> or =65 yr old) during the last 5-yr period. Subjects were separated into euvolemic hyponatremia groups: 13 with hypopituitarism, 8 with syndrome of inappropriate secretion of antidiuretic hormone (SIADH), 8 with mineralocorticoid-responsive hyponatremia of the elderly, and 4 with miscellaneous diseases. Approximately 40% of those with hyponatremia was derived from hypopituitarism, but severe hyponatremia was found in the patients with SIADH and mineralocorticoid-responsive hyponatremia of the elderly. Plasma AVP levels remained relatively high despite hypoosmolality and were tightly linked with exaggerated urinary excretion of AQP-2 and antidiuresis in the 3 groups of patients, except for one miscellaneous one. An acute water load test verified the impairment in water excretion, because the percent excretion of the water load was less than 42% and the minimal urinary osmolality was not sufficiently diluted. Also, plasma AVP and urinary excretion of AQP-2 were not reduced after the water load. The inappropriate secretion of AVP was evident in the patients with SIADH and hypopituitarism, and hydrocortisone replacement normalized urinary excretion of AQP-2 and renal water excretion in those with hypopituitarism. In contrast, the appropriate antidiuresis seemed to compensate loss of body fluid in the patients with mineralocorticoid-responsive hyponatremia of the elderly, who lost circulatory blood volume by 7.3% (mean). Fludrocortisone acetate increased renal sodium handling and body fluid, resulting in the reduction in AVP release and urinary excretion of AQP-2 in mineralocorticoid-responsive hyponatremia of the elderly. These findings indicate that urinary excretion of AQP-2 may be a more sensitive measure of AVP effect on renal collecting duct cells than are plasma AVP levels, and that increased urinary excretion of AQP-2 shows exaggerated AVP-induced antidiuresis in hyponatremic subjects in the elderly. In addition, mineralocorticoid-responsive hyponatremia of the elderly has to be carefully differentiated from SIADH in elderly subjects.

Arginine vasopressin inhibits apoptosis of rat glomerular mesangial cells via V1a receptors.

Arginine vasopressin (AVP) promotes proliferation of glomerular mesangial cells. We examined whether AVP modulates an apoptosis of cultured rat glomerular mesangial cells at 3-17th passages. The agarose gel electrophoresis demonstrated that AVP attenuated a ladder formation stimulated by the serum deprivation. The quantitation of oligonucleosomes by ELISA also showed that AVP suppressed the serum deprivation-induced apoptosis. Such an antiapoptotic effect of AVP was dose-dependent. An AVP V1a receptor antagonist, d(CH2)5Tyr(Me)AVP, abolished the antiapoptotic effect of AVP. The inhibitory effect of AVP on the apoptosis was reduced by staurosporine and mimicked by phorbol-12-myristate-13-acetate. These results suggest that AVP inhibits serum deprivation-induced apoptosis of glomerular mesangial cells via V1a receptor-protein kinase C pathway.

Association of hyperthyroidism with serum leptin levels.

The present study was undertaken to determine whether thyroid hormones affect serum leptin levels in patients with hyperthyroidism. In 32 female patients with hyperthyroidism and 30 body mass index (BMI)-matched control subjects, there was no difference in serum leptin concentrations (9.0+/-1.0 v 8.2+/-0.9 microg/L). The serum leptin levels were compared with the expected values for serum leptin derived from the corresponding BMI with the formula for the control subjects (serum leptin [micrograms per liter] = 0.976 x BMI [kilograms per square meter] - 11.933, P < .001) and are expressed as the percent deviation (%leptin). There was a weak but significant positive correlation between serum free thyroxine (T4) levels and %leptin in the combined analysis of patients with hyperthyroidism and control subjects (r = .28, P < .05). Multiple regression analysis also revealed the independent contribution of free T4 levels to serum leptin. Antithyroid therapy ameliorated the hyperthyroidism and increased the BMI by 7.8% and the percent body fat by 9.9% in a subgroup of 21 patients with hyperthyroidism. However, serum leptin levels did not change during the 2-month therapeutic period (from 9.8+/-1.4 to 8.7+/-1.0 microg/L), and accordingly, %leptin significantly decreased from 133%+/-16% to 98%+/-11% (P < .05). A total of 6 months of observation in 12 hyperthyroid patients showed the same alterations in the anthropometric indexes, serum leptin, and %leptin. These results indicate that serum leptin is slightly increased in subjects with moderate hyperthyroidism, possibly due to the direct action of thyroid hormone, and the levels decline in accordance with the attainment of euthyroidism.

Crucial role of insulin in leptin maintenance: profound decrease in serum leptin by octreotide acetate in insulinoma subjects.

To further clarify the relationship between insulin and leptin, time course changes in plasma glucose, serum insulin and leptin levels were analyzed after subcutaneous administration of 100 microg octreotide acetate in two insulinoma subjects. Octreotide acetate induced a prompt decrease in serum insulin level, accompanied with an increase in plasma glucose in both patients. Following the decrease in serum insulin level, serum leptin concentrations were profoundly decreased by 66% and 44%, 8-12 hrs after octreotide injection; that is, the concentrations decreased from 41.1 to 13.8 ng/ml in patient 1, and from 17.5 to 9.8 ng/ml in patient 2. Daily profiles of plasma glucose, serum insulin and leptin without octreotide administration did not show such alterations in these indexes in patient 1. These data show that circulating leptin may be susceptible to decline dependent on the decrease in serum insulin, suggesting that insulin plays a crucial role in the maintenance of leptin secretion in humans.

Serum levels of leptin and changes during the course of recovery from diabetic ketoacidosis.

Secretion of leptin, the obese gene product, is stimulated by insulin and glucocorticoids and reduced by fasting. In subjects with diabetic ketoacidosis (DKA), severe insulinopenia and prolonged fasting might cause a decrease in serum leptin levels, and subsequent insulin therapy would reverse the decrease. Otherwise, some other confounding factors, neither insulin nor fasting, might affect serum leptin levels in patients with DKA. The present study was undertaken to address these issues. Eleven patients with type 1 diabetes mellitus (seven males and four females, aged 44+/-6 years, mean +/- SEM), admitted to Jichi Medical School Hospital presenting DKA, were studied during the therapeutic period. Thirty-five sex-, age- and body mass index-matched healthy subjects served as controls. Serum leptin levels at the hospitalization were significantly greater than those of the matched control subjects (5.5+/-1.0 vs. 3.2+/-0.3 microg/l, P < 0.01). After the start of therapy with a small dose of short-acting insulin and a large volume of fluid infusion, serum leptin concentrations further increased to 10.6+/-3.6 microg/l at 24 h, and thereafter the concentrations gradually decreased and normalized at the discharge (3.3+/-0.7 microg/l, day 24+/-4). The peak levels at 24 h were significantly higher than the levels at the discharge (P < 0.05), and also +77+/-34% higher than those at the hospitalization (P < 0.005). Serum cortisol levels (1830+/-200 nmol/l) were markedly elevated at hospitalization. These results indicate that serum leptin levels are increased even under insulinopenia and fasting in the patients with DKA. Such a finding may be associated with marked hyperglycemia or enhanced secretion of glucocorticoid hormone, although the exact mechanisms remain to be elucidated. We speculate that leptin may serve as a stress peptide in DKA, but further analysis is necessary to explore a physiological role of leptin in DKA.

Patch clamp studies on V-type ATPase of vacuolar membrane of haploid Saccharomyces cerevisiae. Preparation and utilization of a giant cell containing a giant vacuole.

A method for obtaining giant protoplasts of Escherichia coli (the spheroplast incubation (SI) method: Kuroda et al. (Kuroda, T., Okuda, N., Saitoh, N., Hiyama, T., Terasaki, Y., Anazawa, H., Hirata, A., Mogi, T., Kusaka, I., Tsuchiya, T., and Yabe, I. (1998) J. Biol. Chem. 273, 16897-16904) was adapted to haploid cells of Saccharomyces cerevisiae. The yeast cell grew to become as large as 20 micrometer in diameter and to contain an oversized vacuole inside. A patch clamp technique in the whole cell/vacuole recording mode was applied for the vacuole isolated by osmotic shock. At zero membrane potential, ATP induced a strong current (as high as 100 pA; specific activity, 0.1 pA/micrometer(2)) toward the inside of the vacuole. Bafilomycin A(1,) a specific inhibitor of the V-type ATPase, strongly inhibited the activity (K(i) = 10 nM). Complete inhibition at higher concentrations indicated that any other ATP-driven transport systems were not expressed under the present incubation conditions. This current was not observed in the vacuoles prepared from a mutant that disrupted a catalytic subunit of the V-type ATPase (RH105(Deltavma1::TRP)). The K(m) value for the ATP dose response of the current was 159 microM and the H(+)/ATP ratio estimated from the reversible potential of the V-I curve was 3.5 +/- 0.3. These values agreed well with those previously estimated by measuring the V-type ATPase activity biochemically. This method can potentially be applied to any type of ion channel, ion pump, and ion transporter in S. cerevisiae, and can also be used to investigate gene functions in various organisms by using yeast cells as hosts for homologous and heterogeneous expression systems.

Inhibition by transforming growth factor-beta1 of the cellular action of arginine vasopressin in cultured rat glomerular mesangial cells.

The present study was undertaken to determine whether transforming growth factor (TGF)-beta1 modulates the cellular actions of arginine vasopressin (AVP) in cultured rat glomerular mesangial cells. AVP increased cytosolic free calcium ([Ca2+]i), and TGF-beta1 dose-dependently reduced the AVP-mobilized [Ca2+]i. Such an inhibition by exogenous TGF-beta1 was abolished by liposomal transfection of antisense oligodeoxynucleotide for the TGF-beta type II receptor. AVP activated mitogen-activated protein (MAP) kinase, which was significantly reduced by 1 ng/ml TGF-beta1. AVP increased [3H]thymidine incorporation into mesangial cells in a dose-dependent manner, and 1 ng/ml TGF-beta1 significantly reduced the AVP-stimulated [3H]thymidine incorporation. However, 10 microM antisense oligodeoxynucleotide for the TGF-beta type II receptor seemed to attenuate the inhibition by TGF-beta1. 1 X 10(-7) M AVP significantly increased inositol 1,4,5-trisphosphate (IP3) production by 1.8-fold, but this production was totally blunted by 1 ng/ml TGF-beta1. TGF-beta1 did not affect [3H]AVP receptor binding. 1 X 10(-6) M AVP concentration stimulated TGF-beta1 production in mesangial cells by 4-fold. These results indicate that TGF-beta1 inhibits the cellular signaling of AVP at steps beyond the AVP receptors and prior to the phospholipase C activation, and that TGF-beta1 may participate in a negative feedback regulation on the cellular action of AVP in glomerular mesangial cells.

Inverse distribution of serum sodium and potassium in uncontrolled inpatients with diabetes mellitus.

It has been reported that there is an inverse relationship between serum sodium (Na) and potassium (K) levels in patients with diabetic coma. The present study was undertaken to determine whether such an inverse relation depends upon plasma glucose levels in diabetic patients for their glycemic control. We examined two hundred and fifty-two patients with diabetes mellitus admitted to our hospital during the one-year period to control their plasma glucose levels, except for those having nephropathy or liver dysfunction. Serum Na and K, plasma glucose, and serum and urinary C-peptide levels were determined. There was a negative correlation between serum Na levels and fasting plasma glucose (FPG), and, conversely, a positive correlation between serum K levels and FPG. The changes were more evident in the patients with insulin-dependent diabetes mellitus than those with non-insulin-dependent diabetes mellitus. There was an inverse relation between serum Na and K levels and it was profoundly dependent upon plasma glucose levels in all the diabetic patients before tight control of their glycemic levels. The disorder may be based on the movement of electrolytes between intra- and extracellular spaces, dependent on the impaired insulin action as well as hyperosmolality.

Endogenous glucose production and glucose effectiveness in type 2 diabetic subjects derived from stable-labeled minimal model approach.

Insulin sensitivity, glucose effectiveness, and endogenous glucose production (EGP) during stable-labeled, frequently sampled insulin-modified intravenous glucose tolerance test (FSIGT) were evaluated by a single-and two-compartment minimal model combined with nonparametric deconvolution in eleven nonobese Japanese type 2 diabetic patients. Four patients were treated with sulfonylureas, and the remaining seven with diet therapy alone. None had diabetic retinopathy and microalbuminuria. Their fasting glucose level was 117+/-7 mg/dl (mean +/- SE), and HbA1c was 6.6+/-0.3%. Age-, sex-, and BMI-matched subjects with normal glucose tolerance served as control subjects. Plasma insulin response to the stimuli and insulin sensitivity indexes (S(I), S(I)*, and S(I)2* were derived from a minimal model and single- and two-compartment-labeled minimal models) were impaired in the type 2 diabetic patients. The combined ability of glucose, per se, to increase its own uptake and suppress EGP (glucose effectiveness [SG]), which was derived from kinetic analysis of plasma glucose by a minimal model, was significantly lower in the type 2 diabetic patients (0.0132+/-0.0015 vs. 0.0203+/-0.0022; P<0.05). However, the ability of glucose, per se, to stimulate glucose uptake, assessed as S(G)* and S(G)2* from the kinetic analysis of labeled glucose by single- and two-compartment minimal model, was not impaired in those patients. EGP of the type 2 diabetic patients as a whole was suppressed to the level similar to that of the control subjects despite a higher plasma glucose level throughout FSIGT. When EGP in the diabetic subjects was analyzed, considering their recent glycemic control, the initial suppression was blunted in the patients with higher HbA1c levels. In conclusion, glucose mass action to stimulate glucose uptake remains near-normal in the lean Japanese type 2 diabetic patients of this study, whereas ability of glucose to suppress EGP is impaired in the patients with recent hyperglycemia. This blunted suppression of EGP might be one of the conspirators for decreased S(G) in subjects with type 2 diabetes.

Association of CTLA-4 polymorphism with positive anti-GAD antibody in Japanese subjects with type 1 diabetes mellitus.

OBJECTIVE: CTLA-4, expressed on activated T cells, is thought to be a negative regulator of T cell function. Its gene (2q33) may confer genetic susceptibility to type 1 diabetes mellitus (IDDM12). The present study was undertaken to clarify the role of CTLA-4 gene polymorphism in Japanese subjects with type 1 diabetes and its effect on their clinical features. SUBJECTS AND METHODS: In 117 Japanese subjects with type 1 diabetes, the CTLA-4 exon 1 polymorphism (49 A/G) was defined by PCR-RFLP analysis. Anti-GAD antibodies (GAD-Ab) and fasting serum C-peptide were also determined. 141 healthy age- and sex-matched subjects served as controls. RESULTS: The frequency of each polymorphism was not different between the type 1 diabetic subjects and the controls; AA 21, AG 42 and GG 54 for the diabetic subjects, and AA 22, AG 47 and GG 72 for the controls. The frequency of the GG genotype was higher in the diabetic subjects with positive GAD-Ab (greater than 8 U/ml) (67%) than in the GAD-Ab negative subjects (39%) (P < 0.05). The prevalence of positive GAD-Ab declined with the duration of diabetes. In the diabetic subjects with disease duration of less than 5 years (n = 40), the frequency of the GG genotype was also higher in the GAD-Ab positive subjects (71%) (P < 0.05). In the analysis of all the diabetic subjects, there was a strong association between positive GAD-Ab and beta cell function (P < 0.0001). CONCLUSIONS: There was no evidence that the CTLA-4 exon 1 polymorphism (49 A/G) confers genetic susceptibility to type 1 diabetes mellitus in our case-control study in Japanese subjects. However, the frequency of positive GAD-Ab was higher in the GG subjects. CTLA-4 polymorphism might contribute to the clinical heterogeneity of type 1 diabetes mellitus in Japanese subjects.

Immunologically-related or incidental coexistence of diabetes mellitus and Graves' disease; discrimination by anti-GAD antibody measurement.

Coexistence of diabetes mellitus and Graves' disease may be classified into either an immunologically-related or an incidental phenomenon. It has been reported that anti-GAD antibody (GAD-Ab) persists at high levels for longer duration in subjects with type 1 diabetes and Graves' disease, whereas the prevalence of positive GAD-Ab (1.5%) in 131 non-diabetic subjects with Graves' disease was comparable to that in normal subjects (0.3%, P=0.2012). Thus, GAD-Ab might be a marker of the immunologically-related coexistence of the two diseases. To test this hypothesis, we investigated characteristics of Japanese subjects having both diseases according to the presence or absence of GAD-Ab. Sixty-one patients having diabetes mellitus and Graves' disease (24 men, 37 women, aged 53+/-2 years old, mean +/- SE) were consecutively registered between 1993-1997. The patients were divided into two groups of 14 GAD-Ab positive and 47 negative subjects. In the GAD-Ab positive subjects, earlier (32+/-3 years old) and abrupt onset (86%) of diabetes and insulin dependency (64%) were documented, as would be expected from the features of type 1 diabetes. Graves' disease often preceded diabetes (57%), presenting typical manifestations (79%). In contrast, older (45+/-2 years old, P=0.0031) and gradual onset (87%, P<0.0001) of diabetes, non-insulin dependency (74%, P<0.0001), and masked manifestations of Graves' disease (57%, P=0.0214) were common in the negative subjects. Precedence of diabetes dominated in these subjects (43%, P=0.0109). Immunological studies showed less frequent HLA-DR 2 locus (0%, P<0.02) in the GAD-Ab positive subjects. There was also a trend of higher frequency of HLA-DQA1*03 allele and of lower frequency of DQA1*01 allele in these subjects. Allelic frequency of cytotoxic T lymphocyte antigen 4 (CTLA-4) differed between the positive and negative subjects (P=0.0432). There were distinct clinical and immunological differences between the GAD-Ab positive and negative subjects having both diabetes mellitus and Graves' disease. The present results indicate that GAD-Ab measurement could draw a distinction between the immunologically-related and incidental coexistence of the two diseases.