Edaravone inhibits collagen-induced arthritis possibly through suppression of nuclear factor-kappa B.

OBJECTIVE: Rheumatoid arthritis (RA) is a chronic autoimmune disease which is induced by proinflammatory cytokines or oxidative stress. The activation of nuclear factor-kappa B (NF-kappaB) that contributed to imbalance between apoptosis and proliferation of rheumatoid synovial cells (SC). Edaravone, clinically available free radical scavenger in Japan, is confirmed to be beneficial in the acute stage of stroke. We aimed to investigate the suppressive effect of edaravone on collagen-induced arthritis (CIA) mice and on the activated molecules in SC stimulated by interleukin-1beta (IL-1beta). METHODS: Edaravone was administrated intravenously at a dose of 3mg/kg of body weight to CIA mice. The progression of CIA was evaluated by the macroscopic arthritis scoring system of paws. Interleukin-6 (IL-6) and matrix metalloproteinase-3 (MMP-3) concentrations in culture medium of human SC were measured by enzyme linked immunosorbent assays. Caspase-3/7 activity and nuclear factor-kappa B (NF-kappaB) protein level of cultured human SC were estimated by fluorometric assay and Western blot analysis, respectively. RESULTS: Edaravone significantly decreased macroscopic arthritis score in CIA mice. Acceleration of IL-6 and MMP-3 productions and attenuation of caspase-3/7 activity in IL-1beta-stimulated SC were abated by edaravone. Activated NF-kappaB in IL-1beta-stimulated SC was suppressed by edaravone. CONCLUSION: Edaravone, antioxidants available for clinical use, appears to have therapeutic effect on RA. We suggest that the inhibitory effect of edaravone on RA might be exerted, at least in part, through suppression of activated NF-kappaB. Therefore, we expect therapeutical use of edaravone as an anti-rheumatic agent.

Variants in the BACH2 and CLEC16A gene might be associated with susceptibility to insulin-triggered type 1 diabetes.

AIM/INTRODUCTION: Insulin administration was found to trigger type 1 diabetes in six Japanese type 2 diabetes patients with type 1 diabetes high-risk human leukocyte antigen class II and the class I allele of the insulin gene variable number tandem repeat genotype. The objective of the present study was to assess the contribution of non-human leukocyte antigen single-nucleotide polymorphisms (SNPs) to the risk of developing insulin-triggered type 1 diabetes. MATERIALS AND METHODS: We genotyped 13 type 1 diabetes susceptible SNPs in six patients and compared them with those in Japanese controls (Hap Map3-JPT). The SNPs that showed statistically significant results were further analyzed using non-diabetic control participants and participants with type 2 diabetes at the Ehime University Hospital. RESULTS: The risk allele frequency of BACH2 rs3757247 in the six patients was significantly more frequent than that in 86 Japanese controls (P = 0.038). No significant difference in the allele frequency was observed in the other SNPs. This result was confirmed by the findings that the risk allele frequency of BACH2 in the six patients was significantly higher than that in the non-diabetic control participants (n = 179) and type 2 diabetes with or without insulin treatment (n = 154 or n = 152; P = 0.035, 0.034 or 0.037, respectively). Despite being statistically not significant, the six patients were all homozygous for the CLEC16A rs12708716 risk allele and five were homozygous for the CLEC16A rs2903692 risk allele. CONCLUSIONS: In addition to type 1 diabetes high-risk human leukocyte antigen class II and the class I allele of the insulin gene variable number tandem repeat genotype, the possibility that the risk variants of BACH2 and CLEC16A could contribute to the development of insulin-triggered type 1 diabetes cannot be excluded.

Glucose enhances protein tyrosine phosphatase 1B gene transcription in hepatocytes.

Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of the insulin receptor signal transduction pathway. We investigated the effects of glucose on PTP1B transcription in the human hepatocyte cell line Huh7. Using a reporter gene assay, we found that D-glucose dose-dependently enhanced the PTP1B promoter activity. Real-time PCR demonstrated that D-glucose also increased PTP1B mRNA expression. Protein kinase C (PKC) inhibitors partially but significantly inhibited the transactivation by D-glucose. Mithramycin, a Sp1 inhibitor, completely abrogated this transactivation. The deletion of three possible Sp1 sites in the promoter region of PTP1B significantly reduced the basal promoter activity and transactivation by D-glucose. Sp1 activation by PKC is one of the key mechanisms in the regulation of several gene expressions. Our data suggested that glucose enhanced PTP1B transcription through Sp1 activation by PKC. Increased hepatic PTP1B expression may partly explain glucose toxicity in diabetes.

Muscle strength is a marker of insulin resistance in patients with type 2 diabetes: a pilot study.

To examine the effect of muscle strength on insulin resistance, we investigated the association between quantitative lower-extremity muscle strength and insulin resistance index as evaluated by homeostasis model assessment (HOMA-IR) in patients with type 2 diabetes (20 men and 20 women, mean age SD: 53.3 12.7 years). By simple linear regression analyses, the knee extension force normalized for body weight (%KEF) was found to be significantly correlated with HOMA-IR in both male (r = -0.510, P<0.05) and female patients (r = -0.462, P<0.05). Stepwise regression analysis also showed that %KEF was an independent determinant of HOMA-IR (beta = -0.331, F = 5.400, P<0.005), as was BMI (beta = 0.409, F = 8.260, P<0.05). Our data suggest that lower-extremity muscle strength is independently associated with insulin resistance, which seems to be consistent with previous reports that resistance training improves glycemic control in type 2 diabetic patients. Further studies based on a larger study population will be required to confirm this possibility.

Edaravone inhibits rheumatoid synovial cell proliferation and migration.

Rheumatoid arthritis (RA) is characterized by synovial proliferation and migration which is induced by proinflammatory cytokines or oxidative stress, followed by joint destruction. Edaravone, clinically available free radical scavenger in Japan, is confirmed to be beneficial in the acute stage of cerebral infarction. We aimed to investigate whether edaravone suppressed in vitro proliferation and migration of synovial cells (SC) induced by IL-1beta. SC proliferation and migration induced by IL-1beta were dose-dependently suppressed by edaravone at the clinically available concentration. These data suggest that edaravone has potential effects to suppress SC proliferation and migration, followed by suppression of synovial proliferation in RA. Therefore, edaravone, an antioxidant agent, might be a novel therapeutic agent which develops the new strategy for treatment of RA, and more detailed studies are required to establish the therapeutic effect of edaravone on RA in vivo.

Pleural dissemination of thymoma showing tumor regression after combined corticosteroid and tacrolimus therapy.

We present the case of an elderly woman with myasthenia gravis who had pleural dissemination of thymoma reduced by treatments with a moderate dose of corticosteroids and a conventional dose of tacrolimus. A maintenance dose of prednisolone for myasthenia gravis could not shrink the size of the disseminated thymoma, but prednisolone (>30 mg daily) succeeded in reducing the size of the tumor. Moreover, a combination with tacrolimus enabled the daily dose of prednisolone to be tapered off without recurrence of myasthenia gravis, and the disseminated thymoma almost disappeared. A moderate or higher dosage of corticosteroids with tacrolimus may, in some cases, be an effective procedure for pleural dissemination of thymoma. Treatment should be undertaken on a trial basis for patients not indicated for surgery, radiotherapy, or chemotherapy.

Effect of pitavastatin on transactivation of human serum paraoxonase 1 gene.

Hepatic hydroxymethyl glutary coenzyme A HMG-CoA reductase inhibitors (statins) have various anti atherosclerosis pleiotropic effects that are independent of cholesterol reduction. Human serum paraoxonase 1 (PON1) is associated with high-density lipoprotein (HDL) and inhibits the oxidative modification of low-density lipoprotein (LDL). We investigated the effects of statins on PON1 gene transcription using a reporter gene assay. Promoter activity of the PON1 gene was estimated by measuring luciferase activity of plasmids with a PON1 promoter region transfected into human hepatoma HepG2 cells and human embryonic kidney (HEK) 293 cells. Pitavastatin, simvastatin, and atorvastatin each significantly increased PON1 promoter activity, and the transactivation by pitavastatin was abrogated by mevalonic acid and farnesyl pyrophosphate (FPP), however, not by geranylgeranyl pyrophosphate. Further, PON1 promoter activity was enhanced by farnesyl transferase inhibitor (FTI), but not by geranylgeranyl transferase inhibitor (GGTI). PON1 gene transcription has been reported to be dependent on Sp1 and the transactivation by pitavastatin was completely abrogated by mithramycin, an inhibitor of Sp1. Our results suggest that pitavastatin activates transcription of the PON1 gene through the FPP pathway, which may play an important role in the anti atherosclerotic effects of statins.

Pioglitazone prevents reactive hypoglycemia in impaired glucose tolerance.

A 42-year-old woman with hypoglycemic symptoms that occurred several hours after a meal visited our hospital. The hypoglycemic symptoms appeared when she was 37 years old, and her plasma glucose level had been assessed as less than 60 mg/dL when she experienced the symptoms. One year before, she had been diagnosed with reactive hypoglycemia by 75 g-oral glucose tolerance test (OGTT), which showed a normal glucose tolerance (NGT) pattern, and had begun taking an alpha-glucosidase inhibitor and nutritional treatment. A 75 g-OGTT on admission showed hypoglycemia at 240 min after glucose loading, excessive insulin secretion and an impaired glucose tolerance (IGT) pattern. A euglycemic-hyperinsulinemic clamp study demonstrated decreased insulin sensitivity. Therefore, we suspected that she had reactive hypoglycemia associated with insulin resistance and treated her with 15 mg/day pioglitazone. Her hypoglycemic symptoms completely disappeared after treatment with pioglitazone; insulin sensitivity in a euglycemic-hyperinsulinemic clamp study improved. Another 75 g-OGTT revealed that the excessive insulin secretion and hypoglycemia at 240 min after glucose loading had disappeared, and glucose tolerance was normalized from an IGT pattern to an NGT pattern. Thus, we believe that pioglitazone is effective for reactive hypoglycemia and aggravated glycemic metabolism associated with insulin resistance.

Type 1 diabetes developed in a type 2 diabetic patient with severe insulin resistance.

We report a 38-year-old female with severe insulin resistance who developed type 1 diabetes after being diagnosed with type 2 diabetes. At the initial examination, BMI was 31.8 kg/m(2) and HbA1c 10.8%. Her insulin secretion was sufficient (urinary CPR 80 microg/day) and the GAD antibody was negative. Following treatment with insulin and glimepiride, HbA1c decreased to 6.3%, though diabetic control deteriorated after 1 year (HbA1c, 11.0%) and her body weight was reduced in a short period, from 78 to 67 kg. Re-examination revealed that the GAD antibody was high (1870 U/mL, normal <1.5) and the anti-islet cell antibody positive, and insulin secretion decreased (urinary CPR 18 microg/day). Further, a hyperinsulinemic-euglycemic cramp study using an artificial pancreas showed that the patient had severe insulin resistance [glucose infusion rate 1.8 mg/(min kg); normal, 7.4+/-2.4 (mean+/-S.D.)]. An HLA-analysis showed that she was a homozygote of haplotype DRB1*0901-DQB1*0303. In spite of strict insulin therapy, glucose control was not improved. Pioglitazone could not be used because of side effects, however, metformin was effective for glucose control. The accumulation of case reports of patients with type 1 diabetes and insulin resistance is important for studying the relationship between the onset of the disease and insulin resistance, and for developing an effective treatment strategy.

Roles of Sp1 and protein kinase C in regulation of human serum paraoxonase 1 (PON1) gene transcription in HepG2 cells.

Human serum paraoxonase 1 (PON1) is associated with high-density lipoprotein, and inhibits oxidative modification of low-density lipoprotein in vitro. Therefore, PON1 is supposed to protect against atherosclerosis in vivo. In this study, we investigated the direct effect of Sp1 on PON1 transcription in HepG2 cells using a reporter gene assay. A deletion analysis of the PON1 upstream region revealed that dominant promoter elements were present within a sequence between -269 and -97bp, which contained a consensus binding site for Sp1, and an electrophoretic mobility shift analysis (EMSA) indicated the Sp1 binding to the upstream sequence. In accordance with this, overexpression of Sp1 dramatically enhanced PON1 promoter activity, and the Sp1 inhibitor mithramycin inhibited Sp1-induced promoter activation in a dose-dependent manner. The basal promoter activity was also enhanced by phorbol 12-myristate 13-acetate (PMA), and synergistic promoter activation was observed when Sp1-transfected cells were treated with PMA. The PMA-induced promoter activation was inhibited by mithramycin. In addition, overexpression of the dominant negative version of PKCalpha or zeta, significantly reduced PON1 promoter activity. These data suggest that Sp1 acts as a positive regulator of PON1 transcription, and that an interaction between Sp1 and PKC is a key mechanism for the effect of Sp1 on PON1 transcription.

Regulation of human glucocorticoid receptor gene transcription by Sp1 and p53.

The human glucocorticoid receptor (hGR) gene has several GC boxes in the promoter 1C region. We studied the effects of Sp1 and p53 on promoter 1C in HepG2 and HEK293 cells using luciferase (Lu) reporter assay. The results showed that the first GC box upstream of the transcription site activated the hGR promoter and over-expression of Sp1 obviously enhanced the activity. A mutant Lu-hGR vector, whose first GC box was defective, lost promoter activity nearly completely. Further, over-expression of p53 strongly suppressed the stimulating effect of Sp1 on hGR promoter activity. We concluded that Sp1 activates the hGR gene promoter, at least in part, by acting on the first GC box in promoter 1C, while p53 suppresses the transactivation by Sp1. These phenomena, demonstrated in cultured cells, may be important for the expression of hGR in vivo.

Acute-phase, but not constitutive serum amyloid A (SAA) is chemotactic for cultured human aortic smooth muscle cells.

The human serum amyloid A (SAA) protein family is subclassified as acute phase SAA (A-SAA), which comprises the SAA1 and SAA2 allelic variants, and constitutive SAA (C-SAA), which is the SAA4 isoform. Extrahepatic production of A-SAA occurs in many organs and tissues of the body, including smooth muscle cells (SMC) of the aorta. A-SAA has been shown to act locally as a chemoattractant for neutrophils, monocytes and lymphocytes via the N-formyl peptide receptor-like (fPRL1). In order to gain further understanding of the physiological significance of local production of A-SAA by SMC, the effect of exogenous A-SAA on the in vitro migration of human aortic SMC was investigated. Increased SMC migration in the presence of A-SAA was detectable after six hours and continued to increase up to 24 hours after incubation. The increased migration was dose-dependent over the concentration range 10 to 100 micrograms/ml. The mode of A-SAA stimulated SMC migration was by chemotaxis not chemokinesis. Exogenous constitutive SAA (C-SAA) did not affect SMC migration. Stimulation of SMC migration by A-SAA was inhibited by both polyclonal and monoclonal antibodies to human SAA1 and also by the inhibitors of fPRL1 signaling, wortmannin, bisindolylmaleimide and pertussis toxin. The results herein indicate that A-SAA, but not C-SAA, may serve as an autocrine factor to influence SMC migration in situations of aortic tissue injury and inflammation.

Proinflammatory cytokines but not acute phase serum amyloid A or C-reactive protein, downregulate paraoxonase 1 (PON1) expression by HepG2 cells.

The expression of paraoxonase1 (PON1) during inflammation has been investigated in vitro. The alteration of steady state PON1 mRNA in HepG2 cells by interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), was investigated relative to acute-phase serum amyloid A (A-SAA) mRNA. PON1 mRNA expression by HepG2 cells was decreased within three hours of stimulation by IL-1beta or TNF-alpha. Relative to PON1 mRNA expression, the pattern of steady state A-SAA mRNA expression was altered reciprocally and inversely by IL-1beta. These findings suggested that the decrease in serum PON activity after abdominal surgery in our previous clinical study may be ascribed to a decrease in steady state PON1 mRNA expression by liver with proinflammatory cytokines.

Correlation of plasma oxidized low-density lipoprotein levels to vascular complications and human serum paraoxonase in patients with type 2 diabetes.

The oxidative modification of low-density lipoprotein (LDL) plays a central role in the initiation and acceleration of atherosclerosis. Human serum paraoxonase (PON1) is associated with high-density lipoprotein (HDL) and has been shown to reduce the susceptibility of LDL to lipid peroxidation. We investigated whether circulating oxidized LDL (Ox-LDL) levels were associated with diabetic vascular complications, and whether the enzymatic activity and gene polymorphisms of PON1 influenced Ox-LDL concentrations in vivo. There was no difference in the plasma Ox-LDL concentrations between diabetic patients with and without macrovascular diseases. However, Ox-LDL concentrations corrected by LDL-cholesterol (OxLDL/LDL-C) or apolipoprotein B (apoB) concentrations (Ox-LDL/apoB), which probably reflect the proportion of oxidatively modified LDL to total LDL particles, were significantly higher in patients with macrovascular diseases than in those without. In addition, patients with peripheral neuropathy had a significantly higher Ox-LDL/apoB ratio than patients without this complication. The genotype TT of -108C/T polymorphism in the promoter region of the PON1 gene, which is associated with decreased PON1 expression, showed a significantly higher Ox-LDL/apoB ratio than genotypes TC or CC (TT: 0.60 +/- 0.15, CT + CC: 0.55 +/- 0.11, P =.02). Stepwise multiple regression analysis for Ox-LDL concentration revealed that the -108C/T polymorphism, subsequently to apoB concentration, was identified as a significant contributor. In summary, the Ox-LDL/apoB ratio was associated with macrovascular disease and peripheral neuropathy in Japanese patients with type 2 diabetes. Increased Ox-LDL/apoB may result, at least partly, from reduced serum antioxidant capacity in the diabetic state, including the attenuation of PON1 action. Increased Ox-LDL/apoB could be a significant marker for susceptibility to vascular complications in diabetic patients.

Serum paraoxonase (PON1) concentration in patients undergoing hemodialysis.

Cardiac death from atherosclerosis is common in patients undergoing hemodialysis. Although the enzymic activity of human serum paraoxonase (PON1) has been reported to be decreased in such patients, serum PON1 concentrations have not been measured. We investigated serum PON1 concentrations in 81 patients undergoing hemodialysis and 103 age-matched healthy subjects using an enzyme immunoassay. The PON1 concentration was significantly lower in the patient group than the control group (mean +/- SD: 6.78 +/- 3.56 vs 18.01 +/- 4.55 U/ml, respectively. p < 0.0001). There were no significant relationships between serum PON1 concentrations and the PON1 genetic polymorphisms, 55Leu/Met (L/M) and 192Gln/Arg (Q/R). The concentration adjusted for HDL-cholesterol or apolipoprotein A-I was also lower in the patient group. However, the specific activities (enzyme activity divided by the PON1 concentration) of paraoxonase and arylesterase were increased in the patient group compared with the control group. In the male patients, but not the female patients, PON1 concentrations were significantly lower in subjects with than without coronary heart disease (CHD) (mean +/- SD: 4.48 +/- 2.77 vs 7.34 +/- 3.22 U/ml, respectively. p < 0.01). In conclusion, the serum PON1 concentration in hemodialyzed patients was significantly decreased, resulting in an attenuation of PON1 enzymic activity. This decrease may be in part involved in the development of cardiovascular disease.

Relationships between polymorphisms of the human serum paraoxonase gene and insulin sensitivity in Japanese patients with type 2 diabetes.

Human serum paraoxonase (PON1), which is associated with HDL, is an esterase and has been shown to reduce the susceptibility of LDL to lipid peroxidation. The objective of the study was to determine whether genetic polymorphisms of the PON1 gene are associated with insulin sensitivity. Forty-eight Japanese patients with type 2 diabetes were recruited, and euglycemic hyperinsulinemic clamp was performed to assess insulin sensitivity. The PON1 promoter polymorphism C(-108)T was determined by direct sequencing, and the coding region polymorphism Q192R was determined by polymerase chain reaction and digestion of the amplified fragments. No association was observed between the Q192R polymorphism and the glucose infusion rate (GIR), whereas GIR increased with the following order of genotypes: -108TT < -108CT < and -108CC (4.2+/-1.6, 5.1+/-2.5, and 6.9+/-2.5 mg kg(-1) min(-1), respectively; P<0.02, ANCOVA). Stepwise regression analysis revealed that the C(-108)T polymorphism significantly contributed to the GIR. It has been reported that oxidative stress attenuates insulin signaling in vitro. The PON1 promoter polymorphism C(-108)T may influence insulin sensitivity by modulating serum antioxidant capacity.

Human paraoxonase-1 gene expression by HepG2 cells is downregulated by interleukin-1beta and tumor necrosis factor-alpha, but is upregulated by interleukin-6.

Recent studies have demonstrated that human paraoxonase-1 (PON1) associated with HDL, plays a role for anti-atherosclerotic effects of HDL, however, the relationships between PON1 and inflammatory cytokines remain unclear. To clarify this point, we evaluated the transcriptional regulation of PON1 gene by IL-1beta, IL-6 and TNF-alpha in HepG2 cells using luciferase reporter gene assay. We determined the nucleotide sequence of upstream of PON1 gene, and constructed plasmids containing various lengths of upstream region. In the plasmid constructs of U39 (PON1 upstream -1232/-6), U682 (-589/-6), U797 (-472/-6) and U953 (-318/-6), U953 showed a stepwise upregulation in basal promoter activity. The relative promoter activities using U682 plasmid were generally downregulated by IL-1beta and TNF-alpha, but were upregulated by IL-6. By the combination of IL-1beta, IL-6 and/or TNF-alpha, the promoter activities were proportionally regulated. The result of PON1 transcriptional regulation by cytokines in HepG2 cells was confirmed to be concordant with that of regulation of PON1 mRNA expression by cytokines. These results suggest that PON1 mRNA expression by hepatocytes is regulated by proinflammatory cytokines and that proinflammatory cytokines secreted in a disease state, may play a role in the development of atherosclerotic lesion via modification of PON1 mRNA expression affecting on the anti-oxidative property of HDL.

Serum paraoxonase activity decreases in rheumatoid arthritis.

OBJECTIVE: To estimate the alterations of paraoxonase 1 (PON1) and high-density lipoprotein (HDL) in rheumatoid arthritis (RA). DESIGN AND METHODS: We investigated the serum enzyme activity and concentration of PON1 and their relationship with serum lipids, high-density lipoprotein (HDL) parameters, and acute phase reactants of serum amyloid A (SAA) and C-reactive protein (CRP) in patients with RA. RESULTS: Serum paraoxonase (PON) activity was significantly decreased in RA patients (n = 64, 131 +/- 53 micro mol/min/L) compared with healthy subjects (n = 155, 164 +/- 59) despite the absence of any difference in serum lipid levels between the two groups. This decrease of serum PON activity in RA patients was found in every genotype (Q/Q, Q/R, R/R) of PON1 at 192 Q/R. There was a different distribution in PON1 Q/R genotypes between RA patients and healthy subjects, and RA patients exhibited less (44%) positive PON1-Q than did the healthy subjects (66%). In a further investigation of age- and gender-matched subgroups of RA (n = 25) and healthy subjects (n = 25), not only serum PON activity, but also lecithin-cholesterol acyltransferase (LCAT) was found to be significantly decreased in RA patients (125 +/- 61 micro mol/min/L, 63.2 +/- 17.2 nmol/ml/hr/37 degrees C) than in healthy subjects (169 +/- 67, 74.7 +/- 19.5), respectively. PON1 and LCAT as well as HDL constituent apolipoprotein (apo) AI and apo AII, were altered significantly in RA patients. CONCLUSIONS: Acute-phase HDL, which is remodeled structurally and functionally in RA, might be less anti-atherogenic due to the impairment of original HDL function. These alterations of HDL in RA patients may explain in part the reported increase in cardiovascular mortality in patients with RA.

Diabetes mellitus associated with Klinefelter's syndrome: a case report and review in Japan.

We report a case of Klinefelter's syndrome in a 48-year-old man who had diabetes mellitus associated with severe insulin resistance. We diagnosed him with Klinefelter's syndrome from his atrophic testicles, primary hypogonadism in hormonal examination, and a chromosomal aberration of 47,XXY. He showed severe decreased insulin sensitivity in a hyper-insulinemic euglycemic clamp test. He had injected over 100 units of insulin per day, however, testosterone replacement and administration of pioglitazone improved his glycemic control, which resulted in a decrease of insulin dose to less than 50 units per day. Here, we discuss the characteristics of diabetes mellitus associated with Klinefelter's syndrome in Japanese patients including this case.

Insulin administration may trigger type 1 diabetes in Japanese type 2 diabetes patients with type 1 diabetes high-risk HLA class II and the insulin gene VNTR genotype.

CONTEXT: Insulin administration causes various types of immune responses to insulin. We previously reported three cases of type 1 diabetes mellitus (T1DM) triggered by insulin administration in Japanese type 2 diabetes mellitus patients. OBJECTIVE: The objective of this study was to collect information and characterize insulin-triggered T1DM immunologically and genetically. METHODS: Data for six patients (four men and two women) with insulin-triggered T1DM aged 59.5 ± 12.8 years were collected. Serum or urinary C-peptides, islet-related autoantibodies, insulin antibody, human leukocyte antigen, or the insulin gene variable number of tandem repeat genotype were analyzed. Th1- or Th2-associated responses were evaluated using an Enzyme-Linked ImmunoSpot assay. RESULTS: None of the subjects had received insulin therapy or had an autoantibody to the 65-kDa isoform of glutamic acid decarboxylase before insulin administration. After insulin administration blood glucose control deteriorated acutely without any apparent cause, whereas C-peptide levels rapidly decreased to insulin-deficient levels. The mean duration of insulin administration to the development of T1DM was 7.7 ± 6.1 months. Islet-related autoantibodies became positive, whereas insulin allergy or a high titer of insulin antibody was observed in several cases. All had T1DM high-risk human leukocyte antigen class II (IDDM1) and the insulin gene variable number of tandem repeats genotype (IDDM2). GAD-reactive and insulin peptide-reactive Th1 cells, but not Th2 cells, were identified in two of four cases. CONCLUSIONS: The findings suggest that insulin administration may have triggered TIDM in patients with type 2 diabetes mellitus. IDDM1 and IDDM 2 as well as autoreactive T cells may contribute to the development of T1DM. Developing insulin-triggered T1DM if a patient's blood glucose control acutely deteriorates after insulin administration should be carefully considered.