Effect of exposure to non-esterified fatty acid on progressive deterioration of insulin secretion in patients with Type 2 diabetes: a long-term follow-up study.

AIM: The aim of the study was to determine whether fasting serum non-esterified fatty acid (NEFA) could be associated with long-term progressive deterioration of insulin secretion in patients with Type 2 diabetes. METHODS: Seventy-seven Japanese patients with Type 2 diabetes (mean BMI 23.3 kg/m(2) ) were followed for 10 years. We measured fasting C-peptide level every 1-2 years. By using the slope of regression line between fasting C-peptide level and duration, we calculated its individual annual decline as an index of insulin secretion. During the follow-up periods of C-peptide, the patients were evaluated for fasting serum non-esterified fatty acid, LDL cholesterol, HDL cholesterol and HbA(1c) levels for the last 8 years. We excluded patients who had renal dysfunction or anti-insulin antibodies from among the insulin-treated patients. Association between the individual annual decline of fasting C-peptide level and related factors were evaluated. RESULTS: The mean individual annual decline of fasting serum C-peptide level was -0.013 ± 0.027 nmol/l/year. Fasting serum non-esterified fatty acid level had no significant difference between the first and the last 2 years of the 8-year observation period of non-esterified fatty acid. Using multiple regression analysis, mean fasting serum non-esterified fatty acid level was associated with the individual annual decline of fasting serum C-peptide level (standardized regression coefficient -0.358, P=0.0056), although other related factors, including HbA(1c) level, were not associated. CONCLUSIONS: Mean fasting serum non-esterified fatty acid level during an 8-year observation was independently associated with long-term progressive deterioration of insulin secretion in Japanese patients with Type 2 diabetes.

A missense mutation of Pax4 gene (R121W) is associated with type 2 diabetes in Japanese.

Pax4 is one of the transcription factors that play an important role in the differentiation of islet beta-cells. We scanned the Pax4 gene in 200 unrelated Japanese type 2 diabetic patients and found a missense mutation (R121W) in 6 heterozygous patients and 1 homozygous patient (mutant allele frequency 2.0%). The mutation was not found in 161 nondiabetic subjects. The R121W mutation was located in the paired domain and was thought to affect its transcription activity through lack of DNA binding. Six of seven patients had family history of diabetes or impaired glucose tolerance, and four of seven had transient insulin therapy at the onset. One of them, a homozygous carrier, had relatively early onset diabetes and slowly fell into an insulin-dependent state without an autoimmune-mediated process. This is the first report of a Pax4 gene mutation that exhibits loss of function and seems to be associated with type 2 diabetes. This work provides significant implications for the Pax4 gene as one of the predisposing genes for type 2 diabetes in the Japanese.

Single nucleotide polymorphism (D68D, T to C) in the syntaxin 1A gene correlates to age at onset and insulin requirement in Type II diabetic patients.

AIM/HYPOTHESIS: Syntaxin 1A is a candidate gene for Type II (non-insulin-dependent) diabetes mellitus, because it plays an important role in insulin secretion from the islet beta cells. We aimed to scan this gene for mutations or genetic markers that correlate with Type II diabetes. METHODS: We identified and characterized coding exons of the syntaxin 1A gene and scanned the newly identified 10 exons using direct sequencing. RESULTS: In the single nucleotide polymorphism (SNP) of exon 3 (D68D, T to C) among three newly identified SNPs, genotype frequency of the homozygote of C allele (CC) occurred more frequently in a Type II diabetic group than in a non-diabetic group (16.48 %, n = 182, vs 11.05 %, n = 181, p = 0.0499). Among the diabetic patients, age of onset in patients with CC genotype was lower than that in patients with the TT and TC genotypes [40.10 +/- 1.50 years old (means +/- SEM) vs 44.20 +/- 0.58, p = 0.005]. Patients with the CC genotype had a higher frequency of insulin treatment (78.30 % vs 46.80 %, p = 0.006) with a duration equal to, or longer than, 10 years. Multiple regression analysis confirmed that the genotype was significantly and independently associated with age at onset and mode of treatment, respectively. CONCLUSION/INTERPRETATION: These data indicate that the SNP in the syntaxin 1A gene (D68D, T to C) correlates to the age of onset and insulin requirements of Type II diabetic Japanese patients.

The renal metabolism of insulin: urinary insulin excretion in patients with mutant insulin syndrome (insulin Wakayama).

Many studies have shown that the kidney plays an important role in the metabolism of many proteins and small peptides. To understand insulin handling in the kidney, we examined urinary insulin excretion under several conditions in patients with mutant insulin syndrome (MIS; insulin Wakayama). Urinary excretion of insulin was studied using high-performance liquid chromatography analysis in patients with MIS. In these patients, most of the insulin extracted from a 24-hour urine collection and from urine collected after stimulation of insulin secretion by glucose or glucagon was normal insulin, whereas 90% of serum insulin is structurally abnormal (Leu-A3 insulin). On the other hand, arginine, which is known as an inhibitor of renal tubular reabsorption, increased urinary excretion of Leu-A3 insulin. The ratio of Leu-A3 and normal insulin in urine after arginine was similar to that in serum. A large amount of Leu-A3 insulin is excreted in urine when reabsorption of insulin at renal tubules is inhibited by arginine. These data indicate that normal and Leu-A3 insulin are filtered through the glomerulus with relatively little restriction. Using the fact that basal urine has a high concentration of normal insulin and an extremely low concentration of Leu-A3 insulin, which has less receptor-binding affinity, we speculated some possibilities. One possibility is that both forms of insulin are reabsorbed by the tubular cells, but with different efficiencies. Leu-A3 insulin absorption in more complete, and this suggests differences in the uptake pathways that may account for the differences in response to arginine infusions. Another possibility is that only normal insulin is secreted from tubules into urine which is mediated by receptors. Our results provide new insight into renal metabolism of insulin and showed that MIS is a useful model for studying it.

The Pro12 -->Ala substitution in PPAR-gamma is associated with resistance to development of diabetes in the general population: possible involvement in impairment of insulin secretion in individuals with type 2 diabetes.

The allele frequencies for a Pro12-->Ala substitution in peroxisome proliferator-activated receptor-gamma differ among ethnic groups, and its relationship with diabetes and associated diseases is controversial. The prevalence of this polymorphism and its effects on clinical characteristics have now been evaluated with a large number of Japanese individuals with type 2 diabetes (n = 2,201) and normal control subjects (n = 1,212) recruited by 10 institutions located in seven different cities in Japan. The allele frequency for the Ala12 variant was significantly lower in the type 2 diabetic group than in the control group (2.39 vs. 4.13%, P = 0.000054). However, compared with subjects without the Ala12 variant, the diabetic subjects with this variant exhibited a significantly higher serum concentration of total cholesterol (P = 0.001), manifested a reduced capacity for insulin secretion as evaluated by homeostasis model assessment (P = 0.007), and tended to possess a higher level of HbA1c. These data suggest that the Ala12 variant is associated with a reduced risk for the development of diabetes in the general population, but that it may be also a risk factor for insulin deficiency and disease severity in individuals with type 2 diabetes.

[Non-autoimmune fulminant type 1 diabetes].

A new classification for diabetes mellitus based on ethiology was proposed by the Japan Diabetes Association in 1999. According to the classification, type 1 diabetes was subclassified as type 1A(autoimmune based) and type 1B(idiopathic). However, the latter is considered to be heterogeneous. Recently, a novel subtype of type 1B diabetes mellitus has been reported. In this review, a summary account of this new subtype is given. The patients in this subgroup have a non-autoimmune, fulminant disorder characterized by the absence of insulitis and of diabetes-related antibodies, a remarkably abrupt onset, and high serum pancreatic enzyme concentrations.

Enhanced in vitro production of amyloid-like fibrils from mutant (S20G) islet amyloid polypeptide.

UNLABELLED: Islet amyloid polypeptide (IAPP, "amylin") is the amyloid-fibril-forming polypeptide in the islets of Langerhans associated with type 2 diabetes mellitus. A missense mutation in the IAPP gene associated with early-onset type 2 diabetes has been identified in the Japanese population. This mutation results in a glycine for serine substitution at position 20 of the mature IAPP molecule. Whether or not formation of islet amyloid with resulting destruction of islet tissue is the cause of this diabetes is yet not known. The present in vitro study was performed in order to investigate any influence of the amino acid substitution on the fibril formation capacity. Synthetic full-length wild type (IAPPwt) and mutant (IAPPS20G) as well as corresponding truncated peptides (position 18-29) were dissolved in dimethylsulfoxide (DMSO) or in 10% acetic acid at a concentration of 10 mg/mL and their fibril forming capacity was checked by Congo red staining, electron microscopy, a Congo red affinity assay and Thioflavine Tfluorometric assay. It was found that full-length and truncated IAPPS20G both formed more amyloid-like fibrils and did this faster compared to IAPPwt. The fibril morphology differed slightly between the preparations. CONCLUSION: The amino acid substitution (S20G) is situated close to the region of the IAPP molecule implicated in the IAPP fibrillogenesis. The significantly increased formation of amyloid-like fibrils by IAPPS20G is highly interesting and may be associated with an increased islet amyloid formation in vivo and of fundamental importance in the pathogenesis of this specific form of diabetes.

S20G mutant amylin exhibits increased in vitro amyloidogenicity and increased intracellular cytotoxicity compared to wild-type amylin.

Human amylin, a major constituent of pancreatic amyloid deposits, may be a pathogenetic factor for noninsulin-dependent diabetes mellitus (NIDDM). We demonstrated that the human amylin S20G gene mutation (S20G) was associated with a history of early onset, more severe type of NIDDM, linking the amylin gene to this disease. Also, we demonstrated that expression of human wild-type (WT) amylin in COS-1 cells leads to intracellular amyloidogenesis and induction of apoptosis, suggesting a possible mechanism for disease induction. Therefore we compared the abilities of S20G and WT amylin to induce apoptosis in transfected COS-1 cells and form amyloid in vitro. We transfected the rat (RAT), mutated human (MUT), WT, and S20G amylin genes into COS-1 cells and measured apoptosis using fluorescent-activated cell sorting analysis at 48, 72, and 96 hours. At 96 hours apoptosis increased significantly (P < 0.01) in cells transfected with WT and S20G over RAT or MUT (WT, 19%; S20G, 25%; RAT, 13%; and MUT, 12%) and the difference between WT and S20G was significant (P < 0.05). Synthetic WT and S20G monomeric peptides were used to generate amyloid fibrils in vitro as measured by the thioflavin T binding assay. The S20G amylin formed approximately twofold more amyloid at a rate approximately threefold higher than WT. Electron micrography indicated that the in vitro amyloid generated by WT and S20G amylins were morphologically indistinguishable. The results suggest that increased cytotoxicity by S20G is because of increased amyloidogenicity, which may be a causative factor in the early development of NIDDM, possibly through loss of ss cell mass.

Nonsense mutation of islet-1 gene (Q310X) found in a type 2 diabetic patient with a strong family history.

Islet-1 (Isl-1) is one of the transcription factors that play an important role for the formation of the islet cells. We scanned the Isl-1 gene in 77 Japanese type 2 diabetic patients with a family history and found a heterozygous nonsense mutation (Q310X) in 1 diabetic patient. The mutation was not found in 180 nondiabetic subjects. This mutation is located in the putative transactivation domain and deletes 40 amino acids of the COOH-terminal lesion. The Q310X mutant exhibited a 50% reduction in activity compared with the wild-type when tested for stimulation of transcription of a human amylin promoter-linked luciferase reporter gene in betaTC3 cells. The patient was a 49-year-old nonobese man who was diagnosed as having type 2 diabetes at 32 years of age and has been treated with sulfonylureas. The mutation was found in his mother, who has type 2 diabetes, and in his 14-year-old daughter, who has normal glucose tolerance but a relatively low insulin response. This is the first reported finding of Isl-1 gene mutation in type 2 diabetes. Although Isl-1 is not a common predisposing gene for Japanese type 2 diabetes, the mutation in this gene may be a rare cause of diabetes in isolated families.

Genetics of type 2 diabetes mellitus.

Type 2 diabetes mellitus is a heterogeneous disease that is caused by both genetic and environmental factors. Only a minority of cases of type 2 diabetes are caused by a single-gene defect, such as maturity-onset diabetes of youth (mutated MODY gene), syndrome of insulin resistance (insulin receptor defect), and maternally inherited diabetes and deafness (mitochondrial gene defect). The genetic component of the more common form of type 2 diabetes is probably complex and involves the interactions of multiple genes and environmental factors. The candidate gene approach has identified several genes that regulate insulin signalling and secretion, but their contributions to diabetes are small. Recent genome scan studies have been conducted to identify major susceptibility loci that are linked with type 2 diabetes. This information would provide new insights into the identification of novel genes and pathways that lead to this complex disease.

[Methodology and clinical implication of the identification of clonality in malignant lymphoma].

A variety of methods is now available to identify clonality of lymphoid neoplasia by recent progress in immunology and molecular biology. The target molecules for these include physiologically or pathologically altered genes/their products and virus-derived DNAs. Identification of clonality is important for more accurate diagnosis of lymphoma and, especially, related borderline lesions. It is also of considerable value in staging and follow-up study of the patients because higher sensitivity of polymerase-chain reaction made it possible to detect submicroscopic lesions including minimal residual disease after treatment. However, many lesions and phenomena which are clonal, but do not appear to be malignant, have been characterized. These may prompt an update of the conventional concept of "malignant lymphoma".

A polymorphic marker in the leptin gene associated with Japanese morbid obesity.

The prevaleance of morbid obesity (body mass index of 35.0 or greater) is low in Japan (0.2-0.3%), and little systematic investigation of its cause in this population has been carried out. Leptin plays a central role in regulation of body weight; mice deficient in leptin develop marked obesity. We sought mutations in the leptin gene in 53 morbidly obese Japanese (maximum body mass index 35-60) including 46 with type 2 diabetes. Direct DNA sequencing was performed following polymerase chain reaction amplification. Apart from a silent mutation at codon 25 (CAA/CAG, glutamine) detected in eight subjects, no mutations were detected. We found a significantly higher prevalence of the variant leptin 25CAG allele among the 53 obese subjects (0.085) studied than in 132 nonobese control subjects (0.011, P<0.001). In Japanese populations mutations in the protein coding sequence of the leptin gene are unlikely to be a major cause of morbid obesity. However, the leptin 25CAG allele may be linked to morbid obesity in this population. Specifically, genetic variation located near the leptin gene may be involved in pathogenesis. The leptin polymorphism 25CAG appears to be a new genetic marker for obesity susceptibility, at least in Japanese.

[NIDDM susceptibility genes].

Non-insulin dependent diabetes mellitus (NIDDM) is a heterogeneous disorder and both genetic and nongenetic factors are associated with the development of diabetes. Until now five genes (HNF-4 alpha, glucokinase, HNF-1 alpha, IPF-1 and HNF-1 beta), whose mutation can result in MODY, insulin and insulin receptor genes, and mitochondria DNA have been reported to be responsible for diabetes. Furthermore the mutations in some genes which work for insulin secretion or action also have been reported. This review discusses our current knowledge of these NIDDM susceptibility genes.

Idiopathic CD4+ T lymphocytopenia disclosed by the onset of empyema thoracis.

A 56-year-old man was admitted to our hospital in December 1996 due to empyema thoracis. A laboratory examination revealed lymphocytopenia and CD4+ T lymphocytopenia (<300 cells/ microl). No evidence for a human immunodeficiency virus (HIV) infection was found. No malignant, hematological or autoimmune disease was detected. We thus diagnosed this case as being idiopathic CD4+ T lymphocytopenia (ICL). During his hospital treatment, he was affected with cytomegaloviral retinitis and cured by therapy. His subsequent treatment went well without a recurrence of severe infection although a low CD4+ T lymphocyte count continued after the recovery from empyema thoracis.

Molecular scanning for mutations in the melanocortin-4 receptor gene in obese/diabetic Japanese.

Decreased function of the melanocortin-4 receptor (MC4R) was reported to cause late-onset obesity and insulin resistance in rodents. Thus mutations in the MC4R gene drew strong attention as a possible cause of obesity and diabetes. We screened for mutations in the MC4R gene in extremely obese [body mass index (BMI) > or = 35 kg/m2] Japanese with diabetes by direct sequencing. A heterozygous mutation (V103I) was detected in one case (2.0 %), however the frequency was not significantly different from that in non-obese (BMI < or = 24 kg/m2) and non-diabetic subjects (2.7 %). No other mutations were detected. These results suggest that mutations including V103I in the MC4R gene are not a major cause of obesity or diabetes in Japanese.

Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes with deterioration during pregnancy.

We report a 31-year-old woman who developed myopathy and neuropathy during pregnancy. She was diagnosed as having mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS). A T-to-C transition mutation at nucleotide position 3271 was detected in the mitochondrial gene. Her symptoms subsided spontaneously and she delivered a male infant at 38 weeks of gestation. Prior reports on mitochondrial diseases with pregnancy are very rare, probably because of the early onset of the disease. The metabolic changes during pregnancy increase the stress on the mitochondrial function, particularly in patients with impaired mitochondrial function. Therefore pregnancy can aggravate mitochondrial diseases.

Organization of the human carboxypeptidase E gene and molecular scanning for mutations in Japanese subjects with NIDDM or obesity.

Insulin is synthesized in the pancreatic beta cell as a larger precursor molecule proinsulin which is converted to insulin and C-peptide by the concerted action of prohormone convertase 2 (PC2), prohormone convertase 3 (PC3) and carboxypeptidase E (CPE). One of the features of non-insulin-dependent diabetes mellitus (NIDDM) is an elevation in the proinsulin level and/or proinsulin/insulin molar ratio suggesting that mutations in these three proinsulin processing enzymes might contribute to the development of NIDDM. The identification of a mutation in the CPE gene of the fat/fat mouse which leads to marked hyperproinsulinaemia and late-onset obesity and diabetes is consistent with a possible role for mutations in CPE in the development of diabetes and obesity in humans. In order to test this hypothesis, we have isolated and characterized the human CPE gene and screened it for mutations in a group of Japanese subjects with NIDDM and obesity. The human CPE gene consists of 9 exons spanning more than 60 kb. Primer extension analysis identified the transcriptional start site at -141 bp from the translational start site. Single strand conformational polymorphism analysis and nucleotide sequencing of the promoter and entire coding region of the CPE gene in 269 Japanese subjects with NIDDM, 28 nondiabetic obese subjects and 104 nonobese and nondiabetic controls revealed three nucleotide changes, a G-to-T substitution at nucleotide -53, a G-to-A substitution at nucleotide -144 (relative to start of transcription) in the promoter region and a silent G-to-A substitution in codon 219. None of the nucleotide substitutions were associated with NIDDM or obesity. Thus, genetic variation in the CPE gene does not appear to play a major role in the pathogenesis of NIDDM or obesity in Japanese subjects.