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A prospective, multicentre, open‐label, blinded‐endpoint, randomized controlled study was conducted to evaluate the efficacy of treatment with ipragliflozin (sodium‐dependent glucose transporter‐2 inhibitor) versus metformin for visceral fat reduction and glycaemic control among Japanese patients with type 2 diabetes treated with sitagliptin, HbA1c levels of 7%‐10%, and body mass index (BMI) ≥ 22 kg/m2. Patients were randomly assigned (1:1) to receive ipragliflozin 50 mg or metformin 1000‐1500 mg daily. The primary outcome was change in visceral fat area as measured by computed tomography after 24 weeks of therapy. The secondary outcomes were effects on glucose metabolism and lipid metabolism. Mean percentage reduction in visceral fat area was significantly greater in the ipragliflozin group than in the metformin group (−12.06% vs. −3.65%, P = 0.040). Ipragliflozin also significantly reduced BMI, subcutaneous fat area, waist circumference, fasting insulin, and homeostatic model assessment (HOMA)‐resistance, and increased HDL‐cholesterol levels. Metformin significantly reduced HbA1c and LDL‐cholesterol levels and increased HOMA‐beta. There were no severe adverse events. The use of ipragliflozin or metformin in combination with dipeptidyl peptidase‐4 inhibitors, widely used in Japan, may have beneficial effects in ameliorating multiple cardiovascular risk factors.

Introduction Diabetic macular edema (DME) threatens daily life activities such as reading and driving and reduces the patients’ quality-of-life. Recently, anti-vascular endothelial growth factor (VEGF) agents have become a first-line therapy in DME. However, therapy with anti-VEGF agents has several problems: repeated invasive injections are required; medical costs are high; and a certain proportion of patients with DME are resistant to treatment with anti-VEGF agents. While sodium-glucose co-transporter 2 (SGLT2) inhibitors have been widely used for the treatment of type 2 diabetes mellitus (T2DM), the effects of a combination therapy with anti-VEGF agent and SGLT2 inhibitor on DME are not yet known. Methods This study enrolls subjects with T2DM and DME, randomizes them into either a study agent treatment group (treated with ranibizumab as anti-VEGF agent and luseogliflozin as SGLT2 inhibitor) or a control group (treated with ranibizumab and glimepiride), and observes the subjects for 52 weeks after initiation of treatment. Planned outcomes: The primary endpoint is intergroup difference in the number of intravitreal anti-VEGF injections to the study eye from baseline to week 48. Secondary and exploratory endpoints include safety and ophthalmologic and internal medical clinical parameters. Registration This study is registered at the University Hospital Medical Information Network Clinical Trial Registry (UMIN000033961) and Japan Registry of Clinical Trials (jRCTs031180210).

Aims/Introduction Recent randomized clinical trials have suggested that sodium–glucose cotransporter 2 inhibitors might reduce cardiovascular events and heart failure, and have renal protective effects. Despite these remarkable benefits, the effects of sodium–glucose cotransporter 2 inhibitors on bone and muscle are unclear. Materials and Methods A subanalysis of a randomized controlled study was carried out to evaluate the effects of the sodium–glucose cotransporter 2 inhibitor, ipragliflozin, versus metformin on bone and muscle in Japanese patients with type 2 diabetes mellitus (baseline body mass index ≥22 kg/m2 and hemoglobin A1c 7–10%) who were already receiving sitagliptin. These patients were randomly administered ipragliflozin 50 mg or metformin 1,000–1,500 mg daily. The effects of these medications on the bone formation marker, bone alkali phosphatase; the bone resorption marker, tartrate‐resistant acid phosphatase 5b (TRACP‐5b); handgrip strength; abdominal cross‐sectional muscle area; and bone density of the fourth lumbar vertebra were evaluated. Results After 24 weeks of treatment, the changes in bone density of the fourth lumbar vertebra, handgrip strength and abdominal cross‐sectional muscle area were not significantly different between the two groups. However, TRACP‐5b levels increased in patients treated with ipragliflozin compared with patients treated with metformin (median 11.94 vs −10.30%, P, Ipragliflozin in combination with sitagliptin did not affect bones or muscles adversely compared to metformin. However, ipragliflozin increased the levels of the bone resorption marker, tartrate‐resistant acid phosphatase 5b.

Fumio Shimada,Yoshiyuki Ohira,Yusuke Hirota,Akiko Ikegami,Takeshi Kondo,Kiyoshi Shikino,Shingo Suzuki,Kazutaka Noda,Takanori Uehara,Masatomi Ikusaka Department of General Medicine, Chiba University Hospital, Chiba, Japan Background and objectives: Patients who come for a consultation at a general practice clinic as outpatients often suffer from background anxiety and depression. The psychological state of such patients can alleviate naturally; however, there are cases when these symptoms persist. This study investigated the realities and factors behind anxiety/depression becoming prolonged.Methods: Participants were 678 adult patients, who came to Department of General Medicine at Chiba University Hospital within a 1-year period starting from April 2012 and who completed the Hospital Anxiety and Depression Scale (HADS) during their initial consultation. Participants whose Anxiety or Depression scores in the HADS, or both, were 8 points or higher were defined as being within the anxiety/depression group, with all other participants making up the control group. A telephone interview was also conducted with participants. Furthermore, age, sex, the period from the onset of symptoms to the initial consultation at our department, the period from the initial department consultation to the telephone survey, and the existence of mental illness at the final department diagnosis were investigated.Results: A total of 121 patients (17.8% response rate) agreed to the phone survey. The HADS score during the phone survey showed that the anxiety/depression group had a significantly higher score than the control group. The HADS scores obtained between the initial consultation and telephone survey showed a positive correlation. Logistic regression analysis extracted “age” and the “continuation of the symptoms during the initial consultation” as factors that prolonged anxiety/depression.Conclusion: Anxiety and depression in general practice outpatients have the possibility of becoming prolonged for an extended period of time. Being aged 65 years or over and showing a continuation of symptoms past the initial consultation are the strongest factors associated with these prolonged conditions. When patients with anxiety and depression exhibit these risk factors, they should be further evaluated for treatment. Keywords: anxiety, depression, outpatients, general practice

Une femme de 78 ans a consulte au service des urgences pour une douleur femorale droite presente depuis 2 semaines. Les resultats d’imagerie par resonance magnetique (IRM) laissant soupconner des tumeurs osseuses, la patiente a ete dirigee vers notre hopital. La douleur, qui empirait en

Telediagnosis (TD), which uses information and communications technology, has recently undergone rapid development. Since no studies have compared the diagnostic precision of TD to that of face-to-face diagnosis (FD), we examined and compared the diagnostic accuracy of these diagnostic approaches among general medicine outpatients.Data of 97 patients (45 men and 52 women with a mean age of 52 years) who underwent initial examinations at a regional hospital were analyzed. Two fully trained general medicine physicians were selected from a group of three physicians to perform FD and TD. Levels of agreement (as κ coefficients) were determined between TD and FD diagnoses as well as between final diagnoses and TD and FD diagnoses.The κ coefficients were 0.75 for TD and FD and 0.81 for both, the final diagnoses and the TD and FD diagnoses, revealing a sufficiently high level of diagnostic agreement.TD can provide the same level of diagnostic accuracy as FD among general medicine outpatients for adults. The help of medical assistants and the utilization of physical examination devices might enable medical staff to provide TD care similar in quality to FD. TD could be a useful diagnostic tool when medical work force is limited (e.g., in remote areas, during natural disasters, and in at-home care).

Objective: To determine the effects of SGLT2 inhibitor or metformin on reducing visceral fat in Asian patients with type 2 diabetes inadequately controlled with DPP-4 inhibitor, sitagliptin. Methods: A prospective, multicenter, blinded-endpoint, randomized controlled trial was conducted to evaluate the efficacy of treatment with either SGLT2 inhibitor (ipragliflozin) or metformin added on to sitagliptin for visceral fat reduction and glucose control in patients with type 2 diabetes. Patients who met the eligibility criteria were randomly selected (1:1) to receive ipragliflozin (Ipr; 50 mg daily) or metformin (Met; 1000 mg daily). The primary outcome is the change rate in visceral fat area, measured using computed tomography, after 24 weeks of therapy. Two radiologists, blinded to the information, analyzed the images. Results: A total of 51 patients were in enrolled in the Ipr group and 52 in the Met group. The reduction rate in visceral fat area was significantly greater in the Ipr group (-12.06% vs. -3.65%, group difference of -8.40%; 95% CI of -16.4 to -3.38, P Conclusion: Ipr significantly reduced the visceral fat area compared with Met as the secondary agent used in combination with DPP-4 inhibitors. As the fasting insulin level decreased, the reduction in visceral fat associated with Ipr administration may have contributed to the improvement of insulin resistance. Disclosure M. Koshizaka: None. K. Ishikawa: None. R. Ishibashi: None. Y. Maezawa: None. K. Sakamoto: None. D. Uchida: Speaker's Bureau; Self; Takeda Pharmaceuticals Japan, Tanabe Mitsubishi Pharmaceuticals Japan, Sanofi, MSD K.K., Novo Nordisk Inc. S. Nakamura: None. H. Yokoh: None. A. Kobayashi: None. S. Onishi: None. K. Kobayashi: None. J. Ogino: None. H. Tokuyama: None. F. Shimada: None. E. Ohara: None. T. Ishikawa: None. M. Shoji: None. K. Ide: None. S. Ide: None. Y. Baba: None. T. Horikoshi: None. R. Shimofusa: None. S. Takahashi: None. K. Nagashima: None. M. Takemoto: None. K. Yokote: Research Support; Self; Astellas Pharm Ltd, MSD K.K..

Background: Visceral fat accumulation is associated with insulin resistance and various metabolic complications. We aimed to determine the effect of ipragliflozin (sodium-dependent glucose transporter-2 inhibitor) versus metformin with a dipeptidyl peptidase-4 (DPP-4) inhibitor (sitagliptin) in reducing visceral fat in Asian patients with insufficiently controlled type 2 diabetes. Methods: A prospective, multicenter, blinded-endpoint, randomized controlled study was conducted to evaluate the efficacy of treatment with either ipragliflozin or metformin combined with sitagliptin on visceral fat reduction and glycemic control among Japanese patients with type 2 diabetes, HbA1c 7-10%, and BMI ≥22 kg/m2. Patients who met the eligibility criteria were randomly assigned (1:1) to receive ipragliflozin 50 mg (n=51) or metformin 1000-1500 mg daily (n=52). The primary outcome was rate change in visceral fat area measured by computed tomography at 24 weeks of therapy. Two radiologists, blinded to patients' clinical information and randomized treatment assignment, analyzed the images. Secondary outcomes included effects on blood glucose, fasting insulin level, and lipids. The full analysis set was used for analysis. Findings: Mean percent reduction in visceral fat area was significantly greater in the ipragliflozin group than in the metformin group (-12·06% vs. -3·65%, group difference [95% CI] -8·40%; [-16·4 to -3·38], P=0·040). The ipragliflozin group also showed significant lowering of fasting insulin (-20·73% vs. 0·85%, group difference [95% CI] -21·58%, [2·80 to 34·20], P=0·018), while HbA1c decreased in the metformin group compared with ipragliflozin group. Interpretation: Ipragliflozin significantly reduced the visceral fat area compared with metformin, as the secondary agent used in combination with DPP-4 inhibitor. Since the fasting insulin level decreased, the reduction in visceral fat associated with ipragliflozin administration might have contributed to the improvement in insulin resistance. Trial Registration Number: Trial registration: UMIN 000015170. Funding Statement: To conduct this study, an agreement was signed between Chiba University and Astellas Pharma Inc. (Tokyo, Japan). Astellas Pharma Inc. funded this work. Declaration of Interests: KY received research grants from Astellas Pharma Inc. and MSD K.K. (Tokyo, Japan), and received a lecture fee from Astellas Pharma Inc. and Sumitomo Dainippon Pharma (Tokyo, Japan). No conflicts of interest are declared for other authors. Ethics Approval Statement: The study protocol was approved by the Institutional Review Boards (IRBs), Ethics Review Committees, or Ethics Committees of the following institutions: Chiba University Hospital (ID number: G26009), Asahi General Hospital (ID number: 2014091602), National Hospital Organization Chiba Medical Center, Seirei Sakura Citizen Hospital, Chiba Rosai Hospital (ID number: 26-21), Toho University Sakura Medical Center (ID number: 2014-077), Tokyo Women’s Medical University Yachiyo Medical Center (ID number: 150303), Chiba Aoba Municipal Hospital, Kimitsu Chuo Hospital, Funabashi Central Hospital (ID number: H27-1), and Chiba Kaihin Municipal Hospital. In other facilities, approval was provided at Chiba University Hospital (which had the centralized IRB).

The correlation between the autoignition characteristics of n-butane/di-methyl ether/air mixtures and the knock intensity in a rapid compression and expansion machine was studied. The propagation velocity was obtained from the images of propagating flames at the moment of autoignition by a high-speed camera. The dimensionless parameters, ε and ξ, were used in the investigation, which are based on the theories of Zeldovich and Bradley. To obtain these dimensionless parameters, the numerical calculation using CHEMKIN-PRO with AramcoMech1.3 was carried out. The smaller gradient of autoignition delay time for n-butane with the higher amounts of di-methyl ether addition caused the higher flame propagation velocity and the resultant higher knock intensity, while significant differences of excitation times for autoignition heat release could not be seen. As a result, the gradient of autoignition delay time was concluded to have a strong influence on the knock intensities.

A potential adverse effect of dipeptidyl peptidase-4 inhibitors (DPP-4i) on the pancreas remains controversial. We evaluated the DPP-4i effects on pancreatic amylase and lipase activity in patients with type 2 diabetes. These enzymes were slightly but significantly increased, suggesting DPP-4i cause a low-grade inflammatory change in the exocrine pancreas.

Tyrosine phosphorylation of the insulin receptor is the initial event following receptor binding to insulin, and it induces further tyrosine phosphorylation of various intracellular molecules. This signaling is countered by protein tyrosine phosphatases (PTPases), which reportedly are associated with insulin resistance that can be reduced by regulation of PTPases. Protein tyrosine phosphatase 1B (PTP1B) and leukocyte antigen-related PTPase (LAR) are the PTPases implicated most frequently in insulin resistance and diabetes mellitus. Here, we show that PTP1B and LAR are expressed in human fibroblasts, and we examine the regulation of PTPase activity in fibroblasts from patients with an insulin receptor gene mutation as an in vitro model of insulin resistance. Total PTPase activity was significantly lower in the cytosolic and membrane fractions of fibroblasts with mutations compared with controls (p

Experimental studies are conducted on extinction of non-premixed dimethyl ether (DME) flames stabilized in the counterflow configuration. Studies are carried out by injecting a fuel stream made up of fuel and inert gas (N2 or CO2 or Ar or He) from one duct an oxidizer stream made up of O2 and inert gas (N2 or CO2 or Ar or He) from the other duct. Critical conditions of extinction are measured by increasing the flow rate of the counterflowing streams until the flame extinguishes. Numerical studies are also performed using detailed chemistry at conditions corresponding to those used in the experiments and compared with measurements. The present study highlights the examination of the influence of equilibrium flame temperature, flame structure and kind of inert gas on extinction properties of DME. Results are compared and discussed in terms of Lewis number effect.

Recently there have been many problems related with automotive engineering, such as environmental issues and energy problems. These social circumstances motivate the adoption of urgent measures such as alternative fuel and new combustion techniques for the internal combustion engine. In this context, dimethyl ether (DME) is thought to be a potential alternative to diesel fuel due to lower overall pollutant emissions and more excellent fuel economy. DME has no carbon-carbon bonds and lowest possible carbon to hydrogen ratio after methane. The main features that make DME so attractive are : 1) the high oxygen content of 35% by weight, which provides smokeless combustion and high exhaust gas recycle tolerance ; and 2) the combination of a cetane number of 55 to 60 and a boiling point of-25°C, which provides fast mixture formation, reduced ignition delay, and excellent cold starting. Several recent publications have already presented results from diesel engines operated on pure DME. These experiments showed that DME is an excellent diesel fuel with a high cetane number. This fuel produces very low particulate emissions, while the NOx emissions are similar to those from current diesel fuel under the same engine operating conditions. This allows the engine operating conditions to be adjusted to reduce NOx without an accompanying increase in particular emissions. The purpose of this study is to clarify the combustion properties of DME/Air flames, which should be useful data for combustion modeling and simulation, using the spherical bomb that can embody the real engine conditions.

An 80-year-old woman presented with low-grade fever, weight loss and shoulder ache persistent over 6 months. She had developed headache with scalp allodynia, though these had improved spontaneously within 2 months. Physical examination revealed an absent temporal artery pulse and decreased range of active shoulder motion. Laboratory findings showed a high erythrocyte sedimentation rate (73 mm/h). Temporal artery ultrasonography revealed no halo sign, stenosis or occlusion. Fluorodeoxyglucose positron emission tomography (FDG-PET) showed increased uptake in the thoracic aorta and carotid …

The purpose of this paper is to investigate the combustion properties of CH4/O2 mixtures diluted by CO2 compared to those of CH4/O2/N2 mixtures to study the feasibility of the new combustion method, which is expected to be effective in the NOx reduction and the improvement of combustion. Both experimental and numerical studies were conducted to scrutinize the effects of flame stretch on the burning velocity and the effects of flame instability which may play an important role in combustion performance not only for laminar flames but also for turbulent flames. In this study, experiments were conducted by using spherical combustion bomb for applications to internal combustion engines. First the effect of flame stretch was investigated experimentally and numerically and quantitative discussion was made in terms of Markstein number which means not-dimensional sensitivity of the burning velocity to flame stretch. As a result it has been found that Markstein numbers for both methane mixtures decrease with decreasing equivalence ratio. In addition CH4/O2/ CO2 mixtures have the low values of Markstein number compared with CH4/O2/N2 mixtures over the whole equivalence ratio.

The effects of flame stretch on an outwardly propagating flame are considered experimentally for both laminar and turbulent flames. Although the thermo-diffusive mechanism caused by the flame/stretch interaction is well known for laminar flames or for weakly stretched flames, recent studies have shown that it influences the local and global flame structures of turbulent premixed flames which consist of strongly stretched flamelets. In this study, outwardly propagating flames of methane and propane mixtures, which are representative of light and heavy hydrocarbons, respectively, over a wide range of equivalence ratio, are used in the application to engine combustion. The primary objective of the present study is to investigate both laminar and turbulent burning velocities experimentally and to discuss the underlying mechanisms in consideration of interaction between flame stretch and the Lewis number.

Mutations of the hepatocyte nuclear factor 4 alpha (HNF-4alpha) gene have been demonstrated in maturity-onset diabetes of the young (MODY) 1 families. To investigate the possibility that the HNF-4alpha gene contributes to the onset of non-insulin-dependent diabetes mellitus (NIDDM) in Japanese patients, we screened all exons and flanking introns of this gene for mutations in 100 patients with NIDDM diagnosed after 25 years of age. We identified two missense mutations: M49V in exon 1c and T1301 in exon 4; and two nucleotide substitutions in introns: cytosine to thymidine at -5 nt in intron 1b and adenine to thymidine at -21 nt in intron 5. We screened an additional 220 diabetic subjects for the polymorphism in intron 1b. The c/t substitution in intron 1b was associated with NIDDM. This substitution in the polypyrimidine tract, an important cis-acting element directing intron removal, is likely to influence pre-mRNA splicing of this gene. T1301 in exon 4 was observed in only two diabetic subjects. This mutation could influence the conformation of this peptide, resulting in changes in ligand binding domain function. M49V in exon 1c was found in both diabetic and non-diabetic subjects; isoforms HNF-4alpha 4, 5, and 6 with this mutation may impair glucose metabolism in tissue. In contrast to the primary cause of nonsense and missense mutations of the HNF-4alpha gene in MODY1, the nucleotide substitution in intron 1b may partially contribute to development of NIDDM in combination with other genetic and environmental factors.

Although there are reports of hyperintense lesions in hyperglycemic hemichorea-hemiballism by brain magnetic resonance imaging (MRI) (1, 2), such findings by magnetic resonance angiography (MRA) are rare. An 82-year-old man presented with a 1-month history of left-sided involuntary movements of his face and limbs. Laboratory data revealed a blood glucose level of 20.8 mmol/L, glycosylated hemoglobin of 11.5%, and serum C-peptide of 2.20 ng/mL. There were no urinary ketones or metabolic acidosis. T1-weighed brain MRI showed a hyperintense lesion in the putamen and caudate nucleus, whereas cranial MRA showed an irregular perivascular hyperintense lesion and an arteriostenosis in the middle cerebral artery (MCA) (Figure 1A, arrows). After a diagnosis of hyperglycemic hemichorea-hemiballism, he was treated with insulin. Upon correction of blood glucose levels, the ballistic movements improved. At the 1-month follow-up visit, the abnormal hyperintense areas had improved slightly (Figure 1B), and they had disappeared completely at the 6-month follow-up visit (Figure 1C). His glycosylated hemoglobin improved to 7.6%. Hyperglycemic hemichorea-hemiballism is more common among patients with type 2 diabetes, Asians, and the elderly. Possible underlying mechanisms include microhemorrhages due to blood-brain-barrier failure (3), reactive astrocytes associated with ischemia (4), and decreased -aminobutyric acid production after dehydration (5), and they present as a high-intensity signal on T1-weighed MRI. We observed high perivascular signal intensity on MRA, along with stenosis of the MCA at the bifurcation of the lenticulostriate arteries, which supply the putamen and caudate nucleus. This suggests that these lesions have vascular fragility and they are vulnerable to ischemia and metabolic disorders associated with hyperglycemia.

Cyclic adenosine 5′diphosphate-ribose (cADPR) is thought to have a second messenger role in insulin secretion through mobilisation of Ca2 +. As human lymphocyte antigen CD38 has both ADP-ribosyl cyclase and cADPR hydrolase activity, it may be important in glucose-induced insulin secretion in islets. Thirty one randomly selected Japanese patients with Type II diabetes mellitus who had first-degree and/or second-degree relative(s) with Type II diabetes mellitus were screened for mutations of this gene using single-stranded conformation polymorphism. Two variant patterns in exon 3 and exon 4 of the CD38 gene were identified. The variant in exon 3 resulted in an amino acid substitution from Arg140 (CGG) to Trp (TGG). The Arg140Trp mutation was observed in 4 of 31 patients, and allele frequencies were significantly different in patients and the control subjects (p = 0.004). One patient with this mutation has two missense mutations on beta cell/liver glucose transporter (GLUT2) gene; her mother, who has impaired glucose tolerance, also has this mutation on the CD38 gene and one missense mutation on the GLUT2 gene. Enzyme activity studies using COS-7 cells expressing the Arg140Trp mutation showed a reduction in ADP-ribosyl cyclase and cADPR hydrolase activity of around 50 %. The Arg140Trp mutation on CD38 thus appears to contribute to the development of Type II diabetes mellitus via the impairment of glucose-induced insulin secretion in the presence of other genetic defects. [Diabetologia (1998) 41: 1024–1028]

Yagui K, Yamaguchi T, Kanatsuka A, Shimada F, Huang CI, Tokuyama Y, Ohsawa H, Yamamura K, Miyazaki J. Mikata A, Yoshida S, Makino H. Formation of islet amyloid fibrils in beta-secretory granules of transgenic mice expressing human islet amyloid polypeptide/amylin. Eur J Endocrinol 1995:132:487–96. ISSN 0804–4643 To investigate the relationship between human islet amyloid polypeptide (IAPP)/amylin expression and islet amyloid deposits in the pathogenesis of human non-insulin-dependent diabetes mellitus (NIDDM), we developed transgenic mice using a human IAPP cDNA connected to an insulin promoter. Ribonucleic acid blotting and immunohistochemistry revealed the expression of the transgene in the pancreatic beta cells. Immunogold electron microscopy showed that beta-secretory granules contained the human C-terminal flanking peptide of the IAPP precursor. Reverse-phase HPLC demonstrated human and mouse IAPP amide in the pancreas. Electron microscopy showed the accumulation of fibril-like material in a considerable number of beta-secretory granules. These results suggest that in transgenic mice, the human IAPP precursor is expressed in beta cells and becomes normally sorted into beta-secretory granules in which normal conversion to mature human IAPP takes place. The human IAPP molecules, because of their amyloidogenesis, aggregate into amyloid fibrils in secretory granules. Glucose tolerance was normal at 7 months old and islet amyloid was not observed. A longer time may be required for islet amyloid deposits and hyperglycemia to develop in mice. Our working hypothesis is that in human NIDDM, IAPP aggregates into amyloid fibrils in beta-secretory granules, and that the fibrils are released into the extracellular space and islet amyloid deposits become substantial with time. Azuma Kanatsuka, Second Department of Internal Medicine, Chiba University School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba 260, Japan

Mutations in the insulin receptor gene may lead to insulin resistance and diabetes mellitus in some patients. We have studied an insulin-resistant patient with leprechaunism. Insulin binding to the patient's fibroblasts was markedly decreased. Determination of the nucleotide sequence of the patient's insulin receptor gene revealed heterozygosity for a 2-basepair deletion in exon 15. If the premessenger ribonucleic acid (pre-mRNA) is spliced normally, it causes a replacement of codon 970 in the beta-subunit with a premature chain termination codon, thereby deleting most of the intracellular domain of the receptor. The mRNA transcribed from the allele with a 2-base-pair deletion is likely to be unstable because mRNA transcripts from this allele could not be detected by complementary DNA sequencing. Northern blot analysis showed that the patient's insulin receptor mRNA was decreased by 90% compared with that of a control subject, thus suggesting that the patient is a compound heterozygote for two mutations that decrease levels of insulin receptor mRNA. This deletion mutation in exon 15 seems to be a de novo mutation, because it was not detected in either parent. Investigation of the inheritance of a silent sequence polymorphism in exon 17 provided that the deletion occurred in the maternal allele. Furthermore, linkage analysis suggests that the second mutation is derived from the patient's father, although we could not directly identify it by sequencing the coding region of the insulin receptor gene. Therefore, it is possible that this mutation is present in a regulatory domain of the insulin receptor gene, acting in cis-dominant fashion to reduce the levels of insulin receptor mRNA. Analyses of the hypervariable region in the myoglobin and pMCT118 loci were consistent with the assumption that the father and mother studied here are indeed the biological parents of the diseased patient. We hereby conclude that the patient is a compound heterozygote for two mutant alleles, both of which are responsible for the reduced levels of insulin receptor mRNA and insulin binding.

The beta-cell/liver glucose transporter (GLUT2) gene was screened for mutations using single-strand conformation polymorphism analysis (SSCP) in 30 Japanese subjects with non-insulin dependent diabetes mellitus (NIDDM). Analysis of all exons and adjacent intron regions identified six SSCP polymorphisms, three of which resulted in amino acid substitutions: V101I, T110I and G519E. The V101I and G519E, substitutions represent new polymorphisms in this gene. The six polymorphisms were observed in both NIDDM and control groups and there were no significant differences in allele frequencies between groups. A portion of the insulin receptor substrate 1 gene in 30 NIDDM subjects and in normal control subjects was also screened for mutations. Two SSCP variants that change the sequence of the protein, delta S686/687 (deletion of the codons for serine-686 and 687) and G972R, were identified in two different NIDDM subjects, both whom were also heterozygous for the V101I polymorphisms in GLUT2. The GLUT2 and IRS1 amino acid polymorphisms did not show a simple pattern of co-inheritance with NIDDM in the families of these subjects suggesting that neither polymorphism is sufficient to cause NIDDM but may increase diabetes-susceptibility through their interaction with other loci and environmental factors.

Mutations were screened for in the glucokinase gene of 25 Japanese patients with Type 2 (non-insulin-dependent) diabetes mellitus. Each exon was scanned by electrophoresis of enzymatically amplified DNA segments under non-denaturing conditions and variants were sequenced. A variant pattern was detected in exon 5 of one patient. Direct sequencing of this exon revealed a single nucleotide substitution in codon 188 (GCT-->ACT) of one of two alleles resulting in the mutation of Ala188-->Thr, an invariant residue in the sequence of all mammalian glucokinases and hexokinases. This mutation was not found in 40 normal control subjects. The proband had been diagnosed with Type 2 diabetes at the age of 62 years. Four other members of her family have the same mutation and all have Type 2 diabetes or impaired glucose tolerance. The youngest age at diagnosis of Type 2 diabetes in these other members was 13 years, suggesting that her pedigree was maturity-onset diabetes of the young (MODY). All subjects with the Thr188 mutation show a decreased insulin secretory response during oral glucose tolerance testing. Mutations in the glucokinase gene associated with Type 2 diabetes have been previously identified in Caucasian (French and British) subjects. This study indicates that mutations in this gene are also implicated in the development of Type 2 diabetes in Asians. Further studies are required to determine the frequency of mutations in glucokinase among Japanese patients with Type 2 diabetes.

In a previous report on a 16-year-old Japanese girl with type A insulin resistance, we found that one allele of the insulin receptor gene was inherited from her mother and contained a 1.2 kilobase pair deletion which removed the 14th exon in the beta subunit. We extended investigation of the proband and found the deletion between two Alu sequences. To determine the effect of the deletion on the level of transcription and the splicing pattern of messenger ribonucleic acid (mRNA), we synthesized the complimentary DNA and used the polymerase chain reaction to amplify the region which included the deleted area. The deletion shifted the reading frame, resulting in a termination codon after amino acid 867 (Glu), thereby producing a truncated insulin receptor without a transmembrane region and cytoplasmic domain. We also sequenced each of 22 exons of the insulin receptor gene but found no mutation in exons of the insulin receptor gene, except for deletion of exon 14 of the maternal allele. Thus, the proband is a heterozygote for a single mutant allele. Abnormal mRNA transcribed from the mutant allele resulted in a decrease in insulin binding.

Fragments of 5'-flanking sequences of the human insulin receptor gene were analyzed in transient expression assays after transfection of cell lines with expression assays after transfection of cell lines with an improved low background chloramphenicol acetyl-transferase vector system pSVOOCAT (Araki, E., Shimada, F., Shichiri, M., Mori, M., and Ebina, Y. (1988) Nucleic Acids Res. 16, 1627). Transfection of chimeric chloramphenicol acetyltransferase plasmids containing various deletions and insertions of the promoter of HIR gene into CHO and COS cells indicated that the region between -629 and -1 (initiator ATG is +1) is sufficient for maximal promoter activity. The DNA element of the cluster of four G-C boxes (-593 to -618) enhanced the transcription, examined by the low background pSVOOCAT vector system in vivo. DNase I footprinting and gel retardation experiments using partially purified LacZ-Sp1 hybrid proteins showed that the transcription factor Sp1 can bind to the cluster of the four G-C boxes of the promoter. Thus, the efficient expression of the human insulin receptor gene possibly requires the binding of transcriptional factor Sp1 to four G-C boxes located -593 to -618 base pairs upstream of the ATG translation initiation codon.

We have applied phase imaging on digital mammography to investigate adequate contrast of printed images for digital phase contrast mammography using a practical molybdenum X-ray tube. Phase contrast mammography procedures were performed with defined air gap (e.g., 0.6 m) configuration using customized mammography equipment and a computed radiography (CR) system. Magnified (x2) phase contrast images acquired with 0.0875mm per pixel were mapped onto the laser imager resolution at 0.04375mm per pixel for printing life-size object on wet processing silver halide recording film. For contact mammography of screen-film system, we used MinR2000 system as a baseline method. ACR 156 phantom printed images with contrasts of 2.8, 3.7, 4.9, 5.7 and 6.7 were evaluated by five radiologists. The ACR scores for the life-size image based on the 2 times magnified phase contrast image acquired by the computed radiography were higher than the scores of MinR2000 image, when the contrast of printed images for both methods was 3.7. The ACR scores were lower in the low contrast images (i.e., 2.8) than its higher contrast counterparts (i.e., >= 3.7) for all techniques used. The detectability improvement should be due to higher spatial resolution and lower noise in the phase contrast images.

Phase contrast x-ray imaging has been studied intensively using x-rays from synchrotron radiation and micro-focus x- ray rubes. However, these studies have revealed the difficulty of this technique's application to practical medical imaging. We have created a phase contrast imaging technique using practical x-ray tubes with small focal spot sizes. In a previous study, we identified the radiographic conditions for phase contrast magnification mammography with screen-film systems, where the edge effect due to phase contrast outweighs the geometrical unsharpness introduced by the 0.1mm-focal spot of a molybdenum-anode x-ray tube. In the present study, phase contrast images of a hand and a chest phantoms were obtained using computed radiography and a geometrical magnification of two using a tungsten-anode x- ray tube with a 0.1mm-focal spot. The digital images were printed so that objects were reproduced at their original size. Compared to conventional digital contact images obtained at the same x-ray dose, the life-sized digital phase contrast x-ray images displayed improved sharpness and resolution, and reduced grain. We describe the empirical results of digital phase contrast imaging, and discuss these performance improvements through simulation including x-ray scatter.