Sarcopenia in elderly patients with type 2 diabetes mellitus: prevalence and related clinical factors.

AIMS: Sarcopenia, which shortens healthy life expectancy, has recently been attracting attention because the Japanese population is rapidly aging. In this preliminary study, we estimated the prevalence of elderly diabetic patients who were complicated with sarcopenia and searched for any related clinical factors. METHODS: Elderly (≥65 years of age) Japanese patients with type 2 diabetes mellitus were recruited by asking doctors to supply candidates for the study. The prevalence of sarcopenia was estimated based on the criteria proposed by the Asian Working Group for Sarcopenia in 2014. RESULTS: Two hundred eighty-eight patients (151 males) were accepted for the study. The prevalence of sarcopenia was 15.2% in males and 15.3% in females. Multiple logistic regression analysis indicated that sarcopenia was significantly correlated with serum high-sensitivity C-reactive protein in females, in addition to age and body mass index. Female patients were then classified into four groups according to the presence or absence of impaired muscle mass and/or impaired strength. Serum high-sensitivity C-reactive protein was significantly higher in the sarcopenia group (those with impaired muscle mass and impaired strength) than in the other three groups. CONCLUSIONS: After clarifying the prevalence of sarcopenia in elderly Japanese patients with type 2 diabetes mellitus, we found that serum high-sensitivity C-reactive protein was significantly higher in female patients with sarcopenia than in female patients without sarcopenia. Elevated serum high-sensitivity C-reactive protein requires impaired muscle mass and impaired strength.

Cognitive impairment in elderly patients with type 2 diabetes mellitus: prevalence and related clinical factors.

AIM: Diabetes mellitus is reported to be a risk factor for dementia. We evaluated the cognitive function in elderly diabetic patients and estimated the prevalence of patients with cognitive impairment and looked for any related clinical factors. SUBJECTS AND METHODS: Using 281 elderly (65 years of age or older) Japanese patients with type 2 diabetes mellitus who were free of clinically evident cognitive impairment, we evaluated their cognitive function with the Mini Mental State Examination (MMSE). RESULTS: The MMSE score of all the participants was 27.3 ± 2.4 with 31.3% of them being in the abnormal range (tentatively defined normal range as having an MMSE score of 27-30). Multiple regression analysis disclosed that fasting serum non-esterified fatty acid (NEFA), estimated glomerular filtration ratio (eGFR) and insulin treatment were significantly related factors for the MMSE score, in addition to age and schooling history, which are extremely strong factors. CONCLUSIONS: We revealed that approximately one-third of elderly type 2 diabetic patients who were free of clinically evident cognitive impairment had impaired cognitive function, demonstrating that the MMSE score was significantly correlated with fasting NEFA level, renal function, insulin treatment, age and schooling history.

Quantitative LC-MS/MS Analysis of Proteins Involved in Metastasis of Breast Cancer.

The purpose of this study was to develop quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods for the analysis of proteins involved in metastasis of breast cancer for diagnosis and determining disease prognosis, as well as to further our understand of metastatic mechanisms. We have previously demonstrated that the protein type XIV collagen may be specifically expressed in metastatic tissues by two dimensional LC-MS/MS. In this study, we developed quantitative LC-MS/MS methods for type XIV collagen. Type XIV collagen was quantified by analyzing 2 peptides generated by digesting type XIV collagen using stable isotope-labeled peptides. The individual concentrations were equivalent between 2 different peptides of type XIV collagen by evaluation of imprecise transitions and using the best transition for the peptide concentration. The results indicated that type XIV collagen is highly expressed in metastatic tissues of patients with massive lymph node involvement compared to non-metastatic tissues. These findings were validated by quantitative real-time RT-PCR. Further studies on type XIV collagen are desired to verify its role as a prognostic factor and diagnosis marker for metastasis.

Acute-phase glucose fluctuation is negatively correlated with myocardial salvage after acute myocardial infarction.

BACKGROUND: It remains unclear whether glycemic fluctuation immediately after acute myocardial infarction (AMI) can affect myocardial damage. This study investigated the impact of glucose fluctuation on myocardial salvage following successful recanalization of primary AMI. METHODS AND RESULTS: A total of 36 consecutive patients with AMI were studied. Glycemic variability, as indicated by the mean amplitude of glycemic excursion (MAGE), was measured on a continuous glucose monitoring system. Three subsets (CD14(+)CD16(-), CD14(++)CD16(+) and CD14(+-)CD16(+)) were measured on flow cytometry 1, 2, 3, 4 and 5 days after AMI onset. A 2-h oral glucose test was performed in 23 patients who had no previous diagnosis of diabetes and/or glycated hemoglobin <6.5%, after the onset of AMI at 2 weeks. Plasma active glucagon-like peptide (GLP)-1 level was measured in each sample. The extent of myocardial salvage 7 days after AMI was evaluated on cardiovascular magnetic resonance imaging. MAGE and the peak CD14(+)CD16(-) monocyte level were significantly negatively correlated with myocardial salvage index (MSI). MAGE was significantly correlated with peak CD14(+)CD16(-) monocyte level. Of interest, plasma GLP-1 level was significantly positively correlated with MSI and significantly negatively correlated with MAGE. CONCLUSIONS: Glucose fluctuations during the acute phase of AMI affect MSI, indicating that manipulation of glucose variability from peak to nadir might be a potential therapeutic target for salvaging ischemic damage.

Monoclonality of composite large-cell neuroendocrine carcinoma and invasive intestinal-type mucinous adenocarcinoma of the cervix: a case study.

A rare case of mixed carcinoma of the cervix is reported, composed of a large-cell neuroendocrine carcinoma and an invasive intestinal-type mucinous adenocarcinoma. The large-cell neuroendocrine carcinoma was composed of solid nests, sheets, and trabeculae of medium-sized to large-sized cells, and was positive for chromogranin-A and CD56. The invasive intestinal-type mucinous adenocarcinoma showed sparsely scattered immunoreactivity for chromogranin-A. Using an X-chromosome clonality assay, these 2 components showed patterns of monoclonality. These results suggest that the large-cell neuroendocrine carcinoma may have arisen from the invasive mucinous adenocarcinoma.

Combined examination of glyceryl trinitrate-mediated vascular dilation with flow-mediated vascular dilation is essential for assessment of vascular function in type 2 diabetes.

AIMS/INTRODUCTION: In order to characterize the impaired vascular function in type 2 diabetes (DM) patients, we evaluated the flow-mediated vascular dilation (FMD) with glyceryl trinitrate-mediated vascular dilation (NMD) using ultrasonography. MATERIALS AND METHODS: A total of 111 DM patients and 42 healthy control participants were studied. The maximal dilatation of FMD and NMD (%FMD and %NMD, respectively), the beginning time (T) of dilatation after stimulation and the velocity (V) of the vascular dilatation were also measured. RESULTS: Among DM patients, 49% had impaired %NMD, which affects the results of %FMD. In DM patients with normal %NMD, the %FMD was also significantly lower than that in control participants, although the T and the V were not impaired. In contrast, both the T and the V were disturbed in the DM patients with low %NMD. Multiple linear regression analysis showed that %NMD was independently correlated with albuminuria. Our results indicate that the impaired FMD in DM is be affected by low NMD, and impaired endothelial function already exists even in DM patients whose vascular smooth muscle function is still retained, and also albuminuria is the clinical feature of DM with low %NMD. CONCLUSIONS: Examination of NMD, not only FMD, should be carried out as it offers the possibility of clarifying vascular function in DM patients.

Protective role of human insulin against the cytotoxicity associated with human mutant S20G islet amyloid polypeptide.

AIMS/INTRODUCTION: Islet amyloid polypeptide (IAPP) is a main component of islet amyloid in type 2 diabetes and cosecreted from ß-cell with insulin. Clinical evidence from the patients with S20G mutation of the IAPP gene, as well as experimental evidence that insulin could inhibit amyloid formation of IAPP, suggests that a gradual reduction of insulin could be related to the cytotoxicity associated with S20G-IAPP through long-term deterioration of ß-cells in type 2 diabetes. Our objective was to show an effect of human insulin on S20G-IAPP associated cytotoxicity. MATERIALS AND METHODS: We analyzed the cytotoxicity associated with S20G-IAPP by controlling human insulin expression using adenovirus vectors with micro ribonucleic acid specifically against human insulin in endocrine AtT-20ins cells, which express human insulin permanently. Additionally, we carried out a follow-up study of circulating IAPP and insulin in type 2 diabetic patients. RESULTS: S20G-IAPP expression was associated with a decrease in viability and an increase in terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling-positive cells in AtT-20ins cells. Furthermore, downregulation of human insulin enhanced the cytotoxicity associated with S20G-IAPP, and induced the cytotoxicity associated with wild-type (WT)-IAPP. Reduction of ubiquitin carboxy-terminal hydrolase L1 activity enhanced cytotoxicity under the downregulation of human insulin expression in both S20G- and WT-IAPP transduced cells. A 5-year follow up of type 2 diabetic patients showed a disproportionate increase of serum fasting IAPP-to-insulin ratio from baseline. CONCLUSIONS: Human insulin plays a protective role against the cytotoxicity associated with S20G-IAPP, as well as WT-IAPP. The findings could suggest long-term deterioration of insulin secretion associates with IAPP linked cytotoxicity in type 2 diabetes.

Kir6.2 E23K polymorphism is related to secondary failure of sulfonylureas in non-obese patients with type 2 diabetes.

AIMS/INTRODUCTION: The Kir6.2 E23K polymorphism was studied with a special reference to secondary sulfonylurea (SU) failure in non-obese patients with type 2 diabetes. MATERIALS AND METHODS: We recruited 278 non-obese (body mass index ≤30.0 kg/m(2)) Japanese patients with type 2 diabetes who had a history of SU treatment (for 11.2 ± 6.3 years) and compared the frequency of the secondary SU failure among the genotypes of the polymorphism. Genotyping of the Kir6.2 E23K was carried out by polymerase chain reaction-restriction fragment length polymorphism. RESULTS: The genotype frequencies of the polymorphism were similar to those previously reported in Japanese patients with type 2 diabetes. The frequency with which patients deteriorated into secondary SU failure was significantly higher in those with the KK genotype than those with EE or EK genotypes. Among 214 patients who eventually received insulin therapy because of secondary SU failure, the period of SU treatment in those with the KK genotype was significantly shorter than those with the EE or EK genotype, although the period from diagnosis to the start of SU treatment was not significantly different. CONCLUSIONS: These data suggest that the Kir6.2 E23K polymorphism is related to the acceleration of secondary SU failure in non-obese Japanese patients with type 2 diabetes.

[Evaluation of seasonal changes in hemoglobin A1c in diabetic patients].

In order to evaluate seasonal changes in hemoglobin A1c (HbA1c) values, we examined HbA1c values among 34,590 patients in 2010, and calculated the monthly average of HbA1c values through the year. HbA1c values were the highest in March and the lowest in October with a difference of 0.30%. The similar annual pattern was observed in HbA1c values from 2006 to 2009. Then we selected 453 diabetic patients whose treatment did not change through the year, and calculated average HbA1c values in four seasons each. There were also significant seasonal changes in diabetic patients, which were the highest in the spring and the lowest in the autumn, especially found in patients with insulin therapy. These effects may be caused by cold climate, decreased physical activity, over food intake and body weight gain in the winter. These seasonal changes in HbA1c should be concerned in the case of health service research, clinical trials and evaluation of the effects of medical treatment.

Interferon-γ is protective in cisplatin-induced renal injury by enhancing autophagic flux.

The chemotherapeutic agent cisplatin often causes severe renal dysfunction; however, the molecular mechanism causing renal injury remains unclear. In wild-type mice, intrarenal interferon (IFN)-γ gene expression was found to be enhanced while CD3(+) T cells and Ly-6G neutrophils were the main cellular source of IFN-γ following cisplatin injection. Compared to wild-type mice, cisplatin-treated IFN-γ-deficient (IFN-γ(-/-)) mice exhibited exaggerated histopathological changes with higher blood urea nitrogen and creatinine levels. Cisplatin-induced apoptosis was associated with enhanced caspase-3 activation in renal proximal tubular epithelial cells, with effects suppressed by IFN-γ resulting in increased cell viability. IFN-γ significantly reduced the levels of the autophagic markers LC3-II and p62, and enhanced cathepsin D expression in cisplatin-treated renal proximal tubule epithelial cells, implying that IFN-γ can accelerate autophagic flux. Tubular cell apoptosis was more evident with enhanced caspase-3 activation in IFN-γ-deficient compared to wild-type mice. Elevated intrarenal LC3-II and increased p62 accumulation were associated with reduced cathepsin D activation in IFN-γ-deficient mice, implying that the absence of IFN-γ suppressed autophagic flux. Thus, IFN-γ can accelerate autophagic flux by augmenting cathepsin D levels and reciprocally increasing the viability of renal tubular cells, thereby attenuating cisplatin-induced acute renal injury.

Clinicopathological significance of podocalyxin and phosphorylated ezrin in uterine endometrioid adenocarcinoma.

BACKGROUND: Podocalyxin is an anti-adhesive glycoprotein that has been associated with highly aggressive forms of cancers. Podocalyxin increases the migration and invasive properties of cancer cells through its interaction with ezrin, which undergoes an increase in phosphorylation through podocalyxin. AIMS: To study the roles of podocalyxin and phosphorylated ezrin (pEzrin) in uterine endometrioid adenocarcinoma (UEA). METHODS: Using immunohistochemisty the authors investigated the expression of podocalyxin and pEzrin along with some well-known markers of tumour aggressiveness including p53, oestrogen receptor and Ki-67 in UEA. RESULTS: Podocalyxin and pEzrin expression levels were positive in 36.1% (22 of 61 patients) and 50.8% (31 of 61 patients) of UEA cases, respectively. Further, podocalyxin expression was correlated with tumour grade (p=0.0001), FIGO stage (p=0.0062), p53 expression (p=0.0050) and Ki-67 index (p=0.0069). In addition, pEzrin expression was associated with FIGO stage (p=0.0231), p53 expression (p<0.0001) and Ki-67 index (p=0.0010). The expression of podocalyxin was also correlated with that of pEzrin (p=0.0102). CONCLUSIONS: These results suggest that overexpression of podocalyxin and pEzrin may indicate a more aggressive phenotype; thus, evaluation of these proteins may be useful in prediction of disease progression in UEA cases.

Expression of wild-type and mutant S20G hIAPP in physiologic knock-in mouse models fails to induce islet amyloid formation, but induces mild glucose intolerance.

UNLABELLED: Aims/Introduction: Human islet polypeptide S20G mutation (hIAPP(S20G)) is associated with earlier onset type 2 diabetes and increased amyloidogenicity and cytotoxicity in vitro vs wild-type hIAPP (hIAPP(WT)), suggesting that amyloidogenesis may be pathogenic for type 2 diabetes. We compared the contributions of hIAPP(S20G) and hIAPP(WT) toward intra islet amyloid formation and development of type 2 diabetes in a unique physiologic knock-in mouse model. MATERIALS AND METHODS: We replaced the mouse IAPP gene (M allele) with hIAPP(WT) (W allele) and hIAPP(S20G) (G allele) via homologous recombination and backbred transgenic mice against C57Bl/6 strain 5 generations to minimize genetic variation. Mice (3 month old) were maintained on control (CD) or high fat diet (HFD) for 15 months and studied at 3 month intervals by oral glucose tolerance testing (OGTT) and pancreas histology to assess glucose homeostastis, amyloidogeneisis, islet mass, ß cell replication, and apoptosis. RESULTS: IAPP blood levels were indistinguishable in all mice. WW and GW mice maintained on both diets lacked intraislet amyloid at all ages. On both diets relative to MM controls WW and GW mice exhibit glucose intolerance (P < 0.008) with no differences in insulin secretion. However, GW mice secreted significantly more insulin (P < 0.03 that WW mice on both diets throughout the study. By 12 months on the high fat diet all mice increased their ß cell mass about 3-fold and were indistinguishable. CONCLUSIONS: Physiologic expression of hIAPP(WT) and hIAPP(S20G) in C57Bl/6 mice produces mild glucose intolerance with inappropriately normal insulin secretion that is independent of intraislet amyloid formation. (J Diabetes Invest, doi: 10.1111/j.2040-1124.2011.00166.x, 2011).

Neuropilin-2 expression in papillary thyroid carcinoma: correlation with VEGF-D expression, lymph node metastasis, and VEGF-D-induced aggressive cancer cell phenotype.

CONTEXT: Neuropilin-2 (Nrp2) is a coreceptor for vascular endothelial growth factor-D (VEGF-D) that is expressed on the surface of endothelial cells. Recently, Nrp2 was shown to play a role in lymph node metastasis and promotion of cancer cell migration. VEGF-D also promotes lymphangiogenesis, which in turn promotes tumor metastasis. OBJECTIVE: The aim was to study the role of neuropilin-2 in lymph node metastasis in human papillary thyroid carcinoma (PTC). DESIGN: Expression of Nrp2 was studied by immunohistochemistry and the relationship between Nrp2 expression and lymph node metastasis, VEGF-D expression and other established clinicopathological variables were analyzed in PTC. The effects of neutralizing anti-Nrp2 antibody on VEGF-D-induced invasion and migration were assessed in PTC cell lines. RESULTS: Nrp2 expression was observed in 64.3% (36 of 56) of the PTC patients. Nrp2 expression was significantly correlated with lymph node metastasis (P = 0.0216) and VEGF-D expression (P = 0.0034). VEGF-D was shown to promote filopodia formation and cancer cell migration and invasion by K1 and B-CPAP cells. These responses were significantly blocked by neutralizing anti-Nrp2 antibody. CONCLUSION: Nrp2 expression was correlated with lymph node metastasis and VEGF-D expression in PTC. Our data also showed a role for Nrp2 in regulating VEGF-D-induced invasion and migration in vitro.

[Clinical evaluation of insulin measuring kit not cross-react with insulin analogue preparations].

Clinical evaluation of insulin assay system reacting with only human insulin molecule (kit B) was performed by comparing it with conventional insulin assay system (kit A) cross-reacting with insulin analogue as well as human insulin preparation. In vitro, the kit B was confirmed to cross-react with only human insulin, not with insulin analogue preparations such as insulin aspart, lyspro and glargine. In non-insulin treated diabetic patients, postprandial and post-insulin injected serum immunoreactive insulin (IRI) concentrations measured by kit B were almost the same as those measured by the kit A. On the other hand, in diabetic patients treated with insulin analogue preparations, postprandial and post-insulin injected serum IRI levels measured by kit B were obviously low compared with those by kit A. After intravenous injection of insulin analogue preparations (0.1 unit/kg), insulin lyspro or insulin aspart, serum IRI levels measured by the kit B were not increased but gradually decreased in contrast to the obviously increased serum IRI level measured by the kit A. From these results, the kit B was confirmed not to measure the insulin analogue preparations in vitro and in vivo.

[Effects of aging and menopause on women's vascular systems].

AIM: The morbidity of hypertension increases with aging although the exact relationship between hypertension and menopause has not been clearly elucidated. Therefore we set out to clarify the effects of aging and menopause upon women's vascular systems. METHODS: We divided 151 elderly and middle-aged women into 3 groups (premenopause group, menopause group and post-menopause group). We measured height, weight, blood pressure (BP), total cholesterol (T-chol), HDL-cholesterol (HDL-chol), LDL-cholesterol (LDL-chol), high sensitivity C-reactive protein (hsCRP), creatinine (Cr), triglyceride (TG), fasting blood glucose (FPG), IRI, HOMA-R, brachial-ankle pulse wave velocity (baPWV), augmentation index (AI), percentage of flow-mediated dilatation (%FMD), and echocardiography. RESULTS: In the post-menopause and menopause groups systolic BP and AI were significantly higher than those in the pre-menopause group. There was a significant difference in systolic BP between the post-menopause group and menopause group. In the post-menopause group, baPWV, Cr, and hsCRP were significantly higher than those in the pre-menopause group. There was significant difference in baPWV between the post-menopause group and menopause group. In the post-menopause group, %FMD and eGFR were significantly lower than those in other 2 groups. In the post-menopause group and the menopause group, E/A was significantly lower than in the pre-menopause group. There was also a significant difference in E/A between the post-menopause group and the menopause group. CONCLUSIONS: Elevated blood pressure, atherosclerosis and endothelial dysfunction were associated with aging and menopause in their present study. These results suggest that understanding women's cardiovascular changes which accompany aging are important for women's health care and prevention of cardiovascular events.

Autophagy protects against human islet amyloid polypeptide-associated apoptosis.

UNLABELLED: Aims/Introduction: Islets in type 2 diabetes are characterized by deposition of islet amyloid polypeptide (IAPP) as well as ß-cell dysfunction. The unique amyloidogenic character of human (h)IAPP is associated with cytotoxicity. Autophagy is a ubiquitous system of cellular recycling that contributes to cell survival. Thus, we examined whether autophagy could ameliorate hIAPP-associated cytotoxicity. MATERIALS AND METHODS: First, we used a COS-1 cell model, lacking endogenous IAPP that might affect cytotoxicity related to exogenous hIAPP. Next, we used the mouse ß-cell line, MIN-6 cells. Both cells were transfected with hIAPP or rat (r)IAPP expression constructs, or transfected with bicistronic vectors expressing green fluorescent protein (GFP) and either hIAPP or rIAPP for flow cytometry analysis. Cell viability and apoptosis markers were studied in relation to chemical or genetic modulation of autophagy. RESULTS: The viability of cells expressing hIAPP was significantly decreased as compared with those expressing rIAPP and the cleavage of pro-caspase-3 was elevated in hIAPP-transfected cells. The formation of autophagosomes and the conversion of microtubule-associated protein light chain 3B I to II were elevated in hIAPP-expressing cells. The viability of hIAPP-expressing cells was increased after treatment with rapamycin, an inducer of autophagy, and decreased after treatment with 3-methyladenine, an inhibitor of autophagy. In MIN-6 cells, annexin positive cells were increased by 3-methyladenine and decreased by rapamycin using flow cytometry. Knocking down of the autophagy protein 5 gene decreased hIAPP-transfected cell viability. CONCLUSIONS: Autophagy is co-localized with hIAPP expression and it plays a protective role in hIAPP-associated apoptosis. (J Diabetes Invest, doi: 10.1111/j.2040-1124.2010.00065.x, 2010).

Progressive deterioration of insulin secretion in Japanese type 2 diabetic patients in comparison with those who carry the S20G mutation of the islet amyloid polypeptide gene: A long-term follow-up study.

UNLABELLED: Aims/Introduction: In order to clarify the enhanced ß-cell dysfunction in type 2 diabetic patients carrying the S20G mutation of the islet amyloid polypeptide gene (S20G-patients), we first estimated the decline of insulin secretion in Japanese type 2 diabetic patients without the S20G mutation (non-S20G-T2D-patients) by long-term observation, and then compared it with that of the S20G-patients. MATERIALS AND METHODS: We followed 70 non-S20G-T2D-patients (body mass index <30 kg/m(2)) for more than 10 years and six S20G-patients for more than 5 years. We measured fasting C-peptide (F-CP) every 1-2 years and carried out a glucagon test at least once during the follow-up period. F-CP and a 5-min value of C-peptide after glucagon injection (5'-CP) were used as the indices of insulin secretion. We excluded patients who had renal dysfunction and/or anti-insulin antibodies in the insulin-treated patients. The individual annual declines were calculated from the slopes of the regression lines between C-peptide levels and duration (years after diagnosis). RESULTS: The mean individual annual declines of both F-CP and 5'-CP were significantly greater in the S20G-patients than the non-S20G-T2D-patients (F-CP; 0.047 ± 0.026 vs 0.011 ± 0.037 nmol/L/year, P = 0.025, 5'-CP; 0.139 ± 0.055 vs 0.022 ± 0.012 nmol/L/year, P = 0.008). CONCLUSIONS: We established the annual decline of insulin secretion in the Japanese type 2 diabetic patients by the long-term observation. The results show that the decline of insulin secretion is more rapid in the S20G-patients than the non-S20G-T2D-patients. (J Diabetes Invest, doi: 10.1111/j.2040-1124.2011.00102.x, 2011).