SHORT CHAIN FATTY ACIDS UPREGULATE ADIPOKINE PRODUCTION IN TYPE 2 DIABETES-DERIVED HUMAN ADIPOCYTES.

PURPOSE: Short chain fatty acids (SCFAs) play a major regulatory role in adipocyte function and metabolism. The aim of this study was to investigate the effects of SCFAs on adiponectin and leptin expression in adipocytes, and also to determine whether the effects of SCFA treatment in visceral adipocytes obtained from healthy subjects are different relative to the effects in adipocytes from patients with type 2 diabetes. MATERIALS AND METHODS: Human pericardiac preadipocytes and human pericardiac preadipocytes type 2 diabetes were differentiated into adipocytes for 21 days in 48-well plates. After differentiation, two kinds of mature adipocytes, human pericardiac adipocytes (HPAd) and human pericardiac adipocytes-type 2 diabetes (HPAd-T2D) were incubated with or without 1 mM of acetic acid (AA), butyrate acid (BA), and propionic acid (PA). After 48 hours of incubation, intracellular lipid accumulation was measured using oil red staining. In addition, mRNA levels of adiponectin, leptin and Peroxisome Proliferator-Activated Receptor γ (PPARγ) were determined by Real-Time PCR system. RESULTS: In HPAd, SCFA supplementation did not inhibit lipid accumulation. By contrast, both AA (p<0.01) and PA (p<0.01) significantly inhibited lipid accumulation in HPAd-T2D. Regarding mRNA levels of adiponectin, no significant changes were found in HPAd, while all three types of SCFAs significantly increased (p<0.05) adiponectin expression in HPAd-T2D. Leptin mRNA expression levels were significantly increased by treatment with all three types of SCFAs in both HPAd (p<0.05) and HPAd-T2D (p<0.05). CONCLUSION: SCFAs inhibited lipid droplet accumulation and increased mRNA expression of adiponectin and leptin in T2D-derived adipocytes.

Persistent fasting hyperglycaemia is more predictive of type 2 diabetes than transient fasting hyperglycaemia.

AIMS: We investigated the value of persistent fasting hyperglycaemia as assessed by repeated elevated fasting plasma glucose in predicting the progression to diabetes. METHODS: A retrospective cohort study was conducted from 1998 to 2006 inclusive among 7929 persons (37,742 person-years), with a mean age of 53.0 years at baseline. The cumulative incidence of diabetes was measured. A baseline and follow-up fasting plasma glucose were categorized as normal fasting glucose (< 5.56 mmol/l), or impaired fasting glucose (5.56-6.94 mmol/l). RESULTS: The cumulative incidence and incidence density of diabetes were 3.5% (275 cases) and 7.3 per 1000 person-years over a mean follow-up period of 4.8 years. The cumulative incidence of diabetes among subjects with impaired fasting glucose at both previous examinations (persistent impaired fasting glucose) was 30.4% (222/1518) compared with 0.6% (15/5063) of those with normal fasting glucose at both baseline and initial follow-up. The hazard ratios to develop diabetes, adjusted for possible confounders, was 37.10 (95% CI, 21.6-63.7) for persistent impaired fasting glucose versus persistent normal fasting glucose. Persistent impaired fasting glucose predicted diabetes at 80.7% (222/275) sensitivity and 83.1% (6358/7654) specificity, whereas first baseline impaired fasting glucose only predicted diabetes at 86.9% (239/275) sensitivity and 74.9% (5730/7654) specificity. The model using both previous fasting plasma glucose levels had a greater AUROC (area under receiver operating characteristic) than that using first baseline fasting plasma glucose only (0.92 vs. 0.88; P < 0.001). CONCLUSIONS: Repeated measurements of fasting plasma glucose better predicts incidence of diabetes than a single test. In particular, persistent fasting hyperglycaemia adds more substantial precision to the prediction of future diabetes than transient impaired fasting glucose. This combination is cost efficient and may be practical for early detection of high-risk individuals.

Identification of a novel dual angiotensin II/vasopressin receptor on the basis of molecular recognition theory.

The molecular recognition theory suggests that binding sites of interacting proteins, for example, peptide hormone and its receptor binding site, were originally encoded by and evolved from complementary strands of genomic DNA. To test this theory, we screened a rat kidney complementary DNA library twice: first with the angiotensin II (AII) followed by the vasopressin (AVP) antisense oligonucleotide probe, expecting to isolate cDNA clones of the respective receptors. Surprisingly, the identical cDNA clone was isolated twice independently. Structural analysis revealed a single receptor polypeptide with seven predicted transmembrane regions, distinct AII and AVP putative binding domains, a Gs protein-activation motif, and an internalization recognition sequence. Functional analysis revealed specific binding to both AII and AVP as well as AII- and AVP-induced coupling to the adenylate cyclase second messenger system. Site-directed mutagenesis of the predicted AII binding domain obliterates AII binding but preserves AVP binding. This corroborates the dual nature of the receptor and provides direct molecular genetic evidence for the molecular recognition theory.

The threshold for definition of impaired fasting glucose in a Japanese population.

AIMS: We examined whether the cut-off value of fasting plasma glucose (FPG) for diagnosing impaired fasting glucose (IFG) should be lowered, using data from a large Japanese population. METHODS: A retrospective cohort study was conducted from 1998 to 2006. Follow-up (2002-2006) data were merged with baseline (1998-2002) data, yielding 11 129 persons who had participated on both occasions. Among these, 10 475 persons who did not have diabetes (known diabetes or defined as FPG > or = 7.0 mmol/l) or suspected diabetes (glycated haemoglobin > or = 6.4%) were analysed. RESULTS: During follow-up of an average of 5.4 years, 279 (5.2%) out of 5372 men and 98 (1.9%) out of 5103 women developed diabetes. According to the three baseline FPG categories (< 5.6, 5.6-6.1 and 6.2-6.9 mmol/l), 28/3401 (0.8%), 91/1456 (6.3%) and 160/515 (31.1%), respectively, in men and 13/4231 (0.3%), 30/695 (4.3%) and 55/177 (31.1%), respectively, in women developed diabetes. The optimal cut-off FPG value to predict diabetes was 5.7 mmol/l for both men (sensitivity 84.2%, specificity 76.9%) and women (81.6%, 91.0%). However, lowering the cut-off from 6.1 to 5.7 mmol/l increased the prevalence of IFG 2.7-fold in men and 3.0-fold in women. Lowering the value further to 5.6 mmol/l increased the prevalence of IFG 3.8-fold in men and 4.9-fold in women. CONCLUSIONS: It may be reasonable to retain the conventional lower FPG limit for IFG and treat FPG values of 5.6-6.1 mmol/l as non-diabetic hyperglycaemia, considering the four- to fivefold increase in individuals classified as IFG when the new cut-off is applied.

Atypical protein kinase C is involved in the evolutionarily conserved par protein complex and plays a critical role in establishing epithelia-specific junctional structures.

We have previously shown that during early Caenorhabditis elegans embryogenesis PKC-3, a C. elegans atypical PKC (aPKC), plays critical roles in the establishment of cell polarity required for subsequent asymmetric cleavage by interacting with PAR-3 [Tabuse, Y., Y. Izumi, F. Piano, K.J. Kemphues, J. Miwa, and S. Ohno. 1998. Development (Camb.). 125:3607--3614]. Together with the fact that aPKC and a mammalian PAR-3 homologue, aPKC-specific interacting protein (ASIP), colocalize at the tight junctions of polarized epithelial cells (Izumi, Y., H. Hirose, Y. Tamai, S.-I. Hirai, Y. Nagashima, T. Fujimoto, Y. Tabuse, K.J. Kemphues, and S. Ohno. 1998. J. Cell Biol. 143:95--106), this suggests a ubiquitous role for aPKC in establishing cell polarity in multicellular organisms. Here, we show that the overexpression of a dominant-negative mutant of aPKC (aPKCkn) in MDCK II cells causes mislocalization of ASIP/PAR-3. Immunocytochemical analyses, as well as measurements of paracellular diffusion of ions or nonionic solutes, demonstrate that the biogenesis of the tight junction structure itself is severely affected in aPKCkn-expressing cells. Furthermore, these cells show increased interdomain diffusion of fluorescent lipid and disruption of the polarized distribution of Na(+),K(+)-ATPase, suggesting that epithelial cell surface polarity is severely impaired in these cells. On the other hand, we also found that aPKC associates not only with ASIP/PAR-3, but also with a mammalian homologue of C. elegans PAR-6 (mPAR-6), and thereby mediates the formation of an aPKC-ASIP/PAR-3-PAR-6 ternary complex that localizes to the apical junctional region of MDCK cells. These results indicate that aPKC is involved in the evolutionarily conserved PAR protein complex, and plays critical roles in the development of the junctional structures and apico-basal polarization of mammalian epithelial cells.

Risk factors for the progression of endoscopic Barrett's epithelium in Japan: a multivariate analysis based on the Prague C & M Criteria.

PURPOSE: To determine the prevalence and progression of Barrett's epithelium and associated risk factors in Japan. METHODS: The study population comprised 869 cases. Endoscopic Barrett's epithelium was diagnosed based on the Prague C & M Criteria. The correlations of clinical factors with the prevalence and progression of endoscopic Barrett's epithelium were examined. RESULTS: Endoscopic Barrett's epithelium was diagnosed in 374 cases (43%), in the majority of which the diagnosis was short-segment Barrett's esophagus. The progression of Barrett's epithelium was identified in 47 cases. In univariate and multiple logistic regression analyses, aging, smoking habit, and erosive esophagitis were significantly associated with the prevalence of Barrett's epithelium, whereas aging and erosive esophagitis, especially severe erosive esophagitis, were significant contributing factors to the progression of Barrett's epithelium. CONCLUSIONS: Forty-three percent of the total study population was diagnosed as having endoscopic Barrett's epithelium. During the follow-up period, 12.6% of the cases with Barrett's epithelium exhibited progression which was associated with aging and severe erosive esophagitis.

Requirement of atypical protein kinase clambda for insulin stimulation of glucose uptake but not for Akt activation in 3T3-L1 adipocytes.

Phosphoinositide (PI) 3-kinase contributes to a wide variety of biological actions, including insulin stimulation of glucose transport in adipocytes. Both Akt (protein kinase B), a serine-threonine kinase with a pleckstrin homology domain, and atypical isoforms of protein kinase C (PKCzeta and PKClambda) have been implicated as downstream effectors of PI 3-kinase. Endogenous or transfected PKClambda in 3T3-L1 adipocytes or CHO cells has now been shown to be activated by insulin in a manner sensitive to inhibitors of PI 3-kinase (wortmannin and a dominant negative mutant of PI 3-kinase). Overexpression of kinase-deficient mutants of PKClambda (lambdaKD or lambdaDeltaNKD), achieved with the use of adenovirus-mediated gene transfer, resulted in inhibition of insulin activation of PKClambda, indicating that these mutants exert dominant negative effects. Insulin-stimulated glucose uptake and translocation of the glucose transporter GLUT4 to the plasma membrane, but not growth hormone- or hyperosmolarity-induced glucose uptake, were inhibited by lambdaKD or lambdaDeltaNKD in a dose-dependent manner. The maximal inhibition of insulin-induced glucose uptake achieved by the dominant negative mutants of PKClambda was approximately 50 to 60%. These mutants did not inhibit insulin-induced activation of Akt. A PKClambda mutant that lacks the pseudosubstrate domain (lambdaDeltaPD) exhibited markedly increased kinase activity relative to that of the wild-type enzyme, and expression of lambdaDeltaPD in quiescent 3T3-L1 adipocytes resulted in the stimulation of glucose uptake and translocation of GLUT4 but not in the activation of Akt. Furthermore, overexpression of an Akt mutant in which the phosphorylation sites targeted by growth factors are replaced by alanine resulted in inhibition of insulin-induced activation of Akt but not of PKClambda. These results suggest that insulin-elicited signals that pass through PI 3-kinase subsequently diverge into at least two independent pathways, an Akt pathway and a PKClambda pathway, and that the latter pathway contributes, at least in part, to insulin stimulation of glucose uptake in 3T3-L1 adipocytes.

Low serum creatinine is a type 2 diabetes risk factor in men and women: The Yuport Health Checkup Center cohort study.

AIM: Type 2 diabetes (T2D) is a risk factor for muscle loss and subsequent frailty. The reverse association, however, may also happen. This study examined whether serum creatinine level, an indicator of muscle mass, predicted diabetes development. In addition, a role for body mass index (BMI) as an effect modifier of creatinine levels was evaluated. METHODS: This cohort study included 9667 subjects without diabetes or hypertension and with normal creatinine levels at baseline. Multiple-adjusted hazard ratios (HRs) for associations between baseline creatinine and diabetes development were estimated using the Cox proportional-hazards model. Stratified analyses based on BMI were also performed. RESULTS: During the follow-up period (mean: 5.6 years), 287 (5.5%) men and 115 (2.3%) women developed T2D. HR in men with serum creatinine≤0.7mg/dL compared with 0.9-1.2mg/dL was 1.40 (95% CI: 1.05-1.87) after adjusting for age, BMI, blood pressure and fasting plasma glucose at baseline, whereas the adjusted HR in women with creatinine≤0.5mg/dL compared with 0.7-1.1mg/dL was 1.69 (95% CI: 1.04-2.76). In a subgroup analysis stratified by BMI, interactions between BMI and baseline creatinine levels for T2D were statistically significant in women with the lowest creatinine levels (P=0.08 for interaction). CONCLUSION: Low serum creatinine levels, a surrogate marker of muscle mass, predict T2D development in both genders, even after excluding the effect of diabetic and prediabetic glomerular hyperfiltration. BMI modified the association between creatinine and diabetes development in women.

Inhibitory effects of alkyl gallate and its derivatives on fatty acid desaturation.

Alkyl gallate, which is known as an antioxidant, intensively inhibited delta 5 and delta 6 desaturation in both rat liver microsomes and an arachidonic acid-producing fungus Mortierella alpina 1S-4. The rat liver microsomal delta 5 and delta 6 desaturases were inhibited by gallic acid esterified with alcohols with various numbers of carbons, suggesting that the necessary structure in an esterified alcohol for the inhibition is not so strict. Among the three hydroxy groups in gallic acid, the m-hydroxy group was shown to be the necessary structure. Kinetic analyses revealed that propyl gallate is a noncompetitive inhibitor of delta 5 desaturase (Ki = 2.6.10(-5)M) and delta 6 desaturase (Ki = 1.7.10(-4) M). These data indicate that alkyl gallate is a new type of desaturase inhibitor and different from known natural inhibitors, i.e., sesamin and curcumin.

Internal DNA deletion within the BCL-6 gene on untranslocated chromosome in non-Hodgkin's lymphoma with 3q27 abnormality.

Chromosomal translocations involving the band 3q27 are recognized as common specific cytogenetic abnormalities in B cell non-Hodgkin's lymphoma (NHL), and the BCL-6 gene, identified on 3q27 was shown to be disrupted by these translocations. Previously, we have reported biallelic BCL-6 rearrangements occurring in some patients with B cell NHL. In the present study, we describe a NHL patient with t(3;22)(q27;q11) translocation. In this patient, biallelic BCL-6 abnormalities were indicated by Southern blot analysis. Further studies revealed that one of the two independent abnormalities was a juxtaposition to the immunoglobulin (Ig) lambda gene associated with chromosomal translocation, whereas the other was an internal DNA deletion of 1.5 kb area on untranslocated chromosome 3. Deletion junctions were located within the first exon and the 5' region of the first intron. The result provides the evidence that, besides chromosomal translocation, submicroscopic local DNA recombination can cause structural alteration of the BCL-6 gene.

Late persistent expressions of ICAM-1 and VCAM-1 on myocardial tissue in children with lymphocytic myocarditis.

BACKGROUND: Both intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) have been implicated in cardiac allograft rejection. However, there is little information about the relationship between the expression of these adhesion molecules and myocarditis in children. METHODS AND RESULTS: Immunoreactivities of ICAM-1 and VCAM-1 were examined by enzyme immunoassay in 31 biopsy specimens obtained from 11 pediatric patients with biopsy-proven myocarditis or cardiomyopathy. Five of the 11 patients had clear evidence of acute myocarditis. The other 6 had ECG abnormalities identified by mass screening for heart disease, and subsequently had been histologically diagnosed as having non-specific cardiomyopathy. The period between onset of myocarditis or identification of ECG abnormality and immunohistochemical studies was 23 to 60 days and 8 months to 3 years, respectively. Expression of ICAM-1 and VCAM-1 was assessed by counting ICAM-1 and VCAM-1 positive vessels and dividing by the total number of vessels. ICAM-1 was significantly present on 81% (P < 0.01) of myocardial tissue samples in the 5 patients with healing-stage acute myocarditis, and on 45% (P < 0.05) in the remaining 6 patients with non-specific cardiomyopathy, compared with 24% in control specimens obtained from right ventricular muscle resected at surgery for tetralogy of Fallot. VCAM-1 was also present on 50% (P < 0.05) of the samples from the 5 patients with acute myocarditis, but was not present in those with non-specific cardiomyopathy. CONCLUSION: This persistent expression of ICAM-1 suggests that myocardial cell damage may persist immunologically for a long period in myocarditis. In addition, immunostaining for these adhesion molecules may be diagnostic value in clinically silent lymphocytic myocarditis and chronic cardiomyopathy.

Molecular forms of atrial natriuretic peptide in the atrium of patients with cardiovascular disease.

To determine the molecular forms of atrial natriuretic peptide (ANP) in patients with cardiovascular disease, we analyzed the ANP content in the right atrium using reverse phase high performance liquid chromatography and RIA. Tissue from 35 patients, ranging in age from 1 month to 64 yr, was studied. The right atrial ANP content was high in patients with congestive heart failure regardless of age. We found three ANP components (alpha-, beta-, and gamma ANP), as previously found in tissue obtained at autopsy. In patients younger than 20 yr old, the major component was gamma ANP. In older patients, the frequency and content of the two low mol wt forms, especially beta ANP, were increased. The differences in the content and molecular forms of ANP in atrial tissue reflect the pathological state in patients with cardiovascular diseases, as well as the patient's age.

Subchronic methamphetamine treatment enhances methamphetamine- or cocaine-induced dopamine efflux in vivo.

Intracerebral dialysis was used to study the mechanism underlying behavioral sensitization. Rats were divided into two groups: a control group that received intraperitoneal injections of saline and an experimental group that was given methamphetamine (MAP) (4 mg/kg) once a day for 14 days. Seven days after the last injection, dopamine (DA) and its metabolites were measured in striatal dialysates obtained from awake freely moving rats. A challenge injection of MAP (4 mg/kg) caused a marked increase in the extracellular concentrations of DA, and the extent of the increase was significantly greater in MAP-pretreated rats than in the saline-pretreated controls. A challenge injection of cocaine (20 mg/kg) also caused a significantly greater increase in extracellular DA levels in MAP-pretreated rats than in saline-pretreated rats. These results suggest that an enhancement in striatal DA efflux may play an important role in MAP-induced behavioral sensitization and cross-sensitization to cocaine.

Structure-activity relationship of alpha-galactosylceramides against B16-bearing mice.

Agelasphin-9b, (2S,3S,4R)-1-O-(alpha-D-galactopyranosyl)-16-methyl-2- [N-((R)-2- hydroxytetracosanoyl)-amino]- 1,3,4-heptadecanetriol, is a potent antitumor agent isolated from the marine sponge Agelas mauritianus. Various analogues of agelasphin-9b (a lead compound) were synthesized, and the relationship between their structures and biological activities was examined using several assay systems. From the results, KRN7000, (2S,3S,4R)-1-O-(alpha-D- galactopyranosyl)-2-(N-hexacosanoylamino)-1,3,4-octadecanetriol , was selected as a candidate for clinical application.

Apoptosis as a possible cause of wall thinning in end-stage hypertrophic cardiomyopathy.

A 15-year-old boy with hypertrophic cardiomyopathy died of congestive heart failure with progressive left ventricular wall thinning with poor systolic function. Microscopic examination revealed patchy fibrosis in the ventricular myocardium with wall thinning, and immunohistochemical evaluation of apoptosis showed apoptotic cells and bodies in the destroyed myocytes along the border between the fibrotic area and myofibril.

Transcriptional regulation of adrenomedullin in rat vascular smooth muscle cells.

Adrenomedullin (AM), a potent vasorelaxant peptide recently identified in extracts of pheochromocytoma, is actively secreted from vascular smooth muscle cells (VSMC). AM production in rat VSMC is potently stimulated by proinflammatory cytokines including TNF alpha, and glucocorticoids and many vasoactive substances have also been shown to alter AM synthesis. To study AM promoter function, we cloned the 5'-flanking region of the rat AM gene, and AM promoter function was assessed in rat VSMC stably transfected with a construct containing the cloned fragment. This 1.4 kb AM promoter region includes a number of putative transcriptional control elements. The promoter activity in VSMC was stimulated by TNF alpha and dexamethasone, and was suppressed by 8-bromo-cAMP and forskolin. These data indicate that transcriptional regulation is an important mechanism for AM production in VSMC.

Clinical features of aortic arch anomaly with malalignment ventricular septal defect.

The clinical features and outcome after various surgical procedures on 9 patients with coarctation or interruption of the aortic arch and malalignment ventricular septal defect (group 1) were compared with those of 9 patients with the arch anomaly without malalignment ventricular septal defect (group 2). Cardiomegaly and metabolic acidosis were prominent in group 1. Five of the 9 patients in group 1 died in the immediate postoperative period (56% mortality), but no operative deaths occurred among 8 patients in group 2 (p less than 0.01). The ratio of left ventricular outflow tract to ascending aortic diameter was 0.59 +/- 0.09 in group 1 and 1.03 +/- 0.11 in group 2 (p less than 0.01). Three of 4 patients with a ratio of less than 0.6 died, but no operative deaths occurred among the 6 patients who had a palliative operation and in whom the ratio was more than 0.6. These data suggest that left ventricular outflow tract obstruction is critical when the ratio of left ventricular outflow tract to ascending aortic diameter is 0.6 or less. The presence of severe left ventricular outflow tract obstruction necessitates modification of the present surgical strategy.

Subchronic cocaine treatment enhances cocaine-induced dopamine efflux, studied by in vivo intracerebral dialysis.

Repeated administration of cocaine in animals results in behavioral sensitization. In order to investigate the neurochemical mechanism underlying such behavioral sensitization, we designed the following two experiments. In both experiments, rats were pretreated with cocaine (20 mg/kg i.p.) or saline, once daily for 14 consecutive days. Exp. 1: 7 days after withdrawal from the drug, the stereotyped behavioral response to a challenge of cocaine (20 mg/kg i.p.) was measured. Exp. 2: 7 days after withdrawal from the drug, we measured extracellular dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) after the challenge administration of cocaine using an in vivo intracerebral dialysis technique. The rats pretreated with cocaine (20 mg/kg i.p.) exhibited behavioral augmentation in response to a challenge of cocaine. The challenge administration of cocaine caused an increase in DA and a decrease in DOPAC. The DA level in the striatal perfusates of the cocaine-pretreated rats was significantly greater than that in the saline-pretreated rats. These results suggest that the increased extracellular DA concentration in the striatum plays an important role in the cocaine-induced behavioral sensitization.

Enhanced extracellular dopamine level may be the fundamental neuropharmacological basis of cross-behavioral sensitization between methamphetamine and cocaine--an in vivo dialysis study in freely moving rats.

Intracerebral dialysis was used to study the mechanism underlying cross-behavioral sensitization between methamphetamine (MAP) and cocaine. The challenge injection of cocaine caused a significantly greater increase in striatal perfusate dopamine (DA) levels in MAP-pretreated rats than in saline-pretreated rats. Similarly, the challenge injection of MAP caused a significantly greater increase in extracellular DA levels in cocaine-pretreated rats than in control rats. These results suggest that an enhancement in striatal DA efflux may play an important role in cross-behavioral sensitization between MAP and cocaine.