Overtime Work and Prevalence of Diabetes in Japanese Employees: Japan Epidemiology Collaboration on Occupational Health Study

Objective

Epidemiologic evidence on long working hour and diabetes has been conflicting. We examined the association between overtime work and prevalence of diabetes among Japanese workers.

Methods

The subjects were 40,861 employees (35,170 men and 5,691 women), aged 16 to 83 years, of 4 companies in Japan. Hours of overtime were assessed using self-reported questionnaires. Diabetes was defined as a fasting plasma glucose ≥126 mg/dl (7.0 mmol/l), hemoglobin A1c ≥6.5% (48 mmol/mol), or current use of anti-diabetic drug. Multiple logistic regression analysis was used to calculate odds ratio of diabetes for each category of overtime.

Results

After adjustment for age, sex, company, smoking, and BMI, there was a suggestion of U-shaped relationship between overtime work and prevalence of diabetes (P for quadratic trend = 0.07). Compared with those who worked <45 hours of overtime per month, the adjusted odds ratios (95% confidence interval) of diabetes were 0.86 (0.77–0.94), 0.69 (0.53–0.89), and 1.03 (0.72–1.46) for those who worked 45–79, 80–99, and ≥100 hours of overtime per month, respectively. In one company (n = 33,807), where other potential confounders including shift work, job position, type of department, alcohol consumption, sleep duration, leisure time physical activity, and family history of diabetes was additionally adjusted for, similar result was obtained (P for quadratic trend = 0.05).

Conclusions

Long hours of overtime work may not be associated with increased prevalence of diabetes among Japanese workers.     

Gene Silencing Mediated by Endogenous MicroRNAs under Heat Stress Conditions in Mammalian Cells

Heat shock, sudden change in temperature, triggers various responses in cells for protecting the cells from such a severe circumstance. Here we investigated gene silencing mediated by endogenous microRNAs (miRNAs) in mammalian cells exposed to a mild hyperthermia, by means of miRNA activity assay using a luciferase reporter gene as well as miRNA expression analysis using a DNA microarray. Our findings indicated that the gene silencing activities involving miRNAs were enhanced without increasing in their expression levels under heat-stress conditions. Additionally, the gene silencing activity appeared to be independent of the cytoprotective action involving heat shock proteins that are immediately activated in heat-shocked cells and that function as molecular chaperons for restoring heat-denatured proteins to normal proteins. Our current findings suggested the possibility that gene silencing involving endogenous miRNAs might play a subsidiary role in heat-shocked cells for an aggressive inhibition of the expression of heat-denatured proteins.     

Increase in Homeostasis Model Assessment of Insulin Resistance (HOMA-IR) Had a Strong Impact on the Development of Type 2 Diabetes in Japanese Individuals with Impaired Insulin Secretion: The Saku Study

Our aim was to assess the impact of increase in homeostasis model assessment of insulin resistance (HOMA-IR) on the development of type 2 diabetes in Japanese individuals with impaired insulin secretion (IIS). This study included 2,209 participants aged 30–69 without diabetes at baseline who underwent comprehensive medical check-ups between April 2006 and March 2007 at Saku Central Hospital. Participants were classified into eight groups according to the combination of baseline IIS status (non-IIS and IIS) and category of HOMA-IR change between the baseline and follow-up examinations (decrease, no change/small increase, moderate increase, and large increase). Type 2 diabetes was determined from fasting and 2 h post-load plasma glucose concentrations at the follow-up examination between April 2009 and March 2011. At baseline, 669 individuals (30.3%) were classified as having IIS. At follow-up, 74 individuals developed type 2 diabetes. After adjusting for confounding factors including baseline HOMA-IR values, the multivariable-adjusted odds ratios (95% confidence intervals) for type 2 diabetes in the non-IIS with a decrease (mean change in HOMA-IR: −0.47), non-IIS with a moderate increase (mean change in HOMA-IR: 0.28), non-IIS with a large increase (mean change in HOMA-IR: 0.83), IIS with a decrease (mean change in HOMA-IR: −0.36), IIS with no change/small increase (mean change in HOMA-IR: 0.08), IIS with a moderate increase (mean change in HOMA-IR: 0.27), and IIS with a large increase (mean change in HOMA-IR: 0.73) groups, relative to the non-IIS with no change/small increase (mean change in HOMA-IR: 0.08) group were 0.23 (0.04, 1.11), 1.22 (0.26, 5.72), 2.01 (0.70, 6.46), 1.37 (0.32, 4.28), 3.60 (0.83, 15.57), 5.24 (1.34, 20.52), and 7.01 (1.75, 24.18), respectively. Moderate and large increases in HOMA-IR had a strong impact on the development of type 2 diabetes among individuals with IIS in this Japanese population.     

Introducing Micrometer-Sized Artificial Objects into Live Cells: A Method for Cell–Giant Unilamellar Vesicle Electrofusion

Here, we report a method for introducing large objects of up to a micrometer in diameter into cultured mammalian cells by electrofusion of giant unilamellar vesicles. We prepared GUVs containing various artificial objects using a water-in-oil (w/o) emulsion centrifugation method. GUVs and dispersed HeLa cells were exposed to an alternating current (AC) field to induce a linear cell–GUV alignment, and then a direct current (DC) pulse was applied to facilitate transient electrofusion. With uniformly sized fluorescent beads as size indexes, we successfully and efficiently introduced beads of 1 µm in diameter into living cells along with a plasmid mammalian expression vector. Our electrofusion did not affect cell viability. After the electrofusion, cells proliferated normally until confluence was reached, and the introduced fluorescent beads were inherited during cell division. Analysis by both confocal microscopy and flow cytometry supported these findings. As an alternative approach, we also introduced a designed nanostructure (DNA origami) into live cells. The results we report here represent a milestone for designing artificial symbiosis of functionally active objects (such as micro-machines) in living cells. Moreover, our technique can be used for drug delivery, tissue engineering, and cell manipulation.     

Crystal structures of the S6K1 kinase domain in complexes with inhibitors

Ribosomal protein S6 kinase 1 (S6K1) is a serine/threonine protein kinase that plays an important role in the PIK3/mTOR signaling pathway, and is implicated in diseases including diabetes, obesity, and cancer. The crystal structures of the S6K1 kinase domain in complexes with staurosporine and the S6K1-specific inhibitor PF-4708671 have been reported. In the present study, five compounds (F108, F109, F176, F177, and F179) were newly identified by in silico screening of a chemical library and kinase assay. The crystal structures of the five inhibitors in complexes with the S6K1 kinase domain were determined at resolutions between 1.85 and 2.10 Å. All of the inhibitors bound to the ATP binding site, lying along the P-loop, while the activation loop stayed in the inactive form. Compound F179, with a carbonyl group in the middle of the molecule, altered the αC helix conformation by interacting with the invariant Lys123. Compounds F176 and F177 bound slightly distant from the hinge region, and their sulfoamide groups formed polar interactions with the protein. The structural features required for the specific binding of inhibitors are discussed.     

Ribosomes in a Stacked Array: ELUCIDATION OF THE STEP IN TRANSLATION ELONGATION AT WHICH THEY ARE STALLED DURING S-ADENOSYL-l-METHIONINE-INDUCED TRANSLATION ARREST OF CGS1 mRNA*

Expression of CGS1, which codes for an enzyme of methionine biosynthesis, is feedback-regulated by mRNA degradation in response to S-adenosyl-l-methionine (AdoMet). In vitro studies revealed that AdoMet induces translation arrest at Ser-94, upon which several ribosomes stack behind the arrested one, and mRNA degradation occurs at multiple sites that presumably correspond to individual ribosomes in a stacked array. Despite the significant contribution of stacked ribosomes to inducing mRNA degradation, little is known about the ribosomes in the stacked array. Here, we assigned the peptidyl-tRNA species of the stacked second and third ribosomes to their respective codons and showed that they are arranged at nine-codon intervals behind the Ser-94 codon, indicating tight stacking. Puromycin reacts with peptidyl-tRNA in the P-site, releasing the nascent peptide as peptidyl-puromycin. This reaction is used to monitor the activity of the peptidyltransferase center (PTC) in arrested ribosomes. Puromycin reaction of peptidyl-tRNA on the AdoMet-arrested ribosome, which is stalled at the pre-translocation step, was slow. This limited reactivity can be attributed to the peptidyl-tRNA occupying the A-site at this step rather than to suppression of PTC activity. In contrast, puromycin reactions of peptidyl-tRNA with the stacked second and third ribosomes were slow but were not as slow as pre-translocation step ribosomes. We propose that the anticodon end of peptidyl-tRNA resides in the A-site of the stacked ribosomes and that the stacked ribosomes are stalled at an early step of translocation, possibly at the P/E hybrid state.     

DNAX-activating Protein 10 (DAP10) Membrane Adaptor Associates with Receptor for Advanced Glycation End Products (RAGE) and Modulates the RAGE-triggered Signaling Pathway in Human Keratinocytes

The receptor for advanced glycation end products (RAGE) is involved in the pathogenesis of many inflammatory, degenerative, and hyperproliferative diseases, including cancer. Previously, we revealed mechanisms of downstream signaling from ligand-activated RAGE, which recruits TIRAP/MyD88. Here, we showed that DNAX-activating protein 10 (DAP10), a transmembrane adaptor protein, also binds to RAGE. By artificial oligomerization of RAGE alone or RAGE-DAP10, we found that RAGE-DAP10 heterodimer formation resulted in a marked enhancement of Akt activation, whereas homomultimeric interaction of RAGE led to activation of caspase 8. Normal human epidermal keratinocytes exposed to S100A8/A9, a ligand for RAGE, at a nanomolar concentration mimicked the pro-survival response of RAGE-DAP10 interaction, although at a micromolar concentration, the cells mimicked the pro-apoptotic response of RAGE-RAGE. In transformed epithelial cell lines, A431 and HaCaT, in which endogenous DAP10 was overexpressed, and S100A8/A9, even at a micromolar concentration, led to cell growth and survival due to RAGE-DAP10 interaction. Functional blocking of DAP10 in the cell lines abrogated the Akt phosphorylation from S100A8/A9-activated RAGE, eventually leading to an increase in apoptosis. Finally, S100A8/A9, RAGE, and DAP10 were overexpressed in the psoriatic epidermis. Our findings indicate that the functional interaction between RAGE and DAP10 coordinately regulates S100A8/A9-mediated survival and/or apoptotic response of keratinocytes.     

Torque Generation of Enterococcus hirae V-ATPase

V-ATPase (V_oV₁) converts the chemical free energy of ATP into an ion-motive force across the cell membrane via mechanical rotation. This energy conversion requires proper interactions between the rotor and stator in V_oV₁ for tight coupling among chemical reaction, torque generation, and ion transport. We developed an Escherichia coli expression system for Enterococcus hirae V_oV₁ (EhV_oV₁) and established a single-molecule rotation assay to measure the torque generated. Recombinant and native EhV_oV₁ exhibited almost identical dependence of ATP hydrolysis activity on sodium ion and ATP concentrations, indicating their functional equivalence. In a single-molecule rotation assay with a low load probe at high ATP concentration, EhV_oV₁ only showed the “clear” state without apparent backward steps, whereas EhV₁ showed two states, “clear” and “unclear.” Furthermore, EhV_oV₁ showed slower rotation than EhV₁ without the three distinct pauses separated by 120° that were observed in EhV₁. When using a large probe, EhV_oV₁ showed faster rotation than EhV₁, and the torque of EhV_oV₁ estimated from the continuous rotation was nearly double that of EhV₁. On the other hand, stepping torque of EhV₁ in the clear state was comparable with that of EhV_oV₁. These results indicate that rotor-stator interactions of the V_o moiety and/or sodium ion transport limit the rotation driven by the V₁ moiety, and the rotor-stator interactions in EhV_oV₁ are stabilized by two peripheral stalks to generate a larger torque than that of isolated EhV₁. However, the torque value was substantially lower than that of other rotary ATPases, implying the low energy conversion efficiency of EhV_oV₁.