Effect of Metformin vs. Placebo in Combination with Insulin Analogues on Bone Markers P1NP and CTX in Patients with Type 2 Diabetes Mellitus.

Preclinical studies have shown a potential osteoanabolic effect of metformin but human studies of how metformin affects bone turnover are few. A post hoc sub-study analysis of an 18-month multicenter, placebo-controlled, double-blinded trial in type 2 diabetes mellitus (T2DM), randomizing participants to metformin versus placebo both in combination with different insulin analogue regimens (Metformin + Insulin vs. Placebo + Insulin). Patients were not treatment naive at baseline, 83% had received metformin, 69% had received insulin, 57.5% had received the combination of metformin and insulin before entering the study. Bone formation and resorption were assessed by measuring, N-terminal propeptide of type I procollagen (P1NP) and C-terminal telopeptide of type I collagen (CTX) at baseline and end of study. The influence of gender, age, smoking, body mass index (BMI), T2DM duration, glycosylated hemoglobin A1c (HbA1c), c-reactive protein (CRP) and insulin dosage was also included in the analyses. The levels of bone formation marker P1NP and bone resorption marker CTX increased significantly in both groups during the trial. P1NP increased less in the Metformin + Insulin compared to the placebo + insulin group (p = 0.001) (between group difference change), while the increases in CTX levels (p = 0.11) were not different. CRP was inversely associated (p = 0.012) and insulin dosage (p = 0.011) was positively related with change in P1NP levels. BMI (p = 0.002) and HbA1C (p = 0.037) were inversely associated with change in CTX levels. During 18 months of treatment with metformin or placebo, both in combination with insulin, bone turnover increased in both groups. But the pattern was different as the bone formation marker (P1NP) increased less during Metformin + Insulin treatment, while change in bone resorption (CTX) was not significantly different between the two groups.

Occupational noise exposure, psychosocial working conditions and the risk of tinnitus.

PURPOSE: The purpose of this study was to evaluate the influence of occupational noise (current and cumulative doses) and psychosocial work factors (psychological demands and decision latitude) on tinnitus occurrence among workers, using objective and non-self-reported exposure measures to prevent reporting bias. METHODS: In a cross-sectional study, we analyzed data from a Danish survey from 2009 to 2010 that included 534 workers from children day care units and 10 manufacturing trades. Associations between risk factors (current noise exposure, cumulative noise exposure and psychosocial working conditions) and tinnitus were analyzed with logistic regression. RESULTS: We found no statistically significant associations between either current [OR 0.95 (95% CI 0.89; 1.01)] or cumulative [OR 0.93 (95% CI 0.81; 1.06)] occupational noise exposure and tinnitus. Likewise, results for psychosocial working conditions showed no statistically significant association between work place decision latitude [OR 1.06 (95% CI 0.94; 1.13)] or psychological demands [OR 1.07 (95% CI 0.90; 1.26)] and tinnitus. CONCLUSIONS: Our results suggest that current Danish occupational noise levels (in combination with relevant noise protection) are not associated with tinnitus. Also, results indicated that the psychosocial working conditions we observed in this cohort of mainly industrial workers were not associated with tinnitus. Therefore, psychosocial working conditions comparable to those observed in this study are probably not relevant to take into account in the evaluation of workers presenting with tinnitus.

Atherogenic risk factors and hearing thresholds.

The objective of this study was to evaluate the influence of atherogenic risk factors on hearing thresholds. In a cross-sectional study we analyzed data from a Danish survey in 2009-2010 on physical and psychological working conditions. The study included 576 white- and blue-collar workers from children's day care units, financial services and 10 manufacturing trades. Associations between atherogenic risk factors (blood lipids, glycosylated hemoglobin, smoking habits, body mass index (BMI), and ambulatory blood pressure) and hearing thresholds were analyzed using multiple linear regression models. Adjusted results suggested associations between smoking, high BMI and triglyceride level and low high-density lipoprotein level and increased low-frequency hearing thresholds (average of pure-tone hearing thresholds at 0.25, 0.5 and 1 kHz). Furthermore, an increasing load of atherogenic risk factors seemed associated with increased low-frequency hearing thresholds, but only at a borderline level of statistical significance. Associations were generally strongest with hearing levels of the worst hearing ear. We found no statistically significant associations between atherogenic risk factors and high-frequency hearing thresholds (average of pure-tone hearing thresholds at 4, 6 and 8 kHz).

Recent and long-term occupational noise exposure and salivary cortisol level.

Environmental and occupational noise exposure have been related to increased risk of cardiovascular disease, hypothetically mediated by stress-activation of the hypothalamic-pituitary-adrenal (HPA) axis. The objective of this study was to investigate the relation between recent and long-term occupational noise exposure and cortisol level measured off work to assess a possible sustained HPA-axis effect. We included 501 industrial, finance, and service workers who were followed for 24h during work, leisure, and sleep. Ambient occupational noise exposure levels were recorded every 5s by personal dosimeters and we calculated the full-shift LAEq value and estimated duration and cumulative exposure based on their work histories since 1980. For 332 workers who kept a log-book on the use of hearing protection devices (HPD), we subtracted 10 dB from every noise recording obtained during HPD use and estimated the noise level at the ear. Salivary cortisol concentration was measured at 20.00 h, the following day at awakening, and 30 min after awakening on average 5, 14 and 14.5h after finishing work. The mean ambient noise exposure level was 79.9 dB(A) [range: 55.0-98.9] and the mean estimated level at the ear 77.7 dB(A) [range: 55.0-94.2]. In linear and mixed regression models that adjusted for age, sex, current smoking, heavy alcohol consumption, personal income, BMI, leisure-time noise exposure level, time since occupational noise exposure ceased, awakening time, and time of saliva sampling, we observed no statistically significant exposure response relation between recent, or long-term ambient occupational noise exposure level and any cortisol parameter off work. This was neither the case for recent noise level at the ear. To conclude, neither recent nor long-term occupational noise exposure levels were associated with increased cortisol level off work. Thus, our results do not indicate that a sustained activation of the HPA axis, as measured by cortisol, is involved in the causal pathway between occupational noise exposure and cardiovascular disease.

Effects of pre- and postnatal exposure to the UV-filter octyl methoxycinnamate (OMC) on the reproductive, auditory and neurological development of rat offspring.

Octyl Methoxycinnamate (OMC) is a frequently used UV-filter in sunscreens and other cosmetics. The aim of the present study was to address the potential endocrine disrupting properties of OMC, and to investigate how OMC induced changes in thyroid hormone levels would be related to the neurological development of treated offspring. Groups of 14-18 pregnant Wistar rats were dosed with 0, 500, 750 or 1000 mg OMC/kg bw/day during gestation and lactation. Serum thyroxine (T(4)), testosterone, estradiol and progesterone levels were measured in dams and offspring. Anogenital distance, nipple retention, postnatal growth and timing of sexual maturation were assessed. On postnatal day 16, gene expression in prostate and testes, and weight and histopathology of the thyroid gland, liver, adrenals, prostate, testes, epididymis and ovaries were measured. After weaning, offspring were evaluated in a battery of behavioral and neurophysiological tests, including tests of activity, startle response, cognitive and auditory function. In adult animals, reproductive organ weights and semen quality were investigated. Thyroxine (T(4)) levels showed a very marked decrease during the dosing period in all dosed dams, but were less severely affected in the offspring. On postnatal day 16, high dose male offspring showed reduced relative prostate and testis weights, and a dose-dependent decrease in testosterone levels. In OMC exposed female offspring, motor activity levels were decreased, while low and high dose males showed improved spatial learning abilities. The observed behavioral changes were probably not mediated solely by early T(4) deficiencies, as the observed effects differed from those seen in other studies of developmental hypothyroxinemia. At eight months of age, sperm counts were reduced in all three OMC-dosed groups, and prostate weights were reduced in the highest dose group. Taken together, these results indicate that perinatal OMC-exposure can affect both the reproductive and neurological development of rat offspring, which may be a cause of concern, as humans are systematically exposed to the compound through usage of sunscreens and other cosmetics.

Impact of metformin versus repaglinide on non-glycaemic cardiovascular risk markers related to inflammation and endothelial dysfunction in non-obese patients with type 2 diabetes.

OBJECTIVE: In patients with type 2 diabetes mellitus (T2DM), biomarkers reflecting inflammation and endothelial dysfunction have been linked to cardiovascular disease (CVD biomarkers) and metabolic regulation. In T2DM patients, metformin and insulin secretagogues have demonstrated equal anti-hyperglycaemic potency. Here, we report the effect of metformin versus an insulin secretagogue, repaglinide, on CVD biomarkers in non-obese T2DM patients. DESIGN AND METHODS: Single-centre, double-masked, double-dummy, crossover study during 2x4 months involving 96 non-obese (body mass index< or =27 kg/m(2)) insulin-naïve T2DM patients. At enrolment, previous oral hypoglycaemic agents were stopped and the patients entered a 1-month run-in on diet-only treatment. Hereafter, patients were randomized to either 2 mg repaglinide thrice daily followed by 1 g metformin twice daily or vice versa each during 4 months with a 1-month washout between interventions. RESULTS: Levels of tumour necrosis factor-alpha, plasminogen activator inhibitor-1 antigen, tissue-type plasminogen activator antigen, von Willebrand factor, soluble intercellular adhesion molecule-1 and soluble E-selectin were significantly lower during metformin versus repaglinide treatments. In contrast, Amadori albumin and heart rate were higher during metformin versus repaglinide. Levels of interleukin-6, fibrinogen, soluble vascular cell adhesion molecule-1, asymmetric dimethylarginine and advanced glycation end products as well as glycaemic levels (previously reported) and 24-h blood pressure were similar between treatments. Adjustment for known macrovascular disease did not affect the between-treatment effects. CONCLUSIONS: In non-obese T2DM patients, metformin was more effective in reducing selected biomarkers reflecting inflammation and endothelial dysfunction compared with repaglinide despite similar glycaemic levels between treatments.

The risk of tinnitus following occupational noise exposure in workers with hearing loss or normal hearing.

The purpose was to investigate the relationship between noise exposure and tinnitus among workers with normal hearing and hearing loss, respectively. We conducted a cross-sectional survey of 752 workers employed at 91 workplaces, that were investigated by means of full work-shift noise levels, questionnaire data, and bilateral pure-tone audiometry. Tinnitus was not associated with the present noise level, the duration of noise exposure, or the cumulative noise exposure if participants had normal hearing. As expected, such trends were demonstrated if participants had a hearing handicap. Based on these data, we will be cautious in ascribing tinnitus to noise exposure in our patients' workplaces if they have a normal audiogram. Furthermore our data indicates no risk of noise-induced tinnitus at exposure levels where no hearing loss would be expected, e.g. as usually encountered in non-industrial workplaces.

Evidence of Oatp and Mdr1 in cryopreserved rat hepatocytes.

Transport proteins control uptake of drugs into the liver (e.g., organic anion transporting polypeptide (Oatp)) and excretion of drugs from the liver (e.g., multidrug resistance protein 1 (Mdr1)). In this study, cryopreserved rat hepatocytes were used to investigate the effect of different culture conditions (suspension, conventional culture and sandwich culture) on the uptake of [(3)H]-taurocholate+/-probenecid and the efflux of [(3)H]-vinblastine+/-ketoconazole; mRNA levels of Oatp1a1, Oatp1a4, Mdr1a and Mdr1b were determined using real-time reverse transcription polymerase chain reaction (RT-PCR) and protein expression of Mdr was assessed by immunocytochemistry. The uptake of [(3)H]-taurocholate was higher in cryopreserved rat hepatocytes maintained in suspension as compared to hepatocytes in culture. A significant time dependent decline in the uptake of [(3)H]-taurocholate was noticed from day 2 to day 4 in conventional and sandwich cultures. [(3)H]-taurocholate uptake was significantly reduced using the inhibitor probenecid. Oatp mRNA expression in hepatocytes in suspension was similar to that of liver, whereas much lower levels were detected in the cultures; this was in accordance with [(3)H]-taurocholate uptake results. Mdr1 activity was assessed by accumulation of the Mdr1 selective substrate, [(3)H]-vinblastine, in hepatocytes using ketoconazole as an inhibitor. The results showed Mdr1 activity in cryopreserved rat hepatocytes in conventional and sandwich cultures. A time dependent increase in Mdr1 activity was noticed from day 2 to day 4. Mdr1 activity was not found using hepatocytes in suspension. Mdr1 mRNA expression was high in cryopreserved hepatocytes from both culture systems. Immunocytochemistry showed the Mdr protein in membranes of hepatocytes in culture as well as in that of hepatocytes in liver sections. In conclusion, the present study showed that cryopreserved rat hepatocytes maintained canalicular transport activity (Mdr1) and basolateral transport activity. Hepatocytes in suspension had a higher uptake of taurocholate with a high Oatp (1a1 and 1a4) mRNA expression as compared to hepatocytes in culture. The presence of Mdr1 in both conventional and sandwich culture was confirmed at mRNA level, by protein expression as well as transport activity.

The dynamics of the LPS triggered inflammatory response of murine microglia under different culture and in vivo conditions.

Overall, the inflammatory potential of lipopolysaccharide (LPS) in vitro and in vivo was investigated using different omics technologies. We investigated the hippocampal response to intracerebroventricular (i.c.v) LPS in vivo, at both the transcriptional and protein level. Here, a time course analysis of interleukin-6 (IL-6) and monocyte chemotactic protein-1 (MCP-1) showed a sharp peak at 4 h and a return to baseline at 16 h. The expression of inflammatory mediators was not temporally correlated with expression of the microglia marker F4/80, which did not peak until 2 days after LPS injection. Of 480 inflammation-related genes present on a microarray, 29 transcripts were robustly up-regulated and 90% of them were also detected in LPS stimulated primary microglia (PM) cultures. Further in vitro to in vivo comparison showed that the counter regulation response observed in vivo was less evident in vitro, as transcript levels in PM decreased relatively little over 16 h. This apparent deficiency of homeostatic control of the innate immune response in cultures may also explain why a group of genes comprising tnf receptor associated factor-1, endothelin-1 and schlafen-1 were regulated strongly in vitro, but not in vivo. When the overall LPS-induced transcriptional response of PM was examined on a large Affymetrix chip, chemokines and cytokines constituted the most strongly regulated and largest groups. Interesting new microglia markers included interferon-induced protein with tetratricopeptide repeat (ifit), immune responsive gene-1 (irg-1) and thymidylate kinase family LPS-inducible member (tyki). The regulation of the former two was confirmed on the protein level in a proteomics study. Furthermore, conspicuous regulation of several gene clusters was identified, for instance that of genes pertaining to the extra-cellular matrix and enzymatic regulation thereof. Although most inflammatory genes induced in vitro were transferable to our in vivo model, the observed discrepancy for some genes potentially represents regulatory factors present in the central nervous system (CNS) but not in vitro.

Smoking and height as risk factors for prevalence and 5-year incidence of hearing loss. A questionnaire-based follow-up study of employees in Denmark aged 18-59 years exposed and unexposed to noise.

This paper investigated whether smoking and short stature in adulthood were independent risk factors for hearing loss. We reanalyzed data from the Danish Work Environment Cohort Study (an existing cohort study), on prevalence of self-reported hearing loss among 7,221 employees and on five-year incidence among 4,610 employees. We found that smoking predicted hearing loss incidence and prevalence. Smoking did not predict incidence at noise exposure during half or more of a worker's hours. Very short stature predicted prevalence in the total adult population only weakly, but strongly among employees born before 1951. These prospective findings indicate that smoking is an independent risk factor for incidence of hearing loss. Very short stature predicted prevalence of hearing loss only in a subpopulation.

CEP-11004, an inhibitor of the SAPK/JNK pathway, reduces TNF-alpha release from lipopolysaccharide-treated cells and mice.

CEP-11004, a mixed lineage kinase (MLK) inhibitor, was examined for its effects on tumor necrosis factor-alpha (TNF-alpha) production in human THP-1 monocytes, mouse BV-2 microglia, and C57Bl/6 mice. CEP-11004 inhibited TNF-alpha secretion up to 90% in THP-1 cells incubated with 3 mug/ml lipopolysaccharide, with an IC50 of 137+/-14 nM. CEP-11004 also inhibited TNF-alpha production in lipopolysaccharide-stimulated microglial cells, but did not inhibit the initial increase in TNF-alpha mRNA expression as measured by real-time polymerase chain reaction (PCR). The mitogen-activated protein kinases (MAPKs) phospho-c-jun N-terminal kinase (JNK), phospho-p38, and phospho-MAPK kinase 4 (MKK4) levels were increased in THP-1 cells following lipopolysaccharide treatment, and were reduced by CEP-11004 treatment. For in vivo studies, CEP-11004 was injected 2 h prior to lipopolysaccharide (20 mg/kg) administration. CEP-11004 significantly inhibited TNF-alpha production at doses of 1-10 mg/kg as measured by enzyme-linked immunosorbent assay (ELISA). These results suggest that MLK blockade may be useful in inhibiting pro-inflammatory cytokine production in a wide range of diseases.

Inhibition of microglial inflammation by the MLK inhibitor CEP-1347.

CEP-1347 is a potent inhibitor of the mixed lineage kinases (MLKs), a distinct family of mitogen-activated protein kinase kinase kinases (MAPKKK). It blocks the activation of the c-Jun/JNK apoptotic pathway in neurons exposed to various stressors and attenuates neurodegeneration in animal models of Parkinson's disease (PD). Microglial activation may involve kinase pathways controlled by MLKs and might contribute to the pathology of neurodegenerative diseases. Therefore, the possibility that CEP-1347 modulates the microglial inflammatory response [tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and monocyte chemotactic protein-1 (MCP-1)] was explored. Indeed, the MLK inhibitor CEP-1347 reduced cytokine production in primary cultures of human and murine microglia, and in monocyte/macrophage-derived cell lines, stimulated with various endotoxins or the plaque forming peptide Abeta1-40. Moreover, CEP-1347 inhibited brain TNF production induced by intracerebroventricular injection of lipopolysaccharide in mice. As expected from a MLK inhibitor, CEP-1347 acted upstream of p38 and c-Jun activation in microglia by dampening the activity of both pathways. These data imply MLKs as important, yet unrecognized, modulators of microglial inflammation, and demonstrate a novel anti-inflammatory potential of CEP-1347.