The Effect of Interactivity, Tailoring, and Use Intensity on the Effectiveness of an Internet-Based Smoking Cessation Intervention Over a 12-Month Period: Randomized Controlled Trial.

BACKGROUND: eHealth approaches show promising results for smoking cessation (SC). They can improve quit rates, but rigorous research is sparse regarding their effectiveness and the effects of their interactivity, tailoring, and use intensity. OBJECTIVE: We examined the effectiveness of Techniker Krankenkasse Smoking Cessation Coaching (TK-SCC), an internet-based, tailored, and interactive SC intervention. Our hypotheses were as follows: hypothesis 1, in the intervention group (IG; access to TK-SCC), a clinically relevant number of participants will be abstinent at the 12-month follow-up (T3); hypothesis 2, the number of abstinent participants will be significantly greater in the IG than the control group (CG) at T3; and hypothesis 3, in the IG, more intense use of TK-SCC will be positively associated with abstinence. METHODS: Individuals who smoke were randomized into the IG (563/1115, 50.49%) or CG (552/1115, 49.51%), which received a noninteractive, nontailored, and information-only web-based intervention. Data were collected before the intervention, at the postintervention time point (T1), at the 4-month follow-up (T2), and at T3. We tested hypothesis 1 through equivalence tests between the IG's success rate and success rates of comparable effective interventions reported in 2 current meta-analyses. For hypothesis 2, we conducted binary logistic regressions. For hypothesis 3, we assigned the IG participants to 1 of 4 user types and used binary logistic regressions with user types as the independent variable and smoking abstinence as the dependent variable. RESULTS: In the IG, 11.5% (65/563) and 11.9% (67/563) of participants were smoke free at T1 and T3, respectively. These values were statistically equivalent to the effects in the 2 meta-analyses, which reported 9% (z score=0.64, P=.74) and 10.9% (z score=-0.71, P=.24) success rates, respectively. In the CG, 6.2% (34/552) of the participants were smoke free at T1, which increased up to 8.2% (45/552) at T3. The difference between the IG and CG was statistically significant only at T1 (odds ratio [OR] 2.0, 99% CI 1.1 to 3.6; P=.002), whereas the effect was nonsignificant following α error corrections at T3 (OR 1.6, 99% CI 0.9 to 2.7; P=.02). In the IG, constant users of the program became smoke free significantly more often than rare users of the program (T1: OR 15.0, 99% CI 6.1 to 36.9; P<.001; T3: OR 6.5, 99% CI 2.8 to 15.5; P<.001). CONCLUSIONS: TK-SCC is effective for SC. However, its superiority compared with a minimal SC intervention could not be confirmed in the long term. Insufficient implementation of the techniques used and cotreatment bias could explain this outcome. Higher use intensity of TK-SCC was positively related to abstinence. Therefore, additional efforts to motivate users to adhere to intervention use as intended could improve the intervention's effectiveness. TRIAL REGISTRATION: German Clinical Trials Register DRKS00020249, Universal Trial Number U1111-1245-0273; https://drks.de/search/de/trial/DRKS00020249. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): RR2-10.1186/s13063-021-05470-8.

Differential modulation of ROS signals and other mitochondrial parameters by the antioxidants MitoQ, resveratrol and curcumin in human adipocytes.

Mitochondrial reactive oxygen species (ROS) have been demonstrated to play an important role as signaling and regulating molecules in human adipocytes. In order to evaluate the differential modulating roles of antioxidants, we treated human adipocytes differentiated from human bone marrow-derived mesenchymal stem cells with MitoQ, resveratrol and curcumin. The effects on ROS, viability, mitochondrial respiration and intracellular ATP levels were examined. MitoQ lowered both oxidizing and reducing ROS. Resveratrol decreased reducing and curcumin oxidizing radicals only. All three substances slightly decreased state III respiration immediately after addition. After 24 h of treatment, MitoQ inhibited both basal and uncoupled oxygen consumption, whereas curcumin and resveratrol had no effect. Intracellular ATP levels were not altered. This demonstrates that MitoQ, resveratrol and curcumin exert potent modulating effects on ROS signaling in human adipocyte with marginal effects on metabolic parameters.

Susceptibility to simvastatin-induced toxicity is partly determined by mitochondrial respiration and phosphorylation state of Akt.

Statins are widely used to prevent cardiovascular diseases. They are well-tolerated, with side-effects mainly seen in skeletal muscle. How these side-effects are caused is unknown. We compared isolated primary mouse skeletal muscle myocytes, C2C12 myotubes and liver HepG2 cells to detect differences that could uncover why statins are toxic in skeletal muscle but less so in the liver. 10µM simvastatin caused a decrease in mitochondrial respiration in the primary mouse myocytes and C2C12 myotubes, but had no effect in the HepG2 cells. Mitochondrial integrity is maintained by multiple signaling pathways. One of these pathways, Igf-1/Akt signaling, is also heavily implicated in causing statin-induced toxicity by upregulating atrogin-1. We found that phosphorylated Akt was reduced in C2C12 myotubes but not in HepG2 cells. HepG2 mitochondrial respiration became susceptible to simvastatin-treatment after Akt inhibition, and mitochondrial respiration was rescued in Igf-1-treated C2C12 myotubes. These results suggest that disruption of Igf-1/Akt signaling is a causative factor in simvastatin-induced mitochondrial dysfunction in C2C12 myotubes, whereas HepG2 cells are protected by maintaining Igf-1/Akt signaling. We conclude that phosphorylation of Akt is a key indicator of susceptibility to statin-induced toxicity. How statins can disrupt Igf-1/Akt signaling is unknown. Statins reduce geranylgeranylation of small GTPases, such as Rap1. Previous studies implicate Rap1 as a link between cAMP/Epac and Igf-1/Akt signaling. Transient transfection of constitutively active Rap1 into C2C12 myotubes led to a partial rescue of simvastatin-induced inhibition of mitochondrial respiration, providing a novel link between signaling and respiration.

Health Effects of a 12-Week Web-Based Lifestyle Intervention for Physically Inactive and Overweight or Obese Adults: Study Protocol of Two Randomized Controlled Clinical Trials.

Web-based lifestyle interventions have attracted considerable research interest. Available evidence on such interventions suggests health-promoting effects, but further research is needed. Therefore, this study aims to investigate short-, medium-, and long-term health effects of a web-based health program ("TK-HealthCoach", TK-HC) offered by a national statutory health insurance fund (Techniker Krankenkasse, TK). The study comprises two randomized controlled clinical trials to evaluate the health goals "Increasing Fitness" (Fclin) and "Losing and Maintaining Weight" (Wclin). A total of n = 186 physically inactive (Fclin) and n = 150 overweight or obese (Wclin) adults will be randomly assigned to a 12-week interactive (TK-HC) or non-interactive web-based health program using permuted block randomization with a 1:1 allocation ratio. Primary outcomes include cardiorespiratory fitness (Fclin) and body weight (Wclin). Secondary outcomes comprise musculoskeletal fitness (Fclin), physical activity and dietary behavior, anthropometry, blood pressure, blood levels, and vascular health (Fclin, Wclin). All outcomes will be measured before and after the 12-week intervention and after a 6- and 12-month follow-up. Additionally, usage behavior data on the health programs will be assessed. Linear mixed models (LMMs) will be used for statistical analysis. Findings of this study will expand the available evidence on web-based lifestyle interventions.

Effectiveness of an interactive web-based health program for adults: a study protocol for three concurrent controlled-randomized trials (EVA-TK-Coach).

BACKGROUND: A healthy lifestyle can help prevent diseases that impair quality of life and lead to premature death. The Techniker health insurance fund offers a comprehensive online health program to support users in achieving their health goals of Increasing Fitness, Losing and Maintaining Weight, or Smoking Cessation. METHODS: The aim of this study is to test the long-term effectiveness of the web-based TK-HealthCoach with regard to the primary outcomes of increased physical activity, sustainable weight reduction, and smoking abstinence. We are conducting three interconnected, randomized controlled trials (RCT), one for each health goal, within which participants are allocated to an intervention group (interactive online health program) or a control group (non-interactive online health program). The effects of the intervention groups compared to the control groups will be analyzed by multi-level models for change. Participants' data are captured via online questionnaires before the program starts (baseline t0), again when it ends (t1), and later at two follow-up surveys (t2 and t3); the latter 12 months after t1. We are documenting socio-demographic, health-related, and psychological variables as well as usage behavior data of the programs. According to our sample size calculation, we have to enroll 1114 participants in each Losing and Maintaining Weight and Increasing Fitness RCT and 339 participants in the Smoking Cessation RCT. Additionally, 15-20 participants in the interactive smoking-cessation program will be invited to qualitative telephone interviews with the aim to obtain detailed information concerning utilization, compliance, and satisfaction. The online RCTs' inclusion criteria are: adults of each gender regardless of whether they are insured with Techniker health insurance fund. Persons with impairments or pre-existing conditions require a medical assessment as to whether the program is suitable for them. Specific exclusion criteria apply to each program/RCT. DISCUSSION: We assume that study participants will improve their health behavior by using the offered online health programs and that each health goal's intervention group will reveal advantages regarding the outcome variables compared to the control groups. Study enrollment started on January 1, 2020. TRIAL REGISTRATION: German Clinical Trials Register, Universal Trial Number (UTN): U1111-1245-0273 . Registered on 11 December 2019.

Weak functional coupling of the melanocortin-1 receptor expressed in human adipocytes.

The melanocortin (MC) receptor type-1 (MC1-R) is the only one of the five MC receptor subtypes expressed in human adipose tissue explants, human mesenchymal stem cells (MSCs), and MSC-derived adipocytes. Following our recent expression studies (Obesity 2007, 15, 40-49), we now investigated the functional role of MC1-R in these tissues and cells to deduce the coupling state of MC1-R to intracellular output signals in human fat cells and tissue. Expression of MC1-R by undifferentiated and differentiated MSCs was quantified by real-time TaqMan PCR. Intracellular output signals (cAMP, lipolysis, secretion of IL-6, IL-10, and TNF-alpha), as well as effects on the metabolic rate and proliferation of human MSCs were analyzed by standard assays, exposing undifferentiated and differentiated MSCs and, in part, human adipose tissue explants to the potent MC1-R agonist, [Nle(4), D-Phe(7)]-alpha-MSH (NDP-MSH). This agonist induced a weak cAMP signal in MSC-derived adipocytes. However, it did not affect lipolysis in these cells or in adipose tissue explants, nor did it modulate cytokine release and mRNA expression of IL-6, IL-8, and TNF-alpha upon LPS stimulation. In undifferentiated MSCs, NDP-MSH did not alter the metabolic rate, but it showed a significant antiproliferative effect. Therefore, it appears that MC1-R-effector coupling in (differentiated) human adipocytes is too weak to induce a regulatory effect on lipolysis or inflammation; by contrast, MC1-R stimulation in undifferentiated MSCs induces an inhibitory signal on cell proliferation.

Important mitochondrial proteins in human omental adipose tissue show reduced expression in obesity.

Obesity is associated with impaired mitochondrial function. This study compares mitochondrial protein expression in omental fat in obese and non-obese humans. Omental adipose tissue was obtained by surgical biopsy, adipocytes were purified and mitochondria isolated. Using anion-exchange chromatography, SDS-PAGE and mass-spectrometry, 128 proteins with potentially different abundances in patient groups were identified, 62 of the 128 proteins are mainly localized in the mitochondria. Further quantification of 12 of these 62 proteins by immune dot blot analysis revealed four proteins citrate synthase, HADHA, LETM1 and mitofilin being inversely associated with BMI, and mitofilin being inversely correlated with gender.

Important mitochondrial proteins in human omental adipose tissue show reduced expression in obesity.

Impaired mitochondrial function is important in obesity and the development of insulin resistance and diabetes. The aim of this study was to identify human adipocyte-derived mitochondrial proteins associated with obesity. Mitochondrial proteins from 20 abdominal omental adipose tissue biopsies (13 obese and 7 control subjects) were separated by anion-exchange chromatography coupled to SDS-PAGE. Protein contents were compared and identified by MALDI-TOF-TOF mass spectrometry. Proteins of interest were validated, verified and quantified using immuno dot blot assays in a total of 76 mitochondrial preparations from both obese and non-obese patients. Mass spectrometric comparison of 20 mitochondrial proteomes yielded 62 proteins that were differentially expressed in adipose tissue of obese subjects. The immunological quantification of 12 mitochondrial proteins from 76 omental adipose tissue biopsies revealed four proteins, citrate synthase, HADHA, LETM1 and mitofilin inversely being associated with BMI, and mitofilin being inversely correlated with gender. BIOLOGICAL SIGNIFICANCE: The finding that obese human subjects have reduced levels of important mitochondrial proteins in adipocytes of omental adipose tissue as compared to non-obese controls gives new insights in the impairment of mitochondrial function in this specialized compartment of human adipose tissue in obesity and may eventually lead to the definition of valuable obesity markers.

Hepatocellular toxicity of benzbromarone: effects on mitochondrial function and structure.

Benzbromarone is an uricosuric structurally related to amiodarone and a known mitochondrial toxicant. The aim of the current study was to improve our understanding in the molecular mechanisms of benzbromarone-associated hepatic mitochondrial toxicity. In HepG2 cells and primary human hepatocytes, ATP levels started to decrease in the presence of 25-50µM benzbromarone for 24-48h, whereas cytotoxicity was observed only at 100µM. In HepG2 cells, benzbromarone decreased the mitochondrial membrane potential starting at 50µM following incubation for 24h. Additionally, in HepG2 cells, 50µM benzbromarone for 24h induced mitochondrial uncoupling,and decreased mitochondrial ATP turnover and maximal respiration. This was accompanied by an increased lactate concentration in the cell culture supernatant, reflecting increased glycolysis as a compensatory mechanism to maintain cellular ATP. Investigation of the electron transport chain revealed a decreased activity of all relevant enzyme complexes. Furthermore, treatment with benzbromarone was associated with increased cellular ROS production, which could be located specifically to mitochondria. In HepG2 cells and in isolated mouse liver mitochondria, benzbromarone also reduced palmitic acid metabolism due to an inhibition of the long-chain acyl CoA synthetase. In HepG2 cells, benzbromarone disrupted the mitochondrial network, leading to mitochondrial fragmentation and a decreased mitochondrial volume per cell. Cell death occurred by both apoptosis and necrosis. The study demonstrates that benzbromarone not only affects the function of mitochondria in HepG2 cells and human hepatocytes, but is also associated with profound changes in mitochondrial structure which may be associated with apoptosis.

Mechanisms of hepatocellular toxicity associated with dronedarone--a comparison to amiodarone.

Dronedarone is a new antiarrhythmic drug with an amiodarone-like benzofuran structure. Shortly after its introduction, dronedarone became implicated in causing severe liver injury. Amiodarone is a well-known mitochondrial toxicant. The aim of our study was to investigate mechanisms of hepatotoxicity of dronedarone in vitro and to compare them with amiodarone. We used isolated rat liver mitochondria, primary human hepatocytes, and the human hepatoma cell line HepG2, which were exposed acutely or up to 24h. After exposure of primary hepatocytes or HepG2 cells for 24h, dronedarone and amiodarone caused cytotoxicity and apoptosis starting at 20 and 50 µM, respectively. The cellular ATP content started to decrease at 20 µM for both drugs, suggesting mitochondrial toxicity. Inhibition of the respiratory chain required concentrations of ~10 µM and was caused by an impairment of complexes I and II for both drugs. In parallel, mitochondrial accumulation of reactive oxygen species (ROS) was observed. In isolated rat liver mitochondria, acute treatment with dronedarone decreased the mitochondrial membrane potential, inhibited complex I, and uncoupled the respiratory chain. Furthermore, in acutely treated rat liver mitochondria and in HepG2 cells exposed for 24h, dronedarone started to inhibit mitochondrial ß-oxidation at 10 µM and amiodarone at 20 µM. Similar to amiodarone, dronedarone is an uncoupler and an inhibitor of the mitochondrial respiratory chain and of ß-oxidation both acutely and after exposure for 24h. Inhibition of mitochondrial function leads to accumulation of ROS and fatty acids, eventually leading to apoptosis and/or necrosis of hepatocytes. Mitochondrial toxicity may be an explanation for hepatotoxicity of dronedarone in vivo.

Hepatocellular toxicity of clopidogrel: mechanisms and risk factors.

Clopidogrel is a prodrug used widely as a platelet aggregation inhibitor. After intestinal absorption, approximately 90% is converted to inactive clopidogrel carboxylate and 10% via a two-step procedure to the active metabolite containing a mercapto group. Hepatotoxicity is a rare but potentially serious adverse reaction associated with clopidogrel. The aim of this study was to find out the mechanisms and susceptibility factors for clopidogrel-associated hepatotoxicity. In primary human hepatocytes, clopidogrel (10 and 100 µM) was cytotoxic only after cytochrome P450 (CYP) induction by rifampicin. Clopidogrel (10 and 100 µM) was also toxic for HepG2 cells expressing human CYP3A4 (HepG2/CYP3A4) and HepG2 cells co-incubated with CYP3A4 supersomes (HepG2/CYP3A4 supersome), but not for wild-type HepG2 cells (HepG2/wt). Clopidogrel (100 µM) decreased the cellular glutathione content in HepG2/CYP3A4 supersome and triggered an oxidative stress reaction (10 and 100 µM) in HepG2/CYP3A4, but not in HepG2/wt. Glutathione depletion significantly increased the cytotoxicity of clopidogrel (10 and 100 µM) in HepG2/CYP3A4 supersome. Co-incubation with 1 µM ketoconazole or 10mM glutathione almost completely prevented the cytotoxic effect of clopidogrel in HepG2/CYP3A4 and HepG2/CYP3A4 supersome. HepG2/CYP3A4 incubated with 100 µM clopidogrel showed mitochondrial damage and cytochrome c release, eventually promoting apoptosis and/or necrosis. In contrast to clopidogrel, clopidogrel carboxylate was not toxic for HepG2/wt or HepG2/CYP3A4 up to 100 µM. In conclusion, clopidogrel incubated with CYP3A4 is associated with the formation of metabolites that are toxic for hepatocytes and can be trapped by glutathione. High CYP3A4 activity and low cellular glutathione stores may be risk factors for clopidogrel-associated hepatocellular toxicity.

Obesity affects mitochondrial citrate synthase in human omental adipose tissue.

The activities of some key enzymes in mitochondria from 135 human omental adipose tissue samples of obese and nonobese patients were analyzed for potential association with the patients' state of obesity. The activities of respiratory complexes I and II as well as citrate synthase in isolated mitochondria were measured using spectrophotometric enzyme assays. ATP generation of mitochondria was determined with a bioluminescence assay. Protein levels of citrate synthase were quantified by western blot. The rates of ATP generation and the enzymatic activities of complexes I and II did not display associations with age, gender, obesity, or diabetes. By contrast, the enzymatic activities of citrate synthase and its protein levels were significantly reduced in obesity as compared to controls. In diabetic patients, protein levels but not enzymatic activities of citrate synthase were elevated. Thus, this investigation based on enzymatic assay and determination of protein levels revealed that the development of obesity is associated with a significant impact on citrate synthase in mitochondria of human omental adipose tissue. The state of obesity appears to affect mitochondrial function in human omental adipose tissue by limiting this key enzyme of the tricarboxylic acid cycle rather than by limiting the activities of respiratory chain enzymes.

The role of CYP3A4 in amiodarone-associated toxicity on HepG2 cells.

Amiodarone is a class III antiarrhythmic drug with potentially life-threatening hepatotoxicity. Recent in vitro investigations suggested that the mono-N-desethyl (MDEA) and di-N-desethyl (DDEA) metabolites may cause amiodarone's hepatotoxicity. Since cytochrome P450 (CYP) 3A4 is responsible for amiodarone N-deethylation, CYP3A4 induction may represent a risk factor. Our aim was therefore to investigate the role of CYP3A4 in amiodarone-associated hepatotoxicity. First, we showed that 50µM amiodarone is more toxic to primary human hepatocytes after CYP induction with rifampicin. Second, we overexpressed human CYP3A4 in HepG2 cells (HepG2 cells/CYP3A4) for studying the interaction between CYP3A4 and amiodarone in more detail. We also used HepG2 wild type cells (HepG2 cells/wt) co-incubated with human CYP3A4 supersomes for amiodarone activation (HepG2 cells/CYP3A4 supersomes). Amiodarone (10-50µM) was cytotoxic for HepG2 cells/CYP3A4 or HepG2 cells/CYP3A4 supersomes, but not for HepG2 cells/wt or less toxic for HepG2 cells/wt incubated with control supersomes without CYP3A4. Co-incubation with ketoconazole, attenuated cytotoxicity of amiodarone incubated with HepG2 cells/CYP3A4 or HepG2 cells/CYP3A4 supersomes. MDEA and DDEA were formed only in incubations containing HepG2 cells/CYP3A4 or HepG2 cells/CYP3A4 supersomes but not by HepG2 cells/wt or HepG2 cells/wt with control supersomes. Metabolized amiodarone triggered the production of reactive oxygen species, induced mitochondrial damage and cytochrome c release, and promoted apoptosis/necrosis in HepG2 cells/CYP3A4, but not HepG2 cells/wt. This study supports the hypothesis that a high CYP3A4 activity is a risk factor for amiodarone's hepatotoxicity. Since CYP3A4 inducers are used frequently and amiodarone-associated hepatotoxicity can be fatal, our observations may be clinically relevant.

Mitochondrial DNA content in human omental adipose tissue.

BACKGROUND: Impairment of mitochondrial function plays an important role in obesity and the development of insulin resistance. The aim of this project was to investigate the mitochondrial DNA copy number in human omental adipose tissue with respect to obesity. METHODS: The mitochondrial DNA (mtDNA) content per single adipocyte derived from abdominal omental adipose tissue was determined by quantitative RT-PCR in a group of 75 patients, consisting of obese and morbidly obese subjects, as well as non-obese controls. Additionally, basal metabolic rate and fat oxidation rate were recorded and expressed as total values or per kilogram fat mass. RESULTS: MtDNA content is associated with obesity. Higher body mass index (BMI) resulted in a significantly elevated mtDNA count (ratio = 1.56; p = 0.0331) comparing non-obese (BMI < 30) to obese volunteers (BMI >or= 30). The mtDNA count per cell was not correlated with age or gender. Diabetic patients showed a trend toward reduced mtDNA content. A seasonal change in mtDNA copy number could not be identified. In addition, a substudy investigating the basal metabolic rate and the fasting fat oxidation did not reveal any associations to the mtDNA count. CONCLUSIONS: The mtDNA content per cell of omental adipose tissue did not correlate with various clinical parameters but tended to be reduced in patients with diabetes, which may partly explain the impairment of mitochondrial function observed in insulin resistance. Furthermore, the mtDNA content was significantly increased in patients suffering from obesity (BMI above 30). This might reflect a compensatory response to the development of obesity, which is associated with impairment of mitochondrial function.

Induction of murine ras oncogene peptide-specific T cell responses by immunization with plasmid DNA-based minigene vectors.

In a BALB/c mouse model, we have previously identified a ras oncogene peptide that contained both CD8(+) and CD4(+) T cell epitopes in a nested configuration. In this study, we developed several plasmid DNA minigene vectors encoding these determinants and examined whether they could induce antigen (Ag)-specific CD8(+) cytotoxic and CD4(+) lymphoproliferative responses. Furthermore, we compared two different immunization procedures, epidermal gene gun inoculation and intradermal (i.d.) injection of saline plasmid DNA, along with several approaches addressing different aspects of immune modulation. We demonstrated that each DNA plasmid induced the relevant Ag-specific cellular immune response. Gene gun inoculation was superior to that of needle injection for induction of the CTL response. Moreover, DNA plasmids containing both ras epitopes induced the highest CTL response, as compared with vector preparations containing only the CD8(+) epitope. These results suggested that a DNA plasmid expressing nested mutant ras epitope-specific CD4(+) and CD8(+) T cell epitopes can be processed in vivo to induce both subset-specific T cell responses, and that the addition of the helper epitope quantitatively improved the development of the CTL response, which may have implications for DNA-based anti-tumor immunotherapies.