Bidirectional modulation of TCA cycle metabolites and anaplerosis by metformin and its combination with SGLT2i.

BACKGROUND: Metformin and sodium-glucose-cotransporter-2 inhibitors (SGLT2i) are cornerstone therapies for managing hyperglycemia in diabetes. However, their detailed impacts on metabolic processes, particularly within the citric acid (TCA) cycle and its anaplerotic pathways, remain unclear. This study investigates the tissue-specific metabolic effects of metformin, both as a monotherapy and in combination with SGLT2i, on the TCA cycle and associated anaplerotic reactions in both mice and humans. METHODS: Metformin-specific metabolic changes were initially identified by comparing metformin-treated diabetic mice (MET) with vehicle-treated db/db mice (VG). These findings were then assessed in two human cohorts (KORA and QBB) and a longitudinal KORA study of metformin-naïve patients with Type 2 Diabetes (T2D). We also compared MET with db/db mice on combination therapy (SGLT2i + MET). Metabolic profiling analyzed 716 metabolites from plasma, liver, and kidney tissues post-treatment, using linear regression and Bonferroni correction for statistical analysis, complemented by pathway analyses to explore the pathophysiological implications. RESULTS: Metformin monotherapy significantly upregulated TCA cycle intermediates such as malate, fumarate, and α-ketoglutarate (α-KG) in plasma, and anaplerotic substrates including hepatic glutamate and renal 2-hydroxyglutarate (2-HG) in diabetic mice. Downregulated hepatic taurine was also observed. The addition of SGLT2i, however, reversed these effects, such as downregulating circulating malate and α-KG, and hepatic glutamate and renal 2-HG, but upregulated hepatic taurine. In human T2D patients on metformin therapy, significant systemic alterations in metabolites were observed, including increased malate but decreased citrulline. The bidirectional modulation of TCA cycle intermediates in mice influenced key anaplerotic pathways linked to glutaminolysis, tumorigenesis, immune regulation, and antioxidative responses. CONCLUSION: This study elucidates the specific metabolic consequences of metformin and SGLT2i on the TCA cycle, reflecting potential impacts on the immune system. Metformin shows promise for its anti-inflammatory properties, while the addition of SGLT2i may provide liver protection in conditions like metabolic dysfunction-associated steatotic liver disease (MASLD). These observations underscore the importance of personalized treatment strategies.

[Primary and secondary prevention of skin cancer in rural areas : A cross-sectional study in the Bavarian Forest]. / Primär- und Sekundärprävention von Hautkrebs in ländlichen Regionen : Querschnittstudie im Bayerischen Wald.

BACKGROUND: Skin cancer is the most common malignancy of the fair-skinned population worldwide. To reduce skin cancer's burden primary and secondary prevention are critical. However, various studies indicate an inadequate prevention behavior among rural populations. OBJECTIVE: To examine the risk and prevention behavior with respect to skin cancer and to identify subgroups in rural areas with specific need for prevention efforts. MATERIALS AND METHODS: In a cross-sectional study carried out in the first quarter of 2017, patients and their accompanying persons (≥18 years) were interviewed on the subject of primary and secondary prevention in waiting rooms of nondermatological medical practices in the Bavarian Forest, Germany. Data were collected using paper-based questionnaires. Associations were calculated using logistic regression models. RESULTS: In all, 880 persons (57.7% women, mean age = 49.5 years) were included in the analysis, of whom 53.6% had undergone a skin cancer screening at least once before. Sunscreen was the most frequently used sun protection measure. Male sex and being 18-34 years of age were significantly associated with not using prevention measures (depending on the measure: odds ratio [OR]: 1.4-2.4 and 1.8-3.7, respectively). In addition, not using skin cancer screening was associated with UV exposure more than 6 h daily in summer (OR: 1.8, 95%-CI [confidence interval]: 1.14-2.97). CONCLUSION: Future prevention strategies should increasingly focus on young adults, on men and people with high solar UV exposition particularly, to reduce the burden of skin cancer in rural areas.

Physical activity stages of change surveillance data shows that the majority of Hawai'i's Keiki (Children) meet the guidelines.

Targeting Native Hawaiian and other Pacific Islander (NHOPI) children based on their physical activity (PA) stages of change (SOC) may improve intervention effectiveness. No known SOC surveillance system exists for NHOPI jurisdictions. The purpose was to determine the PA SOC prevalence over 5 years in children living in Hawai'i. Self-reported PA SOC from 5 cohorts (3-6 grade students) in Hawai'i were compared between cohorts and sex. The combined PA SOC distribution (n = 1726, 50.7% female) was: Precontemplation, 7.5%; Contemplation, 7.6%; Preparation, 9.9%; Action, 33.4%; Maintenance, 41.5%. There were no significant difference between cohorts 1 and 2 (n = 258), χ2 (16) = 21.75, p = 0.15; 2 and 3 (n = 129), χ2 (16) = 17.51, p = 0.35; 3 and 4 (n = 171), χ2 (16) = 17.28, p = 0.77; 4 and 5 (n = 129), χ2 (16) = 17.51, p = 0.35; and for all cohorts between males and females (p > 0.05). Most participants were in Action and Maintenance. Prevention efforts should emphasize maintaining PA levels. Extending PA behavior surveillance systems to include intention in NHOPI jurisdictions is warranted.

Night work, chronotype and cortisol at awakening in female hospital employees.

To examine the effect of night shift on salivary cortisol at awakening (C1), 30 min later (C2), and on the cortisol awakening response (CAR, the difference between C2 and C1). We compared shift and non-shift workers with a focus on the impact of worker chronotype. Our study included 66 shift-working females (mean age = 37.3 years, SD = 10.2) and 21 non-shift working females (mean age = 47.0 years, SD = 8.9). The shift workers collected their saliva samples at C1 and C2 on each two consecutive day shifts and night shifts. Non-shift workers collected their samples on two consecutive day shifts. We applied linear mixed-effects models (LMM) to determine the effect of night shift on CAR and log-transformed C1 and C2 levels. LMMs were stratified by chronotype group. Compared to non-shift workers, shift workers before day shifts (i.e. after night sleep) showed lower cortisol at C1 (exp [Formula: see text]=0.58, 95% CI 0.42, 0.81) but not at C2. In shift workers, the CARs after night shifts (i.e. after day sleep) were lower compared to CARs before day shifts ([Formula: see text]= - 11.07, 95% CI - 15.64, - 6.50). This effect was most pronounced in early chronotypes (early: [Formula: see text]= - 16.61, 95% CI - 27.87, - 5.35; intermediate: [Formula: see text]= - 11.82, 95% CI - 18.35, - 5.29; late: [Formula: see text]= - 6.27, 95% CI - 14.28, 1.74). Chronotype did not modify the association between night shift and CAR. In our population of shift workers, there was a mismatch between time of waking up and their natural cortisol peak at waking up (CAR) both during day and night shift duties.

Dilution correction for dynamically influenced urinary analyte data.

Urinary analyte data has to be corrected for the sample specific dilution as the dilution varies intra- and interpersonally dramatically, leading to non-comparable concentration measures. Most methods of dilution correction utilized nowadays like probabilistic quotient normalization or total spectra normalization result in a division of the raw data by a dilution correction factor. Here, however, we show that the implicit assumption behind the application of division, log-linearity between the urinary flow rate and the raw urinary concentration, does not hold for analytes which are not in steady state in blood. We explicate the physiological reason for this short-coming in mathematical terms and demonstrate the empirical consequences via simulations and on multiple time-point metabolomic data, showing the insufficiency of division-based normalization procedures to account for the complex non-linear analyte specific dependencies on the urinary flow rate. By reformulating normalization as a regression problem, we propose an analyte specific way to remove the dilution variance via a flexible non-linear regression methodology which then was shown to be more effective in comparison to division-based normalization procedures. In the progress, we developed several, easily applicable methods of normalization diagnostics to decide on the method of dilution correction in a given sample. On the way, we identified furthermore the time-span since last urination as an important variance factor in urinary metabolome data which is until now completely neglected. In conclusion, we present strong theoretical and empirical evidence that normalization has to be analyte specific in dynamically influenced data. Accordingly, we developed a normalization methodology for removing the dilution variance in urinary data respecting the single analyte kinetics.

Night Shift Work Affects Urine Metabolite Profiles of Nurses with Early Chronotype.

Night shift work can have a serious impact on health. Here, we assess whether and how night shift work influences the metabolite profiles, specifically with respect to different chronotype classes. We have recruited 100 women including 68 nurses working both, day shift and night shifts for up to 5 consecutive days and collected 3640 spontaneous urine samples. About 424 waking-up urine samples were measured using a targeted metabolomics approach. To account for urine dilution, we applied three methods to normalize the metabolite values: creatinine-, osmolality- and regression-based normalization. Based on linear mixed effect models, we found 31 metabolites significantly (false discovery rate <0.05) affected in nurses working in night shifts. One metabolite, acylcarnitine C10:2, was consistently identified with all three normalization methods. We further observed 11 and 4 metabolites significantly associated with night shift in early and late chronotype classes, respectively. Increased levels of medium- and long chain acylcarnitines indicate a strong impairment of the fatty acid oxidation. Our results show that night shift work influences acylcarnitines and BCAAs, particularly in nurses in the early chronotype class. Women with intermediate and late chronotypes appear to be less affected by night shift work.

Crystal structure of the tetra-hydro-furan disolvate of a 94:6 solid solution of [N2,N6-bis-(di-tert-butyl-phosphan-yl)pyridine-2,6-di-amine]-dibromido-manganese(II) and its monophosphine oxide analogue.

The MnBr2 complex of N2,N6-bis-(di-tert-butyl-phosphan-yl)pyridine-2,6-di-amine (1·MnBr2) co-crystallizes with 5.69% of the monophosphine oxide analogue (1O·MnBr2) and two tetra-hydro-furan (THF) mol-ecules, namely [N2,N6-bis-(di-tert-butyl-phosphan-yl)pyridine-2,6-di-amine]-dibromido-manganese(II)-[bis-(di-tert-butyl-phosphan-yl)({6-[(di-tert-butyl-phosphan-yl)amino]-pyridin-2-yl}amino)-phosphine oxide]di-bromido-manganese(II)-tetra-hydro-furan (0.94/0.06/2), [MnBr2(C21H41N3P2)]0.94[MnBr2(C21H41N3OP2)]0.06·2C4H8O. The 1·MnBr2 and 1O·MnBr2 complexes are occupationally disordered about general positions. Both complexes feature square-pyramidal coordination of the MnII atoms. They are connected by weak N-H⋯Br hydrogen bonding into chains extending along [001]. The THF mol-ecules are located between the layers formed by these chains. One THF mol-ecule is involved in hydrogen bonding to an amine H atom.

Stability of targeted metabolite profiles of urine samples under different storage conditions.

INTRODUCTION: Few studies have investigated the influence of storage conditions on urine samples and none of them used targeted mass spectrometry (MS). OBJECTIVES: We investigated the stability of metabolite profiles in urine samples under different storage conditions using targeted metabolomics. METHODS: Pooled, fasting urine samples were collected and stored at -80 °C (biobank standard), -20 °C (freezer), 4 °C (fridge), ~9 °C (cool pack), and ~20 °C (room temperature) for 0, 2, 8 and 24 h. Metabolite concentrations were quantified with MS using the AbsoluteIDQ™ p150 assay. We used the Welch-Satterthwaite-test to compare the concentrations of each metabolite. Mixed effects linear regression was used to assess the influence of the interaction of storage time and temperature. RESULTS: The concentrations of 63 investigated metabolites were stable at -20 and 4 °C for up to 24 h when compared to samples immediately stored at -80 °C. When stored at ~9 °C for 24 h, few amino acids (Arg, Val and Leu/Ile) significantly decreased by 40% in concentration (P < 7.9E-04); for an additional three metabolites (Ser, Met, Hexose H1) when stored at ~20 °C reduced up to 60% in concentrations. The concentrations of four more metabolites (Glu, Phe, Pro, and Thr) were found to be significantly influenced when considering the interaction between exposure time and temperature. CONCLUSION: Our findings indicate that 78% of quantified metabolites were stable for all examined storage conditions. Particularly, some amino acid concentrations were sensitive to changes after prolonged storage at room temperature. Shipping or storing urine samples on cool packs or at room temperature for more than 8 h and multiple numbers of freeze and thaw cycles should be avoided.

Acculturation is Not Related to Physical Activity Stage of Change for Children in Hawai'i.

The relationship between acculturation and physical activity stages of change is unexplored. Stages of change conceptualize behavior change as a progression through a series of five stages indicating the readiness to change behavior. The level of acculturation can be assessed using the Ethnocultural Identity Behavioral Index (EIBI) which is based on three factors: Cultural Activities, Social Interaction and Language Opportunities. The purpose of this project was to explore the relationship between parental acculturation and physical activity stages of change in Hawai'i children. Participants (N = 85; 62% female; aged 5-8 years; 22% Native Hawaiian or Other Pacific Islanders, 42% Asian, 25% White, and 11% Other) completed the EIBI and a physical activity stages of change measure. Acculturation factor means were: Cultural Activities = 4 (SD = 1.26), Social Interaction = 3 (SD = 1.04), and Language Opportunities = 4 (SD = 1.29). The physical activity stages of change distribution was Precontemplation = 11 (13%), Contemplation/Preparation = 15 (18%), and Action/Maintenance = 59 (69%). Analysis of covariance (ANCOVA) for Cultural Activities F(3, 81) = 0.77, P = .47, Social Interaction F(3, 81) = 0.93, P = .40; and Language Opportunities F(3, 81) = 1.34, P = .27 showed no significant differences between physical activity stages of change. The results of our study do not show an association between acculturation and readiness to change for physical activity. The lack of differences may be due to participants being moderately acculturated, possibly lessening the differentiation of acculturation by physical activity stages of change.

Nonmelanoma skin cancer in mountain guides: high prevalence and lack of awareness warrant development of evidence-based prevention tools.

AIMS OF THE STUDY: Nonmelanoma skin cancer (NMSC) is the most common cancer in Switzerland and Europe. The main causative factor is exposure to ultraviolet radiation, which puts outdoor workers in general at a higher risk of developing NMSC than indoor workers. However, few studies have clinically examined the risk of developing NMSC to outdoor workers, especially mountain guides. We aimed to investigate the prevalence of NMSC and corresponding precancerous lesions, and the associated risk behaviour of mountain and ski guides in order to develop future prevention programmes. METHODS: We conducted a cross-sectional study including mountain and ski guides from southern Germany, who underwent a full-body skin check-up by a dermatologist. We assessed their NMSC awareness and risk behaviour using a paper-based questionnaire. RESULTS: Of the 62 state-certified mountain and ski guides (55 men, 7 women; mean age 52.9 ± 13.4 years) included in this study, 27 (43.5%) were diagnosed with NMSC or its premalignant stages. In addition, 59.7% of the participants expressed the opinion that their protection from ultraviolet radiation exposure needs to be improved; 83.6% requested further information on NMSC, and 48.5% had never undergone a skin check-up or consulted a dermatologist before. CONCLUSIONS: Mountain and ski guides are at a high risk for developing NMSC. Their unmet medical needs indicate an underestimation of NMSC prevalence, which is usually based on reports by insurance companies, and offer the chance for developing evidence-based awareness and prevention tools that can be promoted to individuals with other outdoor jobs.

Metformin Effect on Nontargeted Metabolite Profiles in Patients With Type 2 Diabetes and in Multiple Murine Tissues.

Metformin is the first-line oral medication to increase insulin sensitivity in patients with type 2 diabetes (T2D). Our aim was to investigate the pleiotropic effect of metformin using a nontargeted metabolomics approach. We analyzed 353 metabolites in fasting serum samples of the population-based human KORA (Cooperative Health Research in the Region of Augsburg) follow-up survey 4 cohort. To compare T2D patients treated with metformin (mt-T2D, n = 74) and those without antidiabetes medication (ndt-T2D, n = 115), we used multivariable linear regression models in a cross-sectional study. We applied a generalized estimating equation to confirm the initial findings in longitudinal samples of 683 KORA participants. In a translational approach, we used murine plasma, liver, skeletal muscle, and epididymal adipose tissue samples from metformin-treated db/db mice to further corroborate our findings from the human study. We identified two metabolites significantly (P < 1.42E-04) associated with metformin treatment. Citrulline showed lower relative concentrations and an unknown metabolite X-21365 showed higher relative concentrations in human serum when comparing mt-T2D with ndt-T2D. Citrulline was confirmed to be significantly (P < 2.96E-04) decreased at 7-year follow-up in patients who started metformin treatment. In mice, we validated significantly (P < 4.52E-07) lower citrulline values in plasma, skeletal muscle, and adipose tissue of metformin-treated animals but not in their liver. The lowered values of citrulline we observed by using a nontargeted approach most likely resulted from the pleiotropic effect of metformin on the interlocked urea and nitric oxide cycle. The translational data derived from multiple murine tissues corroborated and complemented the findings from the human cohort.

Effects of metformin on metabolite profiles and LDL cholesterol in patients with type 2 diabetes.

OBJECTIVE: Metformin is used as a first-line oral treatment for type 2 diabetes (T2D). However, the underlying mechanism is not fully understood. Here, we aimed to comprehensively investigate the pleiotropic effects of metformin. RESEARCH DESIGN AND METHODS: We analyzed both metabolomic and genomic data of the population-based KORA cohort. To evaluate the effect of metformin treatment on metabolite concentrations, we quantified 131 metabolites in fasting serum samples and used multivariable linear regression models in three independent cross-sectional studies (n = 151 patients with T2D treated with metformin [mt-T2D]). Additionally, we used linear mixed-effect models to study the longitudinal KORA samples (n = 912) and performed mediation analyses to investigate the effects of metformin intake on blood lipid profiles. We combined genotyping data with the identified metformin-associated metabolites in KORA individuals (n = 1,809) and explored the underlying pathways. RESULTS: We found significantly lower (P < 5.0E-06) concentrations of three metabolites (acyl-alkyl phosphatidylcholines [PCs]) when comparing mt-T2D with four control groups who were not using glucose-lowering oral medication. These findings were controlled for conventional risk factors of T2D and replicated in two independent studies. Furthermore, we observed that the levels of these metabolites decreased significantly in patients after they started metformin treatment during 7 years' follow-up. The reduction of these metabolites was also associated with a lowered blood level of LDL cholesterol (LDL-C). Variations of these three metabolites were significantly associated with 17 genes (including FADS1 and FADS2) and controlled by AMPK, a metformin target. CONCLUSIONS: Our results indicate that metformin intake activates AMPK and consequently suppresses FADS, which leads to reduced levels of the three acyl-alkyl PCs and LDL-C. Our findings suggest potential beneficial effects of metformin in the prevention of cardiovascular disease.