Sexual Dimorphic Regulation of Body Weight Dynamics and Adipose Tissue Lipolysis

BACKGROUND: Successful reduction of body weight (BW) is often followed by recidivism to obesity. BW-changes including BW-loss and -regain is associated with marked alterations in energy expenditure (EE) and adipose tissue (AT) metabolism. Since these processes are sex-specifically controlled, we investigated sexual dimorphisms in metabolic processes during BW-dynamics (gain-loss-regain). RESEARCH DESIGN: Obesity was induced in C57BL/6J male (m) and female (f) mice by 15 weeks high-fat diet (HFD) feeding. Subsequently BW was reduced (-20%) by caloric restriction (CR) followed by adaptive feeding, and a regain-phase. Measurement of EE, body composition, blood/organ sampling were performed after each feeding period. Lipolysis was analyzed ex-vivo in gonadal AT. RESULTS: Male mice exhibited accelerated BW-gain compared to females (relative BW-gain m:140.5±3.2%; f:103.7±6.5%; p<0.001). In consonance, lean mass-specific EE was significantly higher in females compared to males during BW-gain. Under CR female mice reached their target-BW significantly faster than male mice (m:12.2 days; f:7.6 days; p<0.001) accompanied by a sustained sex-difference in EE. In addition, female mice predominantly downsized gonadal AT whereas the relation between gonadal and total body fat was not altered in males. Accordingly, only females exhibited an increased rate of forskolin-stimulated lipolysis in AT associated with significantly higher glycerol concentrations, lower RER-values, and increased AT expression of adipose triglyceride lipase (ATGL) and hormone sensitive lipase (HSL). Analysis of AT lipolysis in estrogen receptor alpha (ERα)-deficient mice revealed a reduced lipolytic rate in the absence of ERα exclusively in females. Finally, re-feeding caused BW-regain faster in males than in females. CONCLUSION: The present study shows sex-specific dynamics during BW-gain-loss-regain. Female mice responded to CR with an increase in lipolytic activity, and augmented lipid-oxidation leading to more efficient weight loss. These processes likely involve ERα-dependent signaling in AT and sexual dimorphic regulation of genes involved in lipid metabolism

Burden of disease scenarios for 204 countries and territories, 2022–2050: a forecasting analysis for the Global Burden of Disease Study 2021

Background: Future trends in disease burden and drivers of health are of great interest to policy makers and the public at large. This information can be used for policy and long-term health investment, planning, and prioritisation. We have expanded and improved upon previous forecasts produced as part of the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) and provide a reference forecast (the most likely future), and alternative scenarios assessing disease burden trajectories if selected sets of risk factors were eliminated from current levels by 2050. Methods: Using forecasts of major drivers of health such as the Socio-demographic Index (SDI; a composite measure of lag-distributed income per capita, mean years of education, and total fertility under 25 years of age) and the full set of risk factor exposures captured by GBD, we provide cause-specific forecasts of mortality, years of life lost (YLLs), years lived with disability (YLDs), and disability-adjusted life-years (DALYs) by age and sex from 2022 to 2050 for 204 countries and territories, 21 GBD regions, seven super-regions, and the world. All analyses were done at the cause-specific level so that only risk factors deemed causal by the GBD comparative risk assessment influenced future trajectories of mortality for each disease. Cause-specific mortality was modelled using mixed-effects models with SDI and time as the main covariates, and the combined impact of causal risk factors as an offset in the model. At the all-cause mortality level, we captured unexplained variation by modelling residuals with an autoregressive integrated moving average model with drift attenuation. These all-cause forecasts constrained the cause-specific forecasts at successively deeper levels of the GBD cause hierarchy using cascading mortality models, thus ensuring a robust estimate of cause-specific mortality. For non-fatal measures (eg, low back pain), incidence and prevalence were forecasted from mixed-effects models with SDI as the main covariate, and YLDs were computed from the resulting prevalence forecasts and average disability weights from GBD. Alternative future scenarios were constructed by replacing appropriate reference trajectories for risk factors with hypothetical trajectories of gradual elimination of risk factor exposure from current levels to 2050. The scenarios were constructed from various sets of risk factors: environmental risks (Safer Environment scenario), risks associated with communicable, maternal, neonatal, and nutritional diseases (CMNNs; Improved Childhood Nutrition and Vaccination scenario), risks associated with major non-communicable diseases (NCDs; Improved Behavioural and Metabolic Risks scenario), and the combined effects of these three scenarios. Using the Shared Socioeconomic Pathways climate scenarios SSP2-4.5 as reference and SSP1-1.9 as an optimistic alternative in the Safer Environment scenario, we accounted for climate change impact on health by using the most recent Intergovernmental Panel on Climate Change temperature forecasts and published trajectories of ambient air pollution for the same two scenarios. Life expectancy and healthy life expectancy were computed using standard methods. The forecasting framework includes computing the age-sex-specific future population for each location and separately for each scenario. 95% uncertainty intervals (UIs) for each individual future estimate were derived from the 2·5th and 97·5th percentiles of distributions generated from propagating 500 draws through the multistage computational pipeline. Findings: In the reference scenario forecast, global and super-regional life expectancy increased from 2022 to 2050, but improvement was at a slower pace than in the three decades preceding the COVID-19 pandemic (beginning in 2020). Gains in future life expectancy were forecasted to be greatest in super-regions with comparatively low life expectancies (such as sub-Saharan Africa) compared with super-regions with higher life expectancies (such as the high-income super-region), leading to a trend towards convergence in life expectancy across locations between now and 2050. At the super-region level, forecasted healthy life expectancy patterns were similar to those of life expectancies. Forecasts for the reference scenario found that health will improve in the coming decades, with all-cause age-standardised DALY rates decreasing in every GBD super-region. The total DALY burden measured in counts, however, will increase in every super-region, largely a function of population ageing and growth. We also forecasted that both DALY counts and age-standardised DALY rates will continue to shift from CMNNs to NCDs, with the most pronounced shifts occurring in sub-Saharan Africa (60·1% [95% UI 56·8–63·1] of DALYs were from CMNNs in 2022 compared with 35·8% [31·0–45·0] in 2050) and south Asia (31·7% [29·2–34·1] to 15·5% [13·7–17·5]). This shift is reflected in the leading global causes of DALYs, with the top four causes in 2050 being ischaemic heart disease, stroke, diabetes, and chronic obstructive pulmonary disease, compared with 2022, with ischaemic heart disease, neonatal disorders, stroke, and lower respiratory infections at the top. The global proportion of DALYs due to YLDs likewise increased from 33·8% (27·4–40·3) to 41·1% (33·9–48·1) from 2022 to 2050, demonstrating an important shift in overall disease burden towards morbidity and away from premature death. The largest shift of this kind was forecasted for sub-Saharan Africa, from 20·1% (15·6–25·3) of DALYs due to YLDs in 2022 to 35·6% (26·5–43·0) in 2050. In the assessment of alternative future scenarios, the combined effects of the scenarios (Safer Environment, Improved Childhood Nutrition and Vaccination, and Improved Behavioural and Metabolic Risks scenarios) demonstrated an important decrease in the global burden of DALYs in 2050 of 15·4% (13·5–17·5) compared with the reference scenario, with decreases across super-regions ranging from 10·4% (9·7–11·3) in the high-income super-region to 23·9% (20·7–27·3) in north Africa and the Middle East. The Safer Environment scenario had its largest decrease in sub-Saharan Africa (5·2% [3·5–6·8]), the Improved Behavioural and Metabolic Risks scenario in north Africa and the Middle East (23·2% [20·2–26·5]), and the Improved Nutrition and Vaccination scenario in sub-Saharan Africa (2·0% [–0·6 to 3·6]). Interpretation: Globally, life expectancy and age-standardised disease burden were forecasted to improve between 2022 and 2050, with the majority of the burden continuing to shift from CMNNs to NCDs. That said, continued progress on reducing the CMNN disease burden will be dependent on maintaining investment in and policy emphasis on CMNN disease prevention and treatment. Mostly due to growth and ageing of populations, the number of deaths and DALYs due to all causes combined will generally increase. By constructing alternative future scenarios wherein certain risk exposures are eliminated by 2050, we have shown that opportunities exist to substantially improve health outcomes in the future through concerted efforts to prevent exposure to well established risk factors and to expand access to key health interventions

Global burden and strength of evidence for 88 risk factors in 204 countries and 811 subnational locations, 1990–2021: a systematic analysis for the Global Burden of Disease Study 2021

Background: Understanding the health consequences associated with exposure to risk factors is necessary to inform public health policy and practice. To systematically quantify the contributions of risk factor exposures to specific health outcomes, the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2021 aims to provide comprehensive estimates of exposure levels, relative health risks, and attributable burden of disease for 88 risk factors in 204 countries and territories and 811 subnational locations, from 1990 to 2021. Methods: The GBD 2021 risk factor analysis used data from 54 561 total distinct sources to produce epidemiological estimates for 88 risk factors and their associated health outcomes for a total of 631 risk–outcome pairs. Pairs were included on the basis of data-driven determination of a risk–outcome association. Age-sex-location-year-specific estimates were generated at global, regional, and national levels. Our approach followed the comparative risk assessment framework predicated on a causal web of hierarchically organised, potentially combinative, modifiable risks. Relative risks (RRs) of a given outcome occurring as a function of risk factor exposure were estimated separately for each risk–outcome pair, and summary exposure values (SEVs), representing risk-weighted exposure prevalence, and theoretical minimum risk exposure levels (TMRELs) were estimated for each risk factor. These estimates were used to calculate the population attributable fraction (PAF; ie, the proportional change in health risk that would occur if exposure to a risk factor were reduced to the TMREL). The product of PAFs and disease burden associated with a given outcome, measured in disability-adjusted life-years (DALYs), yielded measures of attributable burden (ie, the proportion of total disease burden attributable to a particular risk factor or combination of risk factors). Adjustments for mediation were applied to account for relationships involving risk factors that act indirectly on outcomes via intermediate risks. Attributable burden estimates were stratified by Socio-demographic Index (SDI) quintile and presented as counts, age-standardised rates, and rankings. To complement estimates of RR and attributable burden, newly developed burden of proof risk function (BPRF) methods were applied to yield supplementary, conservative interpretations of risk–outcome associations based on the consistency of underlying evidence, accounting for unexplained heterogeneity between input data from different studies. Estimates reported represent the mean value across 500 draws from the estimate's distribution, with 95% uncertainty intervals (UIs) calculated as the 2·5th and 97·5th percentile values across the draws. Findings: Among the specific risk factors analysed for this study, particulate matter air pollution was the leading contributor to the global disease burden in 2021, contributing 8·0% (95% UI 6·7–9·4) of total DALYs, followed by high systolic blood pressure (SBP; 7·8% [6·4–9·2]), smoking (5·7% [4·7–6·8]), low birthweight and short gestation (5·6% [4·8–6·3]), and high fasting plasma glucose (FPG; 5·4% [4·8–6·0]). For younger demographics (ie, those aged 0–4 years and 5–14 years), risks such as low birthweight and short gestation and unsafe water, sanitation, and handwashing (WaSH) were among the leading risk factors, while for older age groups, metabolic risks such as high SBP, high body-mass index (BMI), high FPG, and high LDL cholesterol had a greater impact. From 2000 to 2021, there was an observable shift in global health challenges, marked by a decline in the number of all-age DALYs broadly attributable to behavioural risks (decrease of 20·7% [13·9–27·7]) and environmental and occupational risks (decrease of 22·0% [15·5–28·8]), coupled with a 49·4% (42·3–56·9) increase in DALYs attributable to metabolic risks, all reflecting ageing populations and changing lifestyles on a global scale. Age-standardised global DALY rates attributable to high BMI and high FPG rose considerably (15·7% [9·9–21·7] for high BMI and 7·9% [3·3–12·9] for high FPG) over this period, with exposure to these risks increasing annually at rates of 1·8% (1·6–1·9) for high BMI and 1·3% (1·1–1·5) for high FPG. By contrast, the global risk-attributable burden and exposure to many other risk factors declined, notably for risks such as child growth failure and unsafe water source, with age-standardised attributable DALYs decreasing by 71·5% (64·4–78·8) for child growth failure and 66·3% (60·2–72·0) for unsafe water source. We separated risk factors into three groups according to trajectory over time: those with a decreasing attributable burden, due largely to declining risk exposure (eg, diet high in trans-fat and household air pollution) but also to proportionally smaller child and youth populations (eg, child and maternal malnutrition); those for which the burden increased moderately in spite of declining risk exposure, due largely to population ageing (eg, smoking); and those for which the burden increased considerably due to both increasing risk exposure and population ageing (eg, ambient particulate matter air pollution, high BMI, high FPG, and high SBP). Interpretation: Substantial progress has been made in reducing the global disease burden attributable to a range of risk factors, particularly those related to maternal and child health, WaSH, and household air pollution. Maintaining efforts to minimise the impact of these risk factors, especially in low SDI locations, is necessary to sustain progress. Successes in moderating the smoking-related burden by reducing risk exposure highlight the need to advance policies that reduce exposure to other leading risk factors such as ambient particulate matter air pollution and high SBP. Troubling increases in high FPG, high BMI, and other risk factors related to obesity and metabolic syndrome indicate an urgent need to identify and implement interventions

Global incidence, prevalence, years lived with disability (YLDs), disability-adjusted life-years (DALYs), and healthy life expectancy (HALE) for 371 diseases and injuries in 204 countries and territories and 811 subnational locations, 1990–2021: a systematic analysis for the Global Burden of Disease Study 2021

Background: Detailed, comprehensive, and timely reporting on population health by underlying causes of disability and premature death is crucial to understanding and responding to complex patterns of disease and injury burden over time and across age groups, sexes, and locations. The availability of disease burden estimates can promote evidence-based interventions that enable public health researchers, policy makers, and other professionals to implement strategies that can mitigate diseases. It can also facilitate more rigorous monitoring of progress towards national and international health targets, such as the Sustainable Development Goals. For three decades, the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) has filled that need. A global network of collaborators contributed to the production of GBD 2021 by providing, reviewing, and analysing all available data. GBD estimates are updated routinely with additional data and refined analytical methods. GBD 2021 presents, for the first time, estimates of health loss due to the COVID-19 pandemic. Methods: The GBD 2021 disease and injury burden analysis estimated years lived with disability (YLDs), years of life lost (YLLs), disability-adjusted life-years (DALYs), and healthy life expectancy (HALE) for 371 diseases and injuries using 100 983 data sources. Data were extracted from vital registration systems, verbal autopsies, censuses, household surveys, disease-specific registries, health service contact data, and other sources. YLDs were calculated by multiplying cause-age-sex-location-year-specific prevalence of sequelae by their respective disability weights, for each disease and injury. YLLs were calculated by multiplying cause-age-sex-location-year-specific deaths by the standard life expectancy at the age that death occurred. DALYs were calculated by summing YLDs and YLLs. HALE estimates were produced using YLDs per capita and age-specific mortality rates by location, age, sex, year, and cause. 95% uncertainty intervals (UIs) were generated for all final estimates as the 2·5th and 97·5th percentiles values of 500 draws. Uncertainty was propagated at each step of the estimation process. Counts and age-standardised rates were calculated globally, for seven super-regions, 21 regions, 204 countries and territories (including 21 countries with subnational locations), and 811 subnational locations, from 1990 to 2021. Here we report data for 2010 to 2021 to highlight trends in disease burden over the past decade and through the first 2 years of the COVID-19 pandemic. Findings: Global DALYs increased from 2·63 billion (95% UI 2·44–2·85) in 2010 to 2·88 billion (2·64–3·15) in 2021 for all causes combined. Much of this increase in the number of DALYs was due to population growth and ageing, as indicated by a decrease in global age-standardised all-cause DALY rates of 14·2% (95% UI 10·7–17·3) between 2010 and 2019. Notably, however, this decrease in rates reversed during the first 2 years of the COVID-19 pandemic, with increases in global age-standardised all-cause DALY rates since 2019 of 4·1% (1·8–6·3) in 2020 and 7·2% (4·7–10·0) in 2021. In 2021, COVID-19 was the leading cause of DALYs globally (212·0 million [198·0–234·5] DALYs), followed by ischaemic heart disease (188·3 million [176·7–198·3]), neonatal disorders (186·3 million [162·3–214·9]), and stroke (160·4 million [148·0–171·7]). However, notable health gains were seen among other leading communicable, maternal, neonatal, and nutritional (CMNN) diseases. Globally between 2010 and 2021, the age-standardised DALY rates for HIV/AIDS decreased by 47·8% (43·3–51·7) and for diarrhoeal diseases decreased by 47·0% (39·9–52·9). Non-communicable diseases contributed 1·73 billion (95% UI 1·54–1·94) DALYs in 2021, with a decrease in age-standardised DALY rates since 2010 of 6·4% (95% UI 3·5–9·5). Between 2010 and 2021, among the 25 leading Level 3 causes, age-standardised DALY rates increased most substantially for anxiety disorders (16·7% [14·0–19·8]), depressive disorders (16·4% [11·9–21·3]), and diabetes (14·0% [10·0–17·4]). Age-standardised DALY rates due to injuries decreased globally by 24·0% (20·7–27·2) between 2010 and 2021, although improvements were not uniform across locations, ages, and sexes. Globally, HALE at birth improved slightly, from 61·3 years (58·6–63·6) in 2010 to 62·2 years (59·4–64·7) in 2021. However, despite this overall increase, HALE decreased by 2·2% (1·6–2·9) between 2019 and 2021. Interpretation: Putting the COVID-19 pandemic in the context of a mutually exclusive and collectively exhaustive list of causes of health loss is crucial to understanding its impact and ensuring that health funding and policy address needs at both local and global levels through cost-effective and evidence-based interventions. A global epidemiological transition remains underway. Our findings suggest that prioritising non-communicable disease prevention and treatment policies, as well as strengthening health systems, continues to be crucially important. The progress on reducing the burden of CMNN diseases must not stall; although global trends are improving, the burden of CMNN diseases remains unacceptably high. Evidence-based interventions will help save the lives of young children and mothers and improve the overall health and economic conditions of societies across the world. Governments and multilateral organisations should prioritise pandemic preparedness planning alongside efforts to reduce the burden of diseases and injuries that will strain resources in the coming decades. Funding: Bill &amp; Melinda Gates Foundation

Metabolic effects of the first non-peptide angiotensin II receptor type 2 agonist

Das Renin-Angiotensin-System (RAS) stellt eines der wichtigsten Systeme zur Regulation der kardiovaskulären Homöostase und des Blutdruckes des Körpers dar. Das Angiotensin II übt dabei seine Effekte über zwei verschiedene Rezeptor-Subtypen, den AT1-Rezptor (AT1R) und den AT2-Rezeptor (AT2R), aus. Die Rolle des AT1R in der Entwicklung der Insulinresistenz (IR) ist weitestgehend untersucht und es ist bekannt, dass AT1R-Blocker (ARBs) die Glukoseintoleranz und Insulinempfindlichkeit verbessern. Einige ARBs sind zusätzlich in der Lage, partiell den Peroxisom-Proliferator-Aktivierten- Rezeptor-gamma (PPARgamma) zu aktivieren, was zusätzliche deren anti- inflammatorische Eigenschaften erklären könnte. Über die metabolische Rolle des AT2R ist relativ wenig bekannt, wobei diese zudem teilweise konträr diskutiert wird. Die vorliegende Arbeit soll daher die funktionelle Signifikanz des AT2R in der Entwicklung der IR und der Fettgewebsinflammation aufklären. Hierfür wurde der erste nicht-peptidische AT2R-Agonist Compound 21 (C21) bei in-vitro Zellmodellen mit 3T3-L1 Adipozyten und THP-1 Makrophagen sowie bei einem Diät-induzierten Adipositas (DIO)-Modell der Maus eingesetzt. Die AT2R-Stimulation mit C21 (1 µM) reduzierte signifikant die TNF-alpha induzierte in-vitro Inflammation durch die Reduktion des IL-6 Proteins in differenzierten Adipozyten. Ausserdem wurde die Adiponektin Protein-Expression durch TNF-alpha Behandlung signifikant reduziert, während die Parallelbehandlung mit C21 die Spiegel des anti-inflammatorischen Adiponektins teilweise wiederherstellte. Die Effekte waren AT2R-spezifisch, da die Blockade des AT2R mit dem AT2R-Blocker PD123319 zum Verlust der C21-Effekte führte. Weiterhin führte die C21-Behandlung zu einer signifikanten Reduktion der basalen pro-inflammatorischen Leptin-Produktion sowie zu einer erhöhten anti- inflammatorischen IL-10 mRNA-Expression. Dabei besaß C21 (0,1 bis 1 µM) keinen Einfluss auf die PPARgamma-Aktivität und induzierte folglich auch nicht die Adipozytendifferenzierung. Die THP-1 Makrophagen wurden für die in-vitro Inflammation mit LPS behandelt. Dabei führte die Parallelbehandlung mit C21 zu einer signifikanten Reduktion von IL-6. Der Effekt blieb unter AT2-Blockade mit PD123319 aus. Weiterhin erhöhte C21 das basale IL-10 und reduzierte die LPS-induzierte MCP-1 mRNA-Expression. Für das DIO-Modell wurden Wild-Typ (WT; C57Bl-6) und AT2R-Knockout (AT2R-KO) Mäuse entweder mit einer Hochfett-Diät (HFD) oder einer Kontroll-Niedrigfett-Diät (LFD) (60% kcal aus Fett bzw. 10% kcal aus Fett) zur Induktion einer Adipositas und metabolischer Veränderungen über 10 Wochen gefüttert. Anschließend wurden die Tiere (n=10 pro Gruppe) nach folgendem Protokoll zusätzlich zur Diät über 4 Wochen behandelt: Der AT2R- Agonist C21 (0,3 mg/kg KG i.p.), der ARB Valsartan (3 mg/kg KG i.p.), die Kombination C21 / Valsartan (0,3 / 3 mg/kg KG i.p.), das Antihypertonikum Hydralazin (250 mg/L im Trinkwasser) oder Vehikel. Die Glukosetoleranz (GT) und Insulinempfindlichkeit (IS) wurde mit Standard-GTTs und -ITTs bestimmt. Die HFD reduzierte signifikant die GT und IS, wobei diese jeweils durch die C21-Behandlung signifikant verbessert werden konnten. Die GT wurde ebenfalls durch die Valsartan-Behandlung verbessert. Des Weiteren führte die C21-Behandlung zu einer signifikanten Reduktion von TNF-alpha, Resistin und der Triglyzeride im Serum sowie zu einer Erhöhung der insulin-sensitivierenden Inkretine GLP-1 und GIP. In Übereinstimmung mit den in-vitro Ergebnissen wurden ebenfalls die Adiponektin und IL-10 Serumspiegel durch C21-Behandlung jeweils signifikant erhöht bzw. die Leptin-Spiegel signifikant reduziert. Dabei waren die Effekte AT2R-spezifisch, denn alle Effekte konnten nicht in den AT2R-KO Mäusen beobachtet werden. Die anti-inflammatorischen Effekte von C21 konnten zudem lokal im Fettgewebe der Mäuse bestätig werden. C21 reduzierte leicht und Valsartan stark den Blutdruck der HFD-Gruppen, während die Effekte in den AT2R-Tieren abgeschwächt waren. Die metabolischen Effekte schienen dabei Blutdruck-unabhängig zu sein, wie durch die Hydralazin- Kontrolle ermittelt werden konnte. Die Ergebnisse der vorliegenden Arbeit zeigen auf, dass die selektive und direkte AT2R-Stimulation zu anti- inflammatorischen Effekten sowie zu einer positiven Modulation von metabolischen Parametern sowohl im DIO-Modell an Mäusen als auch in entsprechenden in-vitro Experimenten an Adipozyten und Makrophagen führte. Demnach deuten diese Ergebnisse sehr stark darauf hin, dass der AT2R eine wichtige positive metabolische Funktion ausübt und ein mögliches pharmakologisches Ziel für die Behandlung von Adipositas-induzierten Erkrankungen darstellen könnte. Hierfür könnten AT2R-Agonisten in Zukunft eine Therapieoption bieten.The renin-angiotensin-system (RAS) is one oft the most important regulator of cardiovascular homeostasis and blood pressure in the body. Angiotensin II exerts its effects through two different receptor subtypes: the AT1-receptor (AT1R) and the AT2-receptor (AT2R). The functional role of the AT1R in the development of insulin-resistance (IR) is well understood and it is known that AT1R-blockers (ARBs) improve glucose-intolerance and IR. Some of the ARBs were shown to activate the peroxisome-proliferator-activated-receptor-gamma (PPARgamma) partially, which may explain their anti-inflammatory properties. The metabolic contribution of the AT2R is still not clear and in parts controversial. Thus, this study aimed to determine the functional significance of the AT2R for the development of IR and adipose-tissue inflammation using the first non-peptide AT2R-agonist Compound 21 (C21) in in-vitro cell-models with 3T3-L1 adipocytes and THP-1 macrophages as well as in a diet-induced obesity (DIO) mouse model. AT2R-stimulation with C21 (1 µM) significantly reduced TNF-alpha induced in-vitro inflammation by IL-6 protein-reduction in mature adipocytes. Anti-inflammatory adiponectin protein-expression was strongly reduced in adipocytes by TNF-alpha treatment, but partly restored by co-treatment with C21. These effects were due to specific AT2R-activation, since treatment with the AT2R-antagonist PD123319 (10 µM) abolished the effect of C21. Treatment with C21 also led to a significant reduction of basal pro- inflammatory leptin-production and increased anti-inflammatory IL-10 mRNA- expression in adipocytes. Importantly, C21 (0,1 to 1 µM) did not induce PPARgamma-activity and consequently did not induce adipocyte differentiation. THP-1 macrophages were treated with LPS for in-vitro inflammation. Co- treatment with C21 significantly repressed LPS-induced IL-6 protein. This effect was again abolished by PD123319 treatment. C21 treatment also increased basal IL-10 and decreased LPS-induced MCP-1 mRNA-expression. For the DIO-model wildtype (WT; C57Bl-6) and AT2R-knockout (AT2R-KO) mice were fed with high fat diet (HFD) or control low fat diet (LFD) (60% kcal from fat or 10% kcal from fat, respectively) for 10 weeks to induce obesity and metabolic changes. Afterwards animals (n=10 per group) were treated according to the following protocol for 4 weeks in addition to the diet: The AT2R-agonist C21 (0.3 mg/kg body weight (BW) i.p.), the ARB Valsartan (3 mg/kg BW i.p.), the combination C21 / Valsartan (0.3 / 3 mg/kg BW i.p.), the antihypertensive agent Hydralazine (250 mg/l drinking water) or vehicle. Glucose tolerance (GT) und insulin sensitivity (IS) were measured by standard ITT and GTT tests. GT and IS were impaired by HFD-feeding but significantly improved in mice treated with C21. GT was also improved by Valsartan. Furthermore, TNF-alpha, resistin and serum triglycerides levels were significantly reduced, and serum levels of insulin-sensitizing incretins GLP-1 and GIP were increased by C21-treatment. Consistent with the in-vitro data, C21 treatment increased adiponectin as well as IL-10, and decreased leptin serum levels in a significant manner. Importantly, all effects were not observed in AT2R-KO animals treated with C21 pointing to the AT2R-specificity of these effects. Additionally the anti- inflammatory effects of C21 could be confirmed locally in the adipose-tissue of the mice. C21 slightly and Valsartan strongly lowered blood-pressure in mice of the HFD groups, while these effects were attenuated in the AT2R-KO animals. However, metabolic effects of C21 and Valsartan were blood pressure- independent as controlled for by the Hydralazine treated group. The present study demonstrates that selective and direct AT2R-stimulation results in anti- inflammatory actions as well as positive modulation of metabolic markers in a DIO-model in mice and related in-vitro experiments in adipocytes and macrophages. Thus, these data strongly indicate that the AT2R has an important positive metabolic function suggesting that the AT2R might be a pharmacological target for treatment of obesity-induced metabolic diseases. AT2R-agonists may be a theurapeutic option in the future

Voice in Parkinson's Disease: A Machine Learning Study

Introduction: Parkinson's disease (PD) is characterized by specific voice disorders collectively termed hypokinetic dysarthria. We here investigated voice changes by using machine learning algorithms, in a large cohort of patients with PD in different stages of the disease, OFF and ON therapy. Methods: We investigated 115 patients affected by PD (mean age: 68.2 ± 9.2 years) and 108 age-matched healthy subjects (mean age: 60.2 ± 11.0 years). The PD cohort included 57 early-stage patients (Hoehn &amp;Yahr ≤ 2) who never took L-Dopa for their disease at the time of the study, and 58 mid-advanced-stage patients (Hoehn &amp;Yahr &gt;2) who were chronically-treated with L-Dopa. We clinically evaluated voices using specific subitems of the Unified Parkinson's Disease Rating Scale and the Voice Handicap Index. Voice samples recorded through a high-definition audio recorder underwent machine learning analysis based on the support vector machine classifier. We also calculated the receiver operating characteristic curves to examine the diagnostic accuracy of the analysis and assessed possible clinical-instrumental correlations. Results: Voice is abnormal in early-stage PD and as the disease progresses, voice increasingly degradres as demonstrated by high accuracy in the discrimination between healthy subjects and PD patients in the early-stage and mid-advanced-stage. Also, L-dopa therapy improves but not restore voice in PD as shown by high accuracy in the comparison between patients OFF and ON therapy. Finally, for the first time we achieved significant clinical-instrumental correlations by using a new score (LR value) calculated by machine learning. Conclusion: Voice is abnormal in early-stage PD, progressively degrades in mid-advanced-stage and can be improved but not restored by L-Dopa. Lastly, machine learning allows tracking disease severity and quantifying the symptomatic effect of L-Dopa on voice parameters with previously unreported high accuracy, thus representing a potential new biomarker of PD

ERα and lipolysis.

<p>A: Ex-vivo lipolysis assay in murine gonadal-AT explants from wild-type (WT) and estrogen receptor alpha knock out mice (KO) expressed as percent of WT FFA-release after stimulation with forskolin. Bonferroni posttest showed a significant difference between WT and KO in females [n = 4−5 mice/group, two-way ANOVA]. <b>B</b>: Analysis of ERα mRNA expression in gonadal-AT from female/male mice before/after weight reduction. Data are presented as <i>x</i>-fold of females (DIO) [n = 9−10 mice/group, two-way ANOVA]. The black/dark grey columns and symbols represent male mice; white/light grey: females. DIO: before weight reduction, –20%: after weight reduction. *p≤0.05 DIO vs. -20% or WT vs. KO.</p

Animal model.

<p>A: Scheme of the feeding protocol to induce body weight changes (DIO =  diet-induced obesity). <b>B</b>: Original BW data of female/male mice throughout the feeding protocol.</p

Weight maintenance and regain.

<p>A: Stability of body weight of female/male mice during 16 days of adaptive feeding. Shown are the means ±SEM of body weight, measured daily. [n = 10 mice/group] <b>B</b>: During adaptive feeding the amount of food was individually adapted to maintain the target weight over 16 days. Shown are the mean amount of given food ±SEM normalized to the BW of female/male mice [n = 10 mice/group, two-way ANOVA with repeated measures] (factor interaction: p_sex/time<0.001). <b>C</b>: Sex-specific differences during weight regain expressed as percent change of body weight before re-feeding. Shown is the BW-development in female and male mice during 6 weeks ad libitum re-feeding. [n = 10 mice/group, two-way ANOVA with repeated measures] (factor interaction: p_sex/time<0.05). Black symbols represent male mice; white: females. *p≤0.05; **p≤0.01; # p<0.001 vs. other sex.</p

Weight reduction phase.

<p>A: Loss of BW in female/male mice during restricted feeding, expressed as percent of DIO-BW. Body weight target: −20% of DIO-BW, for details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037794#s2" target="_blank">method</a> section [n = 10 mice/group, two-way ANOVA with repeated measures] (factor interaction: p_sex/time<0.001). <b>B</b>: Change of lean-mass specific EE measured in female/male mice before weight loss and during caloric restriction. Shown is the mean over 23 h measurement [n = 10 mice/group, two-way ANOVA] (factor interaction: p_{sex/weight loss}<0.001). <b>C</b>: Total locomotor activity of female/male mice before weight loss and during CR. Shown is total activity during 23 h monitoring [n = 10 mice/group, two-way ANOVA]. <b>D</b>: Analysis of body composition (lean and fat mass) in female/male mice calculated as percent reduction (delta: DIO-mass and mass after weight reduction). Lean and fat mass were analyzed separately [n = 10 mice/group, unpaired <i>t</i>-test]. <b>E</b>: Gonadal fat mass as percent of total fat mass in female/male mice before/after weight reduction [n = 10 mice/group, two-way ANOVA] (factor interaction: p_{sex/weight loss}<0.05). <b>F</b>: Liver triglycerides measured in female/male mice before/after weight reduction [n = 8 mice/group, two-way ANOVA]. Black and dark grey columns/symbols represent male mice; white and light grey = female mice. DIO = diet-induced obesity or before weight reduction; restriction: during restrictive feeding phase; −20%: after weight reduction, at target weight. *p≤0.05; **p≤0.01; # p≤0.001 DIO vs. −20% or vs. other sex.</p