A small erythropoietin derived non-hematopoietic peptide reduces cardiac inflammation, attenuates age associated declines in heart function and prolongs healthspan.

Background: Aging is associated with increased levels of reactive oxygen species and inflammation that disrupt proteostasis and mitochondrial function and leads to organism-wide frailty later in life. ARA290 (cibinetide), an 11-aa non-hematopoietic peptide sequence within the cardioprotective domain of erythropoietin, mediates tissue protection by reducing inflammation and fibrosis. Age-associated cardiac inflammation is linked to structural and functional changes in the heart, including mitochondrial dysfunction, impaired proteostasis, hypertrophic cardiac remodeling, and contractile dysfunction. Can ARA290 ameliorate these age-associated cardiac changes and the severity of frailty in advanced age? Methods: We conducted an integrated longitudinal (n = 48) and cross-sectional (n = 144) 15 months randomized controlled trial in which 18-month-old Fischer 344 x Brown Norway rats were randomly assigned to either receive chronic ARA290 treatment or saline. Serial echocardiography, tail blood pressure and body weight were evaluated repeatedly at 4-month intervals. A frailty index was calculated at the final timepoint (33 months of age). Tissues were harvested at 4-month intervals to define inflammatory markers and left ventricular tissue remodeling. Mitochondrial and myocardial cell health was assessed in isolated left ventricular myocytes. Kaplan-Meier survival curves were established. Mixed ANOVA tests and linear mixed regression analysis were employed to determine the effects of age, treatment, and age-treatment interactions. Results: Chronic ARA290 treatment mitigated age-related increases in the cardiac non-myocyte to myocyte ratio, infiltrating leukocytes and monocytes, pro-inflammatory cytokines, total NF-κB, and p-NF-κB. Additionally, ARA290 treatment enhanced cardiomyocyte autophagy flux and reduced cellular accumulation of lipofuscin. The cardiomyocyte mitochondrial permeability transition pore response to oxidant stress was desensitized following chronic ARA290 treatment. Concurrently, ARA290 significantly blunted the age-associated elevation in blood pressure and preserved the LV ejection fraction. Finally, ARA290 preserved body weight and significantly reduced other markers of organism-wide frailty at the end of life. Conclusion: Administration of ARA290 reduces cell and tissue inflammation, mitigates structural and functional changes within the cardiovascular system leading to amelioration of frailty and preserved healthspan.

Empirical versus theoretical power and type I error (false-positive) rates estimated from real murine aging research data.

We assess the degree of phenotypic variation in a cohort of 24-month-old male C57BL/6 mice. Because murine studies often use small sample sizes, if the commonly relied upon assumption of a normal distribution of residuals is not met, it may inflate type I error rates. In this study, 3-20 mice are resampled from the empirical distributions of 376 mice to create plasmodes, an approach for computing type I error rates and power for commonly used statistical tests without assuming a normal distribution of residuals. While all of the phenotypic and metabolic variables studied show considerable variability, the number of animals required to achieve adequate power is markedly different depending on the statistical test being performed. Overall, this work provides an analysis with which researchers can make informed decisions about the sample size required to achieve statistical power from specific measurements without a priori assumptions of a theoretical distribution.

A high-throughput screen for TMPRSS2 expression identifies FDA-approved compounds that can limit SARS-CoV-2 entry.

SARS-CoV-2 (2019-nCoV) is the pathogenic coronavirus responsible for the global pandemic of COVID-19 disease. The Spike (S) protein of SARS-CoV-2 attaches to host lung epithelial cells through the cell surface receptor ACE2, a process dependent on host proteases including TMPRSS2. Here, we identify small molecules that reduce surface expression of TMPRSS2 using a library of 2,560 FDA-approved or current clinical trial compounds. We identify homoharringtonine and halofuginone as the most attractive agents, reducing endogenous TMPRSS2 expression at sub-micromolar concentrations. These effects appear to be mediated by a drug-induced alteration in TMPRSS2 protein stability. We further demonstrate that halofuginone modulates TMPRSS2 levels through proteasomal-mediated degradation that involves the E3 ubiquitin ligase component DDB1- and CUL4-associated factor 1 (DCAF1). Finally, cells exposed to homoharringtonine and halofuginone, at concentrations of drug known to be achievable in human plasma, demonstrate marked resistance to SARS-CoV-2 infection in both live and pseudoviral in vitro models. Given the safety and pharmacokinetic data already available for the compounds identified in our screen, these results should help expedite the rational design of human clinical trials designed to combat active COVID-19 infection.

Modulation of lysosomal function as a therapeutic approach for coronaviral infections.

The endo-lysosomal pathway plays an important role in pathogen clearance and both bacteria and viruses have evolved complex mechanisms to evade this host system. Here, we describe a novel aspect of coronaviral infection, whereby the master transcriptional regulator of lysosome biogenesis - TFEB - is targeted for proteasomal-mediated degradation upon viral infection. Through mass spectrometry analysis and an unbiased siRNA screen, we identify that TFEB protein stability is coordinately regulated by the E3 ubiquitin ligase subunit DCAF7 and the PAK2 kinase. In particular, viral infection triggers marked PAK2 activation, which in turn, phosphorylates and primes TFEB for ubiquitin-mediated protein degradation. Deletion of either DCAF7 or PAK2 blocks viral-mediated TFEB degradation and protects against viral-induced cytopathic effects. We further derive a series of small molecules that interfere with the DCAF7-TFEB interaction. These agents inhibit viral-triggered TFEB degradation and demonstrate broad anti-viral activities including attenuating in vivo SARS-CoV-2 infection. Together, these results delineate a viral-triggered pathway that disables the endogenous cellular system that maintains lysosomal function and suggest that small molecule inhibitors of the E3 ubiquitin ligase DCAF7 represent a novel class of endo-lysosomal, host-directed, anti-viral therapies.

A cross-sectional study of functional and metabolic changes during aging through the lifespan in male mice.

Aging is associated with distinct phenotypical, physiological, and functional changes, leading to disease and death. The progression of aging-related traits varies widely among individuals, influenced by their environment, lifestyle, and genetics. In this study, we conducted physiologic and functional tests cross-sectionally throughout the entire lifespan of male C57BL/6N mice. In parallel, metabolomics analyses in serum, brain, liver, heart, and skeletal muscle were also performed to identify signatures associated with frailty and age-dependent functional decline. Our findings indicate that declines in gait speed as a function of age and frailty are associated with a dramatic increase in the energetic cost of physical activity and decreases in working capacity. Aging and functional decline prompt organs to rewire their metabolism and substrate selection and toward redox-related pathways, mainly in liver and heart. Collectively, the data provide a framework to further understand and characterize processes of aging at the individual organism and organ levels.

Mitochondrial health is enhanced in rats with higher vs. lower intrinsic exercise capacity and extended lifespan.

The intrinsic aerobic capacity of an organism is thought to play a role in aging and longevity. Maximal respiratory rate capacity, a metabolic performance measure, is one of the best predictors of cardiovascular- and all-cause mortality. Rats selectively bred for high-(HCR) vs. low-(LCR) intrinsic running-endurance capacity have up to 31% longer lifespan. We found that positive changes in indices of mitochondrial health in cardiomyocytes (respiratory reserve, maximal respiratory capacity, resistance to mitochondrial permeability transition, autophagy/mitophagy, and higher lipids-over-glucose utilization) are uniformly associated with the extended longevity in HCR vs. LCR female rats. Cross-sectional heart metabolomics revealed pathways from lipid metabolism in the heart, which were significantly enriched by a select group of strain-dependent metabolites, consistent with enhanced lipids utilization by HCR cardiomyocytes. Heart-liver-serum metabolomics further revealed shunting of lipidic substrates between the liver and heart via serum during aging. Thus, mitochondrial health in cardiomyocytes is associated with extended longevity in rats with higher intrinsic exercise capacity and, probably, these findings can be translated to other populations as predictors of outcomes of health and survival.

Study of Longitudinal Aging in Mice: Presentation of Experimental Techniques.

Aging is associated with functional and metabolic decline and is a risk factor for all noncommunicable diseases. Even though mice are routinely used for modeling human aging and aging-related conditions, no comprehensive assessment to date has been conducted on normative mouse aging. To address this gap, the Study of Longitudinal Aging in Mice (SLAM) was designed and implemented by the National Institute on Aging (NIA/NIH) as the mouse counterpart to the Baltimore Longitudinal Study of Aging (BLSA). In this manuscript, we describe the premise, study design, methodologies, and technologies currently employed in SLAM. We also discuss current and future study directions. In this large population mouse study, inbred C57BL/6J and outbred UM-HET3 mice of both sexes are longitudinally evaluated for functional, phenotypic, and biological health, and collection of biospecimens is conducted throughout their life span. Within the longitudinal cohorts, a cross-sectional arm of the study has also been implemented for the well-controlled collection of tissues to generate a biorepository. SLAM and studies stemming from SLAM seek to identify and characterize phenotypic and biological predictors of mouse aging and age-associated conditions, examine the degrees of functional and biomolecular variability that occur within inbred and genetically heterogeneous mouse populations with age, and assess whether these changes are consistent with alterations observed in human aging in BLSA. The findings from these studies will be critical for evaluating the utility of mouse models for studying different aspects of aging, both in terms of interpreting prior findings and designing and implementing future studies.

A high throughput screen for TMPRSS2 expression identifies FDA-approved and clinically advanced compounds that can limit SARS-CoV-2 entry.

SARS-CoV-2 (2019-nCoV) is the pathogenic coronavirus responsible for the global pandemic of COVID-19 disease. The Spike (S) protein of SARS-CoV-2 attaches to host lung epithelial cells through the cell surface receptor ACE2, a process dependent on host proteases including TMPRSS2. Here, we identified small molecules that can reduce surface expression of TMPRSS2 using a 2,700 FDA-approved or current clinical trial compounds. Among these, homoharringtonine and halofuginone appear the most potent agents, reducing endogenous TMPRSS2 expression at sub-micromolar concentrations. These effects appear to be mediated by a drug-induced alteration in TMPRSS2 protein stability. We further demonstrate that halofuginone modulates TMPRSS2 levels through proteasomal-mediated degradation that involves the E3 ubiquitin ligase component DDB1- and CUL4-associated factor 1 (DCAF1). Finally, cells exposed to homoharringtonine and halofuginone, at concentrations of drug known to be achievable in human plasma, demonstrated marked resistance to SARS-CoV-2 pseudoviral infection. Given the safety and pharmacokinetic data already available for the compounds identified in our screen, these results should help expedite the rational design of human clinical trials designed to combat COVID-19 infection.

Muscle cannabinoid 1 receptor regulates Il-6 and myostatin expression, governing physical performance and whole-body metabolism.

Sarcopenic obesity, the combination of skeletal muscle mass and function loss with an increase in body fat, is associated with physical limitations, cardiovascular diseases, metabolic stress, and increased risk of mortality. Cannabinoid receptor type 1 (CB1R) plays a critical role in the regulation of whole-body energy metabolism because of its involvement in controlling appetite, fuel distribution, and utilization. Inhibition of CB1R improves insulin secretion and insulin sensitivity in pancreatic ß-cells and hepatocytes. We have now developed a skeletal muscle-specific CB1R-knockout (Skm-CB1R-/-) mouse to study the specific role of CB1R in muscle. Muscle-CB1R ablation prevented diet-induced and age-induced insulin resistance by increasing IR signaling. Moreover, muscle-CB1R ablation enhanced AKT signaling, reducing myostatin expression and increasing IL-6 secretion. Subsequently, muscle-CB1R ablation increased myogenesis through its action on MAPK-mediated myogenic gene expression. Consequently, Skm-CB1R-/- mice had increased muscle mass and whole-body lean/fat ratio in obesity and aging. Muscle-CB1R ablation improved mitochondrial performance, leading to increased whole-body muscle energy expenditure and improved physical endurance, with no change in body weight. These results collectively show that CB1R in muscle is sufficient to regulate whole-body metabolism and physical performance and is a novel target for the treatment of sarcopenic obesity. -González-Mariscal, I., Montoro, R. A., O'Connell, J. F., Kim, Y., Gonzalez-Freire, M., Liu, Q.-R., Alfaras, I., Carlson, O. D., Lehrmann, E., Zhang, Y., Becker, K. G., Hardivillé, S., Ghosh, P., Egan, J. M. Muscle cannabinoid 1 receptor regulates Il-6 and myostatin expression, governing physical performance and whole-body metabolism.

Of Aging Mice and Men: Gait Speed Decline Is a Translatable Trait, With Species-Specific Underlying Properties.

In the last two decades, great strides were made in our ability to extend the life span of model organisms through dietary and other manipulations. Survival curves provide evidence of altered aging processes but are uninformative on what lead to that increase in life span. Longitudinal assessments of health and function during intervention studies could help in the identification of predictive biomarkers for health and survival. Comparable biomarkers of healthspan are necessary to effectively translate interventions into human clinical trials. Gait speed is a well-established predictive biomarker of healthspan in humans for risk of disability, health outcomes and mortality, and is relatively simple to assess noninvasively in rodents. In this study, we assessed and compared gait speed in males from two species (mice and humans), from young adulthood to advanced old age. Although gait speed decreases nonlinearly with age in both species, the underlying drivers of this change in gait speed were different, with humans exhibiting a shortened step length, and mice displaying a decrease in cadence. Future longitudinal and interventional studies in mice should examine the predictive value of longitudinal declines in gait speed for health and survival.

Health benefits of late-onset metformin treatment every other week in mice.

Chronic 1% metformin treatment is nephrotoxic in mice, but this dose may nonetheless confer health benefits if given intermittently rather than continuously. Here, we examined the effects of 1% metformin given every-other week (EOW) or two consecutive weeks per month (2WM) on survival of 2-year-old male mice fed standard chow. EOW and 2WM mice had comparable life span compared with control mice. A significant reduction in body weight within the first few weeks of metformin treatment was observed without impact on food consumption and energy expenditure. Moreover, there were differences in the action of metformin on metabolic markers between the EOW and 2WM groups, with EOW metformin conferring greater benefits. Age-associated kidney lesions became more pronounced with metformin, although without pathological consequences. In the liver, metformin treatment led to an overall reduction in steatosis and was accompanied by distinct transcriptomic and metabolomic signatures in response to EOW versus 2WM regimens. Thus, the absence of adverse outcomes associated with chronic, intermittent use of 1% metformin in old mice has clinical translatability into the biology of aging in humans.

Pharmacological Strategies to Retard Cardiovascular Aging.

Aging is the major risk factor for cardiovascular diseases, which are the leading cause of death in the United States. Traditionally, the effort to prevent cardiovascular disease has been focused on addressing the conventional risk factors, including hypertension, hyperglycemia, hypercholesterolemia, and high circulating levels of triglycerides. However, recent preclinical studies have identified new approaches to combat cardiovascular disease. Calorie restriction has been reproducibly shown to prolong lifespan in various experimental model animals. This has led to the development of calorie restriction mimetics and other pharmacological interventions capable to delay age-related diseases. In this review, we will address the mechanistic effects of aging per se on the cardiovascular system and focus on the prolongevity benefits of various therapeutic strategies that support cardiovascular health.

Resveratrol supplementation confers neuroprotection in cortical brain tissue of nonhuman primates fed a high-fat/sucrose diet.

Previous studies have shown positive effects of long-term resveratrol (RSV) supplementation in preventing pancreatic beta cell dysfunction, arterial stiffening and metabolic decline induced by high-fat/high-sugar (HFS) diet in nonhuman primates. Here, the analysis was extended to examine whether RSV may reduce dietary stress toxicity in the cerebral cortex of the same cohort of treated animals. Middle-aged male rhesus monkeys were fed for 2 years with HFS alone or combined with RSV, after which whole-genome microarray analysis of cerebral cortex tissue was carried out along with ELISA, immunofluorescence, and biochemical analyses to examine markers of vascular health and inflammation in the cerebral cortices. A number of genes and pathways that were differentially modulated in these dietary interventions indicated an exacerbation of neuroinflammation (e.g., oxidative stress markers, apoptosis, NF-κB activation) in HFS-fed animals and protection by RSV treatment. The decreased expression of mitochondrial aldehyde dehydrogenase 2, dysregulation in endothelial nitric oxide synthase, and reduced capillary density induced by HFS stress were rescued by RSV supplementation. Our results suggest that long-term RSV treatment confers neuroprotection against cerebral vascular dysfunction during nutrient stress.

GH Receptor Deficiency in Ecuadorian Adults Is Associated With Obesity and Enhanced Insulin Sensitivity.

CONTEXT: Ecuadorian subjects with GH receptor deficiency (GHRD) have not developed diabetes, despite obesity. OBJECTIVE: We sought to determine the metabolic associations for this phenomenon. DESIGN: Four studies were carried out: 1) glucose, lipid, adipocytokine concentrations; 2) metabolomics evaluation; 3) metabolic responses to a high-calorie meal; and 4) oral glucose tolerance tests. SETTING: Clinical Research Institute in Quito, Ecuador. SUBJECTS: Adults homozygous for the E180 splice mutation of the GH receptor (GHRD) were matched for age, gender, and body mass index with unaffected control relatives (C) as follows: study 1, 27 GHRD and 35 C; study 2, 10 GHRD and 10 C; study 3, seven GHRD and 11 C; and study 4, seven GHRD and seven C. RESULTS: Although GHRD subjects had greater mean percentage body fat than controls, their fasting insulin, 2-hour blood glucose, and triglyceride levels were lower. The indicator of insulin sensitivity, homeostasis model of assessment 2%S, was greater (P < .0001), and the indicator of insulin resistance, homeostasis model of assessment 2-IR, was lower (P = .0025). Metabolomic differences between GHRD and control subjects were consistent with their differing insulin sensitivity, including postprandial decreases of branched-chain amino acids that were more pronounced in controls. High molecular weight and total adiponectin concentrations were greater in GHRD (P = .0004 and P = .0128, respectively), and leptin levels were lower (P = .02). Although approximately 65% the weight of controls, GHRD subjects consumed an identical high-calorie meal; nonetheless, their mean glucose concentrations were lower, with mean insulin levels one-third those of controls. Results of the 2-hour oral glucose tolerance test were similar. MAIN OUTCOME MEASURES: Measures of insulin sensitivity, adipocytokines, and energy metabolites. CONCLUSIONS: Without GH counter-regulation, GHRD is associated with insulin efficiency and obesity. Lower leptin levels, despite higher percentage body fat, suggest that obesity-associated leptin resistance is GH dependent. Elevated adiponectin levels not correlated with percentage body fat indicate that GH signaling is necessary for their typical suppression with obesity.

The bioavailability and distribution of trans-resveratrol are constrained by ABC transporters.

The ABC proteins are a family of membrane transporters that mediates the extrusion from cells of a wide variety of structurally unrelated substrates. The current review focuses on the role of these efflux pumps located in the intestine on the low oral bioavailability of trans-resveratrol. The enterocytes hold in the apical membrane three transporters, namely, P-glycoprotein (P-gp), multidrug resistance associated protein 2 (MRP2) and breast cancer resistance protein (BCRP), whereas the basolateral membrane contains multidrug resistance associated protein 3 (MRP3). The use of different specific inhibitors of these transporters as well as knockout mice enabled us to conclude that MRP2 and BCRP are involved in the extrusion of trans-resveratrol glucuronide and sulfate to the intestinal lumen without the participation of P-gp. The role of these transporters as a bottleneck in the absorption of trans-resveratrol cannot be undervalued affecting not only the bioavailability of its glucuronide and sulfate but also their distribution in the different organs.

Colorectal cancer chemoprevention by trans-resveratrol.

trans-Resveratrol (trans-3,4',5-trihydroxystilbene) is a natural phytoalexin present in grapes, red wine, berries and peanuts with health protecting properties. The low oral bioavailability indicated for this polyphenol, with the intestine as a bottleneck to its absorption, has promoted the large intestine as a potential target site for its chemopreventive activity. This review recapitulates the current evidence of the effects of trans-resveratrol on colon cancer. First, we describe the studies conducted in vitro which show that the protective activity takes place by inhibition of proliferation and induction of apoptosis. Secondly, the chemopreventive activity in animal models of colon carcinogenesis is revised. trans-Resveratrol not only reduces the number of preneoplastic lesions but also the incidence and multiplicity of tumors. Lastly, the article also reviews the available data on clinical trials. Altogether, the present findings support the hypothesis that the oral administration of trans-resveratrol might contribute to the prevention of colon carcinogenesis.

Population pharmacokinetic modeling of trans-resveratrol and its glucuronide and sulfate conjugates after oral and intravenous administration in rats.

PURPOSE: To develop a population pharmacokinetic (PK) model which allowed the simultaneous modeling of trans-resveratrol and its glucuronide and sulfate conjugates. METHODS: Male Sprague-Dawley rats were administered i.v. and p.o. with 2, 10 and 20 mg·kg(-1) of trans-resveratrol. Blood was collected at different times during 24 h. An integrated PK model was developed using a sequential analysis, with non-linear mixed effect modeling (NONMEM). A prediction-corrected visual predictive check (pcVPC) was used to assess model performance. The model predictive capability was also evaluated with simulations after the i.v. administration of 15 mg·kg(-1) that were compared with an external data set. RESULTS: Disposition PK of trans-resveratrol and its metabolites was best described by a three-linked two-compartment model. Clearance of trans-resveratrol by conversion to its conjugates occurred by a first-order process, whereas both metabolites were eliminated by parallel first-order and Michaelis-Menten kinetics. The pcVPC confirmed the model stability and precision. The final model was successfully applied to the external data set showing its robustness. CONCLUSIONS: A robust population PK model has been built for trans-resveratrol and its glucuronide and sulfate conjugates that adequately predict plasmatic concentrations.

trans-Resveratrol reduces precancerous colonic lesions in dimethylhydrazine-treated rats.

trans-Resveratrol, a natural occurring polyphenol, has been described as an antiproliferative and proapoptotic agent in vitro. Here, we studied the effect of trans-resveratrol administered orally at a dose of 60 mg/kg for 49 days on early preneoplastic markers induced by the intraperitoneal injection of 1,2-dimethylhydrazine (20 mg/kg). We measured trans-resveratrol and its derivates by liquid-liquid extraction followed by high-performance liquid chromatography diode array detection analysis in colon contents. Dihydroresveratrol was the most abundant compound in the colon, followed by trans-resveratrol glucuronide and small amounts of trans-resveratrol and its sulfate. The administration of trans-resveratrol decreased aberrant crypt foci by 52%, and mucin depleted foci by 45% in colon. In conclusion, the correlation between the reduction of precancerous colonic lesions and the availability of trans-resveratrol in the colon provides a new insight into the therapeutical potential of this polyphenol and its metabolites.

Determination of dihydroresveratrol in rat plasma by HPLC.

Dihydroresveratrol is a metabolite of trans-resveratrol formed in the intestine by the hydrogenation of the double bond by microflora. The aim of the present study was to validate a method to measure dihydroresveratrol in rat plasma and then to quantify its plasmatic concentration after the oral administration of 60 mg/kg to Sprague-Dawley rats. Dihydroresveratrol was extracted from acidified plasma with a C18 cartridge, eluted with methanol, and concentrated prior to HPLC analysis with diode-array detection (HPLC-DAD) at 276 nm. The method was validated by spiking blank plasma samples with pure dihydroresveratrol, obtaining a linear correlation and good interday and intraday precisions, expressed as coefficient of variation (<7%). The average recovery was 96.7% and the limit of detection was 275 nM. The oral administration of dihydroresveratrol to rats and its subsequent detection, along with dihydroresveratrol glucuronide and sulfate, provides evidence of its absorption and metabolism.

Involvement of breast cancer resistance protein (BCRP1/ABCG2) in the bioavailability and tissue distribution of trans-resveratrol in knockout mice.

trans-Resveratrol undergoes extensive metabolism in the intestinal cells, which leads to the formation of glucuronide and sulfate conjugates. Given the important role of the breast cancer resistance protein (ABCG2/BCRP) in the efflux of conjugated forms, the present study investigates the bioavailability and tissue distribution of trans-resveratrol and its metabolites after the oral administration of 60 mg/kg in Bcrp1(-/-) mice. trans-Resveratrol and its metabolites were measured in intestinal content, plasma and tissues by HPLC. At 30 min after administration, intestinal content showed decreases of 71% and 97% of resveratrol glucuronide and sulfate, respectively, in Bcrp1(-/-), indicating a lower efflux from the enterocytes. Furthermore, the area under plasma concentration curves (AUC) of these metabolites increased by 34% and 392%, respectively, whereas a decrease in the AUC of trans-resveratrol was found. In conclusion, Bcrp1 plays an important role in the efflux of resveratrol conjugates, contributing to their bioavailability, tissue distribution and elimination.