Plant-based diets and the gut microbiome: findings from the Baltimore Longitudinal Study of Aging.

BACKGROUND: Mounting evidence indicates that although some plant-based diets are healthful, others are not. Changes in the gut microbiome and microbiome-dependent metabolites, such as trimethylamine N-oxide (TMAO), may explain differential health effects of plant-based diets. However, human data are sparse on whether qualitatively distinct types of plant-based diets differentially affect gut microbiome diversity, composition, particularly at the species level, and/or metabolites. OBJECTIVES: We aimed to examine cross-sectional associations of different plant-based indices with adult gut microbiome diversity, composition, and the metabolite TMAO. METHODS: We studied 705 adults in the Baltimore Longitudinal Study of Aging with data for diet, fecal microbiome (shotgun metagenomic sequencing), and key covariates. We derived healthful plant-based diet index (hPDI) and unhealthful plant-based diet index (uPDI) using data from food frequency questionnaires. We examined plant-based diet indices with microbiome α-diversity (richness and evenness measures), ß-diversity (Bray-Curtis and UniFrac measures), composition (species level), and plasma TMAO. We used regression models to determine associations before and after adjustment for age, sex, education, physical activity, smoking status, body mass index, and total energy intake. RESULTS: The analytic sample (mean age, 71.0 years, SD = 12.8 years) comprised 55.6% female and 67.5% non-Hispanic White participants. hPDI was positively and uPDI negatively associated with microbiome α-diversity, driven by microbial evenness (Pielou P < 0.05). hPDI was also positively associated with relative abundance of 3 polysaccharide-degrading bacterial species (Faecalibacterium prausnitzii, Eubacterium eligens, and Bacteroides thetaiotaomicron) and inversely associated with 6 species (Blautia hydrogenotrophica, Doreasp CAG 317, Eisenbergiella massiliensis, Sellimonas intestinalis, Blautia wexlerae, and Alistipes shahii). Furthermore, hPDI was inversely associated with TMAO. Associations did not differ by age, sex, or race. CONCLUSIONS: Greater adherence to a healthful plant-based diet is associated with microbiome features that have been linked to positive health; adherence to an unhealthful plant-based diet has opposing or null associations with these features.

Cross-sectional analysis of healthy individuals across decades: Aging signatures across multiple physiological compartments.

The study of age-related biomarkers from different biofluids and tissues within the same individual might provide a more comprehensive understanding of age-related changes within and between compartments as these changes are likely highly interconnected. Understanding age-related differences by compartments may shed light on the mechanism of their reciprocal interactions, which may contribute to the phenotypic manifestations of aging. To study such possible interactions, we carried out a targeted metabolomic analysis of plasma, skeletal muscle, and urine collected from healthy participants, age 22-92 years, and identified 92, 34, and 35 age-associated metabolites, respectively. The metabolic pathways that were identified across compartments included inflammation and cellular senescence, microbial metabolism, mitochondrial health, sphingolipid metabolism, lysosomal membrane permeabilization, vascular aging, and kidney function.

SARS-CoV-2 infection establishes a stable and age-independent CD8+ T cell response against a dominant nucleocapsid epitope using restricted T cell receptors.

The resolution of SARS-CoV-2 replication hinges on cell-mediated immunity, wherein CD8+ T cells play a vital role. Nonetheless, the characterization of the specificity and TCR composition of CD8+ T cells targeting non-spike protein of SARS-CoV-2 before and after infection remains incomplete. Here, we analyzed CD8+ T cells recognizing six epitopes from the SARS-CoV-2 nucleocapsid (N) protein and found that SARS-CoV-2 infection slightly increased the frequencies of N-recognizing CD8+ T cells but significantly enhanced activation-induced proliferation compared to that of the uninfected donors. The frequencies of N-specific CD8+ T cells and their proliferative response to stimulation did not decrease over one year. We identified the N222-230 peptide (LLLDRLNQL, referred to as LLL thereafter) as a dominant epitope that elicited the greatest proliferative response from both convalescent and uninfected donors. Single-cell sequencing of T cell receptors (TCR) from LLL-specific CD8+ T cells revealed highly restricted Vα gene usage (TRAV12-2) with limited CDR3α motifs, supported by structural characterization of the TCR-LLL-HLA-A2 complex. Lastly, transcriptome analysis of LLL-specific CD8+ T cells from donors who had expansion (expanders) or no expansion (non-expanders) after in vitro stimulation identified increased chromatin modification and innate immune functions of CD8+ T cells in non-expanders. These results suggests that SARS-CoV-2 infection induces LLL-specific CD8+ T cell responses with a restricted TCR repertoire.

Transcriptomes of human primary skin fibroblasts of healthy individuals reveal age-associated mRNAs and long noncoding RNAs.

Changes in the transcriptomes of human tissues with advancing age are poorly cataloged. Here, we sought to identify the coding and long noncoding RNAs present in cultured primary skin fibroblasts collected from 82 healthy individuals across a wide age spectrum (22-89 years old) who participated in the GESTALT (Genetic and Epigenetic Signatures of Translational Aging Laboratory Testing) study of the National Institute on Aging, NIH. Using high-throughput RNA sequencing and a linear regression model, we identified 1437 coding RNAs (mRNAs) and 1177 linear and circular long noncoding (lncRNAs) that were differentially abundant as a function of age. Gene set enrichment analysis (GSEA) revealed select transcription factors implicated in coordinating the transcription of subsets of differentially abundant mRNAs, while long noncoding RNA enrichment analysis (LncSEA) identified RNA-binding proteins predicted to participate in the age-associated lncRNA profiles. In summary, we report age-associated changes in the global transcriptome, coding and noncoding, from healthy human skin fibroblasts and propose that these transcripts may serve as biomarkers and therapeutic targets in aging skin.

Whole-genome methylation analysis of aging human tissues identifies age-related changes in developmental and neurological pathways.

Age-associated changes in the DNA methylation state can be used to assess the pace of aging. However, it is not understood what mechanisms drive these changes and whether these changes affect the development of aging phenotypes and the aging process in general. This study was aimed at gaining a more comprehensive understanding of aging-related methylation changes across the whole genome, and relating these changes to biological functions. It has been shown that skeletal muscle and blood monocytes undergo typical changes with aging. Using whole-genome bisulfite sequencing, we sought to characterize the genome-wide changes in methylation of DNA derived from both skeletal muscle and blood monocytes, and link these changes to specific genes and pathways through enrichment analysis. We found that methylation changes occur with aging at the locations enriched for developmental and neuronal pathways regulated in these two peripheral tissues. These results contribute to our understanding of changes in epigenome in human aging.

The gut microbiome and regional fat distribution: Findings from the Baltimore Longitudinal Study of Aging.

OBJECTIVE: The aim of this study was to examine associations of gut microbiome diversity and composition with directly measured regional fat distribution, including central fat, in a large community-based cohort. METHODS: A cross-sectional investigation was conducted in the Baltimore Longitudinal Study of Aging (N = 815, 55.2% female, 65.9% White). The fecal microbiome was assessed using whole-genome shotgun metagenomic sequencing, and trunk and leg fat was measured using dual x-ray absorptiometry. Multivariable-adjusted associations of regional fat measures, BMI, or waist circumference with microbiome alpha diversity metrics, microbiome beta diversity metrics, and species differential abundance (verified using two compositional statistical approaches) were examined. RESULTS: Trunk fat, leg fat, BMI, and waist circumference all significantly explained similar amounts of variance in microbiome structure. Differential abundance testing identified 11 bacterial species significantly associated with at least one measure of body composition or anthropometry. Ruminococcus gnavus was strongly and consistently associated with trunk fat mass, which is congruent with prior literature. CONCLUSIONS: Microbiome diversity and composition, in particular higher abundance of Ruminococcus gnavus, were associated with greater trunk fat, in addition to other measures of obesity. Longitudinal studies are needed to replicate these findings, and if replicated, randomized trials are needed to determine whether interventions targeting microbiome features such as abundance of Ruminococcus gnavus can lead to reductions in trunk fat and its metabolic sequelae.

Endocrinology of Taste with Aging.

Taste is one of our five primary senses, and taste impairment has been shown to increase with aging. The ability to taste allows us to enjoy the food we eat and to avoid foods that are potentially spoiled or poisonous. Recent advances in our understanding of the molecular mechanisms of taste receptor cells located within taste buds help us decipher how taste works. The discoveries of "classic" endocrine hormones in taste receptor cells point toward taste buds being actual endocrine organs. A better understanding of how taste works may help in reversing taste impairment associated with aging.

Quality Specific Associations of Carbohydrate Consumption and Frailty Index.

Background: The quality of carbohydrate consumed may influence the risk of frailty. In this study, we tested the hypothesis that indices of carbohydrate intake are associated with trajectories of frailty in participants of the Baltimore Longitudinal Study of Aging (BLSA). Methods: Cross sectional and longitudinal analyses were conducted in 1024 BLSA participants to examine the association between usual intake of carbohydrate and frailty index. Seven measures of carbohydrate consumption were estimated using data derived from Food Frequency Questionnaires (FFQs) and examined in association with a 43-item Frailty Index (FI). Results: In cross-sectional analyses, there was a significant, positive association between higher tertiles of total carbohydrate, glycemic load, and non-whole grains and FI. Conversely, higher tertiles of fiber-to-carbohydrate ratio was associated with lower FI. These differences persisted over the follow-up period of up to 13.8 years. Women in the highest tertile of the fiber-to-carbohydrate ratio showed a less steep increase in FI over time. Conclusions: Carbohydrate intake was positively associated with increased frailty risk in the BLSA participants, whereas a higher fiber-to-carbohydrate ratio was related to reduced risk for frailty.

Heterogeneity and transcriptome changes of human CD8+ T cells across nine decades of life.

The decline of CD8+ T cell functions contributes to deteriorating health with aging, but the mechanisms that underlie this phenomenon are not well understood. We use single-cell RNA sequencing with both cross-sectional and longitudinal samples to assess how human CD8+ T cell heterogeneity and transcriptomes change over nine decades of life. Eleven subpopulations of CD8+ T cells and their dynamic changes with age are identified. Age-related changes in gene expression result from changes in the percentage of cells expressing a given transcript, quantitative changes in the transcript level, or a combination of these two. We develop a machine learning model capable of predicting the age of individual cells based on their transcriptomic features, which are closely associated with their differentiation and mutation burden. Finally, we validate this model in two separate contexts of CD8+ T cell aging: HIV infection and CAR T cell expansion in vivo.

Longitudinal analysis reveals age-related changes in the T cell receptor repertoire of human T cell subsets.

A diverse T cell receptor (TCR) repertoire is essential for protection against a variety of pathogens, and TCR repertoire size is believed to decline with age. However, the precise size of human TCR repertoires, in both total and subsets of T cells, as well as their changes with age, are not fully characterized. We conducted a longitudinal analysis of the human blood TCRα and TCRß repertoire of CD4+ and CD8+ T cell subsets using a unique molecular identifier-based (UMI-based) RNA-seq method. Thorough analysis of 1.9 × 108 T cells yielded the lower estimate of TCR repertoire richness in an adult at 3.8 × 108. Alterations of the TCR repertoire with age were observed in all 4 subsets of T cells. The greatest reduction was observed in naive CD8+ T cells, while the greatest clonal expansion was in memory CD8+ T cells, and the highest increased retention of TCR sequences was in memory CD8+ T cells. Our results demonstrated that age-related TCR repertoire attrition is subset specific and more profound for CD8+ than CD4+ T cells, suggesting that aging has a more profound effect on cytotoxic as opposed to helper T cell functions. This may explain the increased susceptibility of older adults to novel infections.

Prior psychosocial profile and perceived impact of the COVID-19 pandemic: insights from the Baltimore Longitudinal Study of Aging.

Stress, social isolation, and changes in health behaviors during the COVID-19 pandemic period may have a lasting influence on health. Here, the correlation between current or prior demographic, social and health related characteristics, including psychosocial factors with perceived impact of the COVID-19 pandemic assessed by questionnaire during the early pandemic period is evaluated among 770 participants of the Baltimore Longitudinal Study of Aging. In multinomial logistic regression models participants with higher pre-pandemic personal mastery, a construct related to self-efficacy, were more likely to report "both positive and negative" impact of the pandemic than a solely "negative" impact (OR: 2.17, 95% CI: 1.29-3.65). Higher perceived stress and frequent contact with family prior to the pandemic were also associated with pandemic impact. These observations highlight the relevance of psychosocial factors in the COVID-19 pandemic experience and identify characteristics that may inform interventions in future public health crises.

Proteomes of primary skin fibroblasts from healthy individuals reveal altered cell responses across the life span.

Changes in the proteome of different human tissues with advancing age are poorly characterized. Here, we studied the proteins present in primary skin fibroblasts collected from 82 healthy individuals across a wide age spectrum (22-89 years old) who participated in the GESTALT (Genetic and Epigenetic Signatures of Translational Aging Laboratory Testing) study of the National Institute on Aging, NIH. Proteins were extracted from lysed fibroblasts and subjected to liquid chromatography-mass spectrometry analysis, and the expression levels of 9341 proteins were analyzed using linear regression models. We identified key pathways associated with skin fibroblast aging, including autophagy, scavenging of reactive oxygen species (ROS), ribosome biogenesis, DNA replication, and DNA repair. Changes in these prominent pathways were corroborated using molecular and cell culture approaches. Our study establishes a framework of the global proteome governing skin fibroblast aging and points to possible biomarkers and therapeutic targets.

Longitudinal phenotypic aging metrics in the Baltimore Longitudinal Study of Aging.

To define metrics of phenotypic aging, it is essential to identify biological and environmental factors that influence the pace of aging. Previous attempts to develop aging metrics were hampered by cross-sectional designs and/or focused on younger populations. In the Baltimore Longitudinal Study of Aging (BLSA), we collected longitudinally across the adult age range a comprehensive list of phenotypes within four domains (body composition, energetics, homeostatic mechanisms and neurodegeneration/neuroplasticity) and functional outcomes. We integrated individual deviations from population trajectories into a global longitudinal phenotypic metric of aging and demonstrate that accelerated longitudinal phenotypic aging is associated with faster physical and cognitive decline, faster accumulation of multimorbidity and shorter survival. These associations are more robust compared with the use of phenotypic and epigenetic measurements at a single time point. Estimation of these metrics required repeated measures of multiple phenotypes over time but may uniquely facilitate the identification of mechanisms driving phenotypic aging and subsequent age-related functional decline.

Longitudinal trajectories and determinants of human fungiform papillae density.

Tongue fungiform papillae contain taste buds crucial for taste and hormone-producing taste receptor cells; therefore, they may be considered as endocrine organs and have important age-associated physiological implications. We examine the cross-sectional and longitudinal trajectories of fungiform papillae density in 1084 participants from the Baltimore Longitudinal Study of Aging using linear regression models and mixed effects models. At baseline, the mean age was 67.86 ± 14.20 years, with a mean follow-up time among those with repeat visits of 4.24 ± 1.70 years. Women (53%) were younger (66.85 ± 13.78 vs. 69.04 ± 14.61 years, p < 0.001) and had a higher fungiform papillae density than men (16.14 ± 9.54 vs. 13.77 ± 8.61 papillae/cm2, p < 0.001). Whites (67%) had a lower fungiform papillae density than non-Whites after adjusting for age and sex. Factors cross-sectionally associated with a lower fungiform papillae density included a higher waist-hip ratio (ß = -8.525, p = 0.029), current smoking status (ß = -5.133, p = 0.014), and alcohol use within the past 12 months (ß = -1.571, p = 0.025). Longitudinally, fungiform papillae density decreased linearly with follow-up time (ß = -0.646, p < 0.001). The rate of decline was not affected by sex, race, BMI, waist-hip ratio, smoking, or alcohol use. The longitudinal decline of fungiform papillae density over time needs to be explored further in order to identify other possible age-associated physiological determinants.

Novel Human Insulin Isoforms and Cα-Peptide Product in Islets of Langerhans and Choroid Plexus.

Human insulin (INS) gene diverged from the ancestral genes of invertebrate and mammalian species millions of years ago. We previously found that mouse insulin gene (Ins2) isoforms are expressed in brain choroid plexus (ChP) epithelium cells, where insulin secretion is regulated by serotonin and not by glucose. We further compared human INS isoform expression in postmortem ChP and islets of Langerhans. We uncovered novel INS upstream open reading frame isoforms and their protein products. In addition, we found a novel alternatively spliced isoform that translates to a 74-amino acid (AA) proinsulin containing a shorter 19-AA C-peptide sequence, herein designated Cα-peptide. The middle portion of the conventional C-peptide contains ß-sheet (GQVEL) and hairpin (GGGPG) motifs that are not present in Cα-peptide. Islet amyloid polypeptide (IAPP) is not expressed in ChP, and its amyloid formation was inhibited in vitro more efficiently by Cα-peptide than by C-peptide. Of clinical relevance, the ratio of the 74-AA proinsulin to proconvertase-processed Cα-peptide was significantly increased in islets from type 2 diabetes mellitus autopsy donors. Intriguingly, 100 years after the discovery of insulin, we found that INS isoforms are present in ChP from insulin-deficient autopsy donors.

Fasting blood glucose as a predictor of mortality: Lost in translation.

Aging leads to profound changes in glucose homeostasis, weight, and adiposity, which are considered good predictors of health and survival in humans. Direct evidence that these age-associated metabolic alterations are recapitulated in animal models is lacking, impeding progress to develop and test interventions that delay the onset of metabolic dysfunction and promote healthy aging and longevity. We compared longitudinal trajectories, rates of change, and mortality risks of fasting blood glucose, body weight, and fat mass in mice, nonhuman primates, and humans throughout their lifespans and found similar trajectories of body weight and fat in the three species. In contrast, fasting blood glucose decreased late in life in mice but increased over the lifespan of nonhuman primates and humans. Higher glucose was associated with lower mortality in mice but higher mortality in nonhuman primates and humans, providing a cautionary tale for translating age-associated metabolic changes from mice to humans.

Skeletal muscle transcriptome in healthy aging.

Age-associated changes in gene expression in skeletal muscle of healthy individuals reflect accumulation of damage and compensatory adaptations to preserve tissue integrity. To characterize these changes, RNA was extracted and sequenced from muscle biopsies collected from 53 healthy individuals (22-83 years old) of the GESTALT study of the National Institute on Aging-NIH. Expression levels of 57,205 protein-coding and non-coding RNAs were studied as a function of aging by linear and negative binomial regression models. From both models, 1134 RNAs changed significantly with age. The most differentially abundant mRNAs encoded proteins implicated in several age-related processes, including cellular senescence, insulin signaling, and myogenesis. Specific mRNA isoforms that changed significantly with age in skeletal muscle were enriched for proteins involved in oxidative phosphorylation and adipogenesis. Our study establishes a detailed framework of the global transcriptome and mRNA isoforms that govern muscle damage and homeostasis with age.

Age-associated expression of p21and p53 during human wound healing.

In mice, cellular senescence and senescence-associated secretory phenotype (SASP) positively contribute to cutaneous wound healing. In this proof-of-concept study, we investigated the expressions of p16, p21, and other senescence-associated biomarkers during human wound healing in 24 healthy subjects using a double-biopsy experimental design. The first punch biopsy created the wound and established the baseline. The second biopsy, concentric to the first and taken several days after wounding, was used to probe for expression of biomarkers by immunohistochemistry and RNA FISH. To assess the effects of age, we recruited 12 sex-matched younger (30.2 ± 1.3 years) and 12 sex-matched older (75.6 ± 1.8 years) subjects. We found that p21 and p53, but not p16, were induced during healing in younger, but not older subjects. A role for Notch signaling in p21 expression was inferred from the inducible activation of HES1. Further, other SASP biomarkers such as dipeptidyl peptidase-4 (DPP4) were significantly induced upon wounding in both younger and older groups, whereas matrix metallopeptidase 9 (MMP9) was induced only in the younger group. Senescence-associated ß-galactosidase (SA-ß-gal) was not detectable before or after wounding. This pilot study suggests the possibility that human cutaneous wound healing is characterized by differential expression of p21 and p53 between younger and older subjects.

The Relationship of Lean Body Mass With Aging to the Development of Diabetes.

CONTEXT: Older adults have the greatest burden of diabetes; however, the contribution of age-related muscle loss to its development remains unclear. OBJECTIVE: We assessed the relationship of lean body mass with aging to incident diabetes in community-dwelling adults. DESIGN AND SETTING: We studied participants in the Baltimore Longitudinal Study of Aging with median follow-up of 7 years (range 1-16). Cox proportional hazard models with age as the time scale were used. Time-dependent lean body mass measures were updated at each follow-up visit available. PARTICIPANTS: Participants included 871 men and 984 women without diabetes who had  ≥ 1 assessment of body composition using dual x-ray absorptiometry. MAIN OUTCOMES: Incident diabetes, defined as self-reported history and use of glucose-lowering medications; or fasting plasma glucose ≥ 126 mg/dL and 2-hour oral glucose tolerance test glucose ≥ 200 mg/dL either at the same visit or 2 consecutive visits. RESULTS: The baseline mean [standard deviation] age was 58.9  [17.3] years. Men and women with a higher percentage of total lean body mass had lower fasting and 2-hour glucose levels, and less prediabetes (all P < 0.01). Among men, comparing highest versus lowest quartiles, percentage of total lean body mass (hazard ratio [HR],  0.46; 95% confidence interval, 0.22-0.97), percentage leg lean mass (HR, 0.38; 0.15-0.96), and lean-to-fat mass ratio (HR, 0.39; 0.17-0.89) were inversely associated with incident diabetes after accounting for race and attenuated after adjustment for height and weight. Conversely, absolute total lean body mass was positively associated with incident diabetes among women, with similar trends in men. No associations were observed with muscle strength or quality. CONCLUSIONS: Relatively lower lean body mass with aging is associated with incident diabetes in men and partially related to anthropometrics, but not so in women.

Incretins in obesity and diabetes.

Incretins are hormones secreted from enteroendocrine cells after nutrient intake that stimulate insulin secretion from ß cells in a glucose-dependent manner. Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are the only two known incretins. Dysregulation of incretin secretion and actions are noted in diseases such as obesity and diabetes. In this review, we first summarize our traditional understanding of the physiology of GIP and GLP-1, and our current knowledge of the relationships between GIP and GLP-1 and obesity and diabetes. Next, we present the results from major randomized controlled trials on the use of GLP-1 receptor agonists for managing type 2 diabetes, and emerging data on treating obesity and prediabetes. We conclude with a glimpse of the future with possible complex interactions between nutrients, gut microbiota, the endocannabinoid system, and enteroendocrine cells.