Rationale:Obesity and type 2 diabetes (T2D) are growing threats to human health, and their genetic basis is complex and not fully understood. Furthermore, the mitochondrial genome has been shown to encode for many microproteins that have a variety of biological effects. In this study we explore a newly discovered mitochondrial-derived microprotein (MDP) that may be responsible for some forms of diabetes in humans. Methods:We have performed a mitochondrial genome wide interaction study (MiWIS) and discovered a SNP that lies within the gene for an MDP and is associated with type 2 diabetes. We then used cell culture to confirm that this MDP has biological activity and used mass spectrometry to detect it. This novel MDP and more potent analogues were then administered in murine, in vivo studies in models of diabetes and obesity to determine the effects. Further analysis of the in vivo studies was performed with transcriptomic and proteomic techniques. Results:Our MiWIS found a SNP associated with type 2 diabetes in 3 independent cohorts that is found within a novel MDP that we have called MENTSH (MDP Encoded in the ND-Two Subunit of Humans). This common SNP is found in populations indigenous to the Americas that interrupts the start codon of MENTSH. Murine in vivo studies demonstrate that MENTSH administration improves insulin signaling, while analogues of MENTSH can potently block weight gain caused by a high fat diet. Mechanistically, our studies show that MENTSH activates AKT signaling in muscle, while reducing AKT signaling in fat. Conclusions:These observations highlight a new cause of metabolic dysfunction in a vulnerable population, suggesting that MENTSH could be an innovative, precision medicine approach to treating T2D.

Suppression of insulin-like growth factor-1 (IGF-1) signaling extends mammalian life span and protects against a range of age-related diseases. Unexpectedly, we found that reduced IGF-1 signaling fails to extend the life span of mitochondrial mutator mice. Most of the longevity pathways that are normally initiated by IGF-1 suppression were either blocked or blunted in the mutator mice. These observations suggest that the prolongevity effects of IGF-1 suppression critically depend on the integrity of the mitochondrial genome, revealing an unexpected hierarchy in the pathways that control mammalian aging. Together, these findings deepen our understanding of the interactions between the hallmarks of aging and underscore the need for interventions that preserve the integrity of the mitochondrial genome.

Pediatric snoring, a common manifestation of obstructive sleep apnea (OSA), can significantly impact children’s development. This study aimed to characterize the alterations in gut microbiota and metabolome associated with pediatric snoring. Fecal samples were collected from 30 snoring children and 30 matched healthy children, and analyzed using 16 S rRNA gene sequencing and untargeted metabolomics. Analysis of the gut microbiota revealed distinct community structures between the two groups. Key genera such as Faecalibacterium and Bacteroides were enriched in snoring children, whereas Bifidobacterium and Akkermansia were more abundant in healthy children. Functional prediction indicated significant perturbations in microbial metabolic pathways, including amino acid and lipid metabolism. Metabolomic profiling identified 214 significantly altered metabolites, with 101 upregulated and 113 downregulated in the snoring group. Notable changes were observed in metabolites such as L-Arginine, Guanosine, and N1-Acetylspermidine. Pathway enrichment analysis highlighted dysregulation in purine metabolism and bile secretion. A panel of the top differential metabolites demonstrated high diagnostic accuracy for distinguishing snoring children from controls. In conclusion, this multi-omics study reveals significant and coordinated disruptions in the gut microbiota and metabolome of children who snore. These findings provide a foundation for understanding the role of the gut-microbiota-metabolite axis in pediatric snoring and identify potential non-invasive biomarkers for early detection and future mechanistic investigations.

Abstract Regular exercise using assistive movement devices, such as running frames, has emerged as a promising strategy to improve cardiorespiratory fitness in individuals with cerebral palsy (CP). However, the molecular pathways underlying these adaptations remain poorly understood. Here, we examined a novel class of signalling molecules, mitochondrial‐derived microproteins (MDPs), and assessed whether individuals with CP exhibit altered circulating levels compared with typically developing (TD) individuals at rest and following an acute bout of endurance exercise. Three groups were included: TD adults (31 ± 6 years), TD adolescents (16 ± 1 years) and adults with CP (25 ± 6 years). Individuals with CP were classified as Gross Motor Function Classification System (GMFCS) levels II−IV and had at least 3 months of frame running experience. Habitual physical activity, ultrasound‐derived muscle thickness, and peak oxygen uptake were assessed. The exercise session consisted of 45 min of frame running for individuals with CP and conventional running for TD participants. Blood samples were obtained before and 1 h after exercise, and plasma MDP concentrations were measured using in‐house enzyme‐linked immunosorbent assay. Adults with CP had reduced muscle mass and maximal oxygen uptake compared to TD individuals. Despite this, they exhibited basal circulating levels of MDPs, including humanin, MOTS‐c and SHMOOSE, comparable to TD adults and adolescents, with no associations with CP subtype or motor impairment severity. Following exercise, circulating MDPs showed no or only modest changes across groups, with no differences between CP and TD individuals. Overall, these findings suggest preserved mitochondrial‐derived signalling via MDPs in individuals with CP.

Background:The health benefits of the Mediterranean Diet (Med-Diet) have been demonstrated in observational studies and randomized controlled trials. Emerging evidence suggests that the biological effects of the Med-Diet may be mediated by the modulation of mitochondrial function. Human mitochondrial DNA (mtDNA) encodes microproteins, which have been shown to regulate aging, cardiometabolic functions, and neuroprotection. Objectives:To investigate Humanin and SHMOOSE (Small Human Mitochondrial ORF Over SErine tRNA), as potential mitochondrial biomarkers of Med-Diet adherence and their associations with markers of oxidative stress. Methods:Cross-sectional analysis of 49 patients (mean age 78.4 ± 8.7 years; 57% female) selected from an observational study of non-valvular atrial fibrillation (AF) conducted at the Atherothrombosis Center of Sapienza University of Rome. Patients were categorized into low-medium (0-6) and high (7-9) adherence to the Med-Diet based on the 9-item Med-Diet questionnaire. Oxidative stress was evaluated by measuring soluble Nox2-derived peptide (sNox2-dp) and plasma 8-iso-prostaglandin F2α (8-iso-PGF2α) using enzyme-linked immunosorbent assays (ELISA). Circulating Humanin and SHMOOSE levels were measured using an in-house sandwich ELISA. Results:High Med-Diet adherence was observed in 20 patients (40.8%), while 29 patients (59.2%) had low-medium adherence. Patients with high adherence exhibited higher plasma levels of SHMOOSE (p = 0.046) and Humanin (p = 0.045). The analysis of the dietary components of the Med-Diet revealed higher levels of SHMOOSE with olive oil consumption (p = 0.020) and low intake of refined bread (p = 0.029), while Humanin positively correlated with olive oil (p = 0.0069), fish (p = 0.038), and legumes (p = 0.0282). Additionally, Humanin was inversely associated with sNox2-dp (p = 0.019), which remained significant after adjusting for sex and BMI (B = -0.010; β = -0.302; p = 0.040), and 8-iso-PGF2α (p = 0.049). Conclusion:This study indicates (i) a positive association between adherence to the Med-Diet and circulating levels of mitochondrial microproteins SHMOOSE and Humanin supporting their role as potential mediators of Med-Diet benefits; (ii) a putative crosstalk between Humanin signaling and Nox2 activity, suggesting a novel cardioprotective mechanism of the Med-Diet. Collectively, these findings support mitochondrial microproteins as promising biomarkers for tailoring nutritional strategies for healthy aging. Further studies are warranted to elucidate the underlying mechanisms and determine the causal nature of these associations.

The use of mitochondrial wide association studies (MiWAS) to link mitochondrial DNA variants (mtSNPs) to phenotypes of interest has uncovered important connections between mitochondrial genes and human health. The recent introduction of a re-annotated mitochondrial genome that accounts for small open reading frames (sORFs) with protein coding potential suggests the existence of mitochondrial-derived microproteins, many of which remain uncharacterized. Thus, considering the re-annotated mitochondrial genome when conducting genomic analyses such as MiWAS facilitates the mapping of mtSNPs back to microprotein-encoding sORFs and uncovers interactions between mitochondrial microproteins and biological systems. Here, we employ MiWAS of venous blood samples from the Health and Retirement Study (HRS) and identify a mtSNP associated with sex-specific changes to immune composition. After accounting for re-annotation, we map the identified mtSNP back to a sORF that encodes a novel microprotein, termed MASL (Mitochondrial Associated Small d-Loop peptide). Complementary phenome-wide association studies (PheWAS) in HRS and and UK Biobank confirm interactions between this mtSNP and immune phenotypes of interest, and our targeted RNA-Seq method (mitoSNP-seq) elucidates sex-differences in gene expression and functional pathways potentially altered by this mtSNP that may be relevant to the associated microprotein. Early characterization of the MASL microprotein shows sex-differences in circulating MASL levels in human plasma, and sex-specific interactions when comparing male and female mice treated with synthesized MASL. Together, the results of this study not only contribute to our understanding of mitochondrial dynamics in immunity, but also provide early characterization of a novel mitochondrial-derived microprotein with sex-specific modulatory effects.

Supplemental Figure S2 shows the comparison of control Mitochondrial DNA copy number levels in White and Black Individuals

Colorectal Cancer (CRC) is the third most prevalent malignancy, leading to significant morbidity and mortality globally. Epidemiological studies suggest that chronological age and diet are among the major contributing factors correlated with the incidence of CRC. Our study aimed to provide insights into the association between age, diet, and gut microbiome in CRC using molecular techniques including RNA sequencing, cytokine analysis, and metagenomic analysis. We used syngeneic MC38 mice model divided into two age groups (old and young) and three diet groups (standard chow, calorie-restricted and high-fat). The major findings of this study are that age and diet impact intratumoral gene signaling (nuclear and mitochondrial), and hub genes we identified are associated with prognosis in CRC. Fecal microbiome analysis showed that old microbiomes have higher alpha diversity compared to young mice. Our results demonstrate that interactions between host (age) and external (diet) factors regulate tumor growth mediated by cytokines, mitochondrial derived proteins, and the gut microbiome. Collectively, our findings advance current understanding of the mechanisms by which aging, diet and gut microbiota impact CRC onset and progression though further investigation is warranted.

Supplemental Figure S3 shows receiver operating characteristic (ROC) curves between univariable models adjusted for PSA (log-transformed) only, Plasma mtDNA only, and WBC mtDNA only, stratified by Black and White individuals.

Supplemental Table S1 contains mtDNA copy numbers used in this study for Black and White individuals.

Supplemental Figure S1 shows a comparison of control Mitochondrial DNA copy number levels by BPH status for Black and White individuals

Black individuals are disproportionately burdened by prostate cancer compared with White individuals. The mitochondrion is an untapped source for prostate cancer biomarkers, and previous work has shown that altered mitochondrial DNA (mtDNA) copy number is linked to mitochondrial dysfunction and tumorigenesis. We assess whether mtDNA copy number is altered in patients with and without prostate cancer in a racially specific manner. Circulating cell-free mtDNA copy number from plasma and mtDNA copy number from white blood cells (WBC) were measured in 199 patients undergoing biopsy (50:50 White cases/controls and 50:49 Black cases/controls). mtDNA copy number was determined via Droplet Digital PCR. Logistic regressions tested associations between mtDNA and prostate cancer by race. The AUC was compared between covariate-only models and models with mtDNA. In both plasma and WBCs, mtDNA copy number was significantly increased in cases compared with controls in White patients, but not in Black patients. Interestingly, Black controls had higher mtDNA copy number levels than White controls. Multivariable analysis revealed significant associations of plasma mtDNA and WBC mtDNA with prostate cancer for White patients only. Elevated mtDNA copy number was more accurate in predicting prostate cancer in White patients than in Black patients. Higher mtDNA copy number levels were associated with prostate cancer in both Black and White patients. Plasma mtDNA may be more accurate than WBC mtDNA in predicting prostate cancer incidence in Black men. Overall, Black controls had higher mtDNA copy number levels than White controls, suggesting mtDNA copy number may be implicated in prostate cancer health disparities.Prevention Relevance: Our study shows that mtDNA copy number is a significant predictor of prostate cancer in White individuals, suggesting its potential use in early detection and prevention strategies. The absence of this association in Black individuals highlights the need for race-specific biomarkers in prostate cancer prevention efforts.

Supplemental Figure S4 shows a comparison of the distribution of Mitochondrial DNA copy number by prostate cancer grade for Black and White Individuals stratified by plasma and WBC.

Introduction & Objective: MOTS-c is a mitochondrial DNA-encoded microprotein that improves impaired glucose metabolism caused by aging and high fat diet. The presence of a naturally occurring genetic variant of MOTS-c, K14Q MOTS-c, increases the susceptibility to type 2 diabetes (T2D) in East Asians. Nevertheless, the precise mechanism of MOTS-c action has not been fully elucidated. Here, we demonstrate that the protein kinase CK2 is a functional and direct target of MOTS-c and that the reduced binding of K14Q MOTS-c to CK2 increases the risk of T2D. Methods: We performed in vitro experiments, including dot blot, kinase activity, and surface plasmon resonance assays, to investigate the direct interaction between MOTS-c and CK2. We also examined the impact of MOTS-c treatment on CK2 activity in skeletal muscle, as well as on muscle glucose uptake in young mice. Subsequently, we investigated the impact of a naturally occurring K14Q MOTS-c variant on the prevalence of T2D in 12,068 Japanese individuals. Results: Dot blot and cell-free kinase activity assays demonstrated that MOTS-c activated CK2 by binding directly to its α subunit, and this binding was confirmed using a surface plasmon resonance assay. Importantly, the binding affinity of K14Q MOTS-c to CK2α was 16-fold less than that of the wild type MOTS-c, and K14Q MOTS-c did not activate CK2 in the cell-free kinase activity assay. Skeletal muscle CK2 activity was lower in old mice and higher after exercise, and was increased by MOTS-c administration, but not K14Q MOTS-c. MOTS-c administration, but not K14Q MOTS-c, significantly enhanced muscle glucose uptake, which was blunted by a CK2 inhibitor. In humans, the K14Q MOTS-c carriers exhibited an increased risk of type 2 diabetes, particularly among individuals aged 60 and above, while this increased risk was mitigated by daily physical activity. Conclusion: Together, these findings provide evidence that CK2 is required for MOTS-c effects and that the MOTS-c/CK2 pathway is a potential therapeutic target for T2D. H. Kumagai: None. S. Kim: None. B. Miller: None. S. Lee: None. H. Zempo: None. T. Natsume: None. J. Wan: None. R. Ramirez II: None. H.H. Mehta: None. Y. Nishida: None. N. Fuku: None. S. Dobashi: None. E. Miyamoto-Mikami: None. H. Naito: None. M. Hara: None. C. Iwasaka: None. Y. Yamada: None. Y. Higaki: None. K. Tanaka: None. K. Yen: None. P. Cohen: None.

MOTS-c is a mitochondrial microprotein that improves metabolism. Here, we demonstrate CK2 is a direct and functional target of MOTS-c. MOTS-c directly binds to CK2 and activates it in cell-free systems. MOTS-c administration to mice prevented skeletal muscle atrophy and enhanced muscle glucose uptake, which were blunted by suppressing CK2 activity. Interestingly, the effects of MOTS-c are tissue-specific. Systemically administered MOTS-c binds to CK2 in fat and muscle, yet stimulates CK2 activity in muscle while suppressing it in fat by differentially modifying CK2-interacting proteins. Notably, a naturally occurring MOTS-c variant, K14Q MOTS-c, has reduced binding to CK2 and does not activate it or elicit its effects. Male K14Q MOTS-c carriers exhibited a higher risk of sarcopenia and type 2 diabetes (T2D) in an age- and physical-activity-dependent manner, whereas females had an age-specific reduced risk of T2D. Altogether, these findings provide evidence that CK2 is required for MOTS-c effects.

MOTS-c, a mitochondrial microprotein, attenuates immobilization-induced skeletal muscle atrophy. MOTS-c treatment improves systemic inflammation and skeletal muscle AKT/FOXOs signaling pathways. Furthermore, unbiased RNA sequencing and subsequent assays revealed that MOTS-c prevents lipid infiltration in skeletal muscle. Since lipid accumulation is one of the common pathologies among other skeletal muscle atrophies induced by aging, obesity, cancer cachexia, and denervation, MOTS-c treatment could be effective in other muscle atrophy models as well.

Supplementary Figure 1 from IGFBP-3 Is a Metastasis Suppression Gene in Prostate Cancer

Genetic mutations affecting β-amyloid (Αβ) production suggest that decreasing Αβ levels in healthy adults could prevent AD. However, evidence for behavioral interventions that decrease Αβ levels is lacking. The current study examined whether heart rate variability (HRV) biofeedback affects plasma Αβ levels. This intervention involves breathing slowly at a pace that maximizes the amplitude of heart rate oscillations. During slow paced breathing, the vagus nerve receives strong signals from stretch receptors in blood vessels and the lungs. These afferent signals stimulate ascending vagus nerve signaling that activate ‘safety’ signaling pathways, suppressing stress and arousal pathways in the brain and body. Experiencing adversity increases production of Aβ and risk of AD. We hypothesized that, in healthy adults, daily sessions involving slow paced breathing during HRV biofeedback could decrease Aβ by suppressing these adversity-related processes. Healthy adults (N = 54 age 18-35; N = 54 age 55-80) practiced HRV biofeedback daily for five weeks. They were randomized to use slow paced breathing and HRV biofeedback to increase heart rate oscillations (Osc+ condition) or to use personalized strategies and HRV biofeedback to try to decrease heart rate oscillations (Osc- condition). Pre- and post-intervention plasma Aβ40 and Aβ42 levels were quantified blind to condition. Gene expression analysis of peripheral blood samples of the 54 younger adults using bioinformatics-based analyses of genome-wide RNA profiles examined activity of pro-inflammatory, neuroendocrine, and antiviral transcription control pathways relevant to the conserved transcriptional response to adversity (CTRA). We predicted that reductions in the CTRA across the 5-week trial would be associated with reduced plasma Aβ. Both younger and older adults showed significant differences between Osc+ and Osc- conditions in change in plasma Aβ40 and Aβ42 levels (Figure 1). The Osc+ condition decreased Αβ while the Osc- condition increased Αβ. Individual changes in plasma Aβ from pre- to post-intervention were associated with changes in expression of the CTRA in the predicted direction. HRV biofeedback affects plasma Aβ and these effects are associated with changes in expression of genes upregulated during adversity. Future longer-term studies should test whether reducing plasma Αβ via the Osc+ intervention reduces risk of developing AD.