Prasenjit Prasad Saha

Artificial sweeteners are widely used sugar substitutes, but little is known about their long-term effects on cardiometabolic disease risks. Here we examined the commonly used sugar substitute erythritol and atherothrombotic disease risk. In initial untargeted metabolomics studies in patients undergoing cardiac risk assessment (n = 1,157; discovery cohort, NCT00590200 ), circulating levels of multiple polyol sweeteners, especially erythritol, were associated with incident (3 year) risk for major adverse cardiovascular events (MACE; includes death or nonfatal myocardial infarction or stroke). Subsequent targeted metabolomics analyses in independent US (n = 2,149, NCT00590200 ) and European (n = 833, DRKS00020915 ) validation cohorts of stable patients undergoing elective cardiac evaluation confirmed this association (fourth versus first quartile adjusted hazard ratio (95% confidence interval), 1.80 (1.18–2.77) and 2.21 (1.20–4.07), respectively). At physiological levels, erythritol enhanced platelet reactivity in vitro and thrombosis formation in vivo. Finally, in a prospective pilot intervention study ( NCT04731363 ), erythritol ingestion in healthy volunteers (n = 8) induced marked and sustained (>2 d) increases in plasma erythritol levels well above thresholds associated with heightened platelet reactivity and thrombosis potential in in vitro and in vivo studies. Our findings reveal that erythritol is both associated with incident MACE risk and fosters enhanced thrombosis. Studies assessing the long-term safety of erythritol are warranted. Metabolomics analyses reported an increased risk of cardiovascular disease associated with the artificial sweetener erythritol, supported by mechanistic studies showing that high levels of erythritol enhanced platelet reactivity and thrombosis formation.

BACKGROUND: The gut microbiota-dependent metabolite phenylacetylgutamine (PAGln) is both associated with atherothrombotic heart disease in humans, and mechanistically linked to cardiovascular disease pathogenesis in animal models via modulation of adrenergic receptor signaling. METHODS: Here we examined both clinical and mechanistic relationships between PAGln and heart failure (HF). First, we examined associations among plasma levels of PAGln and HF, left ventricular ejection fraction, and N-terminal pro-B-type natriuretic peptide in 2 independent clinical cohorts of subjects undergoing coronary angiography in tertiary referral centers (an initial discovery US Cohort, n=3256; and a validation European Cohort, n=829). Then, the impact of PAGln on cardiovascular phenotypes relevant to HF in cultured cardiomyoblasts, and in vivo were also examined. RESULTS: Circulating PAGln levels were dose-dependently associated with HF presence and indices of severity (reduced ventricular ejection fraction, elevated N-terminal pro-B-type natriuretic peptide) independent of traditional risk factors and renal function in both cohorts. Beyond these clinical associations, mechanistic studies showed both PAGln and its murine counterpart, phenylacetylglycine, directly fostered HF-relevant phenotypes, including decreased cardiomyocyte sarcomere contraction, and B-type natriuretic peptide gene expression in both cultured cardiomyoblasts and murine atrial tissue. CONCLUSIONS: The present study reveals the gut microbial metabolite PAGln is clinically and mechanistically linked to HF presence and severity. Modulating the gut microbiome, in general, and PAGln production, in particular, may represent a potential therapeutic target for modulating HF. REGISTRATION: URL: https://clinicaltrials.gov/; Unique identifier: NCT00590200 and URL: https://drks.de/drks_web/; Unique identifier: DRKS00020915.

Mitochondria are organelles indispensable for maintenance of cellular energy homeostasis. Most mitochondrial proteins are nuclearly encoded and are imported into the matrix compartment where they are properly folded. This process is facilitated by the mitochondrial heat shock protein 70 (mtHsp70), a chaperone contributing to mitochondrial protein quality control. The affinity of mtHsp70 for its protein clients and its chaperone function are regulated by binding of ATP/ADP to mtHsp70's nucleotide-binding domain. Nucleotide exchange factors (NEFs) play a crucial role in exchanging ADP for ATP at mtHsp70's nucleotide-binding domain, thereby modulating mtHsp70's chaperone activity. A single NEF, Mge1, regulates mtHsp70's chaperone activity in lower eukaryotes, but the mammalian orthologs are unknown. Here, we report that two putative NEF orthologs, GrpE-like 1 (GrpEL1) and GrpEL2, modulate mtHsp70's function in human cells. We found that both GrpEL1 and GrpEL2 associate with mtHsp70 as a hetero-oligomeric subcomplex and regulate mtHsp70 function. The formation of this subcomplex was critical for conferring stability to the NEFs, helped fine-tune mitochondrial protein quality control, and regulated crucial mtHsp70 functions, such as import of preproteins and biogenesis of Fe-S clusters. Our results also suggested that GrpEL2 has evolved as a possible stress resistance protein in higher vertebrates to maintain chaperone activity under stress conditions. In conclusion, our findings support the idea that GrpEL1 has a role as a stress modulator in mammalian cells and highlight that multiple NEFs are involved in controlling protein quality in mammalian mitochondria.

Biogenesis of the iron-sulfur (Fe-S) cluster is an indispensable process in living cells. In mammalian mitochondria, the initial step of the Fe-S cluster assembly process is assisted by the NFS1-ISD11 complex, which delivers sulfur to scaffold protein ISCU during Fe-S cluster synthesis. Although ISD11 is an essential protein, its cellular role in Fe-S cluster biogenesis is still not defined. Our study maps the important ISD11 amino acid residues belonging to putative helix 1 (Phe-40), helix 3 (Leu-63, Arg-68, Gln-69, Ile-72, Tyr-76), and C-terminal segment (Leu-81, Glu-84) are critical for in vivo Fe-S cluster biogenesis. Importantly, mutation of these conserved ISD11 residues into alanine leads to its compromised interaction with NFS1, resulting in reduced stability and enhanced aggregation of NFS1 in the mitochondria. Due to altered interaction with ISD11 mutants, the levels of NFS1 and Isu1 were significantly depleted, which affects Fe-S cluster biosynthesis, leading to reduced electron transport chain complex (ETC) activity and mitochondrial respiration. In humans, a clinically relevant ISD11 mutation (R68L) has been associated in the development of a mitochondrial genetic disorder, COXPD19. Our findings highlight that the ISD11 R68A/R68L mutation display reduced affinity to form a stable subcomplex with NFS1, and thereby fails to prevent NFS1 aggregation resulting in impairment of the Fe-S cluster biogenesis. The prime affected machinery is the ETC complex, which showed compromised redox properties, causing diminished mitochondrial respiration. Furthermore, the R68L ISD11 mutant displayed accumulation of mitochondrial iron and reactive oxygen species, leading to mitochondrial dysfunction, which correlates with the phenotype observed in COXPD19 patients.