A micro-chamber free digital bio-detection for multiplexed and ultrasensitive immunoassay based on encoded magnetic microbeads and tyramide signal amplification strategy.

Digital bio-detection has become one of the most appealing methods in recent years due to its excellent performance with ultra-sensitivity in detection of low-abundance targets. Traditional digital bio-detection needs the utilization of micro-chambers for physical isolation of targets, while the recently developed beads-based micro-chamber free one is attracting extensive attention, although there exist the disadvantages of overlaps between positive ("1") and negative ("0") signals as well as the decreased detection sensitivity in multiplexed mode. Here we propose a feasible and robust micro-chamber free digital bio-detection for multiplexed and ultrasensitive immunoassay based on encoded magnetic microbeads (EMMs) and tyramide signal amplification (TSA) strategy. An EMMs-based multiplexed platform is constructed by using a fluorescent encoding method, then a puissant signal amplification of positive events in TSA procedure is achieved via systematical revelation of key factors influences. For proof of concept, a three-plexed tumor markers detection is performed to evaluate our established platform. The detection sensitivity is comparable to the corresponding single-plexed assays and is also approximately 30-15,000 times improvement compared to the conventional suspension chip. Therefore, this multiplexed micro-chamber free digital bio-detection paves a promising way to be an ultrasensitive and powerful tool for clinical diagnosis.

MOTS-c interacts synergistically with exercise intervention to regulate PGC-1α expression, attenuate insulin resistance and enhance glucose metabolism in mice via AMPK signaling pathway.

Mitochondrial-derived peptide (MOTS-c) has gained increasing attention as a promising therapeutic or prevention strategy for obesity and diabetes mellitus. MOTS-c targets the folate cycle, leading to an accumulation of 5-aminomidazole-4-carboxamide ribonucleotide (AICAR) as well as AMPK activation. AMPK is a well-known upstream regulator of the proliferation-activated receptor co-activator 1 (PGC-1α), which can improve mitochondrial biogenesis via co-transcriptional modifications. We hypothesized that AMPK can induce the expression of MOTS-c through PGC-1α. Our study aimed to explore whether MOTS-c and/or exercise can regulate MOTS-c expression, attenuate insulin resistance and enhance glucose metabolism both in vitro and in vivo. It was found that C2C12 myotubes exposed to Compound C (an AMPK inhibitor) had deceases in the protein and mRNA expressions of PGC-1α and MOTS-c. PGC-1α knockdown downregulated the protein and mRNA expressions of MOTS-c in C2C12 myotubes, whereas both PGC-1α overexpression and recombinant MOTS-c supplementation upregulated the protein and mRNA expressions of MOTS-c in C2C12 myotubes. Furthermore, the skeletal muscle and plasma levels of MOTS-c were markedly reduced in high-fat diet-induced obese mice. Treadmill training remarkably upregulated the protein levels of MOTS-c, PGC-1α and GLUT4, along with the phosphorylation levels of AMPK and ACC. Altogether, these results indicate that AMPK/PGC-1α pathway can mediate the secretion and/or production of MOTS-c in skeletal muscle, implying the possible roles of exercise intervention and recombinant MOTS-c in treating obesity and diabetes mellitus.

Adiponectin treatment improves insulin resistance in mice by regulating the expression of the mitochondrial-derived peptide MOTS-c and its response to exercise via APPL1-SIRT1-PGC-1α.

AIMS/HYPOTHESIS: Adiponectin stimulates mitochondrial biogenesis through peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α), a major regulator of mitochondrial biogenesis. MOTS-c (mitochondrial open reading frame of the 12S rRNA) is a biologically active mitochondrial-derived peptide encoded by mitochondrial DNA. It influences the mechanisms of obesity and diabetes. We hypothesised that the adiponectin pathway may regulate the production and/or secretion of MOTS-c in skeletal muscle. We aimed to determine whether exercise and adiponectin affect MOTS-c to improve insulin resistance in mice. METHODS: To investigate this hypothesis, we used wild-type C57BL/6 mice subjected to high-fat diet, an exercise regimen, and i.p. injection of recombinant mouse adiponectin (Acrp30) or MOTS-c, and adiponectin knockout (Adipoq-/-) mice (C57BL/6 background) subjected to i.p. injection of Acrp30. C2C12 myotubes were also treated with sirtuin 1 (SIRT1) inhibitor, PGC-1α inhibitor, SIRT1 activator, plasmid-expressed active APPL1 (adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper), pcDNA-SIRT1, or siRNA against APPL1, SIRT1 or PGC-1α. RESULTS: In Adipoq-/- mice, MOTS-c levels in the plasma and skeletal muscle were downregulated. In C2C12 myotubes, adiponectin increased the mRNA expression of MOTS-c. APPL1 protein level following adiponectin treatment positively correlated with MOTS-c protein and mRNA levels in C2C12 myotubes. SIRT1 overexpression increased the adiponectin-induced mRNA and protein expression of MOTS-c, SIRT1 and PGC-1α. Pharmacologic and genetic inhibition of PGC-1α suppressed the increases in MOTS-c mRNA and protein levels induced by SIRT1 overexpression. In mice, plasma and skeletal muscle MOTS-c levels were significantly downregulated following high-fat-diet. Exercise and i.p. Acrp30 or MOTS-c increased MOTS-c levels and adiponectin mRNA and protein expression in the plasma and skeletal muscle. CONCLUSIONS/INTERPRETATION: Our findings showed that the APPL1-SIRT1-PGC-1α pathway regulates the production and/or secretion of skeletal muscle MOTS-c by mediating adiponectin signalling. Our study provides an insight into the cellular and molecular pathways underlying the pathogenesis of diabetes and shows that MOTS-c is a potential novel therapeutic target in the treatment of diabetes. Graphical abstract.