No abstract

Conditions such as acidosis, uremia, and sepsis are characterized by insulin resistance and muscle wasting, but whether the insulin resistance associated with these disorders contributes to muscle atrophy is unclear. We examined this question in db/db mice with increased blood glucose despite high levels of plasma insulin. Compared with control littermate mice, the weights of different muscles in db/db mice and the cross-sectional areas of muscles were smaller. In muscle of db/db mice, protein degradation and activities of the major proteolytic systems, caspase-3 and the proteasome, were increased. We examined signals that could activate muscle proteolysis and found low values of both phosphatidylinositol 3 kinase (PI3K) activity and phosphorylated Akt that were related to phosphorylation of serine 307 of insulin receptor substrate-1. To assess how changes in circulating insulin and glucose affect muscle protein, we treated db/db mice with rosiglitazone. Rosiglitazone improved indices of insulin resistance and abnormalities in PI3K/Akt signaling and decreased activities of caspase-3 and the proteasome in muscle leading to suppression of proteolysis. Underlying mechanisms of proteolysis include increased glucocorticoid production, decreased circulating adiponectin, and phosphorylation of the forkhead transcription factor associated with increased expression of the E3 ubiquitin-conjugating enzymes atrogin-1/MAFbx and MuRF1. These abnormalities were also corrected by rosiglitazone. Thus, insulin resistance causes muscle wasting by mechanisms that involve suppression of PI3K/Akt signaling leading to activation of caspase-3 and the ubiquitin-proteasome proteolytic pathway causing muscle protein degradation.

The third-generation tyrosine kinase inhibitor osimertinib is approved to treat patients with T790M-positive non-small cell lung cancer (NSCLC) who have developed resistance to earlier-generation drugs. Acquired C797S mutation has been reported to mediate osimertinib resistance in some patients. However, the remaining resistance mechanisms are largely unknown. We performed mutation profiling using targeted next-generation sequencing (NGS) for 416 cancer-relevant genes on 93 osimertinib-resistant lung cancer patients' samples, mainly cell-free DNAs (cfDNAs), and matched pretreatment samples of 12 patients. experiments were conducted to functionally study the secondary mutations identified. G796/C797, L792, and L718/G719 mutations were identified in 24.7%, 10.8%, and 9.7% of the cases, respectively, with certain mutations coexisting in one patient with different prevalence. L792 and L718 mutants markedly increased the half inhibitory concentration (IC) of osimertinib , among which the L718Q mutation conferred the greatest resistance to osimertinib, as well as gefitinib resistance when not coexisting with T790M. Further analysis of the 12 matched pretreatment samples confirmed that these mutations were acquired during osimertinib treatment. Alterations in parallel or downstream oncogenes such as , and were also discovered, potentially contributing to the osimertinib-resistance in patients without secondary mutations. We present comprehensive mutation profiles of a large cohort of osimertinib-resistance lung cancer patients using mainly cfDNA. Besides C797 mutations, novel secondary mutations of L718 and L792 residues confer osimertinib resistance, both and , and are of great clinical and pharmaceutical relevance..

With trauma, sepsis, cancer, or uremia, animals or patients experience accelerated degradation of muscle protein in the ATP-ubiquitin-proteasome (Ub-P’some) system. The initial step in myofibrillar proteolysis is unknown because this proteolytic system does not break down actomyosin complexes or myofibrils, even though it degrades monomeric actin or myosin. Since cytokines or insulin resistance are common in catabolic states and will activate caspases, we examined whether caspase-3 would break down actomyosin. We found that recombinant caspase-3 cleaves actomyosin, producing a characteristic, approximately 14-kDa actin fragment and other proteins that are degraded by the Ub-P’some. In fact, limited actomyosin cleavage by caspase-3 yields a 125% increase in protein degradation by the Ub-P’some system. Serum deprivation of L6 muscle cells stimulates actin cleavage and proteolysis; insulin blocks these responses by a mechanism requiring PI3K. Cleaved actin fragments are present in muscles of rats with muscle atrophy from diabetes or chronic uremia. Accumulation of actin fragments and the rate of proteolysis in muscle stimulated by diabetes are suppressed by a caspase-3 inhibitor. Thus, in catabolic conditions, an initial step resulting in loss of muscle protein is activation of caspase-3, yielding proteins that are degraded by the Ub-P’some system. Therapeutic strategies could be designed to prevent these events

With trauma, sepsis, cancer, or uremia, animals or patients experience accelerated degradation of muscle protein in the ATP-ubiquitin-proteasome (Ub-P'some) system. The initial step in myofibrillar proteolysis is unknown because this proteolytic system does not break down actomyosin complexes or myofibrils, even though it degrades monomeric actin or myosin. Since cytokines or insulin resistance are common in catabolic states and will activate caspases, we examined whether caspase-3 would break down actomyosin. We found that recombinant caspase-3 cleaves actomyosin, producing a characteristic, approximately 14-kDa actin fragment and other proteins that are degraded by the Ub-P'some. In fact, limited actomyosin cleavage by caspase-3 yields a 125% increase in protein degradation by the Ub-P'some system. Serum deprivation of L6 muscle cells stimulates actin cleavage and proteolysis; insulin blocks these responses by a mechanism requiring PI3K. Cleaved actin fragments are present in muscles of rats with muscle atrophy from diabetes or chronic uremia. Accumulation of actin fragments and the rate of proteolysis in muscle stimulated by diabetes are suppressed by a caspase-3 inhibitor. Thus, in catabolic conditions, an initial step resulting in loss of muscle protein is activation of caspase-3, yielding proteins that are degraded by the Ub-P'some system. Therapeutic strategies could be designed to prevent these events.