FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W3128780270> ?p ?o ?g. }

• W3128780270 endingPage "100395" @default.
• W3128780270 startingPage "100395" @default.
• W3128780270 abstract "Chronic glucocorticoid exposure causes insulin resistance and muscle atrophy in skeletal muscle. We previously identified phosphoinositide-3-kinase regulatory subunit 1 (Pik3r1) as a primary target gene of skeletal muscle glucocorticoid receptors involved in the glucocorticoid-mediated suppression of insulin action. However, the in vivo functions of Pik3r1 remain unclear. Here, we generated striated muscle-specific Pik3r1 knockout (MKO) mice and treated them with a dexamethasone (DEX), a synthetic glucocorticoid. Treating wildtype (WT) mice with DEX attenuated insulin activated Akt activity in liver, epididymal white adipose tissue, and gastrocnemius (GA) muscle. This DEX effect was diminished in GA muscle of MKO mice, therefore, resulting in improved glucose and insulin tolerance in DEX-treated MKO mice. Stable isotope labeling techniques revealed that in WT mice, DEX treatment decreased protein fractional synthesis rates in GA muscle. Furthermore, histology showed that in WT mice, DEX treatment reduced GA myotube diameters. In MKO mice, myotube diameters were smaller than in WT mice, and there were more fast oxidative fibers. Importantly, DEX failed to further reduce myotube diameters. Pik3r1 knockout also decreased basal protein synthesis rate (likely caused by lower 4E-BP1 phosphorylation at Thr37/Thr46) and curbed the ability of DEX to attenuate protein synthesis rate. Finally, the ability of DEX to inhibit eIF2α phosphorylation and insulin-induced 4E-BP1 phosphorylation was reduced in MKO mice. Taken together, these results demonstrate the role of Pik3r1 in glucocorticoid-mediated effects on glucose and protein metabolism in skeletal muscle. Chronic glucocorticoid exposure causes insulin resistance and muscle atrophy in skeletal muscle. We previously identified phosphoinositide-3-kinase regulatory subunit 1 (Pik3r1) as a primary target gene of skeletal muscle glucocorticoid receptors involved in the glucocorticoid-mediated suppression of insulin action. However, the in vivo functions of Pik3r1 remain unclear. Here, we generated striated muscle-specific Pik3r1 knockout (MKO) mice and treated them with a dexamethasone (DEX), a synthetic glucocorticoid. Treating wildtype (WT) mice with DEX attenuated insulin activated Akt activity in liver, epididymal white adipose tissue, and gastrocnemius (GA) muscle. This DEX effect was diminished in GA muscle of MKO mice, therefore, resulting in improved glucose and insulin tolerance in DEX-treated MKO mice. Stable isotope labeling techniques revealed that in WT mice, DEX treatment decreased protein fractional synthesis rates in GA muscle. Furthermore, histology showed that in WT mice, DEX treatment reduced GA myotube diameters. In MKO mice, myotube diameters were smaller than in WT mice, and there were more fast oxidative fibers. Importantly, DEX failed to further reduce myotube diameters. Pik3r1 knockout also decreased basal protein synthesis rate (likely caused by lower 4E-BP1 phosphorylation at Thr37/Thr46) and curbed the ability of DEX to attenuate protein synthesis rate. Finally, the ability of DEX to inhibit eIF2α phosphorylation and insulin-induced 4E-BP1 phosphorylation was reduced in MKO mice. Taken together, these results demonstrate the role of Pik3r1 in glucocorticoid-mediated effects on glucose and protein metabolism in skeletal muscle. Glucocorticoids are steroid hormones that play important roles in regulating whole body metabolism under stress conditions, mainly by mobilizing energy sources to face severe challenges. In skeletal muscle, glucocorticoids inhibit protein synthesis, facilitate protein degradation, and suppress glucose utilization. Amino acids generated from glucocorticoid-induced protein mobilization are the precursors for hepatic gluconeogenesis and inhibiting glucose utilization raises plasma glucose concentrations (1Kuo T. Harris C.A. Wang J.C. Metabolic functions of glucocorticoid receptor in skeletal muscle.Mol. Cell. Endocrinol. 2013; 380: 79-88Crossref PubMed Scopus (111) Google Scholar, 2Bodine S.C. Furlow J.D. Glucocorticoids and skeletal muscle.Adv. Exp. Med. Biol. 2015; 872: 145-176Crossref PubMed Scopus (70) Google Scholar). While these glucocorticoid effects are critical for metabolic adaptations during stress, chronic/excess glucocorticoid exposure causes hyperglycemia, insulin resistance, and muscle atrophy (1Kuo T. Harris C.A. Wang J.C. Metabolic functions of glucocorticoid receptor in skeletal muscle.Mol. Cell. Endocrinol. 2013; 380: 79-88Crossref PubMed Scopus (111) Google Scholar, 3Rose A.J. Herzig S. Metabolic control through glucocorticoid hormones: An update.Mol. Cell. Endocrinol. 2013; 380: 65-78Crossref PubMed Scopus (93) Google Scholar, 4Kuo T. McQueen A. Chen T.C. Wang J.C. Regulation of glucose homeostasis by glucocorticoids.Adv. Exp. Med. Biol. 2015; 872: 99-126Crossref PubMed Scopus (238) Google Scholar, 5Magomedova L. Cummins C.L. Glucocorticoids and metabolic control.Handb. Exp. Pharmacol. 2016; 233: 73-93Crossref PubMed Scopus (43) Google Scholar). Glucocorticoids convey their functions through the glucocorticoid receptor (GR), which is a transcription factor that binds to genomic glucocorticoid response elements to modulate the transcriptional rate of its target genes. Thus, GR primary target genes initiate the physiological actions of glucocorticoids. To understand the mechanisms underlying glucocorticoid actions in skeletal muscle, we previously used a combination of global gene expression analysis and chromatin immunoprecipitation sequencing to identify a list of potential GR primary target genes in murine C2C12 myotubes (6Kuo T. Lew M.J. Mayba O. Harris C.A. Speed T.P. Wang J.C. Genome-wide analysis of glucocorticoid receptor-binding sites in myotubes identifies gene networks modulating insulin signaling.Proc. Natl. Acad. Sci. U. S. A. 2012; 109: 11160-11165Crossref PubMed Scopus (103) Google Scholar). Phosphoinositide-3-kinase regulatory subunit 1 (Pik3r1, a.k.a. p85α) is one of these potential GR primary target genes (6Kuo T. Lew M.J. Mayba O. Harris C.A. Speed T.P. Wang J.C. Genome-wide analysis of glucocorticoid receptor-binding sites in myotubes identifies gene networks modulating insulin signaling.Proc. Natl. Acad. Sci. U. S. A. 2012; 109: 11160-11165Crossref PubMed Scopus (103) Google Scholar) and encodes a regulatory subunit of phosphoinositide 3-kinase (PI3K), which is composed of a regulatory subunit (Pik3r1, Pik3r2, or Pik3r3) and a catalytic subunit (Pik3ca1 or Pik3ca2) (7Cantley L.C. The phosphoinositide 3-kinase pathway.Science. 2002; 296: 1655-1657Crossref PubMed Scopus (4473) Google Scholar, 8Jean S. Kiger A.A. Classes of phosphoinositide 3-kinases at a glance.J. Cell Sci. 2014; 127: 923-928Crossref PubMed Scopus (167) Google Scholar). When insulin signaling is activated, PI3K is recruited to the activated insulin receptor substrate 1 to convert phosphatidylinositol-4, 5 bisphosphate to phosphatidylinositol-3, 4, 5 triphosphate. Binding to phosphatidylinositol-3, 4, 5 triphosphate at the plasma membrane is required to activate protein kinase Akt (9Hemmings B.A. Restuccia D.F. PI3K-PKB/Akt pathway.Cold Spring Harb. Perspect. Biol. 2012; 4a011189Crossref PubMed Scopus (531) Google Scholar), a key signaling molecule in mediating the metabolic functions of insulin. Though Pik3r1 is a key component of the insulin pathway, overexpression of monomeric Pik3r1 was found to suppress insulin signaling in myotubes and hepatocytes (10Barbour L.A. Mizanoor Rahman S. Gurevich I. Leitner J.W. Fischer S.J. Roper M.D. Knotts T.A. Vo Y. McCurdy C.E. Yakar S. Leroith D. Kahn C.R. Cantley L.C. Friedman J.E. Draznin B. Increased P85alpha is a potent negative regulator of skeletal muscle insulin signaling and induces in vivo insulin resistance associated with growth hormone excess.J. Biol. Chem. 2005; 280: 37489-37494Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 11Taniguchi C.M. Aleman J.O. Ueki K. Luo J. Asano T. Kaneto H. Stephanopoulos G. Cantley L.C. Kahn C.R. The p85alpha regulatory subunit of phosphoinositide 3-kinase potentiates c-Jun N-terminal kinase-mediated insulin resistance.Mol. Cell. Biol. 2007; 27: 2830-2840Crossref PubMed Scopus (59) Google Scholar). Conversely, Pik3r1 deficiency has been shown to improve insulin sensitivity (12Mauvais-Jarvis F. Ueki K. Fruman D.A. Hirshman M.F. Sakamoto K. Goodyear L.J. Iannacone M. Accili D. Cantley L.C. Kahn C.R. Reduced expression of the murine p85alpha subunit of phosphoinositide 3-kinase improves insulin signaling and ameliorates diabetes.J. Clin. Invest. 2002; 109: 141-149Crossref PubMed Scopus (214) Google Scholar, 13Terauchi Y. Tsuji Y. Satoh S. Minoura H. Murakami K. Okuno A. Inukai K. Asano T. Kaburagi Y. Ueki K. Nakajima H. Hanafusa T. Matsuzawa Y. Sekihara H. Yin Y. et al.Increased insulin sensitivity and hypoglycaemia in mice lacking the p85 alpha subunit of phosphoinositide 3-kinase.Nat. Genet. 1999; 21: 230-235Crossref PubMed Scopus (341) Google Scholar). Several mechanisms have been proposed for the inhibitory effect of excess Pik3r1 on insulin signaling. First, monomeric Pik3r1 competes with heterodimeric PI3K for binding to insulin receptor substrate-1 to suppress insulin signaling (14Luo J. Field S.J. Lee J.Y. Engelman J.A. Cantley L.C. The p85 regulatory subunit of phosphoinositide 3-kinase down-regulates IRS-1 signaling via the formation of a sequestration complex.J. Cell Biol. 2005; 170: 455-464Crossref PubMed Scopus (117) Google Scholar). Alternatively, Pik3r1 could enhance the activity of phosphatase and tensin homolog (PTEN) to inhibit PI3K (15Chagpar R.B. Links P.H. Pastor M.C. Furber L.A. Hawrysh A.D. Chamberlain M.D. Anderson D.H. Direct positive regulation of PTEN by the p85 subunit of phosphatidylinositol 3-kinase.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 5471-5476Crossref PubMed Scopus (149) Google Scholar). Another report shows that homodimeric but not monomeric Pik3r1 suppresses PI3K by protecting PTEN from ubiquitin-mediated proteasomal degradation. Further, the p85alpha homodimer enhances lipid phosphatase activity and membrane association of PTEN (16Cheung L.W. Walkiewicz K.W. Besong T.M. Guo H. Hawke D.H. Arold S.T. Mills G.B. Regulation of the PI3K pathway through a p85alpha monomer-homodimer equilibrium.eLife. 2015; 4e06866Crossref PubMed Scopus (44) Google Scholar). We previously found that the overexpression of Pik3r1 in C2C12 myotubes reduced cell diameter while reduction in Pik3r1 expression compromised glucocorticoid suppression of insulin signaling (6Kuo T. Lew M.J. Mayba O. Harris C.A. Speed T.P. Wang J.C. Genome-wide analysis of glucocorticoid receptor-binding sites in myotubes identifies gene networks modulating insulin signaling.Proc. Natl. Acad. Sci. U. S. A. 2012; 109: 11160-11165Crossref PubMed Scopus (103) Google Scholar). To further investigate the role of Pik3r1 in glucocorticoid actions in skeletal muscle, we created striated muscle–specific Pik3r1 knockout mice (MKO) (17Luo J. Sobkiw C.L. Hirshman M.F. Logsdon M.N. Li T.Q. Goodyear L.J. Cantley L.C. Loss of class IA PI3K signaling in muscle leads to impaired muscle growth, insulin response, and hyperlipidemia.Cell Metab. 2006; 3: 355-366Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). We treated MKO and Pik3r1flox/flox mice (will be referred as wildtype, WT, mice in this report) with or without a synthetic glucocorticoid, dexamethasone (DEX), and studied the effects on insulin signaling in metabolic tissues, including liver, gastrocnemius (GA) muscle, and epididymal white adipose tissue. We also examined the effect of striated muscle Pik3r1 deletion on systemic glucose and insulin tolerance. In addition, using stable isotope labeling techniques and tandem mass spectrometry, we analyzed the DEX effects on protein synthesis rates in GA muscle of WT and MKO mice. Finally, we investigated the signaling pathways involved in the regulation of protein synthesis and conducted histological analysis of the GA muscle in WT and MKO mice. We previously showed that Pik3r1 gene expression was elevated in mouse GA muscle upon DEX treatment (6Kuo T. Lew M.J. Mayba O. Harris C.A. Speed T.P. Wang J.C. Genome-wide analysis of glucocorticoid receptor-binding sites in myotubes identifies gene networks modulating insulin signaling.Proc. Natl. Acad. Sci. U. S. A. 2012; 109: 11160-11165Crossref PubMed Scopus (103) Google Scholar). We examined whether Pik3r1 protein expression was indeed increased by DEX treatment. Male WT mice were injected intraperitoneally with DEX or PBS daily for 1, 4, or 7 days. GA muscles were collected to detect the expression of Pik3r1 using immunoblotting. We found that Pik3r1 expression was significantly increased upon DEX treatment for 4 and 7 days (Fig. 1A). To confirm the activation of Pik3r1 gene transcription by GR in vivo, chromatin immunoprecipitation (ChIP) was performed to test the recruitment of GR to the glucocorticoid response element (GRE) of Pik3r1 gene in mouse GA muscle. Eight-week-old male WT mice were injected intraperitoneally with PBS or DEX for 4 days before tissue collection for ChIP given that Pik3r1 had the highest protein expression with 4 days of DEX treatment. The glucocorticoid response element of Pik3r1 has been located between −43,938 and −43,924 upstream of the mouse Pik3r1 gene (6Kuo T. Lew M.J. Mayba O. Harris C.A. Speed T.P. Wang J.C. Genome-wide analysis of glucocorticoid receptor-binding sites in myotubes identifies gene networks modulating insulin signaling.Proc. Natl. Acad. Sci. U. S. A. 2012; 109: 11160-11165Crossref PubMed Scopus (103) Google Scholar). We found that GR was significantly recruited to the Pik3r1 GRE by DEX treatment (approximately 15-fold comparing to IgG ChIP control) (Fig. 1B). Interestingly, GR was also recruited to the Pik3r1 GRE in PBS-treated animals to a lesser degree (approximately 5-fold, Fig. 1B). This suggests that plasma corticosterone levels are enough to activate Pik3r1 gene transcription through GR. Transcriptional activation is associated with elevated histone acetylation in the enhancer regions (18Shahbazian M.D. Grunstein M. Functions of site-specific histone acetylation and deacetylation.Annu. Rev. Biochem. 2007; 76: 75-100Crossref PubMed Scopus (1140) Google Scholar, 19Calo E. Wysocka J. Modification of enhancer chromatin: What, how, and why?.Mol. Cell. 2013; 49: 825-837Abstract Full Text Full Text PDF PubMed Scopus (786) Google Scholar). We performed ChIP to monitor the acetylated histone H3 (AcH3) and H4 (AcH4) and total H3 and H4 at the Pik3r1 GRE in the GA muscle of PBS and DEX-treated WT mice. The ratios of AcH3/H3 and AcH4/H4 represent the degrees of the histone acetylation in H3 and H4, respectively. As shown in Figure 1C, the level of total histone H3 and H4 was not significantly affected by DEX treatment. However, comparing to IgG control, the ratios of AcH3/H3 and AcH4/H4 were significantly increased by 8 and 10 folds with DEX treatment, respectively (Fig. 1C). In PBS treated animals, the ratios of AcH3/H3 and AcH4/H4 were also significantly higher than the IgG ChIP control (approximately 3 and 7 folds, respectively, Fig. 1C). This observation was consistent with the finding that GR was also recruited to the Pik3r1 GRE under PBS treatment. Nonetheless, the ratios of AcH3/H3 and AcH4/H4 were higher in DEX-treated mice than those of PBS-treated mice. This was in agreement with a stronger GR recruitment to the Pik3r1 GRE under DEX treatment (Fig. 1C). We next examined which histone acetyltransferase is recruited to the Pik3r1 GRE using ChIP. We found that p300 but not Tip60 and GCN5 was significantly recruited to the GRE upon DEX treatment (Fig. 1D). These results suggested that p300 accounted for the higher histone acetylation status at the GRE upon DEX treatment. Notably, neither p300, Tip60, nor GCN5 were recruited to the GRE upon PBS treatment (Fig. 1D). These results suggest that histone acetyltransferase(s) other than these three are involved in the acetylation of the Pik3r1 GRE in PBS-treated mice. To test whether p300 is involved in GR-activated Pik3r1 gene transcription, C2C12 myoblasts were infected with lentivirus expressing scramble small hairpin RNA (sh-scrRNA, control) or shRNA against p300 (sh-p300). After puromycin selection, cells were differentiated into myotubes, then treated with DEX or EtOH for 6 h. RNA was isolated from these cells, and qPCR was performed to monitor the expression of Pik3r1. In sh-scrRNA expressing C2C12 myotubes, DEX treatment increased the expression of Pik3r1 approximately 3-fold (Fig. 1E). However, in sh-p300 expressing C2C12 myotubes, such DEX effect was abolished (Fig. 1E). The Western blot showed that p300 was efficiently reduced by RNAi (Fig. 1E). We generated striated muscle–specific Pik3r1 knockout mice (MKO) by crossing Pik3r1flox/flox mice with transgenic mice carrying muscle creatine kinase promoter driving the expression of Cre recombinase (20Bruning J.C. Michael M.D. Winnay J.N. Hayashi T. Horsch D. Accili D. Goodyear L.J. Kahn C.R. A muscle-specific insulin receptor knockout exhibits features of the metabolic syndrome of NIDDM without altering glucose tolerance.Mol. Cell. 1998; 2: 559-569Abstract Full Text Full Text PDF PubMed Scopus (922) Google Scholar). In MKO mice, Pik3r1 expression was indeed depleted in skeletal muscles, including GA muscle, tibialis anterior muscle, and soleus muscle (Fig. 2A). Notably, Pik3r1 expression was similar in the liver of WT and MKO mice (Fig. 2A). These results validated the specific deletion of Pik3r1 in the striated muscle. We also tested the ability of DEX to induce the expression of GR primary target gene in GA muscle of WT and MKO mice. WT and MKO mice were treated with PBS or DEX for 7 days. GA muscle RNA was isolated, and qPCR was performed to examine the DEX induction of two previously identified GR primary target genes, Fkbp5 (21Paakinaho V. Makkonen H. Jaaskelainen T. Palvimo J.J. Glucocorticoid receptor activates poised FKBP51 locus through long-distance interactions.Mol. Endocrinol. 2010; 24: 511-525Crossref PubMed Scopus (87) Google Scholar) and Sesn1 (6Kuo T. Lew M.J. Mayba O. Harris C.A. Speed T.P. Wang J.C. Genome-wide analysis of glucocorticoid receptor-binding sites in myotubes identifies gene networks modulating insulin signaling.Proc. Natl. Acad. Sci. U. S. A. 2012; 109: 11160-11165Crossref PubMed Scopus (103) Google Scholar). DEX treatment increased approximately 3-fold of expression of Sesn1 in WT mice. In MKO mice, similar DEX response was observed (Fig. 2B). DEX treatment elevated the expression of Fkbp5 approximately 5-fold in WT mice (Fig. 2B). The basal Fkbp5 expression was lower in MKO mice, though not statistically significant (p = 0.11). DEX treatment also efficiently increased Fkbp5 expression in MKO mice (Fig. 2B). These results indicated that depleting Pik3r1 expression did not affect general GR activity in GA muscle. WT and MKO mice treated with or without DEX for 7 days were injected with insulin for 10 min. GA muscle, liver, and epididymal white adipose tissue were isolated, and the activity of a key molecule in insulin signaling, Akt, was monitored. For Akt activity, we monitored the levels of phosphorylated Akt (pAkt) at serine 473 residue (22Franke T.F. Yang S.I. Chan T.O. Datta K. Kazlauskas A. Morrison D.K. Kaplan D.R. Tsichlis P.N. The protein kinase encoded by the Akt proto-oncogene is a target of the PDGF-activated phosphatidylinositol 3-kinase.Cell. 1995; 81: 727-736Abstract Full Text PDF PubMed Scopus (1803) Google Scholar) and total Akt. The activity of Akt was represented by the ratio of pAkt and Akt. Insulin treatment elevated pAkt/Akt ratio in GA muscle, liver, and epididymal white adipose tissue of WT mice (Fig. 2B). In MKO mice, insulin treatment significantly increased pAkt/Akt ratio in GA muscle, liver, and epididymal white adipose tissue (Fig. 3A). We found that DEX treatment reduced Akt activity in all three tissues of WT mice (Fig. 3A). In epididymal white adipose tissue and liver of MKO mice, DEX treatment was still able to inhibit Akt activity (Fig. 3A). However, in GA muscle, the ability of DEX to reduce insulin stimulated Akt activity was significantly attenuated (Fig. 3A). These results demonstrate that Pik3r1 deficiency in striated muscle reduced the DEX effect on insulin action in GA muscle but not in other insulin responsive metabolic tissues, such as epididymal white adipose tissue and liver. To examine whether Pik3r1 deletion in striated muscle affects the ability of DEX to modulate insulin sensitivity, WT and MKO mice were treated with or without DEX for 1 week. After 15 h fasting, intraperitoneal glucose tolerance test was performed in these mice. In WT mice, DEX treatment caused glucose intolerance and hyperinsulinemia. (Fig. 3, B and C). In contrast, in MKO mice, although DEX treatment still caused hyperinsulinemia (Fig. 2D), DEX-induced glucose tolerance was significantly improved (Fig. 2D). We also performed insulin tolerance test in WT and MKO mice treated with or without DEX for 1 week. We found that DEX treatment resulted in insulin intolerance in WT mice (Fig. 3D). In MKO mice, DEX treatment still caused insulin intolerance, but to with a lesser degree (Fig. 3D). Thus, DEX effect on insulin tolerance was somewhat reduced in MKO mice (Fig. 3D). Without DEX treatment, MKO and WT mice had similar glucose and insulin tolerance and plasma insulin levels were similar (Fig. 3, B–D). These results are in agreement with the previous report (17Luo J. Sobkiw C.L. Hirshman M.F. Logsdon M.N. Li T.Q. Goodyear L.J. Cantley L.C. Loss of class IA PI3K signaling in muscle leads to impaired muscle growth, insulin response, and hyperlipidemia.Cell Metab. 2006; 3: 355-366Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Overall, our results demonstrate that Pik3r1 depletion in striated muscle attenuates the DEX treatment-induced glucose and insulin intolerance. To determine how DEX treatment in WT and MKO mice leads to muscle atrophy, muscle proteome–wide fractional synthesis rates were measured by LC-MS/MS after 2H2O labeling. Mice were administered DEX or PBS for 10 days followed by 2H2O labeling in drinking water for the last 7 days (23Holmes W.E. Angel T.E. Li K.W. Hellerstein M.K. Dynamic proteomics: In vivo proteome-wide measurement of protein kinetics using metabolic labeling.Methods Enzymol. 2015; 561: 219-276Crossref PubMed Scopus (30) Google Scholar, 24Price J.C. Khambatta C.F. Li K.W. Bruss M.D. Shankaran M. Dalidd M. Floreani N.A. Roberts L.S. Turner S.M. Holmes W.E. Hellerstein M.K. The effect of long term calorie restriction on in vivo hepatic proteostatis: A novel combination of dynamic and quantitative proteomics.Mol. Cell. Proteomics. 2012; 11: 1801-1814Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 25Papageorgopoulos C. Caldwell K. Shackleton C. Schweingrubber H. Hellerstein M.K. Measuring protein synthesis by mass isotopomer distribution analysis (MIDA).Anal. Biochem. 1999; 267: 1-16Crossref PubMed Scopus (58) Google Scholar, 26Busch R. Kim Y.K. Neese R.A. Schade-Serin V. Collins M. Awada M. Gardner J.L. Beysen C. Marino M.E. Misell L.M. Hellerstein M.K. Measurement of protein turnover rates by heavy water labeling of nonessential amino acids.Biochim. Biophys. Acta. 2006; 1760: 730-744Crossref PubMed Scopus (176) Google Scholar). GA muscle protein fractional synthesis criteria for each group (n ≤ 3) filtered the data set to 57 proteins common among each group. Detailed filtering criteria was further explained in the method section (23Holmes W.E. Angel T.E. Li K.W. Hellerstein M.K. Dynamic proteomics: In vivo proteome-wide measurement of protein kinetics using metabolic labeling.Methods Enzymol. 2015; 561: 219-276Crossref PubMed Scopus (30) Google Scholar). Database for Annotation, Visualization, and Integrated Discover ontology analysis was employed to characterize the biochemical function and cellular localization for the proteomics data set. The proteins were grouped as either glucose metabolism (n = 16), mitochondrial (n = 16), cytoplasmic (n = 20), and myofibril proteins, (n = 5) of which, 19 individual proteins were significantly decreased by DEX after Bonferroni correction for multiple comparisons. (Supporting Information 1). Global average GA muscle protein fraction synthesis rates are shown in WT and MKO mice GA muscle treated with PBS or DEX (represented as WT+PBS, WT+DEX, MKO + PBS and MKO + DEX, respectively, Fig. 4A). The mean GA protein fraction synthesis values were 18.0%, 13.5%, 15.2%, and 12.9% for WT+PBS, WT+DEX, MKO + PBS and MKO + DEX mice, respectively. The overall protein synthesis rate in MKO mouse GA muscle was significantly lower than that of WT mouse GA muscle (15.5% reduction (15.2%/18.0%), p ≤ 0.0001). DEX significantly reduced protein synthesis in both WT and MKO mice. However, in WT mouse GA muscle, the overall protein synthesis rate was decreased by an average of 25.1% (13.5%/18.0%), whereas in MKO mouse GA muscle, the overall protein synthesis rate was reduced by only 15.1% (12.9%/15.2%) with DEX treatment. The relative reduction in the change in GA muscle individual protein fractional synthesis rates were also compared among groups. Experimental fractional synthesis (f) values for the proteins common among each model were plotted in dot plot and sorted from low to high, and then by protein (Fig. 4B). Graphically, in WT+DEX (red line), MKO + PBS (green line), and MKO + DEX mice (purple line), most proteins exhibited a decrease in fractional synthesis as compared with WT (blue line). We then compared the percent change in each protein’s fractional synthesis for WT animals treated with or without DEX (Fig. 4C). Statistical significance was assessed by a binomial distribution of the proportion of proteins showing a negative or positive percent change in fractional synthesis with DEX treatment (Fig. 4C). In WT+DEX, 96.5% or 55/57 proteins showed lower fractional synthesis rates as compared with WT mice (Fig. 4C). Only two proteins, electron transfer flavoprotein subunit beta and peroxiredoxin-1, did not have lower fractional synthesis by DEX treatment (Supporting Information 1). Surprisingly, in MKO animals, 48/57 or 84.2% of GA muscle proteins showed lower fractional synthesis compared with WT mice (Fig. 4C). In addition, 48/57 or 84.2% of proteins in MKO mice were reduced by DEX treatment. Thus, seven proteins whose protein synthesis rates were reduced in WT mice by DEX were not affected by DEX in MKO mouse GA muscle, whereas two proteins, alpha-enolase and phosphoglycerate kinase-1, had a higher protein synthesis rate (17% and 9.4%, respectively) in MKO mice treated with DEX (Supporting Information 2). Finally, 37/57 or 64.9% of GA muscle proteins had lower fractional synthesis in WT + DEX mice compared with MKO + DEX mice, whereas 35.1% had higher fractional synthesis (p = 0.016). Overall, these results indicated that DEX effect on lowering protein synthesis was compromised without Pik3r1 in GA muscle. We also performed histological analysis (Fig. 5A) to monitor the cross-section area and fiber count of GA muscle of WT + PBS, MKO + PBS, WT + DEX, and MKO + DEX mice, asking whether DEX induced muscle histological changes are compromised in MKO mice. Cross-sectional area of GA muscle fibers significantly decreased from a mean of 2277 μm2 in WT animals to 1382 μm2 in WT + DEX mice (Fig. 5B). In MKO mice, cross-sectional area of GA muscle fibers was 1709 μm2 (Fig. 5B). This is in agreement with the lower protein synthesis rates in MKO GA muscle shown above. Importantly, there was no reduction in cross-sectional area of GA muscle fibers in MKO + DEX mice (1979 μm2) compared with MKO mice. GA muscle fiber count showed similar results. DEX treatment increased fiber count in WT GA muscle, and MKO had higher fiber count than WT mice, but DEX did not alter fiber count in MKO mice (Fig. 5C). Immunohistochemical staining was used to analyze the fiber types of GA muscle of WT and MKO mice. The antibodies against MHC I, MHC IIa, MHC IIb, and MHC IIx were used. MHC I is encoded by Myh7 and represents slow oxidative fibers, whereas MHC IIa is encoded by Myh2 and represents fast oxidative fibers (27Schiaffino S. Muscle fiber type diversity revealed by anti-myosin heavy chain antibodies.FEBS J. 2018; 285: 3688-3694Crossref PubMed Scopus (49) Google Scholar). MHC IIb is encoded by Myh4, and MHC IIx is encoded by Myh1 and represent fast glycolytic fibers IIb and IIx, respectively (27Schiaffino S. Muscle fiber type diversity revealed by anti-myosin heavy chain antibodies.FEBS J. 2018; 285: 3688-3694Crossref PubMed Scopus (49) Google Scholar). The fiber type composition of GA muscle of WT mice was similar to previous reports (Fig. 5D) (28Kammoun M. Cassar-Malek I. Meunier B. Picard B. A simplified immunohistochemical classification of skeletal muscle fibres in mouse.Eur. J. Histochem. 2014; 58: 2254Crossref PubMed Scopus (59) Google Scholar). It appears that GA muscle of MKO mice had more fast oxidative fibers, as MHC IIa levels were higher than those of WT mice (Fig. 5D). We further analyzed the DEX effect on signaling processes that regulate protein synthesis. As described above, WT and MKO mice were treated with or without DEX and were injected with insulin for 10 min before GA muscle was isolated. We first monitored the phosphorylation at threonine 389 of p70S6 kinase (S6K) (29Pullen N. Thomas G. The modular phosphorylation and activation of p70s6k.FEBS Lett. 1997; 410: 78-82Crossref PubMed Scopus (478) Google Scholar), which is critical for S6K activity, using ELISA. S6K phosphorylates the S6 protein of the 40S ribosomal subunit and is involved in translational control of 5' oligopyrimidine tract mRNAs (29Pullen N. Thomas G. The modular phosphorylation and activation of p70s6k.FEBS Lett. 1997; 410: 78-82Crossref PubMed Sc" @default.
• W3128780270 created "2021-02-15" @default.
• W3128780270 creator A5027229757 @default.
• W3128780270 creator A5034964059 @default.
• W3128780270 creator A5037568402 @default.
• W3128780270 creator A5038787801 @default.
• W3128780270 creator A5046185218 @default.
• W3128780270 creator A5047114493 @default.
• W3128780270 creator A5055037534 @default.
• W3128780270 creator A5065992735 @default.
• W3128780270 creator A5075902955 @default.
• W3128780270 creator A5076562111 @default.
• W3128780270 creator A5081964520 @default.
• W3128780270 creator A5081991449 @default.
• W3128780270 creator A5091496107 @default.
• W3128780270 date "2021-01-01" @default.
• W3128780270 modified "2023-10-09" @default.
• W3128780270 title "The role of striated muscle Pik3r1 in glucose and protein metabolism following chronic glucocorticoid exposure" @default.
• W3128780270 cites W1023891580 @default.
• W3128780270 cites W1523062737 @default.
• W3128780270 cites W1590930560 @default.
• W3128780270 cites W1876389666 @default.
• W3128780270 cites W1947165807 @default.
• W3128780270 cites W1981001162 @default.
• W3128780270 cites W1981463574 @default.
• W3128780270 cites W1986586739 @default.
• W3128780270 cites W1988674210 @default.
• W3128780270 cites W2004078164 @default.
• W3128780270 cites W2016712537 @default.
• W3128780270 cites W2025483501 @default.
• W3128780270 cites W2026424902 @default.
• W3128780270 cites W2035145212 @default.
• W3128780270 cites W2053127486 @default.
• W3128780270 cites W2062089569 @default.
• W3128780270 cites W2063593894 @default.
• W3128780270 cites W2067753190 @default.
• W3128780270 cites W2070732456 @default.
• W3128780270 cites W2071180802 @default.
• W3128780270 cites W2072780644 @default.
• W3128780270 cites W2073530412 @default.
• W3128780270 cites W2079936381 @default.
• W3128780270 cites W2085100642 @default.
• W3128780270 cites W2087835012 @default.
• W3128780270 cites W2091535117 @default.
• W3128780270 cites W2098957689 @default.
• W3128780270 cites W2099051331 @default.
• W3128780270 cites W2113561514 @default.
• W3128780270 cites W2115826043 @default.
• W3128780270 cites W2122327012 @default.
• W3128780270 cites W2123933189 @default.
• W3128780270 cites W2126784007 @default.
• W3128780270 cites W2133313557 @default.
• W3128780270 cites W2147652719 @default.
• W3128780270 cites W2152599381 @default.
• W3128780270 cites W2158217645 @default.
• W3128780270 cites W2158747282 @default.
• W3128780270 cites W2160079255 @default.
• W3128780270 cites W2160315579 @default.
• W3128780270 cites W2163236262 @default.
• W3128780270 cites W2164443997 @default.
• W3128780270 cites W2165718181 @default.
• W3128780270 cites W2169058447 @default.
• W3128780270 cites W2283563215 @default.
• W3128780270 cites W2299795484 @default.
• W3128780270 cites W2409704225 @default.
• W3128780270 cites W2538175099 @default.
• W3128780270 cites W2596687088 @default.
• W3128780270 cites W2611398367 @default.
• W3128780270 cites W2796317691 @default.
• W3128780270 cites W280207433 @default.
• W3128780270 cites W2804710503 @default.
• W3128780270 cites W2903870788 @default.
• W3128780270 cites W3014838072 @default.
• W3128780270 cites W3019679086 @default.
• W3128780270 cites W4233120011 @default.
• W3128780270 cites W4243898144 @default.
• W3128780270 cites W4249273120 @default.
• W3128780270 doi "https://doi.org/10.1016/j.jbc.2021.100395" @default.
• W3128780270 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/8010618" @default.
• W3128780270 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/33567340" @default.
• W3128780270 hasPublicationYear "2021" @default.
• W3128780270 type Work @default.
• W3128780270 sameAs 3128780270 @default.
• W3128780270 citedByCount "6" @default.
• W3128780270 countsByYear W31287802702022 @default.
• W3128780270 countsByYear W31287802702023 @default.
• W3128780270 crossrefType "journal-article" @default.
• W3128780270 hasAuthorship W3128780270A5027229757 @default.
• W3128780270 hasAuthorship W3128780270A5034964059 @default.
• W3128780270 hasAuthorship W3128780270A5037568402 @default.
• W3128780270 hasAuthorship W3128780270A5038787801 @default.
• W3128780270 hasAuthorship W3128780270A5046185218 @default.
• W3128780270 hasAuthorship W3128780270A5047114493 @default.
• W3128780270 hasAuthorship W3128780270A5055037534 @default.
• W3128780270 hasAuthorship W3128780270A5065992735 @default.
• W3128780270 hasAuthorship W3128780270A5075902955 @default.
• W3128780270 hasAuthorship W3128780270A5076562111 @default.
• W3128780270 hasAuthorship W3128780270A5081964520 @default.

• next