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Muscle-specific knock-out of NUAK family SNF1-like kinase 1 (NUAK1) prevents high fat diet-induced glucose intolerance.

Inazuka F, Sugiyama N, Tomita M, Abe T, Shioi G, Esumi H - J. Biol. Chem. (2012)

Bottom Line: Quantitative phosphoproteome analysis of soleus muscle was performed to understand the molecular mechanisms underlying the knock-out phenotype.Nuak1 mRNA was preferentially expressed in highly oxidative tissues such as brain, heart, and soleus muscle.Phosphoproteome analysis revealed that phosphorylation of IRS1 Ser-1097 was markedly decreased in NUAK1-deficient muscle.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa 277-8561, Japan.

ABSTRACT
NUAK1 is a member of the AMP-activated protein kinase-related kinase family. Recent studies have shown that NUAK1 is involved in cellular senescence and motility in epithelial cells and fibroblasts. However, the physiological roles of NUAK1 are poorly understood because of embryonic lethality in NUAK1 mice. The purpose of this study was to elucidate the roles of NUAK1 in adult tissues. We determined the tissue distribution of NUAK1 and generated muscle-specific NUAK1 knock-out (MNUAK1KO) mice. For phenotypic analysis, whole body glucose homeostasis and muscle glucose metabolism were examined. Quantitative phosphoproteome analysis of soleus muscle was performed to understand the molecular mechanisms underlying the knock-out phenotype. Nuak1 mRNA was preferentially expressed in highly oxidative tissues such as brain, heart, and soleus muscle. On a high fat diet, MNUAK1KO mice had a lower fasting blood glucose level, greater glucose tolerance, higher insulin sensitivity, and higher concentration of muscle glycogen than control mice. Phosphoproteome analysis revealed that phosphorylation of IRS1 Ser-1097 was markedly decreased in NUAK1-deficient muscle. Consistent with this, insulin signaling was enhanced in the soleus muscle of MNUAK1KO mice, as evidenced by increased phosphorylation of IRS1 Tyr-608, AKT Thr-308, and TBC1D4 Thr-649. These observations suggest that a physiological role of NUAK1 is to suppress glucose uptake through negative regulation of insulin signaling in oxidative muscle.

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Phosphoproteome of soleus muscles from MNUAK1KO and control mice. A total of 1,229 phosphopeptides were quantitatively detected in soleus muscle. KO/control ratio of phosphopeptides are shown by blue dots (mean, n = 4). Phosphopeptides with KO/control ratio of more than 2.0 or less than 0.5 are listed in Table 1. An IRS1 phosphopeptide containing Ser-1097 is indicated by an arrow.
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Figure 6: Phosphoproteome of soleus muscles from MNUAK1KO and control mice. A total of 1,229 phosphopeptides were quantitatively detected in soleus muscle. KO/control ratio of phosphopeptides are shown by blue dots (mean, n = 4). Phosphopeptides with KO/control ratio of more than 2.0 or less than 0.5 are listed in Table 1. An IRS1 phosphopeptide containing Ser-1097 is indicated by an arrow.

Mentions: To obtain a comprehensive understanding of the molecular mechanism underlying the observed MNUAK1KO phenotype, a quantitative phosphoproteome analysis was performed on soleus muscle isolated from HFD-fed MNUAK1KO and control mice in random fed state. Total protein was enzymatically digested, differentially labeled with stable isotopes, and then subjected to simultaneous LC-MS/MS analysis, which allowed for precise comparison between the two samples. To ensure reproducibility, four independent experiments were performed. All of the mass spectra and a detailed list of all proteins identified in this study are provided in the supplemental mass spectra and supplemental Table 3, respectively. Table 1 shows proteins that were found to be differentially phosphorylated more than 1.5-fold in all four experiments and more than 2-fold in average between MNUAK1KO and control mice. Among the 1,229 phosphopeptides quantitatively detected, the abundance of 27 phosphopeptides, corresponding to 21 proteins, decreased as a result of the Nuak1 deletion, whereas the abundance of six phosphopeptides, corresponding to six proteins, increased. Most of the differences in phosphorylation status were also observed under NC conditions (supplemental Table S2). It should be noted that differential regulation of phosphoproteins involved in glucose metabolism was clearly shown. In particular, the phosphorylation of IRS1 at Ser-1097 was changed most drastically (KO/control ratio = 0.10 ± 0.03) (Fig. 6). This serine phosphorylation (Ser-1101 in the human isoform), together with the phosphorylation of its upstream regulator PKCθ at Ser-676 (the same residue in the human isoform), is known to mediate negative feedback regulation of insulin signaling through blocking of IRS1 tyrosine phosphorylation (41). Phosphorylation of glycogen synthase 1 (GYS1) at the C-terminal serine residues including Ser-653 and Ser-657 (both same in the human isoform) leads to its inactivation and decreases glycogen synthesis (42, 43). Therefore, the hypophosphorylation of these sites in NUAK1-deficient soleus muscle can enhance insulin sensitivity and glycogen synthesis, which is consistent with the observed decreased blood glucose, improved glucose tolerance and insulin sensitivity, and increased muscle glycogen content phenotype. Besides glucose metabolism, biological processes shown to be affected include cell motility. Components of the actin-myosin cytoskeleton, such as various myosin isoforms, titin, and plectin, were also hypophosphorylated in NUAK1-deficient soleus muscle. Although it is unclear how changes in the phosphorylation status of these cytoskeletal proteins affect glucose metabolism and muscle contraction, the data suggest that NUAK1 has a role in regulation of muscle cytoskeletal structure.


Muscle-specific knock-out of NUAK family SNF1-like kinase 1 (NUAK1) prevents high fat diet-induced glucose intolerance.

Inazuka F, Sugiyama N, Tomita M, Abe T, Shioi G, Esumi H - J. Biol. Chem. (2012)

Phosphoproteome of soleus muscles from MNUAK1KO and control mice. A total of 1,229 phosphopeptides were quantitatively detected in soleus muscle. KO/control ratio of phosphopeptides are shown by blue dots (mean, n = 4). Phosphopeptides with KO/control ratio of more than 2.0 or less than 0.5 are listed in Table 1. An IRS1 phosphopeptide containing Ser-1097 is indicated by an arrow.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3351321&req=5

Figure 6: Phosphoproteome of soleus muscles from MNUAK1KO and control mice. A total of 1,229 phosphopeptides were quantitatively detected in soleus muscle. KO/control ratio of phosphopeptides are shown by blue dots (mean, n = 4). Phosphopeptides with KO/control ratio of more than 2.0 or less than 0.5 are listed in Table 1. An IRS1 phosphopeptide containing Ser-1097 is indicated by an arrow.
Mentions: To obtain a comprehensive understanding of the molecular mechanism underlying the observed MNUAK1KO phenotype, a quantitative phosphoproteome analysis was performed on soleus muscle isolated from HFD-fed MNUAK1KO and control mice in random fed state. Total protein was enzymatically digested, differentially labeled with stable isotopes, and then subjected to simultaneous LC-MS/MS analysis, which allowed for precise comparison between the two samples. To ensure reproducibility, four independent experiments were performed. All of the mass spectra and a detailed list of all proteins identified in this study are provided in the supplemental mass spectra and supplemental Table 3, respectively. Table 1 shows proteins that were found to be differentially phosphorylated more than 1.5-fold in all four experiments and more than 2-fold in average between MNUAK1KO and control mice. Among the 1,229 phosphopeptides quantitatively detected, the abundance of 27 phosphopeptides, corresponding to 21 proteins, decreased as a result of the Nuak1 deletion, whereas the abundance of six phosphopeptides, corresponding to six proteins, increased. Most of the differences in phosphorylation status were also observed under NC conditions (supplemental Table S2). It should be noted that differential regulation of phosphoproteins involved in glucose metabolism was clearly shown. In particular, the phosphorylation of IRS1 at Ser-1097 was changed most drastically (KO/control ratio = 0.10 ± 0.03) (Fig. 6). This serine phosphorylation (Ser-1101 in the human isoform), together with the phosphorylation of its upstream regulator PKCθ at Ser-676 (the same residue in the human isoform), is known to mediate negative feedback regulation of insulin signaling through blocking of IRS1 tyrosine phosphorylation (41). Phosphorylation of glycogen synthase 1 (GYS1) at the C-terminal serine residues including Ser-653 and Ser-657 (both same in the human isoform) leads to its inactivation and decreases glycogen synthesis (42, 43). Therefore, the hypophosphorylation of these sites in NUAK1-deficient soleus muscle can enhance insulin sensitivity and glycogen synthesis, which is consistent with the observed decreased blood glucose, improved glucose tolerance and insulin sensitivity, and increased muscle glycogen content phenotype. Besides glucose metabolism, biological processes shown to be affected include cell motility. Components of the actin-myosin cytoskeleton, such as various myosin isoforms, titin, and plectin, were also hypophosphorylated in NUAK1-deficient soleus muscle. Although it is unclear how changes in the phosphorylation status of these cytoskeletal proteins affect glucose metabolism and muscle contraction, the data suggest that NUAK1 has a role in regulation of muscle cytoskeletal structure.

Bottom Line: Quantitative phosphoproteome analysis of soleus muscle was performed to understand the molecular mechanisms underlying the knock-out phenotype.Nuak1 mRNA was preferentially expressed in highly oxidative tissues such as brain, heart, and soleus muscle.Phosphoproteome analysis revealed that phosphorylation of IRS1 Ser-1097 was markedly decreased in NUAK1-deficient muscle.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa 277-8561, Japan.

ABSTRACT
NUAK1 is a member of the AMP-activated protein kinase-related kinase family. Recent studies have shown that NUAK1 is involved in cellular senescence and motility in epithelial cells and fibroblasts. However, the physiological roles of NUAK1 are poorly understood because of embryonic lethality in NUAK1 mice. The purpose of this study was to elucidate the roles of NUAK1 in adult tissues. We determined the tissue distribution of NUAK1 and generated muscle-specific NUAK1 knock-out (MNUAK1KO) mice. For phenotypic analysis, whole body glucose homeostasis and muscle glucose metabolism were examined. Quantitative phosphoproteome analysis of soleus muscle was performed to understand the molecular mechanisms underlying the knock-out phenotype. Nuak1 mRNA was preferentially expressed in highly oxidative tissues such as brain, heart, and soleus muscle. On a high fat diet, MNUAK1KO mice had a lower fasting blood glucose level, greater glucose tolerance, higher insulin sensitivity, and higher concentration of muscle glycogen than control mice. Phosphoproteome analysis revealed that phosphorylation of IRS1 Ser-1097 was markedly decreased in NUAK1-deficient muscle. Consistent with this, insulin signaling was enhanced in the soleus muscle of MNUAK1KO mice, as evidenced by increased phosphorylation of IRS1 Tyr-608, AKT Thr-308, and TBC1D4 Thr-649. These observations suggest that a physiological role of NUAK1 is to suppress glucose uptake through negative regulation of insulin signaling in oxidative muscle.

Show MeSH
Related in: MedlinePlus