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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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Phosphorylation of insulin signaling proteins are up-regulated in NUAK1-deficient muscle. Immunoblot analysis of phosphorylation of IRS1 at Tyr-608 (black arrowhead) and AKT at Thr-308 in soleus muscle of HFD-fed control and MNUAK1KO mice. A white arrowhead denotes a nonspecific band. Graphs show the intensities of phospho-protein bands normalized to total protein levels in each case. The data are expressed relative to those from control mice. *, p < 0.05; **, p < 0.01 (Student's t test). A, mice under fed conditions. The data are the means ± S.E. (n = 3). B, mice were fasted overnight and administered water or glucose (1 g/kg of body weight). Soleus was excised 40 min after the administration. The data are the means ± S.E. (n = 6). C, mice were fasted overnight and intraperitoneally injected with PBS or insulin (1.5 units/kg of body weight). Soleus was excised 20 min after the injection. The data are the means ± S.E. (n = 3). Cont, control; MNKO, MNUAK1KO.
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Figure 7: Phosphorylation of insulin signaling proteins are up-regulated in NUAK1-deficient muscle. Immunoblot analysis of phosphorylation of IRS1 at Tyr-608 (black arrowhead) and AKT at Thr-308 in soleus muscle of HFD-fed control and MNUAK1KO mice. A white arrowhead denotes a nonspecific band. Graphs show the intensities of phospho-protein bands normalized to total protein levels in each case. The data are expressed relative to those from control mice. *, p < 0.05; **, p < 0.01 (Student's t test). A, mice under fed conditions. The data are the means ± S.E. (n = 3). B, mice were fasted overnight and administered water or glucose (1 g/kg of body weight). Soleus was excised 40 min after the administration. The data are the means ± S.E. (n = 6). C, mice were fasted overnight and intraperitoneally injected with PBS or insulin (1.5 units/kg of body weight). Soleus was excised 20 min after the injection. The data are the means ± S.E. (n = 3). Cont, control; MNKO, MNUAK1KO.

Mentions: To validate the increased insulin signaling suggested by the phosphoproteome analysis, phosphorylation of IRS1 at Tyr-608 and AKT at Thr-308 in soleus muscle of mice fed a HFD were analyzed by immunoblotting. The phosphorylation levels of IRS1 and AKT were significantly higher in soleus muscle of MNUAK1KO mice than in that of control mice under fed conditions, confirming the enhancement of insulin signaling by a lack of NUAK1 (Fig. 7A). We also examined the phosphorylation of IRS1 and AKT in acute response to glucose and insulin in soleus muscle of mice fed a HFD. As shown in Fig. 7B, phosphorylation levels of IRS1 Tyr-608 and AKT Thr-308 in response to glucose administration were significantly higher in soleus muscle of MNUAK1KO mice than in that of control mice. The AKT Thr-308 phosphorylation in response to insulin injection was also significantly higher in the soleus muscle of MNUAK1KO mice than in that of control mice (Fig. 7C). No difference was observed in IRS1 phosphorylation in response to insulin injection, probably because of the time point for this analysis. Note that phosphorylation level of AKT under basal (fasted) conditions was significantly lower in soleus muscle of MNUAK1KO mice than in that of control mice (Fig. 7, B and C). An increase in the basal phosphorylation level of AKT is characteristic of insulin resistance induced by a HFD (44, 45). These observations strongly suggest that a HFD-induced insulin resistance was reduced in soleus muscle of MNUAK1KO mice. Therefore, we concluded that NUAK1 is involved in the negative regulation of insulin signal transduction in soleus muscle.


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)

Phosphorylation of insulin signaling proteins are up-regulated in NUAK1-deficient muscle. Immunoblot analysis of phosphorylation of IRS1 at Tyr-608 (black arrowhead) and AKT at Thr-308 in soleus muscle of HFD-fed control and MNUAK1KO mice. A white arrowhead denotes a nonspecific band. Graphs show the intensities of phospho-protein bands normalized to total protein levels in each case. The data are expressed relative to those from control mice. *, p < 0.05; **, p < 0.01 (Student's t test). A, mice under fed conditions. The data are the means ± S.E. (n = 3). B, mice were fasted overnight and administered water or glucose (1 g/kg of body weight). Soleus was excised 40 min after the administration. The data are the means ± S.E. (n = 6). C, mice were fasted overnight and intraperitoneally injected with PBS or insulin (1.5 units/kg of body weight). Soleus was excised 20 min after the injection. The data are the means ± S.E. (n = 3). Cont, control; MNKO, MNUAK1KO.
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Figure 7: Phosphorylation of insulin signaling proteins are up-regulated in NUAK1-deficient muscle. Immunoblot analysis of phosphorylation of IRS1 at Tyr-608 (black arrowhead) and AKT at Thr-308 in soleus muscle of HFD-fed control and MNUAK1KO mice. A white arrowhead denotes a nonspecific band. Graphs show the intensities of phospho-protein bands normalized to total protein levels in each case. The data are expressed relative to those from control mice. *, p < 0.05; **, p < 0.01 (Student's t test). A, mice under fed conditions. The data are the means ± S.E. (n = 3). B, mice were fasted overnight and administered water or glucose (1 g/kg of body weight). Soleus was excised 40 min after the administration. The data are the means ± S.E. (n = 6). C, mice were fasted overnight and intraperitoneally injected with PBS or insulin (1.5 units/kg of body weight). Soleus was excised 20 min after the injection. The data are the means ± S.E. (n = 3). Cont, control; MNKO, MNUAK1KO.
Mentions: To validate the increased insulin signaling suggested by the phosphoproteome analysis, phosphorylation of IRS1 at Tyr-608 and AKT at Thr-308 in soleus muscle of mice fed a HFD were analyzed by immunoblotting. The phosphorylation levels of IRS1 and AKT were significantly higher in soleus muscle of MNUAK1KO mice than in that of control mice under fed conditions, confirming the enhancement of insulin signaling by a lack of NUAK1 (Fig. 7A). We also examined the phosphorylation of IRS1 and AKT in acute response to glucose and insulin in soleus muscle of mice fed a HFD. As shown in Fig. 7B, phosphorylation levels of IRS1 Tyr-608 and AKT Thr-308 in response to glucose administration were significantly higher in soleus muscle of MNUAK1KO mice than in that of control mice. The AKT Thr-308 phosphorylation in response to insulin injection was also significantly higher in the soleus muscle of MNUAK1KO mice than in that of control mice (Fig. 7C). No difference was observed in IRS1 phosphorylation in response to insulin injection, probably because of the time point for this analysis. Note that phosphorylation level of AKT under basal (fasted) conditions was significantly lower in soleus muscle of MNUAK1KO mice than in that of control mice (Fig. 7, B and C). An increase in the basal phosphorylation level of AKT is characteristic of insulin resistance induced by a HFD (44, 45). These observations strongly suggest that a HFD-induced insulin resistance was reduced in soleus muscle of MNUAK1KO mice. Therefore, we concluded that NUAK1 is involved in the negative regulation of insulin signal transduction in soleus muscle.

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