Journal: Diabetes

Article Title: Tissue-specific differences in the development of insulin resistance in a mouse model for type 1 diabetes.

doi: 10.2337/db13-1794

Figure Lengend Snippet: Figure 5—Protein levels of cellular signaling pathway components in gastrocnemius muscle (A–I) and liver (J–R) of WT, N-DM, and A-DM mice. A–F and J–N: Basal and insulin-stimulated expression levels (n = 6 per group) of IR (A and J), IRS1 (B), IRS2 (K), pIRS1-Ser1101 (C and L), pIRS1-Ser307 (D and M), membrane-to-cytosol ratio of Akt (E and N), and GLUT4 (F). G–I and O–R: Fasted conditions: membrane- to-cytosol ratio of PKCu (G) and PKCe (O) (n = 7–8 per group), cytosolic PKCe (P), pJNK (H and Q) (n = 6 per group), and pNFkB (I and R) (n = 6 per group). GAPDH was used as the loading control for all proteins. Data are mean 6 SEM. *P < 0.05, **P < 0.01, ***P < 0.001 by ANOVA with Bonferroni post hoc analysis. AU, arbitrary unit.

Article Snippet: Membranes were blocked with 5% milk in PBS and incubated with antibodies recognizing total Akt isoforms 1 and 2, extracellular signal-regulated kinase phosphorylated at Thr202 and Tyr204 (pERK-Thr202/Tyr204), c-Jun N-terminal kinase phosphorylated at Thr183 and Tyr185 (pJNK-Thr183/Tyr185), nuclear factor k-lightchain-enhancer of activated B cells phosphorylated at Ser536 (pNFkB-Ser536), p38 mitogen-activated protein kinase phosphorylated at Thr180 and Tyr182 (p-p38Thr180/Tyr182), Ser1101 phosphorylation of insulin receptor substrate 1 (IRS1) (pIRS1-Ser1101) and Ser307 phosphorylation of IRS1 (pIRS1-Ser307) (Cell Signaling Technology, Danvers, MA), GLUT4 (Abcam, Cambridge, U.K.), PKCe and PKCu (BD Biosciences, Franklin Lakes, NJ), and the insulin receptor b-subunit (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Expressing, Membrane, Control

Journal: Diabetes

Article Title: Tissue-specific differences in the development of insulin resistance in a mouse model for type 1 diabetes.

doi: 10.2337/db13-1794

Figure Lengend Snippet: Figure 7—Mechanisms of tissue-specific changes in glucose homeostasis occurring in diabetic and nondiabetic NOD mice. Effects occurring at the onset of T1DM (A-DM mice) are shown with red arrows and marks. Effects of the NOD genotype occurring independently of diabetes (both N-DM and A-DM mice) are shown with blue arrows and marks. The progression of T1DM is associated with increased circulating glucose and free FA levels resulting from impaired control of glucose and lipid metabolism by insulin. Intramyocellular lipotoxic intermediates DAGs activate PKCu, which results in enhanced Ser1101 and Ser307 phosphorylation of IRS1 (pIRS1-Ser1101 and pIRS- Ser307). This inhibits IRS1 activity and the membrane translocation of Akt and GLUT4, thereby decreasing muscle glucose disposal. In the liver, increased production of ROS and lipid peroxidation is associated with increased pJNK and IRS1 (pIRS1-Ser307 and pIRS1-Ser1101). This leads to lower translocation of Akt and impaired insulin-mediated suppression of EGP. Furthermore, fetuin A (FetA) levels in serum are increased, which could contribute to the downregulation of the insulin signaling pathway by binding to insulin receptor b-subunit and shutting down the phosphorylation at tyrosine (pTyr) (50) or by increasing pJNK through TLR4 (39). G6P, glucose-6-phosphate; GS3K, glycogen synthase kinase 3; PI3K, phosphatidylinositol 3-kinase.

Article Snippet: Membranes were blocked with 5% milk in PBS and incubated with antibodies recognizing total Akt isoforms 1 and 2, extracellular signal-regulated kinase phosphorylated at Thr202 and Tyr204 (pERK-Thr202/Tyr204), c-Jun N-terminal kinase phosphorylated at Thr183 and Tyr185 (pJNK-Thr183/Tyr185), nuclear factor k-lightchain-enhancer of activated B cells phosphorylated at Ser536 (pNFkB-Ser536), p38 mitogen-activated protein kinase phosphorylated at Thr180 and Tyr182 (p-p38Thr180/Tyr182), Ser1101 phosphorylation of insulin receptor substrate 1 (IRS1) (pIRS1-Ser1101) and Ser307 phosphorylation of IRS1 (pIRS1-Ser307) (Cell Signaling Technology, Danvers, MA), GLUT4 (Abcam, Cambridge, U.K.), PKCe and PKCu (BD Biosciences, Franklin Lakes, NJ), and the insulin receptor b-subunit (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Control, Phospho-proteomics, Activity Assay, Membrane, Translocation Assay, Binding Assay

Journal: Diabetes

Article Title: Tissue-specific differences in the development of insulin resistance in a mouse model for type 1 diabetes.

doi: 10.2337/db13-1794

Figure Lengend Snippet: Figure 5—Protein levels of cellular signaling pathway components in gastrocnemius muscle (A–I) and liver (J–R) of WT, N-DM, and A-DM mice. A–F and J–N: Basal and insulin-stimulated expression levels (n = 6 per group) of IR (A and J), IRS1 (B), IRS2 (K), pIRS1-Ser1101 (C and L), pIRS1-Ser307 (D and M), membrane-to-cytosol ratio of Akt (E and N), and GLUT4 (F). G–I and O–R: Fasted conditions: membrane- to-cytosol ratio of PKCu (G) and PKCe (O) (n = 7–8 per group), cytosolic PKCe (P), pJNK (H and Q) (n = 6 per group), and pNFkB (I and R) (n = 6 per group). GAPDH was used as the loading control for all proteins. Data are mean 6 SEM. *P < 0.05, **P < 0.01, ***P < 0.001 by ANOVA with Bonferroni post hoc analysis. AU, arbitrary unit.

Article Snippet: Membranes were blocked with 5% milk in PBS and incubated with antibodies recognizing total Akt isoforms 1 and 2, extracellular signal-regulated kinase phosphorylated at Thr202 and Tyr204 (pERK-Thr202/Tyr204), c-Jun N-terminal kinase phosphorylated at Thr183 and Tyr185 (pJNK-Thr183/Tyr185), nuclear factor k-lightchain-enhancer of activated B cells phosphorylated at Ser536 (pNFkB-Ser536), p38 mitogen-activated protein kinase phosphorylated at Thr180 and Tyr182 (p-p38Thr180/Tyr182), Ser1101 phosphorylation of insulin receptor substrate 1 (IRS1) (pIRS1-Ser1101) and Ser307 phosphorylation of IRS1 (pIRS1-Ser307) (Cell Signaling Technology, Danvers, MA), GLUT4 (Abcam, Cambridge, U.K.), PKCe and PKCu (BD Biosciences, Franklin Lakes, NJ), and the insulin receptor b-subunit (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Expressing, Membrane, Control

Journal: Diabetes

Article Title: Tissue-specific differences in the development of insulin resistance in a mouse model for type 1 diabetes.

doi: 10.2337/db13-1794

Figure Lengend Snippet: Figure 7—Mechanisms of tissue-specific changes in glucose homeostasis occurring in diabetic and nondiabetic NOD mice. Effects occurring at the onset of T1DM (A-DM mice) are shown with red arrows and marks. Effects of the NOD genotype occurring independently of diabetes (both N-DM and A-DM mice) are shown with blue arrows and marks. The progression of T1DM is associated with increased circulating glucose and free FA levels resulting from impaired control of glucose and lipid metabolism by insulin. Intramyocellular lipotoxic intermediates DAGs activate PKCu, which results in enhanced Ser1101 and Ser307 phosphorylation of IRS1 (pIRS1-Ser1101 and pIRS- Ser307). This inhibits IRS1 activity and the membrane translocation of Akt and GLUT4, thereby decreasing muscle glucose disposal. In the liver, increased production of ROS and lipid peroxidation is associated with increased pJNK and IRS1 (pIRS1-Ser307 and pIRS1-Ser1101). This leads to lower translocation of Akt and impaired insulin-mediated suppression of EGP. Furthermore, fetuin A (FetA) levels in serum are increased, which could contribute to the downregulation of the insulin signaling pathway by binding to insulin receptor b-subunit and shutting down the phosphorylation at tyrosine (pTyr) (50) or by increasing pJNK through TLR4 (39). G6P, glucose-6-phosphate; GS3K, glycogen synthase kinase 3; PI3K, phosphatidylinositol 3-kinase.

Article Snippet: Membranes were blocked with 5% milk in PBS and incubated with antibodies recognizing total Akt isoforms 1 and 2, extracellular signal-regulated kinase phosphorylated at Thr202 and Tyr204 (pERK-Thr202/Tyr204), c-Jun N-terminal kinase phosphorylated at Thr183 and Tyr185 (pJNK-Thr183/Tyr185), nuclear factor k-lightchain-enhancer of activated B cells phosphorylated at Ser536 (pNFkB-Ser536), p38 mitogen-activated protein kinase phosphorylated at Thr180 and Tyr182 (p-p38Thr180/Tyr182), Ser1101 phosphorylation of insulin receptor substrate 1 (IRS1) (pIRS1-Ser1101) and Ser307 phosphorylation of IRS1 (pIRS1-Ser307) (Cell Signaling Technology, Danvers, MA), GLUT4 (Abcam, Cambridge, U.K.), PKCe and PKCu (BD Biosciences, Franklin Lakes, NJ), and the insulin receptor b-subunit (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Control, Phospho-proteomics, Activity Assay, Membrane, Translocation Assay, Binding Assay