1. Volume 11, Issue 8, Pages 1220-1235 (May 2015)
Differential Role of Insulin/IGF-1 Receptor Signaling in Muscle Growth and Glucose Homeostasis 
Brian T. O’Neill, Hans P.M.M. Lauritzen, Michael F. Hirshman, Graham Smyth, Laurie J. Goodyear, C. Ronald Kahn 
Cell Reports 
Volume 11, Issue 8, Pages (May 2015)
DOI: /j.celrep
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2. Cell Reports 2015 11, 1220-1235DOI: (10.1016/j.celrep.2015.04.037)
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3. Figure 1
Deletion of IRs and IGF1Rs in Muscle Dramatically Decreases Muscle Size and Survival
(A) Western blot of insulin receptor-β (IR-β) and IGF-1 receptor-β (IGF1R-β) was measured in quadriceps from mice with muscle-specific deletion of insulin receptor (M-IR−/−), IGF-1 receptor (M-IGF1R−/−), or both IGF-1 receptor and insulin receptor (MIGIRKO).
(B) Representative profile and hindlimb dissection of control and MIGIRKO littermate mice.
(C) Body weight was measured weekly in control and MIGIRKO mice (n = 7–16).
(D) Survival curve of MIGIRKO mice compared to control, M-IR−/−, and M-IGF1R−/−, represented as “All other genotypes” (n = 14–20 per group).
(E) Body weight was measured at time of sacrifice of control, M-IR−/−, M-IGF1R−/−, and MIGIRKO mice (n = 5–8 knockout mice and pooled 20 controls).
(F) Representative muscle dissection from control and MIGIRKO mice.
(G) Dissected muscle weights measured from control, M-IR−/−, M-IGF1R−/−, and MIGIRKO mice (n = 5–9 knockout mice and pooled 22 controls).
(H) Representative cross section of TA muscle stained for SDH to demonstrate oxidative (purple) and glycolytic (gray/white) muscle fibers from control, M-IR−/−, M-IGF1R−/−, and MIGIRKO mice.
(I) Quantification of total number of muscle fibers normalized to cross sectional area of TA sections in mm2 (n = 3–6 per group).
(J) Quantification of total oxidative and glycolytic fibers per TA section (n = 3–6 per group).
∗p < 0.05, ∗∗p < 0.01 versus control (ANOVA). All mice were 11–15 weeks old. Quad, quadriceps; TA, tibialis anterior; EDL, extensor digitorum longus; Sol, soleus; Gast, gastrocnemius; SDH, succinate dehydrogenase. Data are presented as mean ± SEM. See also Figures S1 and S2 and Table S2.
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4. Figure 2
MIGIRKO Mice Display Normal Glucose Tolerance, Fasting Hypoglycemia, and Increased Basal Glucose Uptake into Muscle, Despite Abolished Insulin Signaling in Muscle
(A) Blood glucose levels were measured in 8- to 10-week-old MIGIRKO and control mice fasted overnight or randomly fed (n = 9–10).
(B and C) Insulin (B) and triglyceride (C) levels from 8- to 10-week-old MIGIRKO and control mice fasted overnight or refed for 4 hr (n = 9–10).
(D) Insulin signaling was determined by western blot analysis in quadriceps muscle from 11- to 15-week-old MIGIRKO and control mice fasted overnight and treated with saline or insulin intravenously.
(E and F) Intraperitoneal glucose tolerance test (GTT) (E) and insulin tolerance test (ITT) (F) were performed in 8- to 10-week-old MIGIRKO and control mice (n = 9–10).
(G) In vivo 2-deoxyglucose uptake was performed as described in Experimental Procedures in control and MIGIRKO mice (n = 6–8 per group).
∗∗p < 0.01 versus control (Student’s t test), #p < 0.05 versus control with same treatment, and †p < 0.05 versus basal of same genotype (ANOVA). Quad, quadriceps; Gastroc, gastrocnemius; BAT, brown adipose tissue. Data are presented as mean ± SEM. See also Figure S3.
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5. Figure 3
Deletion of Muscle IRs and IGF1Rs Paradoxically Increases Glucose Transporter Expression and Membrane Localization
(A and B) Ex vivo 2-deoxyglucose uptake was measured in EDL (A) and soleus (B) from 8- to 10-week-old control, M-IR−/−, M-IGF1R−/−, and MIGIRKO mice (n = 5 knockouts and 12 pooled controls).
(C and D) Glut1 (C) and Glut4 (D) total protein measured by western blot in control, M-IR−/−, M-IGF1R−/−, and MIGIRKO quadriceps (n = 4).
(E) Glut1 and Glut4 mRNA levels were measured in quadriceps from control, M-IR−/−, M-IGF1R−/−, and MIGIRKO mice by qRT-PCR (n = 5–8).
(F) Glut4-EGFP was transfected into vastus lateralis muscle and visualized 5 days later as described in Experimental Procedures. Scale bar, 10 μm (n = 2).
(G) Glut1 and Glut4 levels in plasma membrane (PM) isolates from mixed hindlimb muscle (n = 3).
(H) Phosphorylation of AMPKT172 was measured in quadriceps (n = 4).
∗p < 0.05, ∗∗p < 0.01 versus control; †p < 0.05 versus basal of same genotype (ANOVA). Data are presented as mean ± SEM. See also Figure S4 and Tables S1 and S3.
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6. Figure 4
Deletion of IRs and IGF1Rs in Muscle Leads to Suppression of TBC1D1, and Re-expression of TBC1D1 Normalizes Glut4 Localization
(A) AS160 phospho- and total protein, phospho-Akt substrate 160-kDa band (PAS 160), and TBC1D1 total protein were measured by western blot in control and MIGIRKO quadriceps (n = 4).
(B) TBC1D1 and AS160 mRNA levels were measured in TA muscle from control, M-IR−/−, M-IGF1R−/−, and MIGIRKO mice by qRT-PCR (n = 4–8).
(C) Glut4-EGFP was transfected into vastus lateralis muscle along with empty vector (EV) or with TBC1D1, and visualized 5 days later in the fed state. Scale bar, 10 μm.
(D and E) Quantification of average area of all Glut4 depots >1 μm from control and MIGIRKO mice transfected with Glut4-EGFP + EV and MIGIRKO transfected with Glut4-EGFP + TBC1D1 (n = 2 control and n = 3 MIGIRKO mice per group with three to seven fibers each).
∗p < 0.05, ∗∗p < 0.01 versus control (ANOVA); §p < 0.05 versus MIGIRKO + EV (Student’s t test). Data are presented as mean ± SEM. See also Figure S4 and Table S1.
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7. Figure 5
Increased Energy Expenditure in MIGIRKO Mice Is Associated with Browning of Subcutaneous Fat
(A) Daily food and water intake were measured in control and MIGIRKO animals and normalized per mouse or per milligram of lean body weight (LBW) (n = 9–10).
(B) Oxygen consumption (VO2) and carbon dioxide production (VCO2) per kg of LBW were measured using CLAMS metabolic cages (n = 9–10).
(C) Respiratory exchange ratio (RER) was measured in control and MIGIRKO mice (n = 9–10).
(D) Activity was measured as the number of times an animal crossed a horizontal laser (n = 9–10).
(E) H&E staining was performed on inguinal subcutaneous white adipose tissue (sWAT) from control and MIGIRGO animals.
(F) Markers of brown adipose tissue (BAT) were measured by qRT-PCR in sWAT and epididymal WAT (eWAT); (n = 6).
(G) In vivo 2-deoxyglucose uptake under basal or insulin-stimulated conditions was measured in sWAT and eWAT from control and MIGIRKO mice (n = 6–8).
(H) Fgf21 mRNA levels from quadriceps of MIGIRKO and control mice either randomly fed or fasted for 16 hr were measured by qRT-PCR ( n = 4 per group).
(I) Serum FGF21 levels in randomly fed control and MIGIRKO mice (n = 5).
∗p < 0.05, ∗∗p < 0.01 versus control (Student’s t test); #p < 0.05 versus control with same treatment, †p < 0.05 versus basal of same genotype (ANOVA). Data are presented as mean ± SEM. See also Table S1 and S4.
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8. Figure 6
MIGIRKO Mice Are Not Predisposed to Diabetes, Even after High-Fat Diet Feeding
(A) Body weights of control and MIGIRKO mice were measured weekly while on a chow diet (CD) or high-fat diet (HFD) beginning at 6 weeks of age (n = 4–8).
(B) Serum insulin levels from randomly fed mice and triglycerides from overnight fasted control and MIGIRKO mice on a CD or HFD for 4 weeks (n = 4–8).
(C and D) Intraperitoneal glucose tolerance test (GTT) (C) was performed and area under the curve (AUC) (D) was calculated for MIGIRKO and control mice on a CD or HFD for 9 weeks (n = 3–9).
(E and F) Intraperitoneal insulin tolerance test (ITT) (E) was performed and area above the curve (AAC) (F) was calculated for mice on a CD or HFD for 8 weeks (n = 3–9).
(G–I) VO2 (G), VCO2 (H), and RER (I) were measured in animals on a CD or HFD during both light and dark cycles using CLAMS metabolic cages (n = 3–9).
∗p < 0.05 versus control with same diet, †p < 0.05 versus CD of same genotype (Student’s t test). Data are presented as mean ± SEM. See also Figure S5.
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9. Figure 7
Overexpression of a Dominant-Negative, Kinase-Inactive IGF1R in Muscle of MIGIRKO Mice Induces Glucose Intolerance and Impaired Glucose Uptake in Heart
(A) Body weight was measured in 8- to 10-week-old control mice, mice with overexpression of a kinase inactive IGF1R in muscle (MKR), MIGIRKO mice, and MKR-MIGIRKO mice (n = 3–7).
(B) Blood glucose was measured in 8- to 10-week-old control, MKR, MIGIRKO, and MKR-MIGIRKO mice after an overnight fast and after 4 hr of refeeding (n = 3–7).
(C) Glut1 and Glut4 were measured in quadriceps (n = 8–10).
(D and E) Intraperitoneal glucose tolerance test (GTT) (D) was performed and area under the curve (AUC) (E) was calculated for 7- to 15-week-old mice (n = 4–7).
(F) In vivo 2-deoxyglucose uptake was performed during an IV GTT in control, MKR, MIGIRKO, and MKR-MIGIRKO mice (n = 6–11).
(G) Western blots for IGF1R expression and insulin signaling were performed on quadriceps and heart from control, MKR, MIGIRKO, and MKR-MIGIRKO mice after insulin injection via IVC.
∗p < 0.05, ∗∗p < 0.01 versus control (ANOVA), #p < 0.05 versus MIGIRKO (Student’s t test). Data are presented as mean ± SEM. See also Figures S7 and S8.
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