1. Volume 7, Issue 5, Pages 1085-1094 (May 2001)
A Role for AMP-Activated Protein Kinase in Contraction- and Hypoxia-Regulated Glucose Transport in Skeletal Muscle 
James Mu, Joseph T Brozinick, Otto Valladares, Maja Bucan, Morris J Birnbaum 
Molecular Cell 
Volume 7, Issue 5, Pages (May 2001)
DOI: /S (01)

2. Figure 1
Tissue-Specific Expression of Kinase-Dead AMPKα2 in Skeletal and Cardiac Muscles
(A) Mouse tissues were collected and submitted to Western blot analysis using an antibody that recognizes the myc epitope fused to the AMPKα transgene.
(B) Extensor digitorum longus (EDL) muscles were isolated from two lines of KD AMPKα2 transgenic mice (Tg-KD1 and Tg-KD2) and from their nontransgenic littermates (NTg1 and NTg2). Extracts were obtained and processed for Western blot analysis using α-AMPKN antibody.
(C) Extracts from different skeletal muscles and cardiac muscle were used for detection of AMPK using α-AMPKN antibody as above. All results are representative of three or more experiments. Epitrochlearis (Epi); gastrocnemius (gastro)
Molecular Cell 2001 7, DOI: ( /S (01) )

3. Figure 2
Expression of KD AMPKα in Muscles Blocks the Contraction-, but Not Insulin-, Stimulated Increase in AMPK Activity
(A and B) EDL muscles from nontransgenic (NTg), wild-type AMPKα transgenic (Tg-WT), and kinase dead AMPKα transgenic (Tg-KD1) mice were isolated and submitted to in vitro electrical stimulation as described in Experimental Procedures. Extracts were prepared and processed for Western blot analysis as described in Figure 1. Equal amount of tissue extract (40 μg) was used and the blot was probed with α-p172-AMPK or α-AMPKN, as indicated. The intensity of signal in (B) was quantified, normalized to total AMPKα and displayed in graphical format. Data in the graph are expressed relative to basal phosphorylation in muscle form nontransgenic mice, and the fold increase (mean ± SEM, n ≥ 3) is indicated over the appropriate column.
(C) EDL muscles from NTg or Tg-KD1 mice were excised and incubated in the presence or absence of insulin for 20 min. Extracts were prepared and analyzed by Western blot using α-p473Akt, α-p172AMPK, or α-AMPKN, as indicated. Results are representative of three or more experiments.
(D) EDL muscles from NTg and Tg-KD1 mice were excised and induced to contract in vitro by electrical stimulation. Extracts were prepared, AMPK immunoprecipitated with the α-AMPKN antibody, and kinase assays performed using SAMS peptide as a substrate. Peptide bands were resolved by electrophoresis using a 12% Bis-Tris (Norex) gel. Shown is a typical autoradiogram and quantitation of six experiments. Data are normalized to basal activity in muscle from nontransgenic mice, and the numbers indicate the fold change as the mean ± SEM
Molecular Cell 2001 7, DOI: ( /S (01) )

4. Figure 3
Inactivation of AMPK Blocks AICAR-Stimulated Deoxyglucose Uptake in Skeletal Muscle and Reduces Sensitivity to AICAR In Vivo
(A) Deoxyglucose uptake was performed in vitro in EDL muscle from 2- to 3-month-old mice in the absence or presence of 2 mM AICAR for 20 min. Data are the mean ± SEM of at least seven independent experiments performed in duplicate or triplicate.
(B and C) AICAR (0.25g/kg body weight) in saline was injected into the peritoneal cavity of Tg-KD1 mice or their wild-type littermates (NTg). Blood was collected from tail vein at the times indicated for measurement of glucose. 2- to 4- month-old (B) or 4- to 6-month-old (C) mice were used. Results are expressed as the mean ± SEM of 7–10 experiments
Molecular Cell 2001 7, DOI: ( /S (01) )

5. Figure 4
Inactivation of AMPK Blocks Hypoxia-Stimulated Deoxyglucose Uptake and GLUT4 Translocation to the Cell Surface in Skeletal Muscle
(A) EDL and soleus muscles from 2- to-3-month-old male mice were incubated under hypoxic condition (95%N2/5%CO2) for 45 min followed by measurement of deoxyglucose uptake. Data are the mean ± SEM of seven to eight experiments.
(B) Hypoxia stimulated 3-O-methyl-D-glucose uptake into EDL and soleus was conducted same as described in (A). Results shown are the mean ± SEM of three experiments.
(C) Cell surface GLUT4 was labeled by Bio-LC-ATB-BGPA after incubation under hypoxic and normoxic conditions. Biotinylated molecules were precipitated from solubilized membrane fraction and GLUT4 content determined by Western blot. The graph quantitates the extent of surface GLUT4 labeling expressed as fold change from NTg basal shown; data are the mean ± SEM from three independent experiments
Molecular Cell 2001 7, DOI: ( /S (01) )

6. Figure 5
Inactivation of AMPK in Skeletal Muscle Impairs Activation of Hexose Uptake in Response to Contraction
(A) EDL muscles were excised from 2- to 3-month-old transgenic mice (Tg-KD1) as well as their wild-type littermates (NTg) and stimulated electrically in vitro as indicated. Following this, deoxyglucose uptake was measured. Where indicated, muscle was incubated with insulin for 10 min prior to measurement of hexose uptake. Results are the mean ± SEM of seven experiments. *p < comparing contracted muscle from transgenic to that from wild-type mice.
(B) The sciatic nerves from 2- to-3-month-old transgenic mice (Tg-KD1) as well as their wild-type littermates (NTg) were stimulated electrically in situ as indicated. EDL muscles were excised and deoxyglucose uptake measured. Results are the mean ± SEM of six experiments. *p < 0.05 comparing contracted muscle from transgenic to that from wild-type mice.
(C) Deoxyglucose uptake into soleus after in situ contraction was analyzed as in (B). Results are the mean ± SEM of five experiments. *p < 0.03 comparing contracted muscle from transgenic to that from NTg mice.
(D) Cell surface GLUT4 photolabeling by Bio-LC-ATB-BGPA after in situ contraction. Four pieces of EDL muscles were pooled for each sample. Average fold stimulation comparing to NTg basal from two independent experiments is shown
Molecular Cell 2001 7, DOI: ( /S (01) )

7. Figure 6 Reduced Voluntary Wheel Running Activity in Tg-KD1 Mice
Male Tg-KD1 mice and their wild-type littermates (NTg) from 2 to 3 months of age were housed individually for 3 weeks. The mice had free access to the wheels during this period of time. Their activity was recorded as the times the wheels were turned per unit time. The data are plotted as mean activity ±SEM for each hour in each group of 10 animals. First 12 hr represents the dark phase of the cycle
Molecular Cell 2001 7, DOI: ( /S (01) )

8. Figure 7
Proposed Model for Contraction- and Hypoxia-Stimulated Glucose Transport into Skeletal Muscle
In this model, both contraction and hypoxia or AICAR stimulate the activity of AMPK, which leads to an increase in glucose transport. However, contraction also activates another pathway that independently contributes to the augmentation in hexose uptake. The identity of the other components in the AMPK-independent pathway(s) remains uncertain
Molecular Cell 2001 7, DOI: ( /S (01) )