Methods
Lean (C57Bl) and ob/ob mice (8-10 wks old) were obtained from B&K Universal (Sollentuna, Sweden). The animals were untreated, or dosed by subcutaneous injection with AICAR (1 mg/g body wt per day) or saline administered at 09:00 h. Blood glucose was measured 0.5, 1, 2, and 3 hours after the acute AICAR injection. Thereafter, animals received a daily AICAR or saline injection for 7 days. Blood glucose was determined each morning. Glucose tolerance was performed after 7 days AICAR treatment. Isolated soleus and EDL muscle from lean or ob/ob mice were incubated with insulin (120 nM) or AICAR (2 mM) and 2-deoxyglucose uptake was determined. Muscle and liver were obtained for measurement of glycogen content. GLUT4 and hexokinase II protein expression were assessed in muscle.

Results
The effect of insulin or AICAR was determined in vitro, in isolated skeletal muscle from untreated lean or ob/ob mice. Insulin-stimulated glucose uptake in soleus and EDL was reduced (39 and 22%, respectively) in ob/ob mice (p<0.05), whereas the AICAR-stimulated response was not impaired. The effect of acute or 7 day AICAR-treatment on whole body glucose homeostasis was determined. Acute AICAR treatment (1 mg/g subc.) lowered glucose levels in ob/ob mice within 1 hr (18.7±1.8 to 8.4±1.5 mM; p<0.001), with effects persisting over 4 hr. Long-term daily AICAR treatment for 7 days corrected hyperglycemia in ob/ob mice (9.9 ±1.0 vs 18.3±1.8 for saline vs AICAR-treated; p<0.001), with no change in lean mice. The blood-glucose lowering effect of long-term (7 day) AICAR treatment in ob/ob mice persisted 7 days after AICAR treatment was terminated (13.0±1.3 vs. 22.1±1.4 for treated vs. untreated ob/ob mice; p<0.001). Seven-day AICAR-treatment was associated with a 2-fold increase (p<0.01) in GLUT4 protein expression, a 2-fold increase in hexokinase II protein expression (p<0.01), and a 1.5-fold increase (p<0.01) in glycogen content in gastrocnemius muscle from fasted ob/ob mice. Despite these changes, basal and insulin-stimulated glucose transport in isolated soleus and EDL muscle was not altered in 7 day AICAR- vs. saline-treated animals.

Conclusions
Despite severe insulin resistance, AICAR exposure led to a normal response on glucose transport in isolated skeletal muscle from ob/ob mice. Furthermore, in vivo AICAR-treatment normalized blood glucose and improved glucose tolerance in ob/ob mice. Our findings provide evidence that AICAR-treatment is an effective strategy to improve glucose homeostasis in severely diabetic ob/ob mice.