Insulin resistance is commonly developed in obesity and is a trait of the beginning stage of type 2 diabetes mellitus (DM). It is highly likely that the high plasma fatty acid levels provoke the condition in the obese and insulin resistant state of type 2 DM. Hence, the purpose of this study was to determine if a high concentration of palmitic acid causes insulin resistance and how ceramide content is regulated under the various conditions in the isolated rat soleus muscle. A submaximal insulin stimulus (100 ,aU/ml) increased 3-O-methylglucose transport by -2.7 fold over basal conditions in the soleus (1.90 ± 0.23 µmol•ml"1•hr-1 vs. 5.06 ± 0.38 µmol•ml-1•hr-1, respectively) (P < 0.05). Five hours of palmitic acid preincubation induced a significant decrease in insulin-stimulated glucose transport (3.49 ± 0.11 µmol•ml-1•hr-1) by -31 % (P < 0.05) compared with the control. By contrast, the addition of L-cycloserine, a serine palmitoyltransferase inhibitor, attenuated the palmitic acid response by -20% (4.19 ± 0.27 µmol•ml-1•hr-1) (P < 0.05). A 5 hr preincubation with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), an adenosine analogue that increase AMP activated protein kinase, increased glucose transport (3.29 ± 0.1 µmol•ml-1•hr-1) (P < 0.05) compared with the control group. Moreover, regardless if palmitic acid or L-cycloserine were present, insulin-stimulated glucose uptake was normalized (5.30 ± 0.38 µmol•ml-1•hr-1 and 5.56 ± 0.16 µmol•ml"1•hr-1, respectively) after 5 hr AICAR preincubation. We next measured the ceramide content to investigate whether the reduced glucose uptake results from ceramide accumulation in the soleus. The total ceramide mass in the soleus was increased by -35% in palmitic acid-treated group compared with the control group (122.02 ± 2.07 pmol•mg-1 vs. 90.79 + 1.24 pmol•mg 1, respectively) (P<0.05). Both L-cycloserine and AICAR decreased palmitateinduced ceramide synthesis by -20% and -14%, respectively (97.15 + 2.5 pmol•mg-1 and 105.79 ± 1.94 pmol•mg-1, respectively) (P<0.05) compared with the palmitic acid-treated group. We also measured serine palmitoyltransferase (SPT) to determine if AICAR regulates ceramide synthesis by inhibiting SPT. Total SPT protein level increased by -27% (P<0.05) and SPT activity increased by -44% (P<0.05) compared with the control group. By contrast, after muscles were incubated with L-cycloserine for 5 hr, both SPT protein level and enzyme activity were decreased by -17% and -23%, respectively (P<0.05). In adition, 5 hr AICAR treatment blunted palimitic acid-induced SPT protein level and enzyme activity by 11% (P<0.05) and 20% (P<0.05), respectively, compared with the palmitic acid-treated group. In conclusion, these data suggest that short term exposure (5 hr) to high fatty acid levels appears to cause insulin resistance by increasing ceramide accumulation and that AMPK expression (AICAR treatment) can attenuate the problem by regulating SPT levels. / School of Physical Education, Sport, and Exercise Science