There is considerable interest in defining the processes that lead to the increased accumulation of intramyocellular and extramyocellular lipids (IMCL and EMCL) and lipid unsaturation ratios, and determining the relationships of these changes to the development of insulin resistance (IR) and progression to type 2 diabetes (T2D). Thus, development of novel imaging techniques for lipid and metabolite characterization is needed to allow repeated, non-invasive assessment of these processes in vivo. To achieve this goal, the current proposal will develop and pilot test non-linear reconstruction of a novel accelerated multi- gradient/spin-echo based five-dimensional (5D) MR Spectroscopic Imaging (MRSI) data using non-uniform undersampling (NUS) along one spectral and one spatial dimensions. Recently, a 2nd spectral dimension was added to echo-planar-spectroscopic imaging (EPSI) and named echo-planar correlated spectroscopic imaging (EP-COSI). A multi-echo (ME) version of 5D EP-COSI, named ME-EPCOSI, which is capable of acquiring the data faster than EP-COSI will be implemented on a 3T MRI scanner using a 15- channel knee coil. The sequence will be further optimized for achieving better slice refocusing using the adiabatic full passage 1800 radio-frequency (RF) pulses as the adiabatic RF pulses will improve slice selection profile and reduce chemical shift artifacts better than the commonly used Mao RF pulse. This new sequence will enable better discrimination between the unsaturated and saturated lipid groups in skeletal muscle than the conventional MRS techniques and will allow metabolite imaging of multiple muscle groups simultaneously to better understand the specific relationship between IMCL and insulin sensitivity. Two specific goals are proposed: 1) complete the development of the NUS 5D ME-EP-COSI technology to allow simultaneous determination of IMCL and EMCL levels and lipid unsaturation indices in three calf muscle groups with differing type1/type 2 fiber and IMCL content in 20 young healthy human volunteers and to compare its performance using conventional 3D MRSI; and 2) utilize this new technology to study 20 patients with T2D and 20 matched non-diabetic, healthy subjects. The proposed studies will test the following two hypotheses: 1) a NUS-based 5D MEEP-COSI technique will be more robust than the fully encoding 5D EPCOSI and MRSI sequences, enabling simultaneous recording of 2D COSY spectra in multiple slices of calf skeletal muscle, 2) relative to healthy controls, patients with T2D will exhibit: a) increased skeletal muscle IMCL and EMCL, and b) reduced choline, IMCL and EMCL lipid unsaturation indices. The proposed multi-dimensional MR spectroscopic imaging is expected to facilitate the development and validation of an important new research tool that will significantly advance research in the mechanisms of the development of IR and T2D and may ultimately serve as a convenient methodology to rapidly and non-invasively assess responses to new therapeutic interventions designed to ameliorate IR and treat T2D by normalizing muscle lipid metabolism.