AbstractIn a GM-CSF driven myeloid cell deficient mouse model (Csf2^−/−) that has preserved insulin sensitivity despite increased adiposity, we used unbiased three-dimensional integration of proteome profiles, metabolic profiles, and gene regulatory networks to understand adipose tissue proteome-wide changes and their metabolic implications. Multi-dimensional liquid chromatography mass spectrometry and extended multiplex mass labeling was used to analyze proteomes of epididymal adipose tissues isolated from Csf2^+/+ and Csf2^−/− mice that were fed low fat, high fat, or high fat plus cholesterol diets for 8 weeks. The metabolic health (as measured by body weight, adiposity, plasma fasting glucose, insulin, triglycerides, phospholipids, total cholesterol levels, and glucose and insulin tolerance tests) deteriorated with diet for both genotypes, while mice lacking Csf2 were protected from insulin resistance. Regardless of diet, 30 mostly mitochondrial, branch chain amino acids (BCAA), and lysine metabolism proteins were altered between Csf2^−/− and Csf2^+/+ mice (FDR < 0.05). Lack of GM-CSF driven myeloid cells lead to reduced adipose tissue 2-oxoglutarate dehydrogenase complex (DHTKD1) levels and subsequent increase in plasma 2-aminoadipate (2-AA) levels, both of which are reported to correlate with insulin resistance. Tissue DHTKD1 levels were >4-fold upregulated and plasma 2-AA levels were >2 fold reduced in Csf2^−/− mice (p < 0.05). GM-CSF driven myeloid cells link peripheral insulin sensitivity to adiposity via lysine metabolism involving DHTKD1/2-AA axis in a diet independent manner.