Abstract Disclosure: T.M. Fordham: None. M. Ramos-Roman: None. E.J. Parks: Consulting Fee; Self; Simply Good Foods. Stock Owner; Self; Sagimet Biosciences. Other; Self; Past President of The Obesity Society. Introduction: The mammary produces large amounts of medium-chain saturated fatty acids. Long-chain saturated fatty acids found in human milk primarily derive from the maternal diet or from the mobilization of stored triglycerides. Previous studies have established the importance of insulin in stimulating mammary differentiation, milk synthesis, and lipogenesis. However, no studies have assessed how changes in insulin concentrations alter human mammary de novo lipogenesis (DNL) nor what effect gestational diabetes may have on insulin’s relationship to lipogenesis during lactation. Through the utilization of stable isotopes and the hyperinsulinemic-euglycemic clamp technique, we tested the differing concentrations of insulin to stimulate mammary DNL. Methods: Eight women (32±4 yrs) with (GDM, n=4) or without (NGT, n=4) recent gestational diabetes and overweight or obesity (28.2±2.9 kg/m2) were recruited to participate in this study at 6 weeks postpartum. The night before the study, participants orally consumed 3 doses of 70% deuterated water to label newly made fatty acids. Participants arrived in the morning for IV placement and immediately pumped milk contralaterally for 20 minutes. Milk was pumped two hours later, again at the end of step 1 of the clamp (10 mU/m2/min) and once more after step 2 (either 20 or 40 mU/m2/min). Milk triglycerides were extracted and prepared for analysis by gas chromatography-mass spectrometry. The incorporation of deuterium into milk triglyceride-fatty acids was the primary outcome. Results: Fasting insulin concentrations were significantly associated with newly-made 16:0 in milk triglycerides (r=0.85, P=0.01). Of all measured triglyceride 16:0, 1.6±1.0% were made de novo. This percentage increased 1.5-fold by the end of step 2 of the clamp (P=0.06). Fasting milk DNL 14:0 was 12.3±4.8% which was significantly greater than DNL 16:0 (P<0.01). DNL 14:0 increased 1.7-fold during step 2 (P=0.04). For both milk triglyceride 12:0 and 14:0 derived from DNL, a significant effect across clamp-stage was observed (P<0.001 for both), but not an effect of group (P=0.12, P=0.11, respectively). Bonferroni posthoc analyses revealed that the insulin effect was significant for both groups for 12:0 (GDM, P=0.02; NGT, P <0.01) and 14:0 (GDM, P<0.01; NGT, P=0.01). Interpretation: The present study is the first to assess the acute effect of a hyperinsulinemic-euglycemic clamp on the synthesis of newly-made triglyceride fatty acids in human milk. These results suggest that the increases in mammary DNL may be fatty acid specific, favoring that of lauric and myristic acids, but not longer chain fatty acids like palmitate. Mammary lipogenesis was stimulated by insulin in all subjects regardless of recent gestational diabetes diagnosis. The role mammary DNL plays in overall milk fat content deserves further investigation. Presentation: 6/3/2024