Sirtuin 4 (SIRT4) is a mitochondrial enzyme that can remove several novel post-translational modifications (PTMs) on target proteins and thereby modify their activity. We and others have previously shown that young mice lacking SIRT4 (SIRT4KO mice) have elevated glucose, leucine, and glutamine-stimulated insulin secretion. As these mice aged, they developed insulin resistance and glucose intolerance. We hypothesize that when these mice are young and eat a mixed nutrient diet, they secrete elevated amounts of insulin in response to each meal and this hyperinsulinemia drives the development of insulin resistance in old age. However, while insulin secretion in response to glucose, leucine, and glutamine individually has been reported in these mice, it has not been shown how much insulin these mice secrete in response to combinations of these nutrients, which would more closely resemble a normal meal. Thus, we sought to characterize insulin secretion in SIRT4KO mice in response to mixed nutrient challenges. Indeed, SIRT4KO mice secrete more insulin response to different combinations of glucose, leucine, and glutamine. In most cases, these increases in insulin secretion were synergistic, rather than just an additive effect of each individual nutrient. In particular, when leucine and glucose were administered together in aged SIRT4KO males, plasma insulin levels were synergistically increased more than 3-fold over glucose or leucine alone. Remarkably, treating aged SIRT4KO mice with leucine during a glucose tolerance test resulted in so much insulin secretion that glucose intolerance was normalized. Importantly, despite these extremely high insulin levels, there were no signs of dangerous hypoglycemia. To determine whether these synergistic effects of glucose and leucine on insulin secretion were due to effects of SIRT4 on pancreatic beta-cells, we treated beta-cell specific SIRT4KO mice with glucose and leucine and found no differences in insulin secretion compared to wild-type mice, suggesting that SIRT4 affects insulin secretion by acting indirectly on the beta-cell. To determine whether SIRT4 may affect insulin secretion by altering incretin secretion, we measured GLP-1 secretion in response glucose and leucine in combination and found that GLP-1 secretion was elevated in SIRT4KO mice compared to wild-type controls. Taken together, our data show that SIRT4 may be a key regulator of insulin secretion in response to mixed nutrients via effects on GLP-1 secretion. NIH Support of Competitive Research (SCORE) Pilot Project Award #SC2GM141997 San Jose State University Department of Biological Sciences Start-up Funds. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.