Abstract Body: Elevated serum apolipoprotein C3 (APOC3) predicts cardiovascular disease risk in individuals with diabetes. APOC3 slows the clearance of atherogenic triglyceride-rich lipoproteins (TRLs) and their remnants (RLPs). We assessed the hypothesis that hepatic insulin receptor (IR) deficiency causes increased APOC3 levels, altered lipid profiles, and enhanced atherosclerosis. Liver-specific IR deficiency was induced in adult IR-floxed mice fed a high-fat high-sucrose diet by administering a liver-targeted adeno-associated virus expressing Cre recombinase (TBG-AAV8-Cre). Controls received empty TBG-AAV8 (n=5/group). Two weeks later, a liver-targeted LDL receptor (LDLR) antisense oligonucleotide was injected biweekly for 8 weeks. Mice deficient in hepatic IRs exhibited modestly elevated blood glucose (229 ± 12 [mean ± SEM] vs 167 ± 7 mg/dL in controls; p=0.001) and plasma cholesterol (639 ± 61 vs 409 ± 40 mg/dL; p=0.0004). Plasma insulin levels were markedly elevated (7.2 ± 1.3 vs 0.6 ± 0.3 ng/mL; p=0.008). Plasma triglycerides and free fatty acids did not differ between the groups. However, IR-deficient mice showed reduced hepatic Apoc3 mRNA levels and a dramatic reduction in plasma APOC3 (413 ± 82 vs 1774 ± 183 μg/mL; p=3.68x10^-8). Apolipoprotein kinetics were analyzed by targeted mass spectrometry after ¹³C¹⁵N-lysine injection. Hepatic IR-deficient mice exhibited a strikingly reduced APOC3 production rate (30.5 ± 10.1 vs 146.8 ± 59.6 μg/mL/h; p=0.008) with no difference in fractional clearance rate (FCR). Plasma APOB100 levels were also reduced (155.0 ± 29.6 vs 296.5 ± 38.0 mg/dL, p=0.03) with a lower production rate (0.027 ± 0.006 vs 0.128 ± 0.045 mg/mL/h; p=0.03) and no difference in FCR. APOE production rates and FCRs of APOA1, APOA2, APOA4, APOC1, APOC2, and APOE were also similar between the groups. Despite the exceedingly low APOC3 and APOB100 levels, hepatic IR-deficient mice had increased levels of cholesterol-enriched TRLs/RLPs and a striking increase in both total aortic and aortic sinus atherosclerotic lesions (p=1.73x10^-10). In summary, hepatic IR deficiency in adult LDLR-deficient mice leads to reduced plasma APOC3 and APOB100 levels by suppressing their production rates but paradoxically increases cholesterol-enriched TRLs/RLPs and atherosclerosis. Our study suggests that hepatic IRs regulate lipid metabolism and atherogenesis partly independently of APOC3, and underscores the complexity of IR-mediated regulation of cardiovascular disease risk.