**AWARDS ISSUED PRIOR TO JANUARY 20, 2025, WERE FUNDED UNDER PREVIOUS ADMINISTRATIONS AND MAY NOT REFLECT THE PRIORITIES AND POLICIES OF THE CURRENT ADMINISTRATION.** CALVING AND THE ONSET OF LACTATION LEAD TO A SUDDEN INCREASE IN THE ENERGY REQUIREMENTS OF DAIRY COWS. AT THE SAME TIME, COWS HAVE A DROP IN FEED INTAKE, LOWERING THE AMOUNT OF ENERGY PROVIDED BY FEED AND LEADING TO A STATE OF NEGATIVE ENERGY BALANCE. COWS ADAPT TO THIS STATE BY BREAKING DOWN FAT TISSUES AND RELEASING ENERGY-DENSE FATTY ACIDS IN A PROCESS KNOWN AS LIPOLYSIS. IN FAT TISSUES, LIPOLYSIS INDUCES A REMODELING PROCESS CHARACTERIZED BY INFLAMMATION WITH INFILTRATION OF IMMUNE CELLS, INCLUDING MACROPHAGES (ATM). IN HEALTHY COWS, LIPOLYSIS RATE DECREASES, AND FAT TISSUE INFLAMMATION RESOLVES AS LACTATION PROGRESSES. HOWEVER, WHEN LIPOLYSIS IS NOT REDUCED, FAT INFLAMMATION IS MAINTAINED, MAKING COWS MORE SUSCEPTIBLE TO PERIPARTURIENT DISEASES. BOTH FAT INFLAMMATION AND EXCESSIVE LIPOLYSIS NEGATIVELY IMPACT COWS' HEALTH AND WELL-BEING AND DAIRY FARMERS' SUSTAINABILITY. AN ADDITIONAL FACTOR STRONGLY CONNECTED WITH THE PRESENTATION OF PERIPARTURIENT DISEASES IS THE SHARP INCREASE IN CIRCULATING BACTERIAL REMNANTS OR ENDOTOXINS.WE HYPOTHESIZE THAT HIGH LEVELS OF ENDOTOXIN IN THE BLOOD (ENDOTOXEMIA) TRIGGER EXCESSIVE LIPOLYSIS BY ACTIVATING THE BACTERIA-SENSING TOLL-LIKE RECEPTORS. ENDOTOXEMIA LEADS TO PRO-INFLAMMATORY POLARIZATION OF ATM AND FAT INSULIN RESISTANCE. WE WILL INVESTIGATE THIS HYPOTHESIS BY PURSUING TWO SPECIFIC AIMS AND UTILIZING FAT LIPOLYSIS MODELS COMPLEMENTED WITH FUNCTIONAL ASSAYS AND SINGLE-CELL RNASEQ ANALYSES. AIM 1 WILL DETERMINE HOW ENDOTOXINS ENHANCE FAT CELLS' LIPOLYTIC RESPONSES THROUGH CHANGES IN THEIR INSULIN SENSITIVITY AND ATM INFLAMMATORY PHENOTYPE. IN AIM 2, WE WILL DETERMINE HOW ENDOTOXEMIA ENHANCES FAT LIPOLYSIS DURING NEGATIVE ENERGY BALANCE AND POLARIZES ATM TO INFLAMMATORY PHENOTYPES. THIS PROPOSAL WILL ELUCIDATE THE MECHANISMS BY WHICH ENDOTOXEMIA INDUCES EXCESSIVE LIPOLYSIS AND INCREASES DAIRY COWS' SUSCEPTIBILITY TO DISEASES. OUR LONG-TERM GOAL IS TO DEVELOP NUTRITIONAL AND PHARMACOLOGICAL TOOLS TO PREVENTLIPOLYSIS DYSREGULATION DURING CATABOLIC PERIODS. WE EXPECT THESE TOOLS WILL BENEFIT HERD HEALTH, IMPROVE DAIRY COWS' WELFARE, AND INCREASE THE ECONOMIC SUSTAINABILITY OF DAIRY FARMING.