👤 Kimberly Buhman

Dietary fat absorption by the small intestine is a multistep process that regulates the uptake and delivery of essential nutrients and energy. One step of this process is the temporary storage of dietary fat in cytoplasmic lipid droplets (CLDs). The storage and mobilization of dietary fat is thought to be regulated by proteins that associate with the CLD; however, mechanistic details of this process are currently unknown. In this study we analyzed the proteome of CLDs isolated from enterocytes harvested from the small intestine of mice following a dietary fat challenge. In this analysis we identified 181 proteins associated with the CLD fraction, of which 37 are associated with known lipid related metabolic pathways. We confirmed the localization of several of these proteins on or around the CLD through confocal and electron microscopy, including perilipin 3, apolipoprotein A-IV, and acyl-CoA synthetase long-chain family member 5. The identification of the enterocyte CLD proteome provides new insight into potential regulators of CLD metabolism and the process of dietary fat absorption. Show less

Mammalian enterocytes express apolipoprotein (apo)B-48, which is produced after posttranscriptional RNA editing of the nuclear apoB-100 transcript by the catalytic deaminase apobec-1. Earlier studies in apobec-1-/- mice revealed an apoB-100-only lipoprotein profile but no gross defects in triglyceride absorption. However, subtle defects may have been obscured by the mixed genetic background. In addition, the intrinsic susceptibility to proteolytic degradation of intestinal apoB-100 and apoB-48 has been questioned. Accordingly, we examined triglyceride absorption, intestinal apoB expression, and lipoprotein secretion in apobec-1-/- mice backcrossed into a C57BL/6 background. Inbred apobec-1-/- mice absorb triglyceride normally, yet secrete triglyceride-rich lipoproteins more slowly than wild-type congenic controls. There was comparable induction of apoB synthesis in response to fat feeding in both genotypes, but apoB-100 was preferentially retained and more extensively degraded than apoB-48. By contrast, synthesis, secretion, and content of apo A-IV were indistinguishable in apobec-1-/- and wild-type mice with 100% recovery, suggesting no degradation of this apoprotein in either genotype. Newly secreted lipoproteins from isolated enterocytes of wild-type mice revealed apoB-48 in both high-density lipoproteins and very low-density lipoproteins. By contrast, apobec-1-/- mice secreted apoB-100-containing particles that were almost exclusively in the low and very low-density lipoproteins range with no apoB-100-containing high-density lipoproteins. These studies establish the existence of preferential degradation of intestinal apoB-100 and subtle defects in triglyceride secretion in apobec-1-/- mice, coupled with a shift to the production of larger particles, findings that suggest an important divergence in intestinal lipoprotein assembly pathways with the different isoforms of apoB. Show less