NanoSIMS Analysis of Intravascular Lipolysis and Lipid Movement across Capillaries and into Cardiomyocytes

Abstract

The processing of triglyceride-rich lipoproteins (TRLs) in capillaries provides lipids for vital tissues, but our understanding of TRL metabolism is limited, in part because TRL processing and lipid movement have never been visualized. To investigate the movement of TRL-derived lipids in the heart, mice were given an injection of [H]triglyceride-enriched TRLs, and the movement of H-labeled lipids across capillaries and into cardiomyocytes was examined by NanoSIMS. TRL processing and lipid movement in tissues were extremely rapid. Within 30 s, TRL-derived lipids appeared in the subendothelial spaces and in the lipid droplets and mitochondria of cardiomyocytes. Enrichment ofH in capillary endothelial cells was not greater than in cardiomyocytes, implying that endothelial cells may not be a control point for lipid movement into cardiomyocytes. Remarkably, a deficiency of the putative fatty acid transport protein CD36, which is expressed highly in capillary endothelial cells, did not impede entry of TRL-derived lipids into cardiomyocytes.

NanoSIMS imaging reveals unexpected heterogeneity in nutrient uptake by brown adipocytes

“…The processing of 2 H-TRLs and the subsequent uptake of 2 H-labeled lipids by adipocytes was rapid; 2 H enrichment in the cytosolic lipid droplets of brown adipocytes was easily detectable 1 min after the 2 H-TRL injection. As expected [7], marginated 2 H-TRLs were present along the capillary lumen at the 1-min time point but were much less common at the 15-and 30-min time points (Fig. 1).…”

“…2 H-labeled triglyceride-rich lipoproteins ( 2 H-TRLs) were prepared as described [7]. Briefly, Gpihbp1 –/– mice were given multiple doses of 2 H-labeled mixed fatty acid over 4.5 days.…”

“…In other experiments, 3–4-month-old mice were given 80 μl of [ 13 C]fatty acids (1 mg/μl; Cambridge Isotope Laboratories; two doses 12 h apart) or 75 μl of [ 13 C]glucose (1 mg/μl, Cambridge Isotope Laboratories; three doses 12 h apart) by gastric gavage. BAT was harvested 22 h after the final dose of fatty acids or 4 h after the final dose of glucose, and sections were prepared for NanoSIMS as described [7]. …”

“…Tissue sections were coated with 5 nm of platinum and analyzed with NanoSIMS 50L or NanoSIMS 50 instruments [7, 8]. A 16-KeV 133 Cs + beam was used to bombard the sample, and secondary electrons (SEs) and secondary ions ( e.g., 12 C 14 N–, 1 H–, 2 H–, 12 C–, 13 C–) were collected [7].…”

“…In the current studies, we tested whether the uptake of nutrients in brown adipocytes of mice is uniform or heterogeneous. Rather than using fluorescent fatty acids and adipose tissue explants, we injected mice with stable isotope–labeled nutrients and used NanoSIMS [7–11] to quantify uptake of the stable isotopes by brown adipocytes. An attractive feature of this approach is the ability to quantify the heterogeneity of nutrient uptake in brown adipocytes in living animals.…”