Role of Endothelial Cell Lipoprotein Lipase for Brown Adipose Tissue Lipid and Glucose Handling

FIGURE 1. Endothelial expression of LPL in BAT. (A–B) EndoNuTRAP mice were housed at 22°C, at 6°C or at 30°C for 3d. Expression of Gpihbp1 (A) and Lpl (B) in endothelial cells (TRAP) and total tissue (input) of BAT were analyzed by qPCR (n = 4–5). (C) Transwell system for co-culturing of murine brown adipocytes with murine microvascular endothelial cells. Endothelial cells were cultured in the presence or absence of differentiated brown adipocytes and were incubated without or with the β3-adrenergic agonist CL316,243 at the same time to thermogenically activate the brown adipocytes. (D) Ucp1 expression in brown adipocytes co-cultured without and with endothelial cells (ECs) (n = 4). (E) Lpl expression in endothelial cells co-cultured with brown adipocytes (+adipocytes) or without (-adipocytes), stimulated with CL316,243 or with vehicle (none) (n = 4). (F) Wild type mice were housed at 6°C for 3 days and expression of Lpl was determined in CD31-positive endothelial cells and thermogenic adipocytes of BAT that were isolated by MACS® (n = 3)Results are presented as mean values ±SEM. Statistical significance for (A,B) was determined by one-way ANOVA and for (D,E) by two-way ANOVA. Same letter…

FIGURE 2. Body composition and BAT lipid content in endothelial cell specific LPL knockout mice. (A) Control and EndoLPLko mice received a tamoxifen dosage to generate mice lacking LPL in endothelial cells (Cre+) and controls (Cre-). Mice were exposed to 6°C for 1 day before SVF was isolated from BAT and analyzed for Cre and Lpl expression by qPCR (n = 4). (B–E) EndoLPLko mice and litter mates (n = 6) were fed a western-type diet for 2 weeks, and housed at 6°C (cold) or 30°C (warm) in the second week. (B) Body weight, (C) lean weight and (D) fat weight determined by EchoMRI, (E) plasma cholesterol (chol), (F) triglycerides (TG), (G) non-esterified fatty acids (NEFA) and (H) blood glucose. (I) Hematoxylin eosin staining of BAT. Bars indicate 200 and 50 µm for lower and higher magnification, respectively. (J) Ranges of adipocyte diameters calculated from (I) using NIS-Elements (Nikon®). Results are presented as mean values ±SEM. Statistical significance was determined by Student’s t test (A) or by two-way ANOVA (B–D). Same letter denotes groups that are not significantly different from each other (p ≥ 0.05).

FIGURE 3. Energy expenditure of endothelial cell-specific LPL knockout mice. For indirect calorimetry analysis EndoLPLko (Cre+) and control (Cre-) mice fed a western-type diet were placed in a TSE phenomaster system. (A) Energy expenditure and (B) respiratory exchange ratio were determined by indirect calorimetry (n = 3–4). Results are presented as mean values.

FIGURE 4. Organ lipid and glucose uptake and BAT gene expression in the postprandial state. EndoLPLko (Cre+) mice and Cre-litter mates (n = 6) were fed a western-type diet for 2 weeks, and housed at 6°C (cold) or 30°C (warm) in the second week. Organ and plasma harvest was performed 2 h after a combined oral glucose and fat gavage. (A) Organ uptake of 14C-Triolein and (B) 3H-deoxyglucose (3H-DOG). Plasma levels of (C) triglycerides, (D) cholesterol and (E) Non-esterified fatty acids (NEFA). Expression of (F) thermogenic, (G) lipoprotein and lipid-handling, and (H) glucose-handling genes in BAT of EndoLPLko mice. Results are presented as mean values ±SEM. Statistical significance was determined by two-way ANOVA (p < 0.05). Same letter denotes groups that are not significantly different from each other (p ≥ 0.05).

FIGURE 5. Lipoprotein disposal and BAT gene expression in the fasted state. (A) EndoLPLko, mice lacking LPL in brown adipocytes (BATLPLko), and wild type control mice fed a chow diet were housed at 22°C followed by cold exposure (6°C) for 24 h. To analyze TRL uptake, BODIPY-labeled TRL were injected intravenously into animals fasted for 4 h. BAT was harvested for ex vivo confocal fluorescence microscopy 15 min after injection. The figure shows representative images of TRL uptake. Bar indicates 20 µm. (B–F) EndoLPLko and control mice (n = 5–6) were fed a western-type diet for 2 weeks and housed at 30°C (warm) or 6°C (cold) in the second week. 14C-Triolein labeled TRLs were injected intravenously together with tracer amounts of 3H-DOG. Organs were harvested 15 min after injection. (B) Organ uptake of 14C-triolein, and (C) 3H-DOG in EndoLPLko (Cre+) and control (Cre-) mice. Gene expression of (D) thermogenic, (E) lipoprotein and lipid-handling, and (F) glucose-handling genes in BAT of EndoLPLko and Cre-control mice. Values are shown relative to Cre-warm. Results are presented as mean values ±SEM. Statistical significance was determined by two-way ANOVA. Same letter denotes groups that are not…