Elevated UCP1 levels are sufficient to improve glucose uptake in human white adipocytes

Fig. 1. Comparison of glucose uptake in murine in vitro differentiated white and brown adipocytes. (A) Stromal-vascular cells isolated from inguinal WAT and interscapular BAT were differentiated for 10 days into mature adipocytes. (B) mRNA expression of the BAT-specific markers UCP1 and CIDEA and the WAT-specific marker HOXC8 was measured by qRT-PCR. (C) Basal uptake of 14C-deoxy-glucose was determined in adipocytes after serum starvation overnight, cpm values were normalized to adipocyte number. Data (n = 3) are shown as means + SEM, *p < 0.05, **p < 0.01 (Student's t-test). . (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2. CRISPR/Cas9-mediated UCP1 knockout in human adipocytes. UCP1-deficient cells were generated by co-expressing Cas9 and specific sgRNAs targeting UCP1 in SGBS preadipocytes. (A) The cells were differentiated into mature adipocytes. The knockout of UCP1 and the protein levels of adipogenic markers adiponectin (AdipoQ), peroxisome proliferator-activated receptor gamma (PPARG), and perlipin A (PLIN) were confirmed by Western Blot. NC, non-targeting control. (B) Oxygen consumption rates were determined in a plate-based respirometer. ATP: oligomycin-sensitive OCR; leak: oligomycin resistant OCR (C) Basal glucose uptake was determined by 14C-deoxy-Glucose incorporation. (D) Extracellular acidification rates were determined as in (B). (B-D) Data obtained from both negative controls and UCP1 KO cell types were merged. Data of 3–4 independent experiments are shown as mean + SEM. **, p < 0.01 (Student's t-test).

Fig. 3. Lentivirus-mediated UCP1 overexpression in human preadipocytes. UCP1-overexpressing cells were generation by lentiviral transduction of SGBS preadipocytes. (A) UCP1 overexpression in preadipocytes was confirmed by Western Blot (n = 4 independent experiments). ev, empty vector control. (B) Mitochondrial localization of UCP1 in preadipocytes was confirmed by Western blot of cytosolic (cs) and mitochondrial (mito) subcellular fractions of (n = 3 independent experiments). TIM23 and GAPDH were used as markers for mitochondrial and cytosolic fractions. (C) Cellular respiration of UCP1-overexpressing and control preadipocytes was analyzed in a plate-based respirometer. During the measurement, the cells were sequentially treated with 2 μM oligomycin A, 4 μM FCCP and 1.5 μM antimycin A + 1.5 μM rotenone. (D) Basal, proton leak, and ATP-linked respiration rates were calculated from the respective OCR traces. Data are normalized to JanusGreen staining and are shown as mean + SEM. *, p < 0.05; **, p < 0.01 (Student's t-test).

Fig. 4. Adipogenic differentiation of human preadipocytes overexpressing UCP1. (A) The cells were subjected to adipogenic differentiation for 14 days. (B) UCP1 protein expression was determined by Western Blot. (C) The percentage of differentiated cells was determined by counting preadipocytes and adipocytes using a net micrometer. (D) Lipid accumulation was assessed by measuring the triglyceride content. (E) mRNA expression of key adipogenic marker genes as well as BAT associated genes was determined by qRT-PCR. (F) Oxygen consumption rates (OCR) of UCP1-overexpressing (red color) and control adipocytes (blue color) were determined using plate-based respirometer (media containing 1% BSA). To induce UCP1 activity, subsets of cells were stimulated with 0.5 mM dibutyryl-cAMP (cAMP, closed circles), while control cells were left untreated (open circles) (G) Sequential injections of oligomycin (2 μM), FCCP (4 μM), and antimycin A (1.5 μM) + rotenone (1.5 μM) were used to determine basal, proton leak (striped column part), and ATP-linked respiration rates (open column part). Data were normalized for cellular lipid content by NileRed staining. (H) Mitochondrial membrane potential was measured…

Fig. 5. Glucose uptake and glycolytic flux in adipocytes with elevated UCP1 levels. (A and B) Glycolytic activity of UCP1-overexpressing and control adipocytes were determined by measuring basal cellular 14C-deoxy-glucose incorporation (A) and lactate release (B). (C and D) Extracellular acidification rates (C) and oxygen consumption rates (D) upon inhibition of glycolysis by 50 mM 2-deoxy-glucose were determined in a plate-based respirometer. (E) Expression of glucose transporters 1, 4, 6, and 8 was determined by qRT-PCR. (F) 2-deoxy-glucose uptake was determined after preincubation with Cytochalasin B (CytB) or BAY-876. Data of at least 3 independent experiments are shown as mean + SEM.*, p < 0.05; **, p < 0.01 (Student's t-test).