ApoB100-LDL Acts as a Metabolic Signal from Liver to Peripheral Fat Causing Inhibition of Lipolysis in Adipocytes

International audienceBACKGROUND: Free fatty acids released from adipose tissue affect the synthesis of apolipoprotein B-containing lipoproteins and glucose metabolism in the liver. Whether there also exists a reciprocal metabolic arm affecting energy metabolism in white adipose tissue is unknown. METHODS AND FINDINGS: We investigated the effects of apoB-containing lipoproteins on catecholamine-induced lipolysis in adipocytes from subcutaneous fat cells of obese but otherwise healthy men, fat pads from mice with plasma lipoproteins containing high or intermediate levels of apoB100 or no apoB100, primary cultured adipocytes, and 3T3-L1 cells. In subcutaneous fat cells, the rate of lipolysis was inversely related to plasma apoB levels. In human primary adipocytes, LDL inhibited lipolysis in a concentration-dependent fashion. In contrast, VLDL had no effect. Lipolysis was increased in fat pads from mice lacking plasma apoB100, reduced in apoB100-only mice, and intermediate in wild-type mice. Mice lacking apoB100 also had higher oxygen consumption and lipid oxidation. In 3T3-L1 cells, apoB100-containing lipoproteins inhibited lipolysis in a dose-dependent fashion, but lipoproteins containing apoB48 had no effect. ApoB100-LDL mediated inhibition of lipolysis was abolished in fat pads of mice deficient in the LDL receptor (Ldlr(-/-)Apob(100/100)). CONCLUSIONS: Our results show that the binding of apoB100-LDL to adipocytes via the LDL receptor inhibits intracellular noradrenaline-induced lipolysis in adipocytes. Thus, apoB100-LDL is a novel signaling molecule from the liver to peripheral fat deposits that may be an important link between atherogenic dyslipidemias and facets of the metabolic syndrome

An integrated expression atlas of miRNAs and their promoters in human and mouse

MicroRNAs (miRNAs) are short non-coding RNAs with key roles in cellular regulation. As part of the fifth edition of the Functional Annotation of Mammalian Genome (FANTOM5) project, we created an integrated expression atlas of miRNAs and their promoters by deep-sequencing 492 short RNA (sRNA) libraries, with matching Cap Analysis Gene Expression (CAGE) data, from 396 human and 47 mouse RNA samples. Promoters were identified for 1,357 human and 804 mouse miRNAs and showed strong sequence conservation between species. We also found that primary and mature miRNA expression levels were correlated, allowing us to use the primary miRNA measurements as a proxy for mature miRNA levels in a total of 1,829 human and 1,029 mouse CAGE libraries. We thus provide a broad atlas of miRNA expression and promoters in primary mammalian cells, establishing a foundation for detailed analysis of miRNA expression patterns and transcriptional control regions

MicroRNAs regulate human adipocyte lipolysis: effects of miR-145 are linked to TNF-α.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression and have multiple effects in various tissues including adipose inflammation, a condition characterized by increased local release of the pro-lipolytic cytokine tumor necrosis factor-alpha (TNF-α). Whether miRNAs regulate adipocyte lipolysis is unknown. We set out to determine whether miRNAs affect adipocyte lipolysis in human fat cells. To this end, eleven miRNAs known to be present in human adipose tissue were over-expressed in human in vitro differentiated adipocytes followed by assessments of TNF-α and glycerol levels in conditioned media after 48 h. Three miRNAs (miR-145, -26a and let-7d) modulated both parameters in parallel. However, while miR-26a and let-7d decreased, miR-145 increased both glycerol release and TNF-α secretion. Further studies were focused therefore on miR-145 since this was the only stimulator of lipolysis and TNF-α secretion. Time-course analysis demonstrated that miR-145 over-expression up-regulated TNF-α expression/secretion followed by increased glycerol release. Increase in TNF-α production by miR-145 was mediated via activation of p65, a member of the NF-κB complex. In addition, miR-145 down-regulated the expression of the protease ADAM17, resulting in an increased fraction of membrane bound TNF-α, which is the more biologically active form of TNF-α. MiR-145 overexpression also increased the phosphorylation of activating serine residues in hormone sensitive lipase and decreased the mRNA expression of phosphodiesterase 3B, effects which are also observed upon TNF-α treatment in human adipocytes. We conclude that miR-145 regulates adipocyte lipolysis via multiple mechanisms involving increased production and processing of TNF-α in fat cells

miR-145 increases HSL phosphorylation at activating residues (but does not change total protein content of HSL) and down-regulates PDE3B.

<p>(A) Representative blots of protein expression levels of phosphorylated HSL (Ser-552 and Ser-650) and total HSL in human differentiated adipocytes transfected with miR-145 mimics for 48 h. (B) Relative quantification by densitometry of above depicted blots for p-HSL (Ser-552, dark grey; Ser-650, light grey) and (C) total HSL. Equal amounts of total protein were loaded and separated by SDS-PAGE as indicated in experimental procedures. Total HSL levels were corrected by tubulin expression and p-HSL levels were corrected by total HSL protein content. Values are shown as mean of pooling results from three biological/independent experiments. (D) Relative expression of PDE3B after miR-145 over-expression in human differentiated adipocytes for 48 h. Results are representative of four biological/independent experiments. Values are shown as mean ± SEM. Statistical differences (<i>vs</i>. Neg. Cntl) were analyzed by Student t-test: **p<0.01; ***p<0.001.</p

Transfection screening of microRNAs identifies TNF-α as a key mediator of miRNA effects on human adipocyte lipolysis.

<p>(A) Human differentiated adipocytes were transfected with each individual miRNA Mimics or Negative Control (40 nM) for 48 h as described in experimental procedures. After 48 h, conditioned media was collected and glycerol release and TNF-α secretion were measured. Glycerol levels were evaluated by a bioluminescence method in three to six biological/independent experiments. TNF-α secretion values were detected by ELISA in at least, two biological/independent experiments. Values are shown as mean ± SEM and expressed as relative fold change <i>vs</i>. Neg. Cntl. Statistical differences were analyzed by Student t-test comparing Mimics Neg. Cntl <i>vs</i>. Mimics of each miRNA: *p<0.05; **p<0.01; ***p<0.001. (B) MiR-145 was over-expressed in human differentiated adipocytes in a time-course experiment. Conditioned medium was collected at selected time-points post-transfection (6 h –12 h –24 h –48 h) for determination of glycerol (black line) and TNF-α secretion (black-broken line) levels. Cells were harvested for RNA and TNF-α mRNA expression (grey line) levels were determined. Results are indicative of three biological/independent experiments. Transfection efficiency showed ∼2×10⁴ fold-change up-regulation of individual miR-145 as compared to control (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086800#pone.0086800.s001" target="_blank">Figure S1</a>). Values are shown as mean ± SEM and expressed as relative fold change <i>vs</i>. Neg. Cntl. of each time-point. Statistical differences were analyzed by Student t-test comparing Mimics miR-145 <i>vs</i>. corresponding Neg. Cntl at each indicated time-point: *p<0.05; **p<0.01; ***p<0.001.</p

miR-145 alters TNF-α signaling and induces tightly correlated changes in lipolysis and TNF-α.

<p>(A) Human differentiated adipocytes were transfected with mimics miR-145 or Neg. Cntl (40 nM) for 15 h. After incubation time (15 h), nuclei were isolated and 2.5 µg of nuclear extract was used to perform p65 transactivation assay as described by manufacturer. Figure depicts representative wells of Neg. Cntl and miR-145-treated adipocytes and its relative quantification graph of three biological/independent experiments. Values are shown as mean ± SEM and expressed as relative fold change <i>vs</i>. Neg. Cntl. Statistical differences were analyzed by Student t-test: *p<0.05. (B) TNF-α receptor 1 (TNFR1) was silenced with siRNA for 24 h prior to co-transfection with miRNA Mimics (Neg. Cntl/miR-145) for additional 48 h in human differentiated adipocytes. After incubation time, conditioned medium was collected to measure glycerol release and cells were harvested for (C) TNF-α mRNA measurements. Results are representative of three biological/independent experiments. Values are shown as mean ± SEM. Statistical differences (<i>vs</i>. Neg. Cntl) were analyzed by Student t-test: **p<0.01; ***p<0.001. (D) Correlation between glycerol <i>vs</i>. TNF-α secretion (measured in conditioned medium/supernatant) and <i>vs</i>. (E) TNF-α mRNA levels in miR-145-transfected adipocytes <i>in vitro</i> for 48 h. Values are expressed as relative fold change <i>vs</i>. Neg. Cntl. (in D and E) after correction by transfection efficiency and were compared by linear regression. Each dot represents a technical replicate from 7–8 biological/independent experiments.</p

miR-145 does not affect phosphorylation and protein content of PLIN1.

<p>(A) Representative blot of total protein content of PLIN1 after miR-145 over-expression in human differentiated adipocytes for 48 h. PLIN1 protein content was corrected by tubulin as described in experimental procedures. (B) Relative quantification of PLIN1 <i>vs</i>. tubulin (n = 3).</p

ADAM17 is a miR-145 target that regulates TNF-α processing in human adipocytes.

<p>(A) mRNA expression levels of ADAM17 after miR-145 over-expression at 6 h –12 h –24 h –48 h. Results presented are obtained from three biological/independent experiments. Values are shown as mean ± SEM and expressed as relative fold change <i>vs</i>. Neg. Cntl. at each corresponding time-point. (B) MiR-145 was over-expressed in human differentiated adipocytes for 48 h and whole cell lysates were analyzed by Western blot. TNF-α protein levels are expressed as ratio of TNF-α membrane bound (26 KDa) <i>vs</i>. soluble form (17 KDa). Results are representative of two biological/independent experiments. Values are shown as mean ± SEM. Statistical differences (<i>vs</i>. Neg. Cntl) were analyzed by Student t-test: **p<0.01; ***p<0.001.</p