Pigment epithelium-derived factor regulates lipid metabolism via adipose triglyceride lipase - PubMed (original) (raw)

. 2011 May;60(5):1458-66.

doi: 10.2337/db10-0845. Epub 2011 Apr 4.

Affiliations

Pigment epithelium-derived factor regulates lipid metabolism via adipose triglyceride lipase

Melissa L Borg et al. Diabetes. 2011 May.

Abstract

Objective: Pigment epithelium-derived factor (PEDF) is an adipocyte-secreted factor involved in the development of insulin resistance in obesity. Previous studies have identified PEDF as a regulator of triacylglycerol metabolism in the liver that may act through adipose triglyceride lipase (ATGL). We used ATGL(-/-) mice to determine the role of PEDF in regulating lipid and glucose metabolism.

Research design and methods: Recombinant PEDF was administered to ATGL(-/-) and wild-type mice, and whole-body energy metabolism was studied by indirect calorimetry. Adipose tissue lipolysis and skeletal muscle fatty acid metabolism was determined in isolated tissue preparations. Muscle lipids were assessed by electrospray ionization-tandem mass spectrometry. Whole-body insulin sensitivity and skeletal muscle glucose uptake were assessed.

Results: PEDF impaired the capacity to adjust substrate selection, resulting in a delayed diurnal decline in the respiratory exchange ratio, and suppressed daily fatty acid oxidation. PEDF enhanced adipocyte lipolysis and triacylglycerol lipase activity in skeletal muscle. Muscle fatty acid uptake and storage were unaffected, whereas fatty acid oxidation was impaired. These changes in lipid metabolism were abrogated in ATGL(-/-) mice and were not attributable to hypothalamic actions. ATGL(-/-) mice were also refractory to PEDF-mediated insulin resistance, but this was not related to changes in lipid species in skeletal muscle.

Conclusions: The results are the first direct demonstration that 1) PEDF influences systemic fatty acid metabolism by promoting lipolysis in an ATGL-dependent manner and reducing fatty acid oxidation and 2) ATGL is required for the negative effects of PEDF on insulin action.

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Figures

FIG. 1.

FIG. 1.

ATGL is the major target of PEDF-mediated lipolysis in adipose tissue. A: Immunofluorescence microscopy shows distribution of ATGL in the cytoplasm and around lipid droplets in 3T3-L1 adipocytes. Notably, ATGL is not observed at the plasma membrane (PM) or with endosomal (early endosome antigen 1 [EEA1]: green) or Golgi markers (gm130: blue). B: ATGL colocalizes with the cytoplasmic marker tubulin, but not with membrane markers in a subcellular fractionation. Western blot shows location of various subcellular markers in a fractionation from 3T3-L1 adipocytes (top) and L6 myotubes (below). +PM, plasma membrane–containing fraction. C: Epididymal fat pads were excised from lean ATGL−/−, HSL−/−, or Wt littermates, and lipolysis was assessed as glycerol release into the buffer. PEDF: 100 nmol/L (n = 4 per group). *P < 0.05 vs. vehicle within the same genotype. D: PEDF (100 nmol/L) does not affect β-adrenergic–stimulated lipolysis in epididymal fat pads. Fat pads were incubated for 2 h in isoproterenol (1 μmol/L), and glycerol release was determined (n = 4 per group). E: Immunoblot of plasma PEDF in mice fasted for 16 h or 2 h after refeeding. F: In vivo lipolysis is increased by PEDF. Lean ATGL−/− or Wt mice were injected with PEDF intraperitoneally, blood was obtained after 30 min, and glycerol was assessed in the plasma (n = 6 mice per group). *P < 0.05 vs. vehicle. Data in graphs are mean ± SEM. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 2.

FIG. 2.

PEDF alters whole-body fatty acid metabolism. A–C: Lean C57Bl/6 mice were injected with recombinant PEDF or sterile saline and placed in a metabolic monitoring station. Oxygen uptake (VO2; A) and total activity (B) were similar between groups. C: The respiratory exchange ratio (RER) was assessed during the light and dark cycles and was increased during the early phase of the light cycle in PEDF-treated mice (n = 6 mice per group). *P < 0.05 vs. vehicle at the corresponding time. D–F: VO2, activity, and the RER were assessed in ATGL−/− mice after recombinant PEDF or sterile saline administration (n = 4 per group). #P < 0.05, main effect for treatment. All data are presented as means ± SEM.

FIG. 3.

FIG. 3.

PEDF’s metabolic effects do not depend on central actions. A: The hypothalamus of C57Bl/6 mice was excised, and 18S, PEDF, and ATGL mRNA expression were assessed by qRT-PCR (n = 8). B: C57Bl/6 mice were injected with 0.9 μg PEDF or aCSF and placed in a metabolic monitoring station for assessment of oxygen uptake (VO2). C: RER was also assessed (n = 8 for PEDF and n = 11 for vehicle). D: Mice were injected with 0.9 μg PEDF or aCSF, and the hypothalami were excised after 6 h to assess mRNA of hypothalamic neuropeptides that modulate feeding and energy metabolism. Pro-opiomelanocortin (POMC), agouti-related peptide (AgRP), neuropeptide Y (NPY), and cocaine- and amphetamine-regulated transcript (CART) were measured (n = 8 per group). All data are presented as means ± SEM.

FIG. 4.

FIG. 4.

PEDF modulates fatty acid (FA) metabolism in skeletal muscle. L6 myotubes were treated with 100 nmol/L PEDF or saline (vehicle) for 2 h. Total FA uptake (A), incorporation of FA into triacylglycerol (TAG; B) or diacylglycerol (DAG; C), and FA oxidation (D) were assessed using 0.5 mmol/L [1-14C] palmitate (n = 6–12 for each group). E: Myotube TAG content is shown after 6-h treatment with 0.5 mmol/L oleate in the incubation media (n = 4–6 per group). All data are presented as means ± SEM. *P < 0.05 vs. vehicle. F: PEDF (100 nmol/L) reduces FA oxidation in intact skeletal muscle. Soleus muscles were removed from ATGL−/− and Wt littermates before assessment of FA oxidation using 0.5 mmol/L [1-14C] oleate ex vivo. *P < 0.05 vs. vehicle within the same genotype (n = 6 per group). G: Genes associated with FA oxidation and mitochondrial biogenesis (left) and TAG storage and degradation (right) were assessed in vastus lateralis of mice treated with saline or PEDF for 5 days (n = 6 for each group). *P < 0.05 vs. vehicle.

FIG. 5.

FIG. 5.

PEDF causes skeletal muscle insulin resistance in an ATGL-dependent manner. A: 2-Deoxy-

d

-glucose (2-DG) uptake experiments in primary myotubes. Wt (□) and ATGL−/− (■) myotubes were pretreated with saline or PEDF (100 nmol/L) for 2 h. The media was removed, and basal and insulin-stimulated 2-DG uptake was determined (n = 6 for each group where each experiment was performed in triplicate on two occasions). *P < 0.05 vs. vehicle within the same genotype. Values are means ± SEM. B: Extensor digitorum longus muscles were removed from ATGL−/− (■) and Wt littermates (□) before assessment of insulin-stimulated glucose uptake (n = 4–6 for each group where each experiment was performed on two occasions). *P < 0.05 vs. –PEDF within the same genotype. Values are means ± SEM. Insulin tolerance tests were performed in Wt (C) and ATGL−/− mice (D). PEDF was injected intraperitoneally 2 h before insulin administration. PEDF (n = 6–7 per treatment). *P < 0.05 vs. corresponding time point within the same genotype.

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