Pigment epithelium-derived factor (PEDF) is one of the most abundant proteins secreted by human adipocytes and induces insulin resistance and inflammatory signaling in muscle and fat cells.

OBJECTIVE: Pigment epithelium-derived factor (PEDF) is a multifunctional protein with neurotrophic and anti-angiogenic properties. More recently it became evident that PEDF is upregulated in patients with type 2 diabetes and also contributes to insulin resistance in mice. During characterization of the secretome of in vitro differentiated human adipocytes by two-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization-MS, we found that PEDF is one of the most abundant proteins released by adipocytes. The aim of this study was to investigate the regulation and autocrine function of PEDF in human adipocytes and to determine its paracrine effects on human skeletal muscle cells (hSkMC) and human smooth muscle cells (hSMC). METHODS AND RESULTS: Human primary adipocytes secrete 130 ng ml(-1) PEDF over 24 h from 1 million cells, which is extremely high as compared with adiponectin, interleukin-6 (IL-6) or IL-8. This release of PEDF is significantly higher than from other primary cells, such as adipose-tissue located macrophages (50-times), hSkMC and hSMC (5-times). PEDF protein expression significantly increases during adipogenesis, which is paralleled by increased PEDF secretion. Furthermore, tumor necrosis factor-α and hypoxia significantly downregulate PEDF protein levels. PEDF secretion was significantly reduced by troglitazone and hypoxia and significantly increased by insulin. Treatment of adipocytes and hSkMC with PEDF induced insulin resistance in adipocytes, skeletal and smooth muscle cells at the level of insulin-stimulated Akt phosphorylation, which was dose dependent and more prominent in adipocytes. Furthermore, inflammatory nuclear factor-κB (NF-κB) signaling was induced by PEDF. In hSMC, PEDF induced proliferation (1.7-fold) and acutely activated proliferative and inflammatory signaling pathways (NF-κB, p38 mitogen-activated protein kinase and mammalian target of rapamycin). CONCLUSION: PEDF is one of the most abundant adipokines and its secretion is inversely regulated by insulin and hypoxia. PEDF induces insulin resistance in adipocytes and hSkMC and leads to inflammatory signaling in hSMC. Because of these diverse actions, PEDF is a key adipokine, which could have an important role in diabetes and obesity-related disorders.

Identification and characterization of a novel variant in the highly conserved catalytic center of Rab11a.

Small GTPases of the Rab family regulate vesicular traffic and distribution of proteins in different cell types. Rab11a is a member of this GTP hydrolyzing protein class and acts as a mediator of insulin stimulated translocation of the glucose transporter GLUT4 in peripheral tissues including heart and skeletal muscle. Here we report on Rab11a Q70R, a mutation in the catalytic center of Rab11a, observed in the cardiomyoblast cell line H9c2. Analysis of GTPase activity showed that Rab11a Q70L acts as a classical constitutive active mutant. Interestingly, the GTPase activity of Rab11a Q70R was not significantly different from the enzymatic activity of the Rab11a Q70 wild type protein. We therefore conclude that the glutamine residue of Rab11a at position 70 is not strictly essential for GTPase activity of this protein in contrast to Ras and other Rab proteins.

Hormone-triggered conformational changes within the insulin-receptor ectodomain: requirement for transmembrane anchors.

Interaction between two alphabeta half-receptors within the (alphabeta)(2) holoreceptor complex is required for insulin binding with high affinity and for insulin-triggered changes of size and shape. To understand the underlying structure-function relationship, two truncated receptor constructs have been characterized. Reduction in the Stokes radius and increase in the sedimentation coefficient, which are characteristic for wild-type receptors, were entirely lacking for the recombinant human insulin receptor (HIR) ectodomain (HIR-ED). Stokes radii of about 5.8 nm and sedimentation coefficients of 10.2 S were found for both insulin-bound and free HIR-EDs. However, attaching the membrane anchors to the ectodomain, as with the recombinant membrane-anchored ectodomain (HIR-MAED) construct, was sufficient to restore not only high-affinity hormone binding but also the marked insulin-inducible alterations in hydrodynamic properties. The Stokes radii of HIR-MAED complexes, as assessed by non-denaturing PAGE, decreased upon insulin binding from 9.5 nm to 7.9 nm. In parallel, the sedimentation coefficient was increased from 9.0 S to 9.8 S. CD and fluorescence spectroscopy of HIR-MAED revealed only minor insulin-induced changes in the secondary structure. Similarity with wild-type receptors has also been demonstrated by the differential insertion of insulin-bound and free HIR-MAED complexes into artificial bilayer membranes of Triton X-114. The results are consistent with a model of receptor function that ensures a global insulin-triggered reorientation of subdomains within the ectodomain moieties while the secondary structure is essentially retained. For the rearrangement of such subdomains, the transmembrane anchors confer essential structural constraints on the receptor ectodomain.

Identification of Ser-1275 and Ser-1309 as autophosphorylation sites of the insulin receptor.

We have identified Ser-1275 and Ser-1309 as novel serine autophosphorylation sites by direct sequencing of HPLC-purified tryptic phosphopeptides of the histidine-tagged insulin receptor kinase IRKD-HIS. The corresponding peptides (Ser-1275, amino acids 1272-1292; Ser-1309, amino acids 1305-1313) have been detected in the HPLC profiles of both the soluble kinase IRKD, which contains the entire cytoplasmic domain of the insulin receptor beta-subunit, and the insulin receptor purified from human placenta. In contrast, a kinase negative mutant, IRKD-K1018A, did not undergo phosphorylation at either the tyrosine or serine residues, strongly suggesting that insulin receptor kinase has an intrinsic activity to autophosphorylate serine residues.

Phosphoryl exchange is involved in the mechanism of the insulin receptor kinase.

The cytoplasmic kinase domain of the human insulin receptor (IRKD; M(r) 49 kDa) has been over-expressed in insect cells using the baculovirus expression system. To investigate the kinase mechanism, we have compared the stoichiometry of ADP formation and phosphoryl transfer. After an initial phase of autophosphorylation, ATP is consumed without a stoichiometric increase in incorporated phosphate. During substrate phosphorylation using poly(Glu:Tyr) (4:1) phosphoryl transfer comes close to ATP turnover, which is independent of the presence of the substrate, indicating an increased efficiency (i.e. ATP turnover/phosphate incorporation) of phosphoryl transfer. Autophosphorylation under pulse-chase conditions suggests the existence of a phosphoenzyme intermediate.

Insulin binding to erythrocytes in hyperinsulinemic patients with precirrhotic hemochromatosis and cirrhosis.

This study investigated insulin receptor binding (number and affinity) to erythrocytes in patients with precirrhotic hemochromatosis, patients with cirrhosis, and healthy subjects. To evaluate plasma glucose and insulin levels, an oral glucose tolerance test (OGTT) was performed in all subjects. In the fasting state, all patients exhibited normal glucose levels. Precirrhotic patients showed slight impairment of glucose tolerance while cirrhotic patients were strikingly glucose intolerant. In both patient groups, fasting plasma insulin levels were increased. Following the glucose load, plasma insulin levels were significantly enhanced in precirrhotic patients at 90 and 120 min and increased at all times in cirrhotic patients. In the postabsorptive state (in the presence of hyperinsulinemia) insulin binding and the number and affinity of insulin receptors of erythrocytes were not different in precirrhotic or cirrhotic patients when compared to controls. We conclude that studies of insulin binding on erythrocytes do not contribute to the evaluation of the pathogenesis of insulin resistance in liver disease.

The influence of tracers on insulin binding to human erythrocytes.

We studied insulin binding to human erythrocytes using two different 125I-insulin-tracers. Erythrocytes of 8 normal subjects were examined using [mono-125II-(Tyr A 14)insulin as tracer. Three of these erythrocyte preparations were examined simultaneously using [125I]insulin, which was randomly iodinated by the chloramine-T-method. Data were analysed by a computerized non-linear least-squares procedure both on the basis of one and two class receptor models. Only the one class receptor model yielded consistent results. When the two class receptor model was applied the low affinity branch of the Scatchard plot was not reproducible. On the basis of the one class receptor model the number of receptor sites was lower (R0 = 0.046 +/- 0.006 nmol/l equivalent to 6.3 +/- 0.8 receptors/erythrocyte) with [mono-125I-(Tyr A 14)]insulin as compared to [125I]insulin randomly iodinated by the chloramine-T method (R0 = 0.070 +/- 0.008 nmol/l equivalent to 9.6 +/- 1.1 receptors/erythrocyte). Conversely, the affinity of the [mono-125I-(Tyr A 14)]insulin was higher (Ka = 2.6 +/- 0.3 x 10(9) l . mol-1 vs. 1.9 +/- 0.2 x 10(9) l . mol-1.