Rapid Insulin-Dependent Endocytosis of the Insulin Receptor by Caveolae in Primary Adipocytes

Background: The insulin receptor is localized in caveolae and is dependent on caveolae or cholesterol for signaling in adipocytes. When stimulated with insulin, the receptor is internalized. Methodology/Principal Findings: We examined primary rat adipocytes by subcellular fractionation to examine if the insulin receptor was internalized in a caveolae-mediated process. Insulin induced a rapid, t 1/2,3 min, endocytosis of the insulin receptor in parallel with receptor tyrosine autophosphorylation. Concomitantly, caveolin-1 was phosphorylated at tyrosine(14) and endocytosed. Vanadate increased the phosphorylation of caveolin-1 without affecting insulin receptor phosphorylation or endocytosis. Immunocapture of endosomal vesicles with antibodies against the insulin receptor cocaptured caveolin-1 and immunocapture with antibodies against tyrosine(14)-phosphorylated caveolin-1 co-captured the insulin receptor, demonstrating that the insulin receptor was endocytosed together with tyrosine(14)-phosphorylated caveolin-1. By immunogold electron microscopy the insulin receptor and caveolin-1 were colocalized in endosome vesicles that resembled caveosomes. Clathrin was not endocytosed with the insulin receptor and the inhibitor of clathrin-coated pitmediated endocytosis, chlorpromazine, did not inhibit internalization of the insulin receptor, while transferrin receptor internalization was inhibited. Conclusion: It is concluded that in response to insulin stimulation the autophosphorylated insulin receptor in primar

http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-21319 Rapid Insulin-Dependent Endocytosis of the Insulin Receptor by Caveolae in Primary Adipocytes

Rapid insulin-dependent endocytosis of the insulin receptor by caveolae in primary adipocyte

Phosphorylation and endocytosis of the insulin receptor.

<p>Isolated adipocytes were incubated with 100 nM insulin for the indicated time, when cells were homogenized and plasma membrane (pm) and endosomal (endo) fractions isolated. A. Equal amounts of protein (5 µg) were subjected to SDS-PAGE and immunoblotting with (from the top) antibodies against phosphotyrosine (indicated is the insulin receptor, IR-TyrP, identified by its comigration with insulin receptor protein on the same blot), insulin receptor β-subunit (IR), tyrosine(14)-phosphorylated caveolin-1 (caveolin-TyrP), caveolin-1, and actin. B, C. Time-course for internalization of phosphorylated insulin receptor. Two experiments were quantified and the average of the two is presented as percent of maximal insulin effect on phosphorylation of the insulin receptor at tyrosine (IR-TyrP) (B) or as a ratio of IR-TyrP to the amount of insulin receptor β-subunit protein (IR) (C), in the plasma membrane (open symbols) and endosomal fraction (closed symbols).</p

Cholesterol depletion of the cells blocks insulin receptor endocytosis.

<p>Isolated adipocytes were incubated with or without 10 mM β-cyclodextrin (β−CD) for 50 min, to deplete the plasma membrane of about 50% of its cholesterol, which completely destroys caveolae while leaving the cells intact <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005985#pone.0005985-Parpal1" target="_blank">[23]</a>. Insulin at 100 nM was added for another 5 min. Cells were then homogenized and plasma membrane (pm) and endosomal (endo) fractions isolated. A. Equal amounts of protein were subjected to SDS-PAGE and immunoblotting with (from the top) antibodies against insulin receptor β-subunit (IR) or phosphotyrosine (indicated is insulin receptor, IR-TyrP). B. Quantitation of three experiments. Presented as amount of IR or IR-TyrP in the presence of β-cyclodextrin as % of controls without β-cyclodextrin, mean±SE. *, p<0.05 by Student’s t-test.</p

Localization of insulin receptor and caveolin-1 in endosomes by immuno-gold electron microscopy.

<p>Isolated adipocytes were incubated with insulin at 100 nM for 10 min. Cells were then homogenized and the endosomal fraction isolated. Endosome vesicles were attached to grids, immunogold-labeled against caveolin-1 (6 nm gold particles) and the insulin receptor (15 nm gold particles), lyophilized and sputtered with a 2-nm tungsten film before examination by transmission electron microscopy. C and D are blow-ups from B; arrowheads indicate patches of caveolin-1 labeling; arrows indicate insulin receptor labeling. One experiment of three with similar results is illustrated.</p

Effects of vanadate on endocytosis of the insulin receptor.

<p>Isolated adipocytes were incubated with or without 2 mM Na-ortho-vanadate for 20 min, when insulin at 100 nM was added for another 10 min, as indicated. Cells were then homogenized and plasma membrane (A) and endosomal (B) fractions isolated. Equal amounts of protein were subjected to SDS-PAGE and immunoblotting with (from the top) antibodies against phosphotyrosine (indicated is the insulin receptor, IR-TyrP), insulin receptor β-subunit (IR), and tyrosine(14)-phosphorylated caveolin-1 (caveolin-TyrP). Bar graphs show the quantitation of tyrosine(14)-phosphorylated caveolin-1 as percent of controls without additions, in 7 separate experiments, mean±SE. *, p<0.05 by Student’s t-test.</p

Differential regulation of adipocyte PDE3B in distinct membrane compartments by insulin and the beta(3)-adrenergic receptor agonist CL316243: effects of caveolin-1 knockdown on formation/maintenance of macromolecular signalling complexes

In adipocytes, PDE3B (phosphodiesterase 3B) is an important regulatory effector in signalling pathways controlled by insulin and cAMP-increasing hormones. Stimulation of 3T3-L1 adipocytes with insulin or the beta(3)-adrenergic receptor agonist CL316243 (termed CL) indicated that insulin preferentially phosphorylated/activated PDE3B associated with internal membranes (endoplasmic reticulum/Golgi), whereas CL preferentially phosphorylated/activated PDE3B associated with caveolae. siRNA (small interfering RNA)-mediated KD (knockdown) of CAV-1 (caveolin-1) in 3T3-L1 adipocytes resulted in down-regulation of expression of membrane-associated PDE3B. Insulin-induced activation of PDE3B was reduced, whereas CL-mediated activation was almost totally abolished. Similar results were obtained in adipocytes from Cav-1-deficient mice. siRNA-mediated KID of CAV-1 in 3T3-L1 adipocytes also resulted in inhibition of CL-stimulated phosphorylation of HSL (hormone-sensitive lipase) and perilipin A, and of lipolysis. Superose 6 gel-filtration chromatography of solubilized membrane proteins from adipocytes stimulated with insulin or CL demonstrated the reversible assembly of distinct macromolecular complexes that contained P-32-phosphorylated PDE3B and signalling molecules thought to be involved in its activation. Insulin- and CL-induced macromolecular complexes were enriched in cholesterol, and contained certain common signalling proteins [14-3-3, PP2A (protein phosphatase 2A) and cav-1]. The complexes present in insulin-stimulated cells contained tyrosine-phosphorylated IRS-1 (insulin receptor substrate 1) and its downstream signalling proteins, whereas CL-activated complexes contained beta(3)-adrenergic receptor, PKA-RII [PKA (cAMP-dependent protein kinase)-regulatory subunit] and HSL. Insulin- and CL-mediated macromolecular complex formation was significantly inhibited by CAV-1 KID. These results suggest that cav-1 acts as a molecular chaperone or scaffolding molecule in cholesterol-rich lipid rafts that may be necessary for the proper stabilization and activation of PDE3B in response to CL and insulin

Attenuated mTOR Signaling and Enhanced Autophagy in Adipocytes from Obese Patients with Type 2 Diabetes

Type 2 diabetes (T2D) is strongly linked to obesity and an adipose tissue unresponsive to insulin. The insulin resistance is due to defective insulin signaling, but details remain largely unknown. We examined insulin signaling in adipocytes from T2D patients, and contrary to findings in animal studies, we observed attenuation of insulin activation of mammalian target of rapamycin (mTOR) in complex with raptor (mTORC1). As a consequence, mTORC1 downstream effects were also affected in T2D: feedback signaling by insulin to signal-mediator insulin receptor substrate-1 (IRS1) was attenuated, mitochondria were impaired and autophagy was strongly upregulated. There was concomitant autophagic destruction of mitochondria and lipofuscin particles, and a dependence on autophagy for ATP production. Conversely, mitochondrial dysfunction attenuated insulin activation of mTORC1, enhanced autophagy and attenuated feedback to IRS1. The overactive autophagy was associated with large numbers of cytosolic lipid droplets, a subset with colocalization of perlipin and the autophagy protein LC3/atg8, which can contribute to excessive fatty acid release. Patients with diagnoses of T2D and overweight were consecutively recruited from elective surgery, whereas controls did not have T2D. Results were validated in a cohort of patients without diabetes who exhibited a wide range of insulin sensitivities. Because mitochondrial dysfunction, inflammation, endoplasmic-reticulum stress and hypoxia all inactivate mTORC1, our results may suggest a unifying mechanism for the pathogenesis of insulin resistance in T2D, although the underlying causes might differ