Defective insulin receptor activation and altered lipid rafts in Niemann-Pick type C disease hepatocytes

Abstract

Niemann-Pick type C (NPC) disease is a neuro-visceral cholesterol storage disorder caused by mutations in the NPC-1 or NPC-2 gene. In the present paper, we studied IR (insulin receptor) activation and the plasma-membrane lipid assembly in primary hepatocytes from control and NPC1-/- mice. We have previously reported that, in hepatocytes, IR activation is dependent on cholesterol-sphingolipid rafts [Vainio, Heino, Mansson, Fredman, Kuismanen, Vaarala and Ikonen (2002) EMBO Rep. 3, 95-100]. We found that, in NPC hepatocytes, IR levels were up-regulated and the receptor activation was compromised. Defective IR activation was reproduced in isolated NPC plasma-membrane preparations, which displayed an increased cholesterol content and saturation of major phospholipids. The NPC plasma membranes were less fluid than control membranes as indicated by increased DPH (1,6-diphenyl-1,3,5-hexatriene) fluorescence anisotropy values. Both in NPC hepatocytes and plasma-membrane fractions, the association of IR with low-density DRMs (detergent-resistant membranes) was increased. Moreover, the detergent resistance of both cholesterol and phosphatidylcholine were increased in NPC membranes. Finally, cholesterol removal inhibited IR activation in control membranes but restored IR activation in NPC membranes. Taken together, the results reveal a lipid imbalance in the NPC hepatocyte, which increases lipid ordering in the plasma membrane, alters the properties of lipid rafts and interferes with the function of a raft-associated plasma-membrane receptor. Such a mechanism may participate in the pathogenesis of NPC disease and contribute to insulin resistance in other disorders of lipid metabolism.

Figure 1. Fluorescent images of WT and NPC hepatocytes

(A) Primary hepatocytes from WT and NPC mice were grown on coverslips, fixed with paraformaldehyde and stained with filipin to visualize the distribution of unesterified cholesterol. In NPC cells, the perinuclear cholesterol storage organelles are clearly visible. (B) Serum-starved WT and NPC hepatocytes were fixed with acetone and stained with anti-IRβ and anti-LAMP1 antibodies followed by Alexa Fluor® 488- and Alexa Fluor® 568-conjugated secondary antibodies respectively. Confocal images were captured at the cell-surface level to illustrate the plasma membrane staining of IR (arrowheads). The majority of LAMP1-positive organelles are out of this focal plane.

Figure 2. IR levels and activation in WT and NPC hepatocytes

(A) WT and NPC hepatocytes were stimulated with insulin for 0–20 min and cell lysates were analysed by Western blotting for IR levels (with anti-IRβ antibody) and phosphorylation (with anti-pTyr antibody). Representative immunoblots (IB) after 5 min of insulin stimulation are shown. (B) Quantification of IR levels and (C) IR activation as a function of time. Values are means±S.E.M., n=44 in (B) and n=4–6 in (C). *P<0.05, **P<0.001, P values from a two-tailed Student's t test in all experiments.

Figure 3. Isolation of PM fractions from WT and NPC mouse livers

Plasma membranes were isolated by sequential density-gradient centrifugation as detailed in the Experimental section. (A, B) Depletion of contaminating membranes in the preparation. The distribution of lysosomal and ER marker proteins LAMP1 and calnexin respectively in the gradient fractions and interphases was analysed by Western blotting. Interphases represent the membrane fractions collected from gradient 1 and subjected to gradient 2 (0.8/1.2 M) (A), and the final plasma-membrane fraction was collected from gradient 2 (0.5/0.8 M) (B). Electron micrographs of the interphases are also shown. Arrowheads indicate storage organelles. Suc, sucrose; pel, pellet. Equal volumes of the gradient fractions were used for Western-blot analysis. (C) Enrichment of plasma-membrane proteins in the preparation. Na⁺/K⁺-ATPase and IR were immunoblotted from the starting material (‘Start’; crude liver lysate subjected to gradient 1) and from the final plasma-membrane fraction (PM). To be able to compare the band intensities in the same blot, 40 μg of protein was used for Start and 10 μg for plasma membrane.

Figure 4. IR activation in isolated plasma membranes

(A) Validation of the IR in vitro kinase assay. Under the assay conditions, IR phosphorylation in the isolated membranes is strictly dependent on insulin and ATP. (B) Comparison of IR activation in WT and NPC plasma-membrane preparations. Representative immunoblots are shown. (C) Quantification of the data. Values are means±S.E.M., n=6, *P<0.05.

Figure 5. MS analysis of WT and NPC plasma-membrane lipid composition

(A) Cholesterol/PL ratio. Values are means±S.D. for two independent determinations performed in triplicate. *P<0.05. The total amount of PLs was not significantly different between NPC and WT samples. (B) Acyl chain lengths and saturation of major PL classes. Species constituting a minimum of 5% of total in each PL class are included. Values are means±S.D. for a representative experiment performed in triplicate. PE, phosphatidyl ethanolamine.

Figure 6. Association of IR, cholesterol, SM and PC with DRMs

(A) Isolated plasma membranes from WT and NPC mouse livers were solubilized in 1% Triton X-100 at 4 °C and subjected to Optiprep density-gradient centrifugation. Proteins from the gradient fractions were precipitated with trichloroacetic acid and analysed by Western blotting with anti-IRβ antibodies. OP, Optiprep. (B) For analysing the fraction of ligand-occupied IR in DRMs, WT and NPC hepatocytes were stimulated with ¹²⁵I-insulin, washed and subjected to density-gradient centrifugation. The amount of radioactivity in the fractions was determined and the percentage in DRMs is indicated. (C) The fraction of radiolabelled lipids in DRMs from WT and NPC hepatocytes. Black bars, [³H]PC; white bars, [¹⁴C]cholesterol; grey bars, [³H]SM. The cells were labelled and the lipids were analysed as detailed in the Experimental section. Values are means±S.D. for three to four independent experiments performed in triplicate; *P<0.05, **P<0.001.

Figure 7. Effect of cholesterol removal on IR activation in isolated NPC plasma membranes

Isolated NPC plasma membranes were treated with methyl-β-cyclodextrin (CD; 10 mM for 30 min at 37 °C) and the IR activation assay was performed as in Figure 4. Representative immunoblots (A) with quantification of the data (B) are shown. Values in (B) are means±S.E.M., n=10, ^*P<0.05.