Vimentin is a functional partner of hormone sensitive lipase and facilitates lipolysis

Abstract

Lipolysis involves a number of components including signaling pathways, droplet-associated proteins, and lipases such as hormone-sensitive lipase (HSL). We used surface enhanced laser desorption/ionization time-of-flight mass spectroscopy to identify cellular proteins that might interact with HSL and potentially influence lipolysis. Using recombinant HSL as bait on protein chips, clusters of proteins of 14.7-18.9, 25.8-26.8, 36.1, 44.3-49.1, and 53.7 kDa were identified that interact with HSL, particularly when lysates were examined from beta-agonist treated mouse adipocytes. The ability to detect these interacting proteins was markedly diminished when the adipocytes were treated with insulin. A very similar pattern of proteins was identified when anti-HSL IgG was used as the bait. Following immunocapture, the identification of the prominent 53.7 kDa protein was carried out by tryptic digestion and MS analysis and determined to be vimentin. The interaction of HSL with vimentin, and its hormonal dependence, was confirmed by coimmunoprecipitation. beta-Agonist stimulated lipolysis and the rate of HSL translocation were impaired in vimentin null adipocytes, even though normal amounts of lipases and droplet-associated proteins are expressed. The current studies provide evidence that vimentin participates in lipolysis through direct, hormonally regulated interactions with HSL.

Figure 1

SELDI-TOF-MS of adipose proteins binding to immobilized HSL (upper panel) or immobilized BSA (lower panel). Recombinant HSL (~0.5 μg) or BSA was immobilized on RS100 ProteinChip arrays. Unreacted amine groups were blocked with 0.5 M ethanolamine/phosphate-buffered saline for 1 h. The ProteinChips were washed three times for 5 min with phosphate-buffered saline containing 0.1% Triton X-100. Freshly prepared adipocyte lysates (2–4 mg/ml) were incubated with the arrays, then washed twice for 5 min with phosphate-buffered saline containing 0.1% Triton X-100 and once for 3 min with Tris-HCl buffer (pH 9.0) containing 1 M urea, 2% CHAPS, and 0.5 M NaCl. The ProteinChips were rinsed with 5 mM HEPES (pH 7.2) for 15 s and air-dried. A saturated solution of 3,5-dimethoxy-4-hydroxyinnamic acid (sinapinic acid) in 50% acetonitrile and 0.5% trifluoroacetic acid was applied to the ProteinChips and mass analysis was performed by SELDI-TOF-MS.

Figure 2

SELDI-TOF-MS of proteins binding to immobilized HSL. Cell lysates were obtained from control (C), isoproterenol-treated (Iso, 1 μM) and isoproterenol (1 μM) plus insulin (100 μU/ml) - treated (Iso+Ins) primary wild-type mouse adipose cells. Each treatment was conducted with 3 independent incubations yielding 3 independent cell lysates. Data were obtained as described in Figure 1.

Figure 3

SELDI-TOF-MS of proteins binding to immobilized anti-HSL IgG. Anti-HSL IgG (4μg) or BSA was immobilized on PS10 arrays and then treated as described in Figure 1. Cell lysates were obtained from control (C), isoproterenol-treated (Iso) and isoproterenol plus insulin-treated (Iso+Ins) primary wild-type mouse adipose cells. Each treatment was conducted with 3 independent incubations yielding 3 independent cell lysates.

Figure 4

Purification and Identification of the 53.7 kDa HSL-Interacting Partner. Panel A: Proteins captured on anti-HSL IgG-conjugated beads. Lane 1, wash; lane 2, flow through; lane 3 loading; lane 4, elution 1; lane 5, elution 2. Anti-HSL IgG was conjugated to AminoLink Plus Coupling Resin and 250 μl of packed, anti-HSL IgG-conjugated beads were incubated with freshly prepared adipose cell lysates (3–4 mg/ml) overnight at 4°C. The beads were washed twice with phosphate-buffered saline containing 0.1% Triton X-100, and once with Tris-HCl buffer (pH 9.0) containing 1 M urea, 2% CHAPS, and 0.5 M NaCl. Captured proteins were then eluted with 50% acetonitrile/0.5% trifluoroacetic acid. The eluted proteins were run on SDS-PAGE under nonreducing conditions and stained with coumassie. Panel B. SELDI-TOF-MS of tryptic digests of the 53 kDa protein. Panel C: MS scan of the tryptic digests of the 53.7 kDa protein. Panel D: MS/MS analysis of peptide fragmentation of the 1444 Da peptide highlighted in Panel C.

Figure 5

Co-immunoprecipitation of vimentin and HSL. Cell lysates (250 μg) from adipose cells were immunoprecipitated with anti-HSL IgG and then immunoblotted with anti-vimentin antibody as described in the Materials and Methods. Lane 1, control adipose lysates; lane 2, lysates of adipose cells treated with isoproterenol (1 μM); lane 3, lysates of adipose cells treated with isoproterenol (1 μM) and insulin (100 μU/ml).

Figure 6

Expression of lipases and droplet associated proteins in vimentin null (KO) and wild-type (WT) adipose tissue. Panel A: RT-PCR of mRNA expressed relative to 36B4. n=4 males in each group. Panel B: Immunoblot of total cell lysates. Cell lysates (20 μg) were resolved by 4–15% gradient SDS-PAGE gel, blotted onto nitrocellulose membranes, incubated with specific antibodies, and detected by infrared fluorescent imaging as described in the Materials and Methods. ATGL, adipose triglyceride lipase; HSL, hormone-sensitive lipase; TGH, triacylglycerol hydrolase; CGI-58, comparative gene identification-58; Plin, perilipin; ADRP, adipocyte differentiation related protein; TIP47, tail-interacting protein of 47 kDa. Panel C: Immunoblot of phosphorylated and total ERK in isolated adipocytes. Cell lysates (20 μg) were resolved by 4–15% gradient SDS-PAGE gel, blotted onto nitrocellulose membranes, incubated with specific antibodies, and detected by chemiluminescence as described in the Materials and Methods. Lane 1, basal; lane 2, isoproterenol treated (0.5 μM); lane 3, forskolin treated (20 μM); lane 4, diacylglycerol treated (1 μM). P-ERK, phospho-extracellular signal-regulated kinase. Panel D. Adipose cell size in vimentin null (KO) and wild-type (WT) mice. n=6 WT and 5 KO mice.

Figure 7

Lipolysis in adipose cells from vimentin null mice. Panel A: Dose response of glycerol release to isoproterenol in adipose cells isolated from vimentin null (Vim−/−) and wild-type (WT) mice. Adipocytes isolated from 12–16 wks old vimentin null and wild-type littermates were incubated in the absence or presence of the indicated concentrations of isoproterenol in 120 mM NaCl, 4 mM KH₂PO₄, 1 mM MgSO₄, 1 mM CaCl₂, 10 mM NaHCO₃, 27 mM HEPES (pH 7.4) containing 3% BSA and 2.5 mM glucose for 60 min at 37°C in 95% air-5% CO₂. At the end of the incubation, an aliquot of infranatant was removed for measurement of glycerol concentration. *, p<0.001 Panel B: Time course of HSL association with the fat cake in adipose cells isolated from vimentin null (Vim−/−) and wild-type (WT) mice. Adipocytes isolated from vimentin null and wild-type littermates were incubated with isoproterenol (1 μM) as above for various times at 37°C in 95% air-5% CO₂. At the indicated times the cells were washed, homogenized, the fat cake and cytosol separated by centrifugation, and HSL detected by immunoblotting as described in the Materials and Methods. The results represent 4 independent experiments. †, p<0.05