Human triglyceride-rich lipoproteins impair glucose metabolism and insulin signalling in L6 skeletal muscle cells independently of non-esterified fatty acid levels

Diabetologia, Apr 2005

Aims/hypothesis Elevated fasting and postprandial plasma levels of triglyceride-rich lipoproteins (TGRLs), i.e. VLDL/remnants and chylomicrons/remnants, are a characteristic feature of insulin resistance and are considered a consequence of this state. The aim of this study was to investigate whether intact TGRL particles are capable of inducing insulin resistance. Methods We studied the effect of highly purified TGRLs on glycogen synthesis, glycogen synthase activity, glucose uptake, insulin signalling and intramyocellular lipid (IMCL) content using fully differentiated L6 skeletal muscle cells. Results Incubation with TGRLs diminished insulin-stimulated glycogen synthesis, glycogen synthase activity, glucose uptake and insulin-stimulated phosphorylation of Akt and glycogen synthase kinase 3. Insulin-stimulated tyrosine phosphorylation of IRS-1, and IRS-1- and IRS-2-associated phosphatidylinositol 3-kinase (PI3K) activity were not impaired by TGRLs, suggesting that these steps were not involved in the lipoprotein-induced effects on glucose metabolism. The overall observed effects were time- and dose-dependent and paralleled IMCL accumulation. NEFA concentration in the incubation media did not increase in the presence of TGRLs indicating that the effects observed were solely due to intact lipoprotein particles. Moreover, co-incubation of TGRLs with orlistat, a potent active-site inhibitor of various lipases, did not alter TGRL-induced effects, whereas co-incubation with receptor-associated protein (RAP), which inhibits interaction of TGRL particles with members of the LDL receptor family, reversed the TGRL-induced effects on glycogen synthesis and insulin signalling. Conclusions/interpretation Our data suggest that the accumulation of TGRLs in the blood stream of insulin-resistant patients may not only be a consequence of insulin resistance but could also be a cause for it.

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Human triglyceride-rich lipoproteins impair glucose metabolism and insulin signalling in L6 skeletal muscle cells independently of non-esterified fatty acid levels

M. T. Pedrini . M. Kranebitter . A. Niederwanger . S. Kaser . J. Engl . P. Debbage . L. A. Huber . J. R. Patsch Aims/hypothesis: Elevated fasting and postprandial plasma levels of triglyceride-rich lipoproteins (TGRLs), i.e. VLDL/remnants and chylomicrons/remnants, are a characteristic feature of insulin resistance and are considered a consequence of this state. The aim of this study was to investigate whether intact TGRL particles are capable of inducing insulin resistance. Methods: We studied the effect of highly purified TGRLs on glycogen synthesis, glycogen synthase activity, glucose uptake, insulin signalling and intramyocellular lipid (IMCL) content using fully differentiated L6 skeletal muscle cells. Results: Incubation with TGRLs diminished insulin-stimulated glycogen synthesis, glycogen synthase activity, glucose uptake and insulin-stimulated phosphorylation of Akt and glycogen synthase kinase 3. Insulin-stimulated tyrosine phosphorylation of IRS-1, and IRS-1- and IRS-2-associated phosphatidylinositol 3-kinase (PI3K) activity were not impaired by TGRLs, suggesting that these steps were not involved in the lipoprotein-induced effects on glucose metabolism. The overall observed effects were time- and dose-dependent and paralleled IMCL accumulation. NEFA concentration in the incubation media did not increase in the presence of TGRLs indicating that the effects observed were solely due to intact lipoprotein particles. Moreover, co-incubation of TGRLs Introduction with orlistat, a potent active-site inhibitor of various lipases, Duality of interest All authors declare that there is no duality of interest with a company/organisation that would financially benefit from the publication of the data in our manuscript did not alter TGRL-induced effects, whereas co-incubation with receptor-associated protein (RAP), which inhibits interaction of TGRL particles with members of the LDL receptor family, reversed the TGRL-induced effects on glycogen synthesis and insulin signalling. Conclusions/ interpretation: Our data suggest that the accumulation of TGRLs in the blood stream of insulin-resistant patients may not only be a consequence of insulin resistance but could also be a cause for it. - Insulin resistance represents a major metabolic abnormality in the pathogenesis of type 2 diabetes, one of the leading causes of mortality and invalidity. The factors responsible for the development of insulin resistance and type 2 diabetes have yet to be elucidated. Three lipid abnormalities are characteristic of insulin resistance and type 2 diabetes, i.e. high plasma levels of NEFAs, dyslipidaemia and pronounced postprandial lipaemia. A fourth lipid abnormality suggested to be a determinant of insulin resistance is the increase of intramyocellular lipid (IMCL) content in skeletal muscle [14]. Various experimental settings have been used to study the effects of NEFAs on insulin sensitivity. In a number of human and animal studies, plasma NEFA levels were raised by intravenous infusion of lipid emulsions together with heparin to stimulate lipoprotein lipase (LPL) activity. The rise in plasma levels of NEFAs has been shown to induce insulin resistance [58]. In line with these studies, it has been demonstrated that CD36 knockout mice have improved insulin sensitivity in muscle, implying that fatty acid flux into the cells plays a critical role in insulin resistance [9]. Several studies investigated the effect of various NEFAs on insulin signalling and glucose metabolism in vitro using different cell lines [1013]. Taking together the results of all these studies, elevated NEFA levels have been convincingly shown to play a causal role in the pathogenesis of insulin resistance. In addition, several studies have demonstrated that NEFAs are also capable of inducing beta cell dysfunction [1417], the other major metabolic abnormality of type 2 diabetes. Despite the wealth of data on the contribution of NEFAs to the development of insulin resistance and beta cell dysfunction, very little is known about the role of native lipoproteins in the development of these two processes. Elevated fasting and postprandial plasma levels of triglyceride-rich lipoproteins (TGRLs), i.e. VLDL/remnants and chylomicrons/remnants, decreased HDL levels, and small dense LDL particles, are characteristic of the dyslipidaemia of insulin resistance and type 2 diabetes. According to a widely held view, these characteristic changes in lipoprotein pattern are a consequence of insulin resistance and type 2 diabetes. However, a recent study using mouse pancreatic islets and a transformed insulin-secreting beta cell line demonstrated that purified TGRL and LDL particles may induce insulin-secreting beta cell dysfunction, indicating that the changes in plasma lipoproteins characteristic of type 2 diabetes are not only a consequence but also a cause of this disease [18]. To our knowledge, however, nothing is known as to whether intact lipoprotein particles are also capable of inducing insulin resistance. Intact lipoproteins including TGRL particles have been shown to be able to enter the subendothelial space by whole lipoprotein particle uptake [1925] and in this way can come into contact with cell surfaces of various peripheral tissues. Therefore, the rationale for this study was to investigate whether intact TGRL particles are capable of inducing insulin resistance in skeletal muscle cells, a principal site of peripheral insulin resistance. Materials and methods Materials The L6 rat skeletal muscle cell line was obtained from ATCC (Manassas, VA, USA); -MEM, amyloglucosidase and the L--PI3 standard were purchased from Sigma (St. Louis, MO, USA), FCS from PromoCell (Heidelberg, Germany) and fatty acid-/insulin-free BSA from Valeant Pharmaceuticals (Bryan, OH, USA). Sepharose 2B, [14C]-UDPG and the ECL kit were purchased from Amersham Biosciences (Buckinghamshire, UK), deoxy[3H]-glucose and [32P]-ATP from PerkinElmer (Boston, MA, USA), phosphatidylinositol from Avanti Polar Lipids (Alabaster, AL, USA) and the 415% linear-gradient mini gels from Biorad (Hercules, CA, USA). Anti-IRS-1, antiIRS-2, anti-phosphotyrosine-4G10 and anti-GSK-3 (antiglycogen synthase kinase 3) antibodies were obtained from Upstate (Charlottesville, VA, USA); anti-phospho-GSK-3/ -ser21/9, anti-phospho-Akt-ser473 and anti-Akt antibodies were from Cell Signaling Technology (Beverly, MA, USA). GAPDH antibody was purchased from Abcam (Cambridge, UK). The RAP fusion protein was obtained from Progen (Heidelberg, Germany) and orlistat (Xenical) from Roche (Hertfordshire, UK). Lipoprotein fractionation gels were obtained from LaboMed (Waldkirch, Germany). Cell culture Stock cells of the L6 rat skeletal muscle cell line were stored frozen in liquid nitrogen and a fresh vial of cells was thawed for every experiment. Cells were cultured in -MEM containing 10% FCS to confluency and then switched to the same media containing 2% FCS. Cells, cultured in 60-mm culture dishes or chamber slides (for oil red O staining), were used up to the fifth passage, and experiments were performed with fully differentiated myotubes 1214 days post-confluency. Lipoprotein isolation TGRLs, customarily defined as lipoproteins with a Svedberg flotation rate (Sf) higher than 20, were isolated from young healthy individuals in the postprandial state from blood withdrawn 4 h after ingestion of a standardised fatty meal [26]. Informed consent was obtained from all donors. To prevent bacterial growth, 10 mmol/l NaN3 was added to all solutions for the following centrifugation steps. For isolation of lipoproteins, entire plasma was subjected to an ultracentrifugation step in a Beckman type 42.1 rotor at 40,000g at a plasma density of 1.006 kg/l for 16 h at 15C. The top fraction of the tubes containing TGRLs was then subjected to a zonal ultracentrifugation procedure for analytically defining and preparatively isolating TGRL particle subfractions [27]. A density gradient linear with volume ranging from 1.00 to 1.15 kg/l was formed and ultracentrifugation was performed at 42,000g for 45 min at 15C [28]. Two fractions were obtained by pooling appropriate volume contents of the rotor (Beckman TI-14): firstly, lipoprotein fractions with an Sf higher than 200 corresponding mainly to chylomicrons/remnants; and secondly, a fraction with an Sf of between 20 and 200 corresponding mainly to VLDL/ remnants. The zonally isolated lipoproteins were concentrated by pressure filtration using Amicon cells and subsequently purified further by gel filtration using Sepharose 2B with PBS as an eluent buffer [29]. Purity of TGRL fractions was confirmed by gel electrophoresis using lipoprotein fractionation gels. The purified lipoproteins were stored in the dark at 4C under nitrogen for up to 2 weeks after preparation. The lipoprotein concentration in the media was chosen to correspond to a triglyceride concentration of 456 mol/l. Incubation experiments One day prior to the experiments, the media was replaced by serum-free -MEM containing 0.25% fatty acid- and insulin-free BSA, and cells were incubated in the presence or absence of purified TGRLs. Immediately prior to insulin stimulation, cells were washed with the above starvation media. Cells were incubated without or with insulin for various lengths of time, lysed and analysed for glycogen content, glycogen synthase activity, glucose uptake and insulin signalling as detailed below. Controls for cytotoxicity and cell viability To test whether TGRLs have toxic effects on L6 cells, we measured lactate dehydrogenase (LDH) in the incubation media at the beginning and end of all lipoprotein incubations. We did not observe any rise in LDH concentration upon TGRL incubation. In addition, possible TGRL-induced apoptosis was excluded by DAPI staining of the cells. Cell viability after treatment with TGRLs was assessed by trypan blue exclusion. Viability was found to be equal to that in non-TGRLtreated cells. For all these assays, H2O2 or BSA-deprived cell culture media were used as positive controls. 2-Deoxy-D-glucose uptake After starvation, cells were incubated without or with 1 mol/l insulin for 1 h at 37C, washed with HBS (20 mmol/l HEPES pH 7.4, 140 mmol/l NaCl, 2.5 mmol/l MgSO4, 1 mmol/l CaCl2, 5 mmol/l KCl) and then incubated with HBS-RM (HBS containing 10 mol/l deoxyglucose and 37 MBq/l deoxy-[3H]-glucose for 10 min at room temperature. After washing cells with ice-cold NaCl 0.9%, cells were lysed in 0.05 mol/l NaOH and radioactivity was counted using a Beckman scintillation counter. Glucose uptake was expressed as pmol deoxyglucose per min per mg protein. Cytochalasin B at 10 mol/l was used to estimate carrier-independent glucose uptake. Virtually all cultured skeletal muscle cell lines including L6 cells were found to have low levels of GLUT4 gene expression [33]. Therefore, insulin at a concentration of 1 mol/l was chosen for this experiment to achieve more pronounced effects on glucose uptake. Glycogen content After incubation of cells without or with 100 nmol/l insulin for 3 h at 37C, we determined glycogen content by a modification of the method described Western blot analysis After incubation without or with by Keppler and Decker [30]. Briefly, after washing four 100 nmol/l insulin for 5 min, cells were washed with PBS times with ice-cold PBS, cells were collected in 0.6 mol/l and solubilised in lysis buffer (50 mmol/l HEPES, pH 7.5, HClO4 and homogenised by sonification in ice water. 1% Triton X-100, 150 mmol/l NaCl, 10 mmol/l EDTA, Aliquots of the homogenate were neutralised with 1 mol/l 10% glycerol, 10 mg/l trypsin inhibitor, 1 mmol/l Na3VO4, KHCO3 and incubated with 10 g/l amyloglucosidase in 1 mmol/l PMSF, 5 mg/l pepstatin A, 10 mg/l aprotinin, 0.2 mol/l acetate buffer (pH 4.8) for 2 h at 40C. The 10 mg/l leupeptin, 10 mmol/l NaF and 10 nmol/l sodium reaction was stopped by addition of chilled 2 mol/l HClO4 pyrophosphate). After incubation on ice for 30 min, samand centrifugation at 14,000 g at 4C for 10 min. Glucose ples were centrifuged at 10,000g for 5 min at 4C and the concentration was determined using a Cobas MIRA ana- protein content of the supernatant was determined accordlyser from Roche. Glycogen content was expressed as nmol ing to the method of Bradford. An aliquot was taken for glucose/mg protein. direct blotting, and the remaining supernatant was used for For the experiments with receptor-associated protein immunoprecipitation with an anti-IRS-1- and anti-phos(RAP), this protein was added at a concentration of 1 mol/l photyrosine antibody respectively overnight at 4C. The to the incubation media 15 min prior to the addition of immune complexes were collected on protein A-agarose TGRLs and then co-incubated with TGRLs for 3 h. during 2 h of incubation at 4C. The beads were washed Orlistat was prepared as previously described [31] and four times with lysis buffer including 0.1% instead of 1% co-incubated at a concentration of 250 g/l with TGRLs Triton X-100 and boiled for 5 min in Laemmli buffer. For for 3 h. direct blotting, Laemmli buffer was added to the aliquot of the detergent extract. The solubilised proteins were Glycogen synthase activity For determining glycogen syn- resolved by SDS-PAGE on 415% linear-gradient mini thase activity by a modification of the method described gels and subjected to immunoblotting using respective antiby Thomas et al. [32], cells were incubated without or with bodies. Loading control was performed using an antibody 100 nmol/l insulin for 30 min at 37C, washed with a buffer against GAPDH. For these experiments, the same blot was containing 50 mmol/l TrisHCl pH 7.6 and 100 mmol/l KF stripped several times with stripping buffer (100 mmol/l and collected in the above buffer containing 30% glycerol, 2-mercaptoethanol, 2% SDS, 62.5 mmol/l Tris/HCl, pH 1 mmol/l EDTA, 10 mg/l aprotinin, 10 mg/l leupeptin and 6.7) at 70C for 30 min and reprobed with respective 1 mmol/l PMSF. After sonification and centrifugation, al- antibodies. Upon incubation with secondary antibodies, iquots of the supernatant were added to the reaction mix immunoreactive bands were detected by enhanced chemicontaining 50 mmol/l TrisHCl pH 7.6, 20 mmol/l EDTA, luminescence according to the manufacturers instructions 25 mmol/l KF, 10 g/l glycogen, 7.2 mmol/l UDPG and using Biorads Fluor S Max imager. Intensity of various 1.85 kBq [14C]-UDPG in the presence of 0.3 mmol/l glu- bands was expressed as n-fold stimulation of condition in cose 6-phosphate and 6.7 mmol/l glucose 6-phosphate re- the absence of insulin and TGRLs. spectively for 1 h at 30C. The reaction was terminated by spotting the mixture on filter papers that were extensively IRS-1- and IRS-2-associated phosphatidylinositol 3-kinase washed with ice-cold 70% ethanol overnight and air dried. activity For this purpose, cells were incubated without or Then, radioactivity was counted using a Beckman scintil- with 100 nmol/l insulin for 10 min, washed three times with lation counter, and enzyme activity was expressed as a buffer 1 (20 mmol/l TrisHCl, pH 7.4, 137 mmol/l NaCl, percentage of the glucose 6-phosphate-independent form. 1 mmol/l CaCl2, 1 mmol/l MgCl2 and 1 mmol/l Na3VO4) and then lysed in buffer 1 containing 1% NP-40, 1 mmol/l PMSF, 2 mg/l leupeptin, 20 mmol/l sodium pyrophosphate and 50 mmol/l NaF. After centrifugation at 14,000g for 10 min at 4C, the supernatant was immunoprecipitated using an anti-IRS-1 antibody and anti-IRS-2 antibody respectively for 2 h at 4C. Immunoprecipitates were washed three times with buffer 1 containing 1% NP-40, 1 mmol/l PMSF, 2 mg/l leupeptin, 20 mmol/l sodium pyrophosphate and 50 mmol/l NaF and then washed five times with buffer 2 (25 mmol/l MOPS, pH 7.3, 5 mmol/l MgCl2, 1 mmol/l EGTA, 1 mmol/l Na3VO4, 1 mmol/l PMSF, 2 mg/l leupeptin, 20 mmol/l sodium pyrophosphate and 50 mmol/l NaF). The immunoprecipitate was then resuspended in 50 l buffer 2 and heated to 37C in a heating block. To start the reaction, 10 l of 0.4 mg/l sonicated phosphatidylinositol and 20 l of ATP mix (buffer 2 containing 60 mol/l ATP and 925 kBq [32P]-ATP) were added. After 10 min at 37C, the reaction was stopped by the addition of 37% HCl/methanol (1:1, v/v). Lipids were extracted with chloroform and the organic phase was removed and applied to silica gel thin layer chromatography plates. The plates were developed in chloroform/methanol/water/ammonia (60/47/11. 3/2, v/v) and the PI3-product was identified by its comigration with an L-PI3-standard and quantified on a Packard Cyclone phosphoimager. Wortmannin (100 nmol/l) was used as a negative control. Intensity of various dots was expressed as n-fold stimulation of condition in the absence of insulin and TGRLs. NEFA determination NEFA levels in the cell culture media were determined in the absence of TGRLs and at the beginning and end of all TGRL incubations using a commercial kit from Wako (Neuss, Germany) with the Cobas MIRA system. Determination of intramyocellular lipid content Cells were grown in chamber slides, starved overnight and incubated in the presence or absence of TGRLs as described above. Cells were fixed in 3.7% formaline for 30 min and excess of formaline was removed with tap water. Subsequently, cells were permeabilised using 0.3% Tween 20 for 1 min followed by three washes with tap water. Oil red O staining was carried out according to Koopman [34]. Imaging was performed on a laser scanning microscope (Axiovert 200M/LSM 510, Carl Zeiss) with a Texas red excitation filter at 543 nm and an emission filter (LP 590, Carl Zeiss) at 590 nm. Pictures were preprocessed in MatLab (Mathworks) using an edge-finding algorithm to define the borders of lipid droplets [35]. The area of lipid droplets/ cytoplasmic area was then analysed with QuantityOne (Biorad). Statistical analysis Two-way ANOVA was performed for the factors insulin and TGRLs. Post-hoc comparisons were made using Fishers least significant difference method. For experiments in which results were expressed as n-fold stimulation over basal, t-tests were used: the one-sample t-test was used for comparisons to the condition in the absence of insulin and TGRLs, since this reference condition was set to 1 and therefore has the standard deviation 0. The unpaired t-test was used for the remaining comparisons. For t-tests, significance levels were corrected using the Bonferroni procedure. For calculations of IMCL content in Fig. 7a, logarithmic transformation of the data was performed to achieve approximately normal distribution. All values were expressed as meansSEM and statistical significance was accepted as p being less than 0.05. Reduction of glycogen synthesis, glycogen synthase activity and glucose transport by TGRLs Glycogen synthesis, a sensitive parameter of insulin sensitivity [36], was studied in the presence or absence of TGRLs in a number of pilot experiments. First, we performed a series of doseresponse experiments using TGRLs with a Sf higher than 200, defined in the Materials and methods section. We added this fraction to the incubation mixture for 3 h at triglyceride concentrations of 0, 114, 228 and 456 mol/l respectively. As shown in Fig. 1, there was a progressive reduction of insulin-induced glycogen synthesis with increasing doses of TGRLs, with 456 mol/l triglyceride reducing glycogen synthesis by 80%. Hence, a dose of 456 mol/l lipoprotein triglyceride was chosen for all following experiments. Next, we studied the effect of the TGRL fractions with an Sf higher than 200 on glycogen synthesis as a function of incubation time. In the absence of lipoproteins, the wellknown stimulatory effect of insulin on glycogen synthesis was seen (Fig. 2a, two bars on the left). Presence of TGRLs in the incubation mixture had no significant effects on basal glycogen synthesis but reduced the insulin effects over time resulting in an overall progressive reduction in the insulin-induced effects over basal (Fig. 2a). Fig. 1 Reduction of glycogen synthesis by increasing TGRL concentrations. L6 cells were incubated in the absence or presence of increasing concentrations of TGRLs with an Sf higher than 200 for 3 h, subsequently incubated without or with 100 nmol/l insulin for 3 h and analysed for glycogen content. TGRL concentrations corresponded to triglyceride concentrations of 0, 114, 228 and 456 mol/l incubation media. Bars represent the meansSEM for four experiments and significant differences are indicated as *P<0.05, ***P<0.001 (two-way ANOVA, Fishers least significant difference method). Ins, insulin Fig. 2 Reduction of glycogen synthesis, glycogen synthase activity and glucose uptake by TGRLs over time. L6 cells were incubated in the absence or presence of TGRLs with an Sf higher than 200 (456 mol/l triglyceride) for indicated time periods, subsequently incubated without or with insulin and analysed for glycogen content (a), glycogen synthase activity (b) and glucose uptake (c), as detailed under Materials and methods. Bars represent the means SEM for four to five independent experiments performed in duplicate and significant differences are indicated as *P<0.05, **P<0.01, ***P<0.001 (two-way ANOVA, Fishers least significant difference method). G6P, glucose 6-phosphate; DG, deoxyglucose; Ins, insulin In accordance with the TGRL-induced effects on glycogen synthesis, glycogen synthase activity showed virtually no changes in the basal state and a progressive reduction of insulin effects over time upon incubation with TGRLs with an Sf higher than 200 (Fig. 2b). For glucose uptake, a similar pattern of changes upon incubation with TGRLs with an Sf higher 200 was observed (Fig. 2c). In parallel experiments, we also studied the denser fraction of TGRLs, as defined in the Materials and methods section, containing the lipoproteins with an Sf of between 20 and 200 and compared its effects on glycogen synthesis, glycogen synthase activity and glucose uptake with the fraction with an Sf higher than 200. The TGRL with an Sf of between 20 and 200 showed comparable effects (data not shown). Because of the virtual identity of the two TGRL fractions with an Sf higher than 200 and an Sf of between 20 and 200 on glycogen synthesis, glycogen synthase activity and glucose uptake, for the subsequent experiments on the underlying mechanism(s), only one of the two lipoprotein fractions, namely the Sf higher than 200 fraction, was used. Effects of TGRLs on insulin signalling To answer the question of whether insulin signalling is impaired in a similar fashion to glycogen synthesis, glycogen synthase activity and glucose uptake by TGRLs, we studied several insulin signalling steps that are viewed as important for the regulation of glucose metabolism. First, we examined IRS-1 tyrosine phosphorylation, regarded as a major proximal step in the activation of downstream insulin signalling [37]. In the absence of lipoproteins, the well-known stimulation of IRS-1 phosphorylation by insulin was seen. The insulin-stimulated tyrosine phosphorylation of IRS-1 showed no significant changes upon TGRL incubation (Fig. 3a, b). Next, we examined the levels of the alpha p85 subunit of PI3K in phosphotyrosine immunoprecipitates. As shown in Fig. 4, insulin induced a Fig. 3 Effect of TGRLs on IRS-1 tyrosine phosphorylation. Cells were incubated in the absence or presence of TGRLs with an Sf higher than 200 (456 mol/l triglyceride) for indicated time periods, subsequently incubated without or with 100 nmol/l insulin for 5 min and solubilised in lysis buffer as described in Materials and methods. Lysates were subjected to immunoprecipitation (IP) with an antibody to IRS-1 followed by SDS-PAGE. Immunoblotting (IB) was performed with an antibody to IRS-1 (top panel) and phosphotyrosine (pY) (bottom panel) (a). The bar graph shows the quantification of tyrosine-phosphorylated IRS-1 (b). Bars represent the means SEM for five independent experiments performed in duplicates and significant differences are indicated as *P<0.05 (onesample t-test for comparisons to condition in the absence of insulin and TGRLs, unpaired t-test for remaining comparisons, Bonferroni correction). Ins, insulin marked increase in alpha p85 levels co-immunoprecipitated with tyrosine-phosphorylated proteins. TGRL incubation had no effect on alpha p85 levels in phosphotyrosine precipitates (Fig. 4a, bottom panel, Fig. 4b) and on the expression of alpha p85 (Fig. 4a, top panel). In accordance, both IRS-1associated PI3K activity (Fig. 5a) and IRS-2-associated PI3K activity (Fig. 5b), suggested to be important for insulin-induced translocation of GLUT-4 and thus for glucose transport [38], showed no changes with TGRL incubation. To determine whether more distal steps in the insulin signalling cascade were affected by TGRLs, we next assessed the phosphorylation of Akt at serine 473. In contrast to IRS-1 tyrosine phosphorylation and IRS-1- IRS-2-associated PI3K activity, a progressive decrease in insulininduced phosphorylation over time was observed, resulting in an overall decrease in the insulin effects over basal (Fig. 6a). Activated Akt has been shown to phosphorylate GSK-3 alpha Fig. 4 Effect of TGRLs on the expression of alpha p85 of PI3K and on alpha p85 levels in anti-phosphotyrosine immunoprecipitates. Cells were incubated in the absence or presence of TGRLs with an Sf higher than 200 (456 mol/l triglyceride) for indicated time periods, subsequently incubated without or with 100 nmol/l insulin for 5 min and solubilised in lysis buffer as described in Materials and methods. An aliquot of the lysates was subjected to SDSPAGE for direct blotting (DB) with an antibody to alpha p85 of PI3K (p85) (top panel), and the remaining lysate was used for immunoprecipitation with an antibody to phosphotyrosine (pY) followed by immunoblotting with an antibody to alpha p85 of PI3K (bottom panel) (a). The bar graph shows the quantification of alpha p85 associated with tyrosine-phosphorylated proteins (b). Bars represent the meansSEM for three independent experiments performed in duplicate and significant differences are indicated as *P<0.05 (one-sample t-test for comparisons to condition in the absence of insulin and TGRLs, unpaired t-test for remaining comparisons, Bonferroni correction). Ins, insulin Fig. 5 Effect of TGRLs on IRS-1-and IRS-2-associated PI3K activity. Cells were incubated in the absence or presence of TGRLs with an Sf higher than 200 (456 mol/l triglyceride) for indicated time periods, subsequently incubated without or with 100 nmol/l insulin for 10 min and solubilised in lysis buffer. After immunoprecipitation with an antibody to IRS-1 and IRS-2, IRS-1-associated PI3K activity (a) and IRS-2-associated PI3K activity (b) were determined as described under Materials and methods. Lipid extracts from assays were subjected to thin layer chromatography and phosphorimaging. Bars represent the meansSEM of five independent experiments performed in duplicate and significant differences are indicated as *P<0.05 (one-sample t-test for comparisons to condition in the absence of insulin and TGRLs, unpaired t-test for remaining comparisons, Bonferroni correction). Ins, insulin; PI3P, phosphatidylinositol 3-phosphate; WM, wortmannin (100 nmol/l) at serine 21 and GSK-3 beta at serine 9 [39]. Phosphorylated GSK-3 exhibits reduced inhibitory activity towards glycogen synthase, thus increasing glycogen synthesis [40]. TGRLs showed similar effects on both the alpha isoform (Fig. 6b) and the beta isoform (Fig. 6c) of GSK-3. In agreement with the changes noted for Akt phosphorylation, TGRL induced a reduction of insulin-stimulated phosphorylation of GSK-3 over time. For all blotting experiments, controls were performed to estimate the abundance of the respective signalling molecules. These control experiments indicated that the differences in the extent of phosphorylation of the signalling 3 Fig. 6 Effect of TGRLs on Akt- and GSK-3 phosphorylation. Cells were incubated in the absence or presence of TGRLs with an Sf higher than 200 (456 mol/l triglyceride) for indicated time periods, subsequently incubated without or with 100 nmol/l insulin for 5 min and solubilised in lysis buffer as described under Materials and methods. Lysates were subjected to SDS-PAGE and, subsequently, direct blotting (DB) was performed with an antibody to Akt and phospho-Akt (pAkt) (a) and an antibody to GSK-3 and phosphoGSK-3/ (pGSK3). In b phosphorylation of GSK-3 is shown and in c phosphorylation of GSK-3 is shown. The bar graphs show the quantification of the phosphorylated forms of Akt and GSK-3. Bars represent the meansSEM of five independent experiments performed in duplicate and significant differences are indicated as *P<0.05, **P<0.01 (one-sample t-test for comparisons to condition in the absence of insulin and TGRLs, unpaired t-test for remaining comparisons, Bonferroni correction). Ins, insulin Fig. 7 Effect of TGRLs and Intralipid on IMCL content and of Intralipid on glycogen synthesis in L6 myotubes. For analysis of the IMCL content, L6 cells were incubated in the absence or presence of TGRLs with an Sf higher than 200 (456 mol/l triglyceride) and Intralipid (456 mol/l triglyceride) for various lengths of time, i.e. 10 min, 1 h, 3 h and 16 h. After fixation of cells in formaline and permeabilisation with Tween 20, intracellular lipid droplets were stained with oil red O and detected by laser scan microscopy. Area of lipid droplets was calculated as detailed in the Materials and methods section. Analysis of glycogen synthesis was described in Fig. 2a. Area of lipid droplets per cytoplasmic area upon TGRL and Intralipid incubations (a). Effect of Intralipid incubation on glycogen synthesis (b). Bars represent the meansSEM for five independent experiments performed in duplicates and significant differences are indicated as *P<0.05, **P<0.01, ***P<0.001 (twoway ANOVA, Fishers least significant difference method; in a logarithmic transformation of the data was performed). IL, Intralipid 3 Fig. 8 Effect of RAP and orlistat on TGRL-induced alterations. L6 cells were incubated in the absence or presence of TGRLs with an Sf higher than 200 (456 mol/l triglyceride) for 3 h, subsequently incubated without or with 100 nmol/l insulin for 3 h and analysed for glycogen content (a). TGRL incubation was performed in the absence or presence of RAP and orlistat. To study the effect of RAP on TGRL-induced alterations in the phosphorylation of Akt and GSK-3 (b), L6 cells were incubated in the absence or presence of RAP and TGRLs with an Sf higher than 200 (456 mol/l triglyceride) for 3 h, and subsequently incubated without or with 100 nmol/l insulin for 5 min. Direct blotting was performed with antibodies to phosphoAkt (pAkt) and phospho-GSK-3 (pGSK-3). The bar graph shows the quantification of Akt- and GSK-3 phosphorylation (c). Bars represent the meansSEM for three independent experiments performed in duplicate and significant differences are indicated as *P<0.05, **P<0.01, ***P<0.001. For each data set of inhibitors (no inhibitor, RAP, orlistat) in (a), two-way ANOVA with Fishers least significant difference method was used. For quantification of Akt- and GSK-3 phosphorylation in (c), one sample t-test was used for comparisons to condition in the absence of insulin and TGRLs and the unpaired t-test for all remaining comparisons. Ins, insulin; RAP, receptor-associated protein such that after 16 h IMCL content represented about 20% of the whole cytoplasmic area. Specificity of the observed effects To answer the question of whether the observed effects are lipoprotein specific or whether they are also induced by triglyceride not assembled in native lipoprotein particles, we replaced TGRLs by Intralipid. Intralipid was subjected to gel filtration as described in the Materials and methods section, and the fraction containing the triglyceride micelles was selected and added to incubation mixtures at a triglyceride concentration of 456 mol/l, as used for lipoproteins. In contrast to what was observed with TGRLs (Fig. 2a), no effect on insulin-stimulated glycogen synthesis was seen after 10 min, 1 h and 3 h. Only after 16 h of incubation with Intralipid micelles was insulin-stimulated glycogen content decreased by about 30% (Fig. 7b). We next studied whether the effect of Intralipid on glycogen synthesis is related to IMCL content. There was little, if any, IMCL accumulation over 3 h, but after 16 h a pronounced rise in IMCL content, paralleling closely the reduction in glycogen synthesis, was apparent (Fig. 7a, white bars). Moreover, there is evidence for extrahepatic whole-particle uptake of intact chylomicrons and their remnants. In a series of studies with rabbits and rats, it has been shown that chylomicrons are able to penetrate arteries as efficiently as smaller macromolecules, including LDL, HDL and albumin [20, 21, 23, 49, 50]. Whole-VLDL-particle uptake has also been demonstrated in mice, whereby whole-particle lipoprotein uptake in muscle increases by transgenic expression of catalytically inactive LPL in the presence of active LPL [51, 52]. A receptor-independent process [53], further characterised as endothelial transcytosis [54], has been proposed to represent the molecular mechanism of chylomicron uptake. For humans, there is also evidence for whole-lipoprotein-particle uptake [24, 19]; based on results with arteriovenous concentration differences of lipoprotein particle constituents, Karpe et al. hypothesised that skeletal muscle and adipose tissue are likely to be of importance for removal of chylomicron remnant particles [19]. Therefore, we believe that our observations with TGRLs are of biological relevance. Finally, we would like to comment on how our data fit the published results on the effect of NEFAs on insulin sensitivity. A large number of reports on NEFAs including various experimental settings ranging from cell culture to studies in humans showed that NEFAs cause insulin resistance. We would like to emphasise that our in vitro data do not contradict the large body of evidence for the effect of NEFAs on insulin sensitivity but indicate that, in addition to NEFAs, important biological effects regarding insulin resistance can also be exerted by intact TGRL particles. The notion that in our experimental system, the observed effects are solely due to intact TGRL particles, not to NEFAs present in the incubation media, is supported by our observation that NEFA levels in the media did not increase with lipoprotein incubations and that NEFA levels in the TGRLcontaining media were equal to the NEFA levels in the incubation media with no lipoproteins present. Consistent with NEFA-independent TGRL-particle-induced effects, our experiment with orlistat demonstrated that co-incubation of TGRLs with this potent lipase inhibitor did not alter TGRL-induced impairment of glycogen synthesis, whereas co-incubation with RAP, inhibiting TGRL interaction with members of the LDL receptor family, reversed TGRLinduced effects. In summary, we show for the first time that TGRLs, a lipoprotein fraction accumulating in the fasting and even more severely in the postprandial phase of insulin-resistant and diabetic subjects, are capable of causing insulin resistance as evidenced by an impairment of insulin-induced glycogen synthesis, glycogen synthase activity, glucose uptake and several insulin signalling steps. Our results complement those on lipoprotein-induced beta cell dysfunction [18] to suggest that changes in plasma lipoproteins observed in type 2 diabetes may not only be a consequence of this disease but also a cause of it. Acknowledgements This study was supported by a grant from the Austrian Science Fund (FWF): P15951-B07 to M. T. Pedrini. We are grateful to Karin Salzmann for excellent technical assistance and to Dr Georg Kemmler for assistance with statistical analyses.


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M. T. Pedrini, M. Kranebitter, A. Niederwanger, S. Kaser, J. Engl. Human triglyceride-rich lipoproteins impair glucose metabolism and insulin signalling in L6 skeletal muscle cells independently of non-esterified fatty acid levels, Diabetologia, 2005, 756-766, DOI: 10.1007/s00125-005-1684-8