Lysophosphatidylcholine as an effector of fatty acid-induced insulin resistance.

The mechanism of FFA-induced insulin resistance is not fully understood. We have searched for effector molecules(s) in FFA-induced insulin resistance. Palmitic acid (PA) but not oleic acid (OA) induced insulin resistance in L6 myotubes through C-Jun N-terminal kinase (JNK) and insulin receptor substrate 1 (IRS-1) Ser307 phosphorylation. Inhibitors of ceramide synthesis did not block insulin resistance by PA. However, inhibition of the conversion of PA to lysophosphatidylcholine (LPC) by calcium-independent phospholipase A₂ (iPLA₂) inhibitors, such as bromoenol lactone (BEL) or palmitoyl trifluoromethyl ketone (PACOCF₃), prevented insulin resistance by PA. iPLA₂ inhibitors or iPLA₂ small interfering RNA (siRNA) attenuated JNK or IRS-1 Ser307 phosphorylation by PA. PA treatment increased LPC content, which was reversed by iPLA₂ inhibitors or iPLA₂ siRNA. The intracellular DAG level was increased by iPLA₂ inhibitors, despite ameliorated insulin resistance. Pertussis toxin (PTX), which inhibits LPC action through the G-protein coupled receptor (GPCR)/Gα(i), reversed insulin resistance by PA. BEL administration ameliorated insulin resistance and diabetes in db/db mice. JNK and IRS-1Ser307 phosphorylation in the liver and muscle of db/db mice was attenuated by BEL. LPC content was increased in the liver and muscle of db/db mice, which was suppressed by BEL. These findings implicate LPC as an important lipid intermediate that links saturated fatty acids to insulin resistance.


DAG measurement
DAG was extracted from the cell pellet with chloroform/methanol (2:1, v/v) containing 0.01% butylated hydroxytoluene. A known amount of 1,2-dipentadecanoin was added as an internal standard. After vortexing and centrifugation, the lower phase was collected. Samples were evaporated and dissolved in hexane/ methylene chloride/methyl tert -butyl ether for loading onto a diol-bonded solid phase extraction column (Waters, Inc., Milford, MA) under vacuum. DAG was eluted as described previously ( 12 ). The extracted lipids were dried under N 2 and redissolved in methanol. Then, the DAG content among extracted lipids was measured by LC-MS/MS using a bench-top tandem mass spectrometer (API 4000 Q-trap; Applied Biosystems, Framingham, MA) interfaced with an atmospheric pressure chemical ionization source and Agilent series 1200 micro-pump equipped with an autosampler as described ( 13 ). Five selected DAG species (di-16:0, di-18:1, 16:0-18:1, 18:0-18:2, and 18:0-20:4) were separated by HPLC with a phenyl column and ionized in the positive atmospheric pressure chemical ionization mode.
[M+H-18]/product ions from corresponding DAG were monitored for multiple reaction monitoring quantitation of DAG. The mobile phase was 98% methanol with a fl ow rate of 0.25 ml/min, and 10 m l sample was injected.

TG measurement
TG was measured as previously described ( 9 ). In short, cells were fi xed with 10% formaldehyde for 1 h. After staining with 3 m g/ml Oil Red O solution for 15 min, dye was extracted by isopropanol and A 540 measured.
Despite strong epidemiological and in vivo data suggesting a relationship between FFA and type 2 diabetes, a detailed molecular and cellular mechanism underlying insulin resistance has not been clearly elucidated. Recent studies have dissected the metabolic pathway to unravel the intracellular mechanism of insulin resistance by FFA.
The results of such studies have implicated diacylglycerol (DAG), ceramide, triglyceride (TG), or other metabolites arising from incomplete b -oxidation of fatty acids as the fi nal effector molecules inducing insulin resistance or lipotoxicity (5)(6)(7)(8). However, it remains controversial which metabolites produced from FFA are directly responsible for the insulin resistance or b -cell failure by FFA.
We have studied the mechanism of lipoapoptosis using various pharmacological inhibitors ( 9 ). In this investigation, we employed the same pharmacological strategy to determine the mechanism of insulin resistance by FFA, and we found evidence favoring the role of LPC produced from FFA in insulin resistance in vitro and in vivo.

Cell culture
L6 myoblasts (kindly provided by Dr. Lee Wan, College of Medicine, Dongguk University, Kyungju, Korea) were grown in a -MEM (Gibco-BRL, Grand Island, NY)-10% FBS. For differentiation, 4 × 10 4 /ml L6 myoblasts were plated in 60 mm dishes and cultured in a -MEM 10% FBS for 24 h. The medium was replaced with a -MEM 2% FBS to induce fusion of myoblasts to myotubes ( 10 ). Formation of myotubes was monitored by phase contrast microscopy. Differentiation medium was replenished every 24-48 h. Mature myotubes formed within 6-8 days after seeding.

FFA treatment
The palmitic acid (PA) solution was made according to a previously published protocol with modifi cations ( 11 ). Briefl y, a PA stock solution (50 mM) was prepared by dissolving PA in 70% ethanol and heating at 50°C. The working PA solution was made by diluting stock solution in a -MEM 2% FFA-free BSA 2% FBS, then fi ltered before use.
We next studied the signal transduction related to the PA-induced insulin resistance. We investigated whether PA affects JNK phosphorylation, which is critically involved in obesity-induced insulin resistance ( 19,20 ). Treatment with 800 m M PA for 6-12 h induced a substantial JNK phosphorylation in L6 myotubes as evidenced by increased phospho-p54 and phospho-p46 JNK bands, suggesting a role for JNK activation in PA-induced insulin resistance ( Fig. 1C ). We also examined whether PA induces Ser307 phosphorylation of IRS-1 since previous studies have shown that IRS-1 Ser307 phosphorylation by activated JNK attenuates tyrosine phosphorylation of IRS-1 by insulin ( 19,21 ). Indeed, treatment with 800 m M PA induced a signifi cant phosphorylation of IRS-1 Ser307 ( Fig. 1C ). Furthermore, SP600125, a JNK inhibitor, remarkably attenuated IRS-1 Ser307 phosphorylation by PA ( Fig. 1D ), suggesting that JNK phosphorylation by PA leads to IRS-1 Ser307 phosphorylation and insulin resistance. The effect of PA on the phosphorylation of JNK and IRS-1 Ser307 was observed in a dose-dependent manner between 100 and 800 m M (supplementary Fig. I).
In contrast to PA, OA did not induce JNK or IRS-1 Ser307 phosphorylation ( Fig. 1E ), consistent with the absence of effects on the insulin-induced glucose uptake or Akt phosphorylation ( Fig. 1A, B ). Notably, OA induced a weak JNK activation 15 min after treatment, which might be through a mechanism distinct from PA-induced JNK activation ( Fig. 1E ).

LPC as a mediator of PA-induced insulin resistance
Because PA is converted to diverse lipid intermediates, such as ceramides, intracellularly and ceramides have been reported to activate protein kinase C (PKC) and inhibit insulin signaling ( 4, 5 ), we asked whether intracellular conversion of PA to ceramide is responsible for the insulin resistance induced by PA. We fi rst studied the effect of Fumonisin B1, which blocks ceramide synthesis by inhibiting sphingosine N -acyltransferase (ceramide synthase) ( 22,23 ), on insulin signaling and glucose uptake. Fumonisin B1 did not reverse impaired insulin-induced Akt Ser473 or IRS-1 Tyr612 phosphorylation by PA in L6 myotubes ( Fig. 2A ). Fumonisin B1 also did not affect reduced insulin-induced 2-deoxyglucose uptake by PA Ser473, Thr308 (Cell Signaling, Beverly, MA), anti-phospho-IRS-1 Ser307 (Upstate Biotechnology, Lake Placid, NY), anti-phospho-IRS-1 Tyr612 (Biosource International, Camarillo, CA), and anti-JNK antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

Glucose uptake
Uptake of 2-deoxyglucose was determined as previously described ( 16 ). Briefl y, after starvation of L6 myotubes in serum-free a -MEM for 2 h, cells were incubated in a -MEM containing 100 nM insulin at 37°C for 30 min. After washing in HEPES buffer [20 mM Na-HEPES (pH 7.4) 140 mM NaCl, 5 mM KCl, 2.5 mM MgSO 4 , and 1 mM CaCl 2 ], the cells were incubated in HEPES buffer containing 10 m M 2-deoxyglucose and 0.5 m Ci/ml 2-deoxy-D-[ 3 H]glucose (Amersham, Piscataway, NJ) for 10 min. The reaction was stopped by washing with ice-cold 0.9% NaCl. The cells were solubilized in 1.5 ml 0.05 N NaOH for 30 min to measure [ 3 H]glucose uptake using a scintillation counter. Nonspecifi c glucose uptake was measured in the presence of 10 m M cytochalasin B and subtracted from the total uptake to calculate the specifi c uptake.

In vivo effect of iPLA 2 inhibitors
BEL was dissolved in a solution of 5% ethanol, 40% polyethylene glycerol 400, 15% cremophor EL, and 40% PBS, and then 200 m g/kg of BEL was injected intraperitoneally to C57BL/Ks db/db mice three times a week for four weeks beginning at fi ve weeks of age. Control db/db mice were injected with vehicle only. An intraperitoneal glucose tolerance test (IPGTT) was carried out after overnight fasting by an intraperitoneal injection of 1 gm/kg of glucose ( 17 ). Blood glucose concentrations were determined with an Accu-Chek glucometer (Roche, Mannheim, Germany) before (0 min) and 15, 30, 60, and 120 min after the injection of glucose. The homeostasis model assessment of insulin resistance (HOMA-IR) index was calculated as described ( 18 ). Serum insulin concentrations were determined using a commercial RIA kit for rat/mouse insulin measurement (Linco, St. Charles, MO). An insulin tolerance test (ITT) was conducted by intraperitoneal injection of 1 U/kg regular insulin to fasted mice as previously described ( 15 ). To study insulin signaling in vivo, 5 U/kg regular insulin (Novo Nordisk, Bagsvaerd, Denmark) was injected into the tail vein. At 5 Five min after insulin infusion, the liver and muscle tissue were removed and frozen in liquid nitrogen until use. All animal experiments were conducted in accordance with the Public Health Service Policy in Humane Care and Use of Laboratory Animals, and were approved by the Institutional Review Board of Samsung Medical Center Animal Facility.

␤ -cell mass
The pancreatic b -cell mass was measured by point counting morphometry after insulin immunohistochemistry of pancreatic sections was carried out as previously described ( 15 ).

Statistical analysis
All values are expressed as the means ± SE from two to four independent experiments performed in triplicate to ensure reproducibility. Two-tailed Student's t -test was employed to compare the values between two groups. P values less than 0.05 were considered to represent statistically signifi cant differences.

Insulin resistance by FFA
We fi rst studied the effect of PA, the most abundant saturated FFA, on insulin signaling in differentiated L6 Next, we studied which intracellular lipid intermediates other than ceramide are involved in PA-induced insulin resistance. We have reported that DAG generated from PA could be further converted to phosphatidylcholine (PC), then to LPC, a well-known activator of JNK ( 26 ), by iPLA 2 ( 9 ). Because LPC is able to induce insulin resistance when administered exogenously ( 27 ), we investigated whether conversion of PA to LPC plays a role in the PA-induced JNK activation and insulin resistance. When L6 myotubes were pretreated with PACOCF 3 or BEL that could inhibit conversion of PA to LPC by inhibiting iPLA 2 ( 9 ), JNK phosphorylation by PA was substantially decreased, suggesting that endogenous LPC produced from PA induces JNK activation ( Fig. 3A ). IRS-1 Ser307 phosphorylation by PA was also reduced by PACOCF 3 , which is consistent with the decreased JNK phosphorylation by PACOCF 3 or BEL ( Fig. 3A ). Additionally, PACOCF 3 or BEL reversed the decreased insulin-induced Akt Ser473 and IRS-1 Tyr612 phosphorylation by 800 m M PA ( Fig. 3B ). Consistent with the decreased JNK phosphorylation and increased insulin-( P > 0.05) ( Fig. 2B ). Because these results are in contrast to previous studies reporting an important role of de novo ceramide synthesis in insulin resistance, we next used Myriocin, which inhibits the fi rst step of de novo ceramide synthesis by specifi cally inhibiting serine palmitoyltransferase (SPT) ( 24,25 ). Myriocin did not reverse impaired insulininduced Akt Ser473 or IRS-1 Tyr612 phosphorylation by PA in L6 myotubes ( Fig. 2A ), suggesting that conversion of PA to ceramide is not responsible for insulin resistance induced by PA. Myriocin also did not reverse the reduced insulin-induced glucose uptake by PA ( P > 0.05) ( Fig. 2B ). Further, Fumonisin and Myriocin did not prevent PAinduced JNK activation ( Fig. 2C ). LC-MS/MS showed that the total ceramide content, which was markedly increased after PA treatment ( P < 0.005), was signifi cantly reduced by pretreatment with Fumonisin B1 or Myriocin ( P < 0.005 for both comparisons) ( Fig. 2D ), indicating that ceramide synthesis was suppressed by chemical inhibitors as expected but that insulin resistance was not ameliorated despite successful suppression of ceramide content. resistance in L6 myotubes. Transfection of iPLA 2 b or iPLA 2 g siRNA, which attenuated JNK activation by PA ( Fig.  3D ), also signifi cantly suppressed the increase of LPC content after PA treatment ( P < 0.005) ( Fig. 4C ).
We also measured DAG that could be produced intracellularly from PA and has been implicated as an effector molecule in FFA-induced insulin resistance ( 7 ). Total intracellular DAG content in L6 myotubes was signifi cantly increased by treatment with 600 m M PA ( P < 0.005) ( Fig.  4D ). The addition of PACOCF 3 further increased the total DAG content in L6 myotubes ( P < 0.005) ( Fig. 4D ) despite improved insulin sensitivity ( Fig. 3A-C ). These results are attributable to the inhibition of iPLA 2 by PACOCF 3 at the distal step of DAG synthesis and suggest that amelioration of insulin resistance by PACOCF 3 is probably not related to DAG. The addition of BEL also increased the total intracellular DAG level in L6 myotubes; however, the increase was not signifi cant ( P > 0.05) ( Fig. 4D ), probably because BEL inhibits phosphatidic acid phosphatase (PAP) in addition to iPLA 2 ( 28 ). When individual molecular DAG species were analyzed, the contents of di-16:0 and 16:0-18:1 were most conspicuously increased after PA treatment and induced Akt phosphorylation, PACOCF 3 reversed the reduced insulin-induced 2-deoxyglucose uptake by PA ( P < 0.05) ( Fig. 3C ). Because the pharmacologic inhibitors we used could have off-target effects, we next employed genetic approaches. Transfection of iPLA 2 b or iPLA 2 g siRNA or their combination decreased JNK phosphorylation by PA, suggesting potential involvement of multiple types of iPLA 2 in the conversion of PA to LPC in L6 myotubes ( Fig. 3D ).
Because these results suggested a role for LPC in PAinduced insulin resistance, we examined whether LPC content is increased by PA treatment of L6 myotubes. Indeed, treatment of L6 myotubes with 600-1,000 m M PA signifi cantly increased LPC content ( P < 0.01-0.05), while OA did not affect intracellular LPC content ( P > 0.05) ( Fig. 4A ), which supports the idea that LPC is one of the important lipid metabolites of PA leading to insulin resistance. Treatment with PACOCF 3 or BEL, which ameliorated JNK activation by PA ( Fig. 3A ), markedly attenuated the increase in LPC content by PA ( P < 0.05 and P < 0.05, respectively) ( Fig. 4B ), supporting the fi nding that LPC converted from PA induces JNK activation and insulin Because PA could be converted to LPC in L6 myotubes, we studied whether LPC impairs insulin signaling directly. Exogenous LPC attenuated the phosphorylation of IRS-1 Tyr612 and Akt Ser473 in response to insulin ( Fig. 5A ). LPC also induced IRS-1 Ser307 and JNK phosphorylation ( Fig. 5B ), indicating that exogenous LPC inhibits insulin signaling through JNK phosphorylation. LPC-induced JNK activation was not affected by transfection of iPLA 2 b or iPLA 2 g siRNA, as expected ( Fig. 5C ). Exogenous LPC also decreased insulin-induced 2-deoxyglucose uptake by L6 myotubes ( P < 0.05) ( Fig. 5D ), suggesting that LPC induces insulin resistance through JNK activation. We also tested the effect of blockade of G-protein coupled receptor (GPCR) by pertussis toxin (PTX) on PA-induced insulin resistance because PTX has been reported to inhibit most LPC signaling through GPCR/G a i ( 30,31 ). PTX reversed the decreased insulin-induced IRS-1 Tyr612 and Akt phosphorylation by LPC or PA ( Fig. 5E, F ) and ameliorated JNK activation by PA ( Fig. 5G ), suggesting that LPC further enhanced by combined treatment with PACOCF 3 (supplementary Fig. II). These results are consistent with a previous study showing predominant production of di-16:0 phosphatidic acid after PA treatment ( 29 ) and again suggest attenuation of insulin resistance independent of DAG content. Changes in other DAG species were much less significant or insignifi cant compared with those of di-16:0 and 16:0-18:1 DAG (data not shown). Because changes in lipid droplet size due to increased TG content might lead to the sequestration of DAG or other bioactive lipid metabolites and dissociation of total DAG content versus bioactive DAG involved in lipid signaling, we measured TG content after treatment of L6 myotubes with FFA. TG content was not signifi cantly affected by treatment with 600 m M PA that increased DAG concentration ( Fig. 4D and supplementary   control db/db mice became diabetic during the same observation period ( Fig. 6A ). The body weights were not signifi cantly different between the two groups ( P > 0.05), suggesting that BEL lowers blood glucose without affecting appetite and does not have signifi cant systemic toxicity ( Fig. 6B ). Indeed, food intake was not signifi cantly different between the db/db mice treated with BEL and control db/db mice (supplementary Fig. V). IPGTT showed that BEL treatment for four weeks dramatically improved glucose tolerance in db/db mice ( P < 0.005-0.05) ( Fig. 6C ). The HOMA-IR index was significantly decreased by BEL treatment for four weeks in db/db mice ( P < 0.005) ( Fig. 6D ), supporting the fi nding that BEL treatment improves insulin resistance. The ITT also showed that K ITT , another index of insulin resistance ( 33 ), was significantly improved by the administration of BEL to db/db mice ( 2 0.37 ± 0.15%/min in vehicle-treated db/db mice versus 2.12 ± 0.62%/min in BEL-treated db/db mice; n = 8 each; P < 0.005). In contrast, BEL administration did not affect the blood glucose level, body weight, or HOMA-IR in C57BL/6 mice ( P > 0.05 for all comparisons) ( Fig. 6A-D ).
To elucidate the mechanism underlying the improvement in the glycemic profi le by BEL, we examined whether induces insulin resistance through GPCR/G a i . Because LPC can be incorporated into DAG or other lipid metabolites through autotoxin or other pathways ( 32 ), we measured the content of lipids that can affect insulin signaling. DAG, ceramide, and TG contents were not signifi cantly changed after LPC treatment ( P > 0.1 for all comparisons) (supplementary Fig. IV), indicating that the effect of exogenous LPC on insulin signaling is not related to its conversion to other lipid intermediates potentially involved in the modulation of insulin signaling.

In vivo effect of LPC modulation on diabetes
Because these in vitro results suggested an important role for LPC generated from PA in insulin resistance, we determined whether BEL, an inhibitor of endogenous production of LPC from FFA in vitro, could attenuate insulin resistance and diabetes in vivo. When 200 m g/kg of BEL was administered to db/db mice, a signifi cant decrease in the nonfasting blood glucose level was noted after one week of treatment compared with control db/db mice treated with vehicle alone ( P < 0.005-0.01). Treated mice remained normoglycemic throughout the four-week treatment period, while all of the weeks also attenuated the increased IRS-1 Ser307 phosphorylation in the liver and muscle of db/db mice ( Fig.  7B ). Probably because of the decreased JNK activation and IRS-1 Ser307 phosphorylation, BEL treatment for four weeks restored the diminished IRS-1 Tyr612 and Akt Thr308 phosphorylation after insulin injection in the liver and muscle of db/db mice ( Fig. 7C ).
Concomitant with the ameliorated insulin resistance, the serum insulin level was signifi cantly reduced ( P < 0.05) ( Fig. 7D ) and the pancreatic b -cell mass was signifi cantly increased ( P < 0.01) after BEL administration to db/db mice for four weeks ( Fig. 7E ).

DISCUSSION
We observed that PA induced insulin resistance in L6 myotubes through JNK activation between 6 and 12 h unlike OA, which is consistent with previous studies using BEL could affect LPC content in the insulin target tissues in vivo. Consistent with our hypothesis that LPC is an important mediator of insulin resistance, the LPC content was increased in the liver and muscle of db/db mice compared with C57BL/6 mice ( P < 0.005 and P < 0.01, respectively) ( Fig. 7A ). Furthermore, BEL treatment for four weeks signifi cantly lowered the increased LPC content in both tissues ( P < 0.05 for both comparisons) ( Fig. 7A ), suggesting that BEL improves blood glucose profi les by normalizing LPC content. Next, we studied whether BEL treatment in vivo could affect JNK activation since LPC could be an inducer of JNK activation after FFA treatment ( 26 ). JNK was activated in the liver and muscle of the db/db mice as previously reported, and the increased JNK activation in db/db mice was signifi cantly ameliorated by BEL treatment for four weeks ( Fig. 7B ), consistent with our hypothesis that LPC generated from PA induces insulin resistance through JNK activation. BEL treatment for four After treatment with 100 nM insulin for 15 min, cell lysates were subjected to Western blotting to determine phosphorylation of IRS-1 Tyr612 or Akt Ser473 using specifi c antibodies. G: L6 myotubes were pretreated with PTX for 1 h, then incubated with 800 m M PA for 14 h. Cell lysates were subjected to Western blotting using an antibody specifi c for phospho-JNK. All results shown are representative of two to four independent experiments. * P < 0.05. lin resistance. DAG could be another candidate for the effector in FFA-induced insulin resistance because FFA could be converted to DAG intracellularly through phosphatidic acid ( 7 ), and DAG is a well-known activator of PKC that could mediate insulin resistance ( 40 ). However, we observed that iPLA 2 inhibitors, such as BEL or PA-COCF 3 , signifi cantly inhibited JNK activation and insulin resistance by PA, while increasing intracellular DAG content. Because DAG could be converted to PC and then to LPC, a well-known activator of JNK, by PLA 2 ( 26,41 ), our results, which showed a substantial inhibition of FFAinduced insulin resistance by BEL or PACOCF 3 , suggest the involvement of iPLA 2 and LPC rather than DAG upstream of PLA 2 in FFA-induced JNK activation and insulin resistance. While BEL has been reported to inhibit PAP in addition to iPLA 2 ( 28 ), our fi nding that PACOCF 3 , another iPLA 2 -specifi c inhibitor structurally unrelated to BEL ( 28 ), also strongly inhibited insulin resistance by PA, suggests that iPLA 2 and LPC play a role in FFA-induced JNK activation and insulin resistance. The increase of DAG by BEL was less than that by PACOCF 3 , likely because of concomitant inhibition of PAP by BEL ( 28 ). The biochemical mechanism underlying the preferential increase of LPC by PA but not by OA is not clearly understood. OA might be more prone to be directed to other pathways, such as TG synthesis or b -oxidation, as was shown in the current study (supplementary Fig. III) and previous reports ( 9,42 ).
hepatocytes ( 20,34 ). OA induced a weak activation of JNK at an early time point, which could be through a mechanism, such as FFA receptor binding ( 35 ), distinct from PAinduced JNK activation. In fact, PA also induced a weak JNK activation at the same early time point ( Fig. 1C ), which could be due to binding of PA and OA to the same FFA receptors and might be mechanistically different from JNK activation by PA but not by OA at later time points due to the conversion of PA to other lipid metabolites such as LPC.
In our search for effector molecule(s) in insulin resistance by FFA, we found evidence suggesting that LPC generated from saturated FFA via phosphatidic acid, DAG, and PC, is an effector for JNK activation, IRS-1 Ser307 phosphorylation, and insulin resistance. TG, DAG, or ceramide could be candidates for possible effector molecules in FFA-induced insulin resistance. Evidence supporting the role of TG in obesity-induced insulin resistance is mostly from the association between intracellular TG content and insulin resistance (36)(37)(38). However, biochemical data substantiating the etiological role of TG in obesity-or FFA-induced insulin resistance are lacking. Furthermore, unsaturated FFA led to increased intracellular TG content but did not signifi cantly activate JNK, while saturated FFA did not signifi cantly increase intracellular TG content but was a strong inducer of JNK activation and insulin resistance ( 9,20,39 ). These results suggest that TG is not a direct effector molecule causing JNK activation and insu- Fig. 6. Effect of BEL on diabetes of db/db mice. A: BEL (200 m g/kg) or vehicle (VEH) was administrated intraperitoneally to 5-week-old db/db and C57BL/6 control mice three times a week for four weeks. Nonfasting blood glucose levels were determined using a glucometer. B: Body weight was monitored throughout the treatment period. C: An IPGTT was conducted by injecting 1 mg/kg of glucose intraperitoneally to db/db or C57BL/6 control mice treated with 200 m g/kg BEL or vehicle for four weeks, and blood glucose levels were measured at the indicated time points. D: HOMA-IR index was calculated as described in Materials and Methods. Results pooled from three independent experiments are presented. * P < 0.05; ** P < 0.01; *** P < 0.005. ᭹ , db/db -VEH (A-C, n = 12); ᭺ , db/db -BEL (A-C, n = 12); , C57BL/6-VEH (A-C, n = 8); ᮀ , C57BL/ 6-BEL (A-C, n = 8).
toward PA, and other saturated fatty acids, such as stearic acid or myristic acid, are poor substrates for SPT ( 44 ). Therefore, de novo synthesis of ceramide may play a role in insulin resistance when PA is involved but not when Ceramide is another strong candidate for the effector molecule in obesity-or FFA-induced insulin resistance ( 4,5,43 ). However SPT, the enzyme catalyzing the initial step in de novo synthesis of ceramide from PA, has a specifi city Fig. 7. LPC content in vivo. A: LPC content in the liver and muscle tissues from db/db or C57BL/6 control mice treated with 200 m g/kg of BEL or vehicle (VEH) for four weeks was measured as in Fig. 4A . B: Tissue lysates of the liver and muscle from db/db and C57BL/6 control mice treated with 200 m g/kg of BEL or vehicle for four weeks were subjected to Western blotting to determine phosphorylation of JNK and IRS-1 Ser307. C: An amount of 5 U/kg of regular insulin was injected into the tail vein of the db/db or control C57BL/6 mice treated with 200 m g/kg of BEL or vehicle for four weeks. After 5 min, tissue lysates of the liver and muscle from db/db and C57BL/6 control mice were prepared, and the tissue lysates were subjected to Western blotting to assess phosphorylation of IRS-1 Tyr612 and Akt Thr308. D: Serum was obtained from fasted db/db mice or control C57BL/6 mice treated with 200 m g/kg of BEL or vehicle for four weeks, and insulin levels were measured by RIA. E: After insulin immunohistochemistry of pancreatic sections from db/ db mice treated with 200 m g/kg of BEL or vehicle for four weeks was carried out, point counting morphometry was conducted to measure the relative b -cell volume. Representative values (means ± SE) from three independent experiments are presented in A. Western blots are representative of three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.005. While we focused on the role of LPC as an effector of insulin resistance in the current study, we previously reported that LPC could be an effector in lipoapoptosis ( 9 ). Reports by other investigators have shown that PLA 2 is involved in several cellular injury models and that iPLA 2 inhibitors could decrease tissue damage by various stressors ( 53,54 ). The increase in b -cell mass after BEL administration to db/db mice observed in our study could be due to decreased b -cell stress following reduced insulin resistance or direct protective effect of iPLA 2 inhibitors against lipid injury or lipoapoptosis of b -cells. Hence, LPC could be involved in the pathogenesis of type 2 diabetes by inducing insulin resistance in insulin target tissue and by imposing islet cell injury. Altogether, our results suggest that LPC, in addition to the previously reported metabolites, such as ceramide or DAG, could be an effector molecule in FFAinduced insulin resistance via JNK activation, and that iPLA 2 inhibitors could be employed as potential therapeutic agents against diabetes associated with obesity or lipid injury.
other saturated FFA, such as stearic acid, is involved, while stearic acid or myristic acid well induces JNK activation and insulin resistance ( 20,45 ). Previous studies have also reported that the production of TNF a and IL-6, which are implicated in obesity-or FFA-induced insulin resistance, was not affected by ceramide synthesis inhibitors ( 46,47 ), suggesting that de novo synthesis of ceramide may not play an important role in insulin resistance induced by obesity or FFA.
Our data suggesting the role of endogenous LPC in FFA-induced insulin resistance is supported by the significantly elevated intracellular content of LPC after PA treatment of L6 myotubes that did not occur in the presence of iPLA 2 inhibitors, such as PACOCF 3 or BEL. The mechanism of the increase in LPC content after PA treatment might be related to the induction of genes involved in the conversion of PA to LPC. However, the main cause of the increase of LPC after PA treatment could be the increase in the amount of substrate. In fact, we observed no significant change of iPLA 2 b or iPLA 2 g expression after PA treatment of L6 myotubes (W. Quan et al., unpublished data). Induction of insulin resistance by exogenous LPC, which is consistent with the fi ndings of a previous study ( 27 ), also substantiates the role of endogenous LPC in FFA-induced insulin resistance. Because potential receptors for LPC, such as GPCR, may recognize extracellular ligands, LPC might be released to the extracellular space and bind to GPCR ( 48 ). Or LPC might enter the receptor-binding site in a lateral fashion between transmembrane regions of the receptor without leaving the membrane ( 49 ). However, it is not clear which among several types of PLA 2 is responsible for the production of endogenous LPC after FFA treatment of L6 myotubes. While we observed a signifi cant decrease in FFA-induced insulin resistance by iPLA 2 b or iPLA 2 g siRNA, our data alone cannot eliminate the potential role of other types of PLA 2 . Although our previous results using a lipoapoptosis model ( 9 ) and the current data showing improvement of insulin resistance and diabetes by selective iPLA 2 inhibitors demonstrate important roles of iPLA 2 in lipid injury, further work will be necessary to elucidate the roles for various PLA 2 types in FFA-induced insulin resistance.
Our in vitro data showing the role of endogenous LPC generated from FFA by iPLA 2 in FFA-induced JNK activation and insulin resistance suggest the possibility that modulation of endogenous LPC content could be a strategy to treat obesity-induced diabetes, corroborated by our in vivo data showing that administration of BEL ameliorated JNK activation, insulin resistance, and diabetes in db/db mice by decreasing LPC content in the liver and muscle. A previous study showed that iPLA 2 b -null mice have impaired insulin secretion but improved insulin sensitivity on a high-fat diet ( 50 ), which might be due to decreased LPC content in insulin target tissues. The relatively mild effect of targeted disruption of iPLA 2 b compared with that of BEL treatment could be due to compensatory changes in iPLA 2 b -null mice or potential effects of BEL on iPLA 2 subtypes other than iPLA 2 b . In agreement with this idea, recent studies have shown that targeted disruption of iPLA 2 g leads to prevention of high-fat, diet-induced insulin resistance ( 51,52 ).