P38 Vector Plan Drawings Free

Gratis fatty acids (FFA) are considered as a causative link between obesity and diabetes. In various beast models and in humans FFA can stimulate hepatic gluconeogenesis. Although the in vivo role of FFA in hepatic gluconeogenesis has been clearly established, the intracellular role of FFA and related signaling pathway remain unclear in the regulation of hepatic gluconeogenic factor transcription. In this written report, we have identified p38 mitogen-activated poly peptide kinase (p38) as a disquisitional signaling component in FFA-induced transcription of key gluconeogenic genes. We prove in primary hepatocytes that both mid- and long-chain fat acids (saturated or unsaturated) could activate p38 and increment levels of phosphoenolpyruvate carboxykinase (PEPCK), glucose-six-phosphatase, and peroxisome proliferator-activated receptor γ coactivator α (PGC-1α) gene transcripts. The FFA-induced expression of PEPCK and PGC-1α genes and gluconeogenesis in isolated hepatocytes could be blocked by the inhibition of p38. Furthermore, PGC-1α phosphorylation by p38 was necessary for FFA-induced activation of the PEPCK promoter. Additionally, FFA stimulated phosphorylation of military camp-response element-binding protein (CREB) through p38. The overexpression of the dominant-negative CREB prevented FFA-induced activation of the PEPCK promoter. Finally, we show that FFA activation of p38 requires protein kinase Cδ. Together, our results bespeak that p38 plays a critical role in FFA-induced transcription of gluconeogenic genes, and the known gluconeogenic regulators, PGC-1α and CREB, are also integral parts of FFA-stimulated transcription of gluconeogenic genes.

Hepatic gluconeogenesis, which is the de novo synthesis of glucose from non-hexose carbohydrate precursors, is essential for maintaining blood glucose levels during fasting. However, excessive gluconeogenesis is a major source of hyperglycemia in both type I and type Two diabetes (

,

). Gluconeogenesis is direct controlled past rate-limiting gluconeogenic enzymes, including phosphoenolpyruvate carboxykinase (PEPCK),

 ⁱⁱ

The abbreviations used are: PEPCK, phosphoenolpyruvate carboxykinase; G6Pase, glucose-6-phosphatase; FFA, costless fatty acrid; PGC-1α, peroxisome proliferator-activated receptor γ coactivator α; SB, SB203580; siRNA, small interfering RNA; PKC, poly peptide kinase C; CREB, cAMP-response element-binding poly peptide; DMEM, Dulbecco'south modified Eagle'southward medium; BSA, bovine serum albumin; RT, reverse transcription; GST, glutathione S-transferase; dn-p38, dominant negative p38; GPR, M poly peptide-coupled receptor.

2 The abbreviations used are: PEPCK, phosphoenolpyruvate carboxykinase; G6Pase, glucose-6-phosphatase; FFA, free fatty acrid; PGC-1α, peroxisome proliferator-activated receptor γ coactivator α; SB, SB203580; siRNA, small interfering RNA; PKC, poly peptide kinase C; CREB, campsite-response element-binding protein; DMEM, Dulbecco'south modified Eagle's medium; BSA, bovine serum albumin; RT, opposite transcription; GST, glutathione Southward-transferase; dn-p38, dominant negative p38; GPR, G protein-coupled receptor.

glucose-6-phosphatase (G6Pase), and fructose-1,6-bisphosphatase (come across Ref.

for review). The role of these enzymes is mainly regulated at their transcriptional levels by the pancreatic hormones insulin and glucagon (

). Insulin suppresses, whereas glucagon stimulates, transcription of these gluconeogenic genes in the liver (

). In addition to hormonal control, hepatic gluconeogenesis has been shown to be directly regulated by some nutrients such every bit free fatty acids (FFA) (see Refs.

and

for review).

FFA levels are oftentimes increased in obese individuals in both the fed and fasted states and take been implicated every bit critical players in the progression of obesity to type Two diabetes (

,

,

,

,

,

,

). The regulatory office of FFA in gluconeogenesis is complicated, considering information technology can be directly and indirect. The indirect role of FFA is through insulin secretion and insulin action. FFA-stimulated insulin secretion from pancreatic islets suppresses gluconeogenesis (come across Refs.

and

for review). Merely on the other hand, exposure of the liver to FFA tin desensitize insulin signaling and dampen its suppression of gluconeogenesis, with the net effect of elevating hepatic gluconeogenesis (

,

,

,

,

,

,

,

,

,

,

). In addition, FFA has been proposed to straight regulate hepatic gluconeogenesis independent of hormones in several ways. Commencement, FFA promotes gluconeogenesis by serving as a source of substrates and energy, including acetyl-CoA, NADH, and ATP. 2d, FFA may direct regulate gluconeogenesis through transcription of key gluconeogenic genes (see Ref.

for review). Although this direct part of FFA in hepatic gluconeogenesis has been suggested in previous studies (

,

), the components of the signaling pour through which FFA operates have not been systematically investigated and remain largely unknown.

p38 mitogen-activated poly peptide kinase (p38) has been recently linked to energy metabolism in adipocytes, skeletal muscle, cardiomyocytes, and hepatocytes (

,

,

,

,

,

,

,

). In particular, p38 plays a regulatory role in the office of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α). This coactivator is known to be an important stimulator of hepatic gluconeogenesis (

,

). In this study, nosotros examined the role of p38 in FFA consecration of gluconeogenic gene expression in hepatocytes. Our results show that FFA stimulated phosphorylation of p38 and transcription of PGC-1α, PEPCK, and G6Pase genes. The inhibition of p38 blocked FFA-induced transcription of PGC-1α and PEPCK genes and gluconeogenesis in isolated hepatocytes. Together, nosotros have identified p38 as a mediator of FFA-induced transcription of hepatic gluconeogenic genes, and have provided new insight into understanding of fatty acid regulation of hepatic gluconeogenesis.

MATERIALS AND METHODS

Chemicals and Antibodies—SB203580 (SB) and rottlerin were from Calbiochem. Glucagon was from Sigma. Antibodies against p38, phosphorylated p38 (true cat. no. 9211s), phosphorylated CREB (serine 133, cat. no. 9191), and phosphorylated ATF-2 (cat. no. 9221s) were from Cell Signaling Technology. The β-actin antisera was from Sigma (cat. no. A-5441). The siRNA duplexes against PKCδ were purchased from Super Array Bioscience Corp. (cat. no. RM-0951). The PGC-1α promoter (2 kb) construct was purchased from Addgene (www.addgene.com, plasmid no. 8887). The PEPCK promoter construct was a gift from Dr. Jianhua Shao. The A-CREB construct was a kind gift from Dr. Charles R. Vinson. The expression vectors of wild-type PGC-1α and phosphorylation-deficient mutant of PGC-1α (PGC-1α-A3) were kind gifts from Dr. Bruce Spiegelman.

Isolation of Primary Hepatocytes—Primary hepatocytes were isolated from C57BL/6 mice as previously described (

). (All the mice used for isolation of hepatocytes were fed under normal chow diet and regular schedule unless otherwise noted.) Briefly, under anesthesia with pentobarbital (intraperitoneal, thirty mg/kg body weight), livers were perfused with Ca²⁺-free Hanks' counterbalanced solution (Invitrogen) at v ml/min for eight min, followed by continuous perfusion with serum-free Williams' medium containing collagenase (Worthington, type II, l units/ml, Invitrogen), HEPES (10 mm), and NaOH (0.004 northward) at 5 ml/min for 12 min. Hepatocytes were harvested and purified with Percoll. The viability of hepatocytes was examined with trypan blueish exclusion. Only jail cell isolates with viability >five% were used. Hepatocytes were inoculated into collagen-coated 6-well plates (v × 10⁵/well) in Williams' medium with 10% fetal bovine serum and were incubated for 24 h earlier any experimentation.

Preparation of FFA and Handling of Hepatocytes with FFA—FFA solutions were prepared every bit previously described (

). Briefly, palmitate and capric acids were dissolved with methanol and then diluted with DMEM medium (containing 5.five gm glucose) to 10 mm. Oleate, linoleate, and caproic acid were all purchased as liquid solutions, and were freshly diluted with DMEM medium (containing 5.five mm glucose) to 100 grandm. The serum-costless DMEM media used for the treatment of cells with FFA were supplemented with five.5 gm glucose and 2% FFA-free bovine albumin (BSA).

Transfection of Hepatocytes—Primary hepatocytes were transfected as previously described (

). Briefly, cells were settled down in Williams' medium for 24 h before the transfection. Hepa1c1c7 cells were maintained in minimal essential medium (Invitrogen) with 10% fetal bovine serum. Plasmid DNAs were introduced into chief hepatocytes and Hepa1c1c7 cells past using Lipofectamine 2000 (Invitrogen). The transfection efficiency was monitored past introducing an identical amount of dark-green fluorescent protein plasmid Dna into the command cells simultaneously, followed by observation nether a fluoresce microscope. Approximately l% of cells were transfected.

Measurement of Glucose Product in Primary Hepatocytes—Primary hepatocytes were isolated from mice, which had been fasted for 24 h to deplete glycogen in the liver. Glucose product from the chief hepatocytes was measured as previously described (

). Briefly, cells were washed three times with warm phosphate-buffered saline to remove glucose and pre-treated with v μyard SB (30 min) every bit noted, followed by the treatment with 0.5 gg oleate (iv h) in the glucose-free medium containing gluconeogenic substrates (twenty mg sodium lactate and 2 mm sodium pyruvate). Glucose concentrations were adamant with a glucose analysis kit from Roche Applied Scientific discipline (cat. no. 0716251) and normalized to the cellular protein concentrations. Full glucose production was derived from both glycogenolysis and gluconeogenesis. Glucose production from glycogenolysis was measured in the absence of gluconeogenic substrates. The amount of glucose product past gluconeogenesis is defined as the deviation between total glucose production and glycogenolysis.

Immunoblotting—Immunoblotting analyses were performed as previously described (

,

,

,

). Briefly, cell lysates were prepared by homogenization and sonication, followed by addition of two× Laemmli sample buffer. Aliquots (5 μg/well) were resolved with mini Tris-glycine gradient gels (4–20%, Invitrogen) and transferred to nitrocellulose membranes. Levels of phosphorylated and total p38 or phosphorylated CREB were detected with a 1:ane,000 dilution of each specific antiserum, followed by incubation with a 1:10,000 dilution of goat anti-rabbit immunoglobulin G conjugated with alkaline phosphatase (RPN5783, Amersham Biosciences). Fluorescence bands were visualized with a Typhoon PhosphorImager (Molecular Dynamics).

RNA Isolation, Semi-quantitative RT-PCR, and TaqMan Real-fourth dimension PCR—Total RNAs from hepatocytes were prepared by using RNA purification kits from Qiagen. Semi-quantitative RT-PCR reactions were performed co-ordinate to the manuals from the manufacturer. Existent-time RT-PCR TaqMan probes and reaction agents were purchased from Applied Biosystems. Reactions were performed according to manuals from the manufacturer. Catalogue numbers for the probes were as follows: PEPCK (Mm00440636-m1), G6Pase (Mm00839363-m1), and PGC-1α (Ahs00173304-m1).

Introduction of siRNA Duplexes into Master Hepatocytes—The siRNA duplexes were introduced into master hepatocytes as previously described (

). Briefly, siRNA duplexes as indicated in each experiment were mixed with 4 μl of Lipofectamine 2000 (Invitrogen) in OPTI medium (Invitrogen) and added to the primary hepatocytes, which had been seeded in 6-well plates 24 h earlier.

Measurement of Promoter Activities—The PEPCK or PGC-1α promoter was introduced via transient transfection into Hepa1c1c 7 hepatoma cells together with an expression vector for β-galactosidase every bit an internal control (

,

,

), and cells were treated as noted in each experiment. Promoter activities were measured past luciferase assays and normalized to the β-galactosidase internal command.

Measurements of p38 Activity—Activity of p38 was measured as previously described (

). In brief, cells were done twice with phosphate-buffered saline and lysed with a buffer containing v 1000m β-glycerophosphate, 1 1000m sodium orthovanadate, 25 mone thousand Tris-HEPES, 150 chiliadm NaCl, v mm β-glycerophosphate, 1 mk sodium orthovanadate, v mm sodium pyrophosphate, 5 mm EDTA, 5 mm EGTA, 0.9% Triton 10-100, 0.1% IGEPAL, 10% glycerol, and proteinase inhibitors (Roche Applied Science, cat. no. 11836153001, ane tablet per ten ml). Jail cell lysates (20 μl) were incubated at 37 °C for 30 min with 4 μg of GST-ATF-2 (Cell Signaling Technology, cat. no. 9224) and 200 μk ATP in 100 μl of kinase reaction buffer (20 mchiliad HEPES, 20 mm MgCl₂, 25 mone thousand β-glycerophosphate, two mm dithiothreitol, and 0.1 1000thousand sodium orthovanadate). Glutathione-Sepharose 4B (50 μl/reaction) was used to precipitate GST-ATF-2, which was immunoblotted with antisera against phosphorylated ATF-ii (i:1,000 dilution, Cell Signaling Engineering science, cat. no. 9221). Phosphorylated ATF-2 was finally detected with goat anti-rabbit immunoglobulin G conjugated with alkaline phosphatase (1:10,000 dilution, Amersham Biosciences, cat. no. RPN5783) and visualized with a Draft PhosphorImager.

RESULTS

FFA Stimulates p38 Phosphorylation in Primary Hepatocytes—FFA tin can stimulate hepatic gluconeogenesis in a multifariousness of animal models and in humans (

,

,

,

,

,

). After an overnight fast plasma levels of FFA fluctuate between 350 and 740 μm and tin can reach 1–2.v mm during prolonged fasting and in diabetes, in which hepatic gluconeogenesis is elevated (

,

,

). In cardiac myocytes and endothelial cells FFAs take been previously shown to activate p38 (

,

), and we have recently reported that p38 plays a key office in glucagon-induced hepatic gluconeogenesis (

). Therefore, we postulated that FFA might activate p38 to induce hepatic gluconeogenesis. To test this hypothesis, nosotros get-go examined the effect of unsaturated FFA on p38 activation in principal hepatocytes. As shown in Fig. 1, both oleate (monounsaturated, long chain) and linoleate (double-unsaturated, long concatenation) stimulated p38 phosphorylation in a dose-dependent way. Adjacent, the role of saturated FFA in p38 activation was examined. As shown in Fig. two, palmitate (long chain) and capric acid (mid chain) could both stimulate p38 phosphorylation, whereas caproic acid (brusk concatenation) failed to do so. Together, these results suggest that both mid- and long-chain FFA (saturated or unsaturated) can stimulate phosphorylation of p38 in primary hepatocytes.

Figure one p38 activation by unsaturated FFA in chief hepatocytes. Principal hepatocytes were isolated and cultured as detailed under "Materials and Methods" and were treated for 20 min with increasing amounts of oleate (A and B) or linoleate (C and D) in serum-complimentary DMEM media containing v.5 mm glucose and two% FFA-costless BSA. Levels of p38 in cell lysates were detected by immunoblotting with antisera confronting phospho-p38 (p38-P). Results are normalized to the nonspecific band on the aforementioned absorb and stand for three independent experiments. *, p < 0.01 compared with 0 1000m oleate; #, p < 0.05 compared with 0 1000m linoleate.

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FIGURE 2 p38 activation by saturated FFA in primary hepatocytes. Primary hepatocytes were isolated and cultured equally detailed nether "Materials and Methods" and were treated for 20 min with increasing amounts of palmitate (A and B), capric acid (C and D), or caproic acrid (E and F) in serum-free DMEM media containing 5.5 mm glucose and two% FFA-free BSA. Levels of p38 in cell lysates were detected by immunoblotting with antisera against p38 (p38-T) or phospho-p38 (p38-P). Results of p38-P were normalized to p38-T, and represent three independent experiments. *, p < 0.05 compared with 0 mg palmitate; #, p < 0.01 compared with 0 mm capric acid.

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FFA Induces Gluconeogenesis in a p38-dependent Manner—Although the office of FFA in hepatic gluconeogenesis has been well established (

,

,

,

,

,

), the direct role of FFA on gluconeogenesis has not been adamant in primary hepatocytes. Therefore, we treated isolated primary hepatocytes with oleate in the presence or absence of p38 inhibition with SB. (Oleate is 1 of the most abundant FFA in blood (

).) As shown in Fig. 3, oleate promoted glucose production via gluconeogenesis in master hepatocytes merely was blocked past the inhibition of p38. These results indicate that FFA tin can straight stimulate gluconeogenesis in isolated hepatocytes through activation of p38.

Effigy 3 FFA induces gluconeogenesis in primary hepatocytes in a p38-dependent mode. Isolated hepatocytes were incubated with oleate (0.5 mm) in the presence or absenteeism of SB (5 μm) for 4 h. The treatment with oleate was performed in serum-gratis DMEM media containing 2% FFA-free BSA. Glucose production from gluconeogenesis was quantified equally detailed under "Materials and Methods." Results represent mean ± S.D. of two independent experiments, each in triplicate. *, p < 0.05 compared with all other treatments. #, p > 0.05 compared with treatment with SB lonely.

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To determine the role of FFA in transcription of gluconeogenic genes, isolated hepatocytes were treated with increasing amounts of FFA, followed past measuring transcripts of fundamental gluconeogenic genes. As shown in Fig. 4, the event of oleate on expression of PEPCK and G6Pase genes was biphasic. Specifically, oleate elevated levels of both PEPCK and G6Pase transcripts at 50 μone thousand, but this elevation was reduced to a level, which is withal significantly higher than the basal one (p < 0.05), at 500 μm, followed past another increase at 1000 μm. We repeatedly observed this biphasic effect of oleate, but we have not been able to explain this miracle yet. Capric acrid started to increase levels of PEPCK and G6Pase transcripts at 50 μg, and this increase continued at 100 μk for PEPCK and 500 μm for G6Pase. Elevation of PEPCK and G6Pase transcripts past capric acid started to decrease when concentrations of capric acid reached 500 μm (for PEPCK) or 1000 μg (for G6Pase). The result of palmitate (saturated FFA) on PEPCK and G6Pase genes were significantly milder in comparison to oleate and capric acid. Palmitate started moderately to increase levels of PEPCK and G6Pase transcripts only at high concentrations (≥500 μm). Together, these results indicate that diverse FFAs can direct elevate levels of gluconeogenic gene transcripts at different concentrations in primary hepatocytes.

Effigy 4 FFA induction of gluconeogenic gene expression in master hepatocytes. Chief hepatocytes were isolated and cultured as detailed under "Materials and Methods" and treated with oleate (OA), palmitate (PA), or capric acid (CA) (0.one chiliadm to i thousandg) for 2 h. The treatment with FFA was performed in serum-gratuitous DMEM media containing 5.v gg glucose and 2% FFA-complimentary BSA. Levels of PEPCK and G6Pase transcripts were detected by TaqMan real-time RT-PCR and normalized to the internal control glyceraldehyde-3-phosphate dehydrogenase. Results shown represent ways ± Due south.D. of iii independent experiments. *, p < 0.05 compared with 0 mone thousand OA; **, p < 0.01 compared with 0 chiliadm OA; #, p < 0.05 compared with 0 mthousand CA; &, p < 0.05 compared with 0 mm PA; $, p < 0.05 compared with 0 grandg CA; $$, p < 0.01 compared with 0 mk CA.

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To decide the role of p38 in FFA-induced elevation of gluconeogenic factor transcripts, primary hepatocytes were treated with oleate in the presence or absence of p38 inhibitors. As shown in Fig. 5, oleate increased levels of both PEPCK and G6Pase transcripts. The increase in PEPCK transcripts was blocked by the inhibition of p38 with either SB or ascendant-negative p38 (dn-p38), whereas, interestingly, levels of G6Pase transcripts were not affected by the blockade of p38. Similar results were observed when palmitate instead of oleate was used (data not shown). Together, these results signal that FFA induction of the rate-limiting enzyme, PEPCK, is p38-dependent, whereas FFA induction of the G6Pase factor is independent of p38.

Figure 5 p38 mediates FFA-induced transcription of the PEPCK gene in primary hepatocytes. Primary hepatocytes were isolated and cultured every bit detailed under "Materials and Methods" and pre-treated with p38 inhibitor SB (x μm, 30 min) or overexpression of ascendant-negative p38α (dn-p38α) via transient transfection, followed by the treatment with 0.1 mm oleate (OA) for 2 h. The treatment with oleate was performed in serum-costless DMEM media containing 5.5 m1000 glucose and 2% FFA-free BSA. Levels of PEPCK and G6Pase genes were detected past TaqMan real-time RT-PCR and normalized to the internal control glyceraldehyde-iii-phosphate dehydrogenase. Results shown represent means ± Due south.D. of three contained experiments. *, p < 0.05 compared with basal, SB, OA plus SB, or OA plus dn-p38α.#, p < 0.05 compared with basal, SB, or dn-p38.

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To further investigate the role of p38 in FFA-induced elevation of gluconeogenic gene transcripts, the PEPCK promoter was introduced into Hepa1c1c7 hepatoma cells, which were subsequently treated with oleate in the presence of p38 inhibitor SB or constitutive activator (MKK6E). As shown in Fig. 6, activation of the PEPCK promoter by oleate was suppressed by SB but further enhanced by the overexpression of MKK6E. These results further support a office for p38 in FFA stimulation of PEPCK factor transcription.

Figure vi p38 mediates FFA stimulation of the PEPCK promoter in Hepa1c1c7 cells. The PEPCK promoter was introduced into Hepa1c1c7 cells via transient transfection. Cells were and so pre-treated with p38 inhibitor SB (5 μm, thirty min) or overexpression of constitutive activator of p38 (MKK6E) via transient transfection, followed past treatment with 0.5 mm oleate for 6 h. The treatment with oleate was performed in serum-free minimal essential medium containing 5.5 yardchiliad glucose and 2% FFA-free BSA. Promoter activities were then measured by luciferase assays and normalized to the β-galactosidase activity. Results shown represent mean ± S.D. of four independent experiments, each in triplicate. *, p < 0.01 compared with basal, SB, or oleate plus SB; #, p < 0.05 compared with oleate or MKK6E.

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p38 Mediates FFA-induced Transcription of the PGC-oneα Gene—PGC-1α and FFA both play critical roles in hepatic gluconeogenesis (

,

,

). Levels of PGC-1α cistron transcription and plasma FFA are coincidently increased during fasting and in diabetes (

,

,

,

,

,

,

,

,

). We have recently shown that p38 mediates glucagon-induced transcription of the PGC-1α gene (

). Our results in Figs. ane and ii show that FFA tin actuate p38 in hepatocytes. Therefore, we postulated that FFA activation of p38 might influence expression of the PGC-1α gene. To exam this hypothesis, isolated hepatocytes were treated with oleate in the presence or absence of p38 inhibitors, followed past measurements of PGC-1α transcripts. As shown in Fig. 7A, levels of PGC-1α transcripts were increased by oleate but blocked by the inhibition of p38 with either SB or dominant-negative p38α.

FIGURE seven p38 mediates FFA-induced transcription of the PGC-1α cistron. Master hepatocytes were isolated and cultured as detailed under "Materials and Methods." A, cells were treated with 0.i mchiliad oleate for 2 h in the present or absence of either SB (10 μ1000) for 30 min or overexpression of dn-p38α via transient transfection. The handling with oleate was performed in serum-free DMEM media containing 5.5 one thousandg glucose and 2% FFA-costless BSA. Levels of PGC-1α transcripts were measured past TaqMan existent-time RT-PCR and normalized to the internal control glyceraldehyde-3-phosphate dehydrogenase. Results shown represent means ± Due south.D. of iii contained experiments. *, p < 0.05 compared with basal, SB, OA plus SB, or OA plus dn-p38α. B, the PGC-1α promoter (2 kb) was introduced into Hepa1c1c7 cells via transient transfection. Twenty-iv hours afterward, cells were stimulated with oleate (0.5 yardm) for vi h in the presence or absence of SB (5 μm) or overexpression of MKK6E via transient transfection. The handling with oleate was performed in serum-costless minimal essential medium containing five.five kchiliad glucose and two% FFA-free BSA. Promoter activities were measured by luciferase assays and normalized to the internal control β-galactosidase. Results represent iii independent experiments, each in triplicate. *, p < 0.05 compared with basal; **, p < 0.01 compared with basal; #, p < 0.05 compared with oleate; ##, p < 0.01 compared with oleate.

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To farther written report the issue of p38 on the PGC-1α gene, we introduced the PGC-1α promoter into Hepa1c1c7 cells, which were subsequently stimulated by oleate in the presence of inhibitor or activator of p38. As shown in Fig. 7B, oleate activated the PGC-1α promoter, only this activation was blocked by the inhibition of p38. In contrast, the overexpression of p38 activator MKK6E further enhanced the promoter activity induced by oleate. Together, these results demonstrate that p38 is a mediator of FFA-induced transcription of the PGC-1α gene.

PGC-aneα Phosphorylation by p38 Is a Component of FFA-induced Transcription of the PEPCK Factor—Nosotros and others have previously shown that the co-activating forcefulness of PGC-1α activity is dependent upon p38 mediated-phosphorylation (

,

). Therefore, nosotros introduced the PEPCK promoter together with either the wild-type or phosphorylation-deficient PGC-1α into Hepa1c1c7 hepatoma cells, followed by the treatment with oleate. As shown in Fig. 8, the PEPCK promoter was activated by oleate and further enhanced past the overexpression of the wild-type PGC-1α. Interestingly, the co-expression of the phosphorylation-deficient PGC-1α-A3 not only failed to enhance the activation of the PEPCK promoter but also suppressed the oleate-induced promoter activity. Together, these results suggest that the phosphorylation of PGC-1α by p38 is another element in the regulation of the PEPCK gene by FFA.

Figure viii PGC-1α phosphorylation by p38 is required for FFA stimulation of the PEPCK promoter. The PEPCK promoter was introduced to Hepa1c1c7 cells and stimulated by 0.five m1000 oleate for 6 h in the presence or absence of overexpression of wild-blazon PGC-1α or phosphorylation-deficient PGC-1α (PGC-1α-A3) via transient transfection. Treatment with oleate was performed in serum-free minimal essential medium containing v.5 mchiliad glucose and 2% FFA-gratis BSA. Promoter activities were measured by luciferase assays and normalized to the β-galactosidase. Results shown represent mean ± S.D. of four independent experiments, each in triplicate. *, p < 0.01 compared with basal, PGC-1α-A3, or oleate plus PGC-1α-A3. ##, p < 0.01 compared with oleate alone.

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CREB Is Activated by FFA in Main Hepatocytes through p38, and Activity of CREB Is Required for FFA-induced Transcription of the PEPCK Gene—CREB is some other critical actor in the control of hepatic gluconeogenesis (

). We have previously shown that activation of CREB in the liver by fasting and in isolated hepatocytes by glucagon is p38-dependent (

). It is established that CREB is an indirect substrate of p38 (

,

,

). To determine whether CREB participates in the FFA regulation of gluconeogenesis, primary hepatocytes were treated with oleate in the presence of increasing amounts of SB, followed by measurements of CREB phosphorylation. Phosphorylation of CREB was stimulated by oleate (Fig. 9A) and other fatty acids, including palmitate and capric acid (data not shown), but blocked past the inhibition of p38. These results indicate that FFA can activate CREB in hepatocytes through p38.

Effigy 9 CREB is activated by FFA in master hepatocytes in a p38-dependent manner and is required for FFA activation of the PEPCK promoter. A, isolated hepatocytes were treated with oleate (0.5 mone thousand) for 20 min in the presence or absence of SB (1–x μm) as detailed under "Materials and Methods." The treatment with oleate was performed in serum-free DMEM media containing v.v m1000 glucose and 2% FFA-free BSA. Levels of phosphorylated CREB and β-actin were detected by immunoblotting. B, the PEPCK promoter was introduced to Hepa1c1c7 cells and stimulated with 0.five thousandm oleate for 6 h in the presence or absenteeism of overexpression of dominant-negative CREB (A-CREB) via transient transfection. The handling with oleate was performed in serum-free minimal essential medium containing v.5 thousandm glucose and two% FFA-gratis BSA. Glucagon (x northchiliad) was used as a positive control. Promoter activities were measured past luciferase assays and normalized to the β-galactosidase. Results shown stand for mean ± S.D. of four independent experiments, each in triplicate. *, p < 0.05 compared with basal, A-CREB, or oleate plus A-CREB.

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To determine whether CREB activity is required for FFA-induced expression of gluconeogenic genes, the PEPCK promoter was introduced into Hepa1c1c7 cells and stimulated by oleate in the presence or absence of dominant-negative CREB (A-CREB). Every bit expected, the PEPCK promoter was activated past oleate but inhibited by the overexpression of A-CERB (Fig. 9B). These results suggest that CREB activation is necessary for FFA-induced transcription of the PEPCK gene.

PKCδ Is Required for Fatty Acid Activation of p38 in Chief Hepatocytes—FFA has been previously shown to actuate certain isoforms of PKC in adipocytes (

). Amongst these isoforms, PKCδ is an established activator of p38 (

,

). Therefore, we chose to examine the possible involvement of PKCδ in the activation of p38 by FFA. As shown in Fig 10A, the activation of p38 past oleate was completely blocked by rottlerin, which is a relatively specific inhibitor of PKCδ. To further determine the function of PKCδ, the specific siRNA was used to silence the PKCδ factor. The PKCδ gene was knocked down by ∼seventy% with the siRNA (Fig. 10B), and this reduction of the PKCδ transcripts significantly decreased the level of p38 action induced by oleate in comparison to the control siRNA. Together, these results propose that PKCδ is an upstream activator FFA activation of p38 in hepatocytes.

Effigy 10 Oleate activation of p38 is PKCδ-dependent. A, master hepatocytes were isolated as detailed under "Materials and Methods" and incubated with 0.5 gk oleate for 20 min in the presence of rottlerin, siRNA against PKCδ, or command siRNA (scrambled siRNA) as indicated. The treatment with oleate was performed in serum-costless DMEM media containing 5.v mgrand glucose and 2% FFA-free BSA. Activities of p38 in the cells were measured every bit detailed under "Materials and Methods." Levels of β-actin protein in the cell lysates were measured as a loading control. Results represent three independent experiments. B, levels of PKCδ and β-actin cistron transcripts detected with real-time RT-PCR in main hepatocytes treated with either the siRNA against the PKCδ gene or a scrambled siRNA.

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DISCUSSION

Although FFA accept been firmly established in many in vivo beast models and in humans as a promoter of hepatic gluconeogenesis (

,

,

,

,

,

), the mechanism by which FFAs promote transcription of gluconeogenic genes at cellular and molecular levels remains unclear. In this study, we have identified a critical function for p38 in transcription of hepatic gluconeogenic genes induced by FFA. Our results also indicate that PKCδ is an upstream activator, whereas PGC-1α and CREB are downstream effectors of p38 in FFA regulation of hepatic gluconeogenesis.

Previous studies accept implicated a link between FFA and p38 in the command of hepatic gluconeogenesis nether physiological and diabetic conditions. First, levels of both plasma FFA and p38 phosphorylation in the liver are increased during the post absorption stage or fasting when hepatic gluconeogenesis is elevated (

,

,

,

,

,

,

). 2nd, levels of both FFA and p38 phosphorylation are elevated in obese animals with or without diabetes (

,

,

), which is characterized by exaggerated hepatic gluconeogenesis. Third, FFAs stimulate hepatic gluconeogenesis in vivo (

,

,

,

,

,

). Fourth, FFA can stimulate transcription of the PEPCK gene in adipocytes, although this effect induced by a specific FFA was not observed in a hepatoma jail cell line (come across Ref.

for review). 5th, FFA tin actuate p38 in cardiac myocytes and endothelial cells (

,

). 6th, p38 has recently been shown to mediate glucagon-induced transcription of hepatic gluconeogenic genes (

). In this written report, using isolated primary hepatocytes, we methodically studied the role of FFA on activation of p38 and expression of key gluconeogenic genes. Our study has identified p38 every bit a disquisitional mediator of FFA regulation of gluconeogenic gene transcription. However, the mechanism past which FFA stimulates p38 activity remains largely undetermined.

Several orphan One thousand protein-coupled receptors, including GPR40, -41, -43, and -120, take recently been identified as receptors for FFA with unlike length and saturation (

60

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• Tcheang Fifty.
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• Wigglesworth Grand.J.
• Kinghorn I.
• Fraser N.J.
• Pike N.B.
• Strum J.C.
• Steplewski K.1000.
• Murdock P.R.
• Holder J.C.
• Marshall F.H.
• Szekeres P.M.
• Wilson S.
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). FFA-induced phosphorylation of p38 in hepatocytes was not influenced by the inhibition of Ca²⁺ mobilization in our study (data not shown). Therefore, FFA activation of p38 is unlikely mediated past these GPRs. Our results suggest that PKCδ is ane of the upstream components of FFA-induced activation of p38 in hepatocytes. This observation is consistent with previous reports that PKCδ is an activator of p38 (

,

). However, the signaling components beyond PKCδ remain unknown.

Our results also evidence some other interesting attribute of the regulation of PECK and G6Pase genes. Although the expression of both PEPCK and G6Pase genes are stimulated by FFA in this study, just the expression of the PECK factor is mediated by p38, whereas the FFA induction of the G6Pase gene is independent of p38. Information technology is well known that both PEPCK and G6Pase are rate-limiting enzymes of gluconeogenesis. They share many common features in their gene transcription. For example, their transcriptions are both stimulated by military camp-producing hormones such as glucagon only suppressed past insulin (

,

). Even so, there are some distinctions in the regulation of these two genes. For instance, the signals from the central nervous system regulate the expression of the G6Pase factor just practise non influence the transcription of the PECK gene in the liver (

). Considering PEPCK is the earliest rate-limiting enzyme in the process of gluconeogenesis, it may play a more than of import role in the regulation of gluconeogenesis. Our results appear to support this notion, considering gluconeogenesis is blocked by the inhibition of p38, although simply the expression of the PEPCK factor is suppressed past the inhibition of p38. This notion is also strongly supported by the previous study that the deletion of the PEPCK gene causes early on death of newborn mice due to severe hypoglycemia (

).

In summary, our results in this study demonstrate that both mid- and long-chain FFA (saturated or unsaturated) can stimulate transcription of hepatic gluconeogenic genes. This stimulation is dependent upon p38, PGC-1α, and CREB (Fig. 11). In addition to FFA, glucagon is another established and principle stimulator of hepatic gluconeogenesis. Plasma levels of both FFA and glucagon are increased during fasting and in diabetes (reviewed in Ref.

). Our previous (

) and current studies show that p38 plays a critical role in regulation of hepatic gluconeogenesis induced by either glucagon or FFA. Therefore, futurity studies on the signaling pathways by which glucagon and FFA activate p38 may shed new calorie-free into understanding of glucose homeostasis and provide new intervening targets for the prevention and treatment of diabetes.

Acknowledgments

We thank Drs. Sheila Collins, Jamie Bonner, and Jacques Robidoux for their instrumental advice and critical reading of the manuscript. Nosotros also thank Drs. Bruce Spiegelman, Charles Vinson, and Jianhua Shao for providing some of the constructs used in this report.

Supplementary Material

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Article Info

Publication History

Published online: June 27, 2006

Received in revised grade: June 22, 2006

Received: March 7, 2006

Identification

DOI: https://doi.org/ten.1074/jbc.M602177200

Copyright

© 2006 ASBMB. Currently published by Elsevier Inc; originally published by American Guild for Biochemistry and Molecular Biology.

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Source: https://www.jbc.org/article/S0021-9258(18)95121-5/abstract

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