Kupffer cell cytokines interleukin-1β and interleukin-10 combine to inhibit phosphoenolpyruvate carboxykinase and gluconeogenesis in cultured hepatocytes

The International Journal of Biochemistry & Cell Biology 36 (2004) 1462–1472

Kupffer cell cytokines interleukin-1␤ and interleukin-10 combine to inhibit phosphoenolpyruvate carboxykinase and gluconeogenesis in cultured hepatocytes Stephanie T. Yerkovich a , Paul J. Rigby b , Paul A. Fournier c , John K. Olynyk d,e , George C. T. Yeoh a,e,∗ a

e

Biochemistry and Molecular Biology, University of Western Australia, Nedlands, 35 Stirling Highway, Crawley 6009, WA, Australia b Department of Pharmacology, University of Western Australia, Nedlands, Crawley 6009, WA, Australia c School of Human Movement and Exercise Science, University of Western Australia, Nedlands, Crawley 6009, WA, Australia d School of Medicine & Pharmacology, University of Western Australia, Nedlands, Crawley 6009, WA, Australia Western Australian Institute of Medical Research and Centre for Medical Research, University of Western Australia, Nedlands, Crawley 6009, WA, Australia Received 17 July 2003; received in revised form 17 October 2003; accepted 20 October 2003

Abstract Background and aims: Recent evidence suggests that inflammatory cytokines may mediate reduced hepatic glucose production and reduced blood glucose concentrations in sepsis. Therefore the aim of this study is to provide direct evidence of a cytokine-mediated interaction between Kupffer cells and hepatocytes by characterising the effects of lipopolysaccharidestimulated Kupffer cells on hepatocyte gluconeogenesis, and the activity of key regulatory enzymes of this pathway. Methods and results: Primary isolates of hepatocytes co-cultured with lipopolysaccharide-stimulated Kupffer cells in Transwell inserts showed a 48% inhibition of gluconeogenesis (P < 0.001). RNase protection assay and ELISA of Kupffer cells and the culture media following exposure to lipopolysaccharide showed increased levels of interleukin-1 alpha and beta, tumour necrosis factor alpha and IL-10. The addition of IL-1␤ and IL-10 to hepatocyte cultures inhibited gluconeogenesis by 52% (P < 0.001), whereas each cytokine alone was ineffective. To determine whether altered production or activity of phosphoenolpyruvate carboxykinase or pyruvate kinase was responsible for the reduced glucose synthesis, their mRNA, protein levels and enzyme activities were measured. Primary hepatocytes co-cultured with lipopolysaccharide-stimulated Kupffer cells or cultured with a combination of IL-1␤ and IL-10 displayed reduced levels of phosphoenolpyruvate carboxykinase mRNA, protein and enzyme activity. In contrast the mRNA, protein levels and enzyme activity of pyruvate kinase were not altered; suggesting that gluconeogenesis was suppressed by downregulation of phosphoenolpyruvate carboxykinase. Conclusions: Therefore, hypoglycaemia, which is often observed in sepsis, may be mediated by Kupffer cell-derived IL-1␤ and IL-10. In addition this study suggests these cytokines inhibit phosphoenolpyruvate carboxykinase production and thereby hepatic gluconeogenesis. © 2003 Elsevier Ltd. All rights reserved. Keywords: Kupffer cells; Cytokines; Gluconeogenesis; Phosphoenolpyruvate carboxykinase

Abbreviations: Bt2 cAMP, dibutyrylcyclic AMP; EGF, epidermal growth factor; FBPase, Fructose bisphosphatase; G-6-Pase, glucose-6-phosphatase; GK, glucokinase; IFN␥, interferon gamma; IL, interleukin; ITS, insulin-transferrin-selenious acid; LPS, lipopolysaccharide; LPK, L-type pyruvate kinase; PEPCK, phosphoenolpyruvate carboxykinase; PFK, phosphofructokinase; RPA, RNase protection assay; TNF␣, tumour necrosis factor alpha ∗ Corresponding author. Tel.: +61-8-9380-2986; fax: +61-8-9380-1148. E-mail address: [email protected] (G.C.T. Yeoh). 1357-2725/$ – see front matter © 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2003.10.022

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1. Introduction Bacterial infection can lead to septic shock, which is characterised by the development of hypotension, multiorgan failure, and in severe cases, death. The development of hypoglycaemia in association with sepsis was first described following Gram-negative infection in rats (Lanoue, Mason, & Daniels, 1968). Subsequent studies investigating the possibility that pro-inflammatory cytokines may mediate this response reported that hypoglycaemia occurred following the administration of IL-1 or TNF␣ to rodents (Chajek-Shaul et al., 1990; Del Ray & Besedovsky, 1987). However, studies examining the role of cytokines in glucose metabolism in vitro have produced conflicting results. Some show no effect of either IL-1␤ or TNF␣ on glucose production by adult hepatocytes in vitro (Blumberg, Hochwald, Brennan, & Burt, 1995; Christ & Nath, 1996; Goto, Yoshioka, Battelino, Ravindranath, & Zeller, 2001; Rofe, Conyers, Bais, Gamble, & Vadas, 1987; Stadler et al., 1995), while others report that TNF␣, alone (Dahn, Hsu, Lange, & Jefferson, 1994) or in combination with IFN␥, IL-1␤ and LPS (Ceppi, Smith, & Titheradge, 1996), reduces glucose production by as much as 50%. Furthermore, it has been observed that individually, TNF␣ or IL-1␤ (Christ & Nath, 1996) and the combination of TNF␣, IL-1␤, IFN␥ and LPS (Stadler et al., 1995) inhibit glucagon-mediated stimulation of gluconeogenesis. Similarly, several enzymes of gluconeogenesis or glycolysis in the liver are altered following in vivo administration of LPS or cytokines. These include glucose-6-phosphatase (G-6-Pase) (Chajek-Shaul et al., 1990; Maitra, Gestring, El-Maghrabi, Lang, & Henry, 1999; Maitra, Wang, Brathwaite, & El-Maghrabi, 2000; Metzger et al., 1997; Yasmineh & Theologides, 1992), phosphoenolpyruvate carboxykinase (PEPCK) (Chajek-Shaul et al., 1990; Hill & McCallum, 1992; Horton, Knowles, & Titheradge, 1994; Metzger et al., 1997) and phosphofructokinase (PFK) (Ceppi, Knowles, Carpenter, & Titheradge, 1992; Knowles, McCabe, Beevers, & Pogson, 1987). Whilst cultured hepatocytes have been widely used to study the effects of cytokines on glucose production, there are limited reports documenting alterations to gluconeogenic or glycolytic enzymes following hepatocyte exposure to cytokines in culture (Christ, Nath,

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Heinrich, & Jungerman, 1994; Christ & Nath, 1996; Christ, Yazici, & Nath, 2000; Goto et al., 2001). To obtain evidence of a direct role of cytokines on hepatic glucose production, this study investigated the effect of cytokines on hepatic glucose production and the enzymes involved in this process in cultured hepatocytes. Kupffer cells are the resident macrophage of the liver and are pivotal in the clearance of LPS (Fox, Thomas, & Broitman, 1989) and the production of cytokines. There is currently no evidence to indicate whether these cells have the capacity to inhibit glucose production by hepatocytes. A second aim of this study was to demonstrate the existence of a direct link between the two cell types by showing that medium conditioned by Kupffer cells, and specific cytokines produced by Kupffer cells, inhibits glucose production and enzymes involved in gluconeogenesis.

2. Material and methods 2.1. Animals Male Wistar albino rats of the strain Rattus norvegicus (purchased from Animal Resources Centre, Western Australia, Australia) were housed in a 12 h light dark cycle and fed ad libtum. Rats were anaesthetised by an intraperitoneal injection of pentobarbitone. All animal studies were performed in accordance with the code of practice of the National Health and Medical Research Council of Australia. 2.2. Preparation and culture of hepatocytes Hepatocytes were isolated from 200 ± 50 g rats as described previously (Seglen, 1972). The cells were plated at a density of 1.5 × 105 cells in 35 mm diameter collagen coated culture dishes in Williams’ E medium pH 7.4, supplemented with 10 mM nicotinamide, 2 mM glutamine, 10−7 M dexamethasone, 1X ITS+ (Collaborative Biomedical Products, USA), 0.2 mM ascorbic acid, 20 mM HEPES, 1 mM sodium pyruvate, 0.15% sodium bicarbonate (w/v), 14 mM glucose, 0.013 mM penicillin, 0.46 mM streptomycin, fungizone, 100 ␮g/ml EGF (hereafter referred to as Williams’ E medium) and 10% FCS. The following day, the cultures were washed to remove non-adherent

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cells and replaced with fresh Williams’ E medium without FCS. 2.3. Preparation and culture of Kupffer cells Kupffer cells were isolated from rats (280–390 g) as described previously (Olynyk & Clarke, 1998) and plated at a density of 3 × 106 cells on TranswellTM inserts (0.4 ␮M pore, 24 mm diameter, Corning Costar, USA) in Williams’ E medium with 10% FCS. The following day the inserts were washed to remove non-adherent cells and cell debris, and the medium was replaced with Williams’ E medium without FCS. 2.4. Experimental design: co-culture Hepatocytes and Kupffer cells were isolated on the same day and the cells allowed to interact the following day by placing the TranswellTM insert (Kupffer cells) into the chamber (hepatocytes). LPS (10 ␮g/ml) was added to the TranswellTM and left for 24 hs. Hepatocytes cultured with cytokines—recombinant IL-1␤ (Pharmingen, USA), IL-10 (Biosource International, USA) and TNF␣ (Gibco BRL, Australia) were used at a concentration of 50 ng/ml (Christ & Nath, 1996), while dibutyryl cyclic AMP (Bt2 cAMP) was used at a concentration of 0.5 mM. Bt2 cAMP was included as a control to ensure that hepatocyte preparations were responsive to external stimuli (Johnson, Das, Butcher, & Fain, 1972). Cytokines or Bt2 cAMP were added to cell cultures for 24 h on the day following hepatocyte isolation. 2.5. Measurement of glucose production Culture medium was removed and the hepatocytes washed three times in BSS (0.15 mM NaH2 PO4 , 1.5 mM Na2 HPO4 , 60 mM NaHCO3 , 135 mM NaCl, 2.5 mM KCl, 1 mM CaCl2 and 1 mM MgCl2 ). A 5 min incubation between each wash was included to ensure the complete removal of glucose prior to assay. The hepatocytes were then incubated in 1 ml of glucose free DMEM supplemented with 20 mM HEPES, 1.5 mM lactate and 1 ␮l [U-14 C] lactate (5.70 GBq/mmol, Amersham Pharmacia Biotech, England). After 90 min (glucose production was shown to be linear for 120 min) the medium was

removed and glucose production determined. The hepatocyte monolayer was collected, sonicated and the DNA content assayed (Hinegardner, 1971). The newly synthesised glucose was separated from the precursor (lactate) by ion-exchange chromatography (Ali & Jois, 1997). A 500 ␮l aliquot of the medium was placed on a column (1 cm×1 cm) with 1 g of resin (AG1-X8 resin 100-200 mesh, acetate form, Bio-Rad Laboratories, USA) pre-equilibrated with 3 ml of H2 O. Glucose was eluted with 5 ml of H2 O mixed with scintillation fluid (Emulsifier Safe, Packard) and radioactivity determined in a LS 6500 model Scintillation counter (Beckman Instruments, USA). Blanks (medium with 14 C lactate) were incorporated and subtracted for each sample. 2.6. Isolation of RNA and Ribonuclease Protection Assay RNA was extracted from Kupffer cells by the method of Chomczynski and Sacchi (1987). A 2.5 ␮g sample of RNA was used to determine rat cytokine transcripts using the RiboquantTM Multi-Probe RNase Protection Assay with the rCK-1 template set (RPA, Pharmingen, USA) according to manufacturer’s directions. Bands were visualised using a BAS-1000 Bio Imaging Analyser (Fuji, Japan). 2.7. ELISA TNF␣ protein produced over 24 h was measured in the medium of LPS-stimulated Kupffer cells by ELISA (Genzyme, USA) according to manufacturer’s directions. IL-1␤ and IL-10 were also measured by ELISA using antibodies and substrate solutions from R&D Systems (USA) according to the suggested protocol. 2.8. Northern analysis Total RNA was isolated from cultured cells by the TRIzol method according to manufacturer’s instructions (Invitrogen, USA), and separated on a 1.2% agarose gel prepared in 2.2 M formaldehyde and 0.1% ethidium bromide (w/v) in 20 mM MOPS pH 7.0. The RNA was transferred onto a nylon membrane (Hybond N+, Amersham, UK) by capillary transfer

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(Thomas, 1980). cDNA probes were labelled using the Random Prime DNA labelling system according to manufacturer’s directions (Invitrogen, USA). The membranes were hybridised for 24 h at 42 ◦ C in 5× SSPE, 50% deionised formamide, 10% dextran sulphate, 1% SDS, 0.25 mg/ml salmon sperm DNA and 5× Denhardts solution with probes for fructose bisphosphatase (FBPase) (Stein, Liehr, & Eschrich, 2001), PFK (A kind gift from Dr Tomoyuki Yamasaki, Laboratory of Molecular and Metabolic Research, Faculty of Medicine, Osaka University), G-6-Pase (Shelly et al., 1993), PEPCK (Ruppert et al., 1990), glucokinase (GK) (Matsuda, Noguchi, Yamada, Takenaka, & Tanaka, 1990) and GAPDH (Fort et al., 1985). For LPK (Noguchi, Inoue, Chen, Matsubara, & Tanaka, 1983), hybridisation was performed at 55 ◦ C for 24 h with the omission of dextran sulphate. Following hybridisation, the membranes were exposed to a Fuji Phosphorimager Imaging plate, visualised using a Fuji 2500 Phosphorimager and the Image Reader V1 software package. Image Gauge software was used to quantitate the membrane transcript levels relative to the ubiquitously expressed GAPDH level. 2.9. PEPCK and LPK enzyme activity Cells were harvested from two 100 mm culture dishes in BSS, pelleted and resuspended in 10 mM Tris–HCl pH 7.4, 1 mM EDTA and 1 mM dithiotheitol (DTT). PEPCK and LPK activity were determined as described previously (Mahoud, Wang, & Chaudry, 1997). One unit of PEPCK or LPK activity is equivalent to 1 ␮mol of product formed per min. 2.10. Immunofluorescent staining Hepatocytes cultured on collagen coated glass coverslips were washed with PBS and fixed in 50% methanol/50% acetone (v/v) for 2 min at room temperature. Coverslips were blocked for 1 h at room temperature in 0.5% non-immune serum, then incubated in rabbit anti-rat LPK or goat anti-rat PEPCK antibodies for 1 h at room temperature, washed and incubated for 1 h in goat anti-rabbit IgG FITC (Molecular Probes, USA) or donkey anti-goat IgG FITC (Santa Cruz, USA), respectively, and mounted with anti-fade mounting medium (100 mg p-phenylenediamine in 10 ml PBS and 90 ml glycerol pH 8.0). The signal

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was visualised and quantitated with a Bio-Rad MRC 1024 UV Laser Scanning Confocal Microscope and analysed with Confocal Assistant software (Version 4.02). A Z series was collected every 1 ␮m through the entire cell and microscope settings (iris, gain, black level and the number of Kelmans) remained constant for each antibody. At least 500 cells were counted for each antibody and staining in individual cells was quantitated from the average projection of the Z images. 2.11. Statistical analysis All results are presented as the mean ± S.E.M. The number of samples (n) obtained from duplicate or triplicate cultures is presented with the mean value. Statistical analysis was performed using Student’s t-test with P < 0.05 accepted as significant. 3. Results 3.1. Kupffer cells decrease glucose production by hepatocytes in co-cultures Control hepatocyte cultures produce 53.6 ± 4.0 ␮g glucose/ng DNA/90 min (n = 5), and this was decreased by 48% (P < 0.001) to 28.1 ± 1.6 ␮g glucose/ng DNA/90 min (n = 5) when they were co-cultured with LPS-stimulated Kupffer cells. The ability of each preparation of hepatocytes to regulate glucose production was verified by measuring its response to Bt2 cAMP. All preparations responded by increasing glucose production (151.7 ± 13.5 ␮g glucose/ng DNA/90 min n = 5, P < 0.005). In separate experiments (Table 1), the addition of LPS to hepatocyte cultures or co-culturing hepatocytes with unstimulated Kupffer cells did not substantially alter the level of glucose production, while stimulating the Kupffer cells with LPS significantly (P < 0.05) lowered glucose production. 3.2. Basal and LPS-stimulated cytokine production by Kupffer cells Cytokine transcripts detected by the rCK-1 template set (Pharmingen, USA) are not observed in control Kupffer cells not stimulated with LPS (data not

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Table 1 Effect of LPS, LPS-stimulated and non-stimulated Kupffer cells on glucose production in cultured hepatocytes Glucose production (␮g glucose/(␮g DNA/90 min)) Control +LPS +Unstimulated Kupffer cells +LPS-stimulated Kupffer cells

46.7 40.5 39.7 26.3

± ± ± ±

0.9 1.5 3.9 2.3a

Rat hepatocytes were cultured for 24 h in the presence of LPS or co-cultured with either non-stimulated or LPS-stimulated Kupffer cells, after which glucose production was measured. The results are the mean ± S.E.M. of three different cell preparations each measured in duplicate. a Significantly different from control at P < 0.05.

shown). Transcripts for IL-1␣, IL-1␤, TNF␣ and IL-6 were detected in LPS-stimulated Kupffer cells (Fig. 1) and the intensity of each band determined (Table 2). When Kupffer cells cultured in the presence of dexamethasone were stimulated with LPS, the transcript for IL-6 was no longer detectable, and the intensity of transcripts for IL-1␣, IL-1␤ and TNF␣ was reduced (Fig. 1). The transcript for IL-10 was only detected when dexamethasone was present. Transcripts for IL-3, IL-4, IL-5, TNF␤, IL-2 or IFN␥ were not detected in LPS-stimulated Kupffer cells. There was no difference between the cytokine profiles 4 h or 24 h after LPS stimulation. TNF␣, IL-1␤ and IL-10 protein levels measured by ELISA showed LPS-stimulated Kupffer cells produced 1941 ± 238.7 pg/ml of TNF␣ compared with 55.7 ± 9.8 pg/ml 24 h after stimulation when dexamTable 2 Density of cytokine transcripts produced by LPS-stimulated Kupffer cells Cytokine

IL-1␣ IL-1␤ IL-6 IL-10 TNF␣

4 h post-LPS

24 h post-LPS

−Dex

+Dex

−Dex

+Dex

16.6 32.0 5.0 0.0 8.6

0.8 3.8 0.0 0.3 0.6

4.7 16.5 0.2 0.0 1.9

0.6 3.6 0.0 0.4 0.6

Cytokine transcripts from Kupffer cells cultured in the presence or absence of dexamethasone (Dex) (Fig. 1), were quantified by densitometry and normalised against the density of GAPDH mRNA to account for loading differences. The results are presented as arbitrary units.

Fig. 1. Identification of cytokine transcripts from LPS-stimulated Kupffer cells. Kupffer cells were stimulated with 10 ␮g/ml LPS the day following isolation, and cultured for a further 4 h or 24 h in the presence or absence of dexamethasone. RNA was isolated and cytokine transcripts were identified by ribonuclease protection assay.

ethasone was present (n = 3, P < 0.01), a finding consistent with the decrease observed by RPA analysis. Transwell co-cultures showed high levels of IL-10 (1538 ± 386 pg/ml) 24 h after LPS-stimulation compared to control hepatocyte-only levels of 64 ± 12 pg/ml (n = 5, P < 0.01). The high levels of IL-10 in the co-cultures could come from Kupffer

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Table 3 Effect of cytokines on glucose production in cultured hepatocytes Culture condition

Glucose production (␮g glucose/(␮g DNA/90 min))

Control TNF␣ IL-1␤ IL-10 IL-1␤/IL-10 IL-1␤/IL-10/TNF␣ IL-1␤/IL-10/LPS

55.0 57.0 45.2 57.7 26.2 29.5 25.7

± ± ± ± ± ± ±

4.9 15.2 8.0 8.4 2.7∗ 2.3∗ 1.4∗

Rat hepatocytes were cultured for 24 h in the presence of cytokines and glucose production measured. The results are the mean ± S.E.M. of 3–9 different cell preparations. ∗ Significance is indicated as P < 0.001 from the control.

cells as well as hepatocytes. IL-1␤ levels were also elevated in the supernatants from LPS-stimulated co-cultures with 257 ± 29 pg/ml compared to control cultures which ranged from undetectable amounts (detection limit 50 pg/ml) to 130 pg/ml (n = 6). 3.3. A combination of IL-1β and IL-10 reduces glucose production TNF␣, IL-1␤ or IL-10-alone did not affect hepatocyte glucose production, whereas the combination of IL-1␤ and IL-10 depressed glucose production by 52% (P < 0.001, Table 3). There was no further reduction when either TNF␣ or LPS was added to this combination. 3.4. PEPCK is reduced following culture with cytokines or LPS-stimulated Kupffer cells

Fig. 2. Northern blotting to determine mRNA expression of gluconeogenic and glycolytic enzymes. Northern analysis was performed on cultured hepatocytes after exposure to Kupffer cell conditioned medium or a combination of IL-1␤ and IL-10 and the mRNA levels of PEPCK, LPK, FBPase, PFK, G-6-Pase, GK and GAPDH were measured in four different cultures and a representative membrane is shown. Table 4 Quantification of immunofluorescent staining of PEPCK and LPK in hepatocytes cultured under different conditions Culture condition

Expression of PEPCK mRNA (Fig. 2) was reduced by 38 ± 2% (n = 4, P < 0.005) of the control value when hepatocytes were cultured with LPS-stimulated Kupffer cell conditioned medium and reduced by 38± 4% (n = 4, P < 0.005) when cultured with a combination of IL-1␤ and IL-10. There were no alterations in the mRNA expression levels for the other gluconeogenic enzymes studied, FBPase and G-6-Pase, nor for the glycolytic enzymes LPK, PFK or GK after culturing hepatocytes with LPS-stimulated Kupffer cell conditioned medium or IL-1␤ and IL-10. Immunofluorescent staining showed there was heterogeneity of hepatocytes expressing PEPCK and LPK (Fig. 3). The level of fluorescence indi-

Immunofluorescent units (×103 per cell) PEPCK

Control Bt2 cAMP LPS-stimulated Kupffer cells IL-1␤/IL-10

307 413 227 138

± ± ± ±

3.8 6.4∗ 4.2∗ 4.0∗

LPK 176 181 183 173

± ± ± ±

5.2 3.4 3.5 4.2

Hepatocytes were cultured either alone (control), in the presence of Bt2 cAMP, with LPS-stimulated Kupffer cells, or with IL-1␤ and IL-10. Immunofluorescent staining was visualised by confocal microscopy and the amount of fluorescence per cell expressed as arbitrary immunofluorescent units × 103 . The results represent the mean ± S.E.M. of five fields of view per experiment, with four different experiments performed for Bt2 cAMP and LPS-stimulated Kupffer cells. The results for hepatocytes cultured with IL-1␤ and IL-10 are from three experiments. ∗ P < 0.0001 vs. control.

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S.T. Yerkovich et al. / The International Journal of Biochemistry & Cell Biology 36 (2004) 1462–1472 Table 5 Activity of PEPCK and PK following culture under different conditions Culture condition

PEPCK activity (mU/mg protein)

PK activity (U/mg protein)

Control Bt2 cAMP Kupffer cell conditioned medium IL-1␤ and IL-10

19.2 ± 4.0 28.0 ± 4.5∗ 11.0 ± 2.8∗

0.28 ± 0.04 0.19 ± 0.03 0.25 ± 0.03

9.3 ± 2.0∗

0.31 ± 0.05

Hepatocytes were cultured for 24 h either alone (control), in the presence of Bt2 cAMP, LPS-stimulated Kupffer cell conditioned medium, IL-1␤ and IL-10 and PEPCK and PK enzyme activities were measured. The results are shown as the mean ± S.E.M. of four different cell preparations each measured in duplicate. ∗ P < 0.05 vs. control.

zyme activity by 53% (P < 0.05). In contrast, the activity of L-PK was not altered by any of the culture conditions.

4. Discussion

Fig. 3. PEPCK and LPK immunofluorescent staining. Hepatocytes were cultured alone (control), in the presence of Bt2 cAMP, with LPS-stimulated Kupffer cells or with IL-1␤ and IL-10 and PEPCK and LPK were detected by immunofluorescent staining. Immunofluorescence was visualised by confocal microscopy, and a representative sample of the projection from a series of Z images, each separated by 1 ␮m is shown. Areas of white represent the brightest staining, while dark grey is the least intense staining. Magnification bar represents 50 ␮m.

cates that PEPCK expression was increased 35% (P < 0.0001) when the hepatocytes were cultured with Bt2 cAMP (Table 4). Co-culturing hepatocytes with LPS-stimulated Kupffer cells or culturing in the presence of IL-1␤ and IL-10 reduced the staining of PEPCK by 26% (P < 0.0001) and 55% (P < 0.0001), respectively. There was no difference in the average intensity of staining for LPK in hepatocytes cultured under different conditions (Table 4). The activity of PEPCK was significantly affected by all culture conditions (Table 5). Addition of Bt2 cAMP medium increased the activity by 47% (P < 0.05), while culturing with Kupffer cell conditioned medium reduced PEPCK enzyme activity by 42% (P < 0.05). The combination of IL-1␤ and IL-10 reduced the en-

The maintenance of a constant blood glucose level is fundamental to the survival of humans and other mammals. Sepsis is a condition where bacteria, or more specifically LPS, initiates a hypoglycaemic state via inhibition of hepatic glucose production. Cytokines are thought to be mediators of this response due to their greatly increased production during sepsis. This study was undertaken to establish if Kupffer cells, via cytokine production, are capable of suppressing hepatic glucose production in vitro. Kupffer cells are the resident macrophages and major cytokine producing cells of the liver. Kupffer cells have previously been shown to reduce albumin secretion (Itoh et al., 1994) and total protein synthesis (West et al., 1988) in cultured hepatocytes. This precedence of Kupffer cells mediating a metabolic response, together with their proximity to hepatocytes, prompted our interest in their role in altering hepatic glucose production. In the current study, we have shown that LPS-stimulated Kupffer cells directly inhibit hepatocellular glucose production through reduction of PEPCK activity. This effect is mediated by the combined effects of IL-1␤ and IL-10 on PEPCK gene expression and protein production. The majority of reports investigating the role of cytokines on gluconeogenesis have measured glucose

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production resulting from gluconeogenesis and glycogenolysis. Since IL-1␤ (Kanemaki et al., 1998) and IL-6 (Kanemaki et al., 1998; Ritchie, 1990) have been reported to initiate glycogenolysis in cultured rat hepatocytes, glucose levels do not always reflect the rate of gluconeogenesis. To overcome this limitation, we measured gluconeogenesis by the production of radio-labelled glucose from its radio-labelled precursor lactate. We show that co-culture of LPS-stimulated Kupffer cells with hepatocytes results in a 48% inhibition of gluconeogenesis. There was no effect on glucose production from lactate when LPS was added directly to hepatocyte cultures, or when hepatocytes were co-cultured with unstimulated Kupffer cells, suggesting that products of LPS-stimulated Kupffer cells were responsible for the reduced glucose production. IL-1␤ and IL-10 were identified as the major active agents produced by Kupffer cells at both the RNA and protein level, and their combination in culture fully accounted for the reduction in gluconeogenesis observed after LPS-stimulated Kupffer cell were co-cultured with hepatocytes (Table 3). This confirms and extends results from previous studies which suggest that a combination of cytokines is required to reduce either basal glucose production (Ceppi et al., 1996) or glucagon-stimulated glucose production (Stadler et al., 1995), while the addition of a single cytokine has no effect (Ceppi et al., 1996; Christ & Nath, 1996; Roh et al., 1986; Stadler et al., 1995). This may explain why single cytokines can lead to reduced gluconeogenesis in vivo but not in vitro, as there is a complex interaction between cytokines and it is possible that in vivo administered cytokines combine with other endogenous cytokines to elicit an effect. RPA analysis revealed that Kupffer cells expressed the pro-inflammatory cytokines IL-1␣, IL-1␤, TNF␣ and IL-6 in the absence of dexamethasone (Fig. 1). In this study, it was necessary to maintain the co-cultures in dexamethasone to retain hepatocyte viability and differentiation. Early sepsis is characterised by a hyperdynamic state with production of pro-inflammatory cytokines such as IL-1 and TNF␣, while more prolonged sepsis leads to enhanced IL-10 levels (Gogos, Drosou, Bassaris, & Skoutelis, 1991). Cytokines such as IL-1␤, and to a lesser extent IL-6 and TNF␣, can stimulate the hypothalamic–pituitary–adrenal axis resulting in increased glucocorticoid production in rats, mice and humans (Imure, Fukata, & Mori,

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1991; Naito et al., 1989). Glucocorticoids, such as dexamethasone, can then inhibit the production of IL-6 (Tobler et al., 1992), TNF␣ (Hoffman, Grewe, Estler, Schulze-Specking, & Decker, 1994) and IL-1␣ (Chensue, Terebuh, Remick, Scales, & Kunkel, 1991). Furthermore, IL-10 can inhibit the production of IL-6 (Knolle et al., 1997) and TNF␣ (de Waal-Malefyt, Abrams, Bennett, Figdor, & de Vries, 1991). This may have resulted in the detection of RNA transcripts only for IL-␣, IL-1␤, TNF␣ and IL-10, with IL-1␤ and IL-10 present at the protein level. This is the first report that an anti-inflammatory cytokine, in combination with a pro-inflammatory cytokine, decreases hepatic glucose production. IL-10 is an anti-inflammatory cytokine that has a variety of effects. In experimental sepsis, IL-10 has been shown to have a beneficial role (Emmanuilidis et al., 2001; Rongione et al., 2000) opposing the inflammatory responses, thereby decreasing the severity of sepsis and increasing survival. This has prompted several investigations into the use of this cytokine (Rogy et al., 1995; Xing, Ohkawara, Jordana, Graham, & Gauldie, 1997) as a potential therapy in sepsis. Our results suggest that caution should be exercised with this approach as increased levels of IL-10 is linked to reduced glucose production in vitro and could potentially lead to hypoglycaemia. Northern blotting identified PEPCK as the only enzyme with altered mRNA expression following culture with LPS-stimulated Kupffer cell conditioned medium or a combination of IL-1␤ and IL-10. Northern analysis and immunofluorescent staining demonstrated that down regulation of PEPCK gene expression and protein synthesis was responsible for the reduced PEPCK enzyme activity and glucose production induced by IL-1␤ and IL-10. Concurrently, there was no alteration to the opposing glycolytic enzyme, LPK. It is not surprising that PEPCK is implicated as the site of inhibition in the gluconeogenic pathway, as it is a key regulatory enzyme of this pathway, and similar findings in response to LPS have been reported in vivo (McCallum, Seale, & Stith, 1983) and in vitro (Ceppi et al., 1992; Horton et al., 1994). The mechanism underlying the effect of IL-1␤ and IL-10 on PEPCK gene expression is unclear. It is possible that one or both of the cytokines stimulate NF-␬B which in turn combines with cAMP response element (CRE) binding protein (CREB) and blocks

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PEPCK gene transcription. The CRE is critical in mediating both basal and cAMP-induced transcription (Liu, Park, Gurney, Roesler, & Hanson, 1991) with the CREB coordinating the action of these factors in regulating PEPCK transcription (Leahy, Crawford, Grossman, Gronostajski, & Hanson, 1999). NF-␬B has been linked with CREB (Waltner-Law, Daniels, Sutherland, & Granner, 2000) in inhibiting the increase in PEPCK gene transcription after treatment with glucocorticoids. This is especially relevant as both CREB and NF-␬B are increased during sepsis (Ye & Liu, 2001). This is a possible mechanism by which cytokines down regulate hepatic PEPCK expression and activity. Adding credence to this proposal is the fact that IL-1␤ can stimulate NF-␬B in liver derived cells (Hellerbrand, Jobin, Licato, Sartor, & Brenner, 1998). An alternative mechanism to explain the fall in PEPCK expression is the induction of PI3-kinase by IL-1␤ and IL-10. There is precedence for both IL-10 (Zhou et al., 2001) and IL-1␤ (Funakoshi, Sonoda, Tago, Tominaga, & Kasahara, 2000) in PI3-kinase stimulation. This pathway is capable of mediating the insulin-inhibition of PEPCK expression. Christ et al. (2000) have suggested that PI3-kinase and PKC are involved in the IL-6 mediated depression of gluconeogenesis and PEPCK expression after treatment with glucagon. Interestingly, IL-1␤ stimulates PI3-kinase which can subsequently activate NF-␬B (Funakoshi et al., 2000; Reddy, Huang, & Liao, 1997; Sizemore, Leung, & Stark, 1999). This highlights possible cross-talk in the signalling pathways and suggests several factors and pathways may be involved. In conclusion, we show that LPS stimulates Kupffer cells to produce a range of cytokines. In the context of glucose production, IL-1␤ and IL-10 are important, for they combine to depress hepatic PEPCK levels. This depressed enzyme expression is one of several possible mechanisms which result in impaired glucose production which results in hypoglycaemia observed during sepsis.

Acknowledgements The authors would like to thank Barbara Akhurst, Belinda Knight and Sharon Clarke for their technical assistance. This work was supported by grants from

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