Splanchnic Balance of Free Fatty Acids, Endocannabinoids, and Lipids in Subjects With Nonalcoholic Fatty Liver Disease

GASTROENTEROLOGY 2010;139:1961–1971

Splanchnic Balance of Free Fatty Acids, Endocannabinoids, and Lipids in Subjects With Nonalcoholic Fatty Liver Disease JUKKA WESTERBACKA,* ANNA KOTRONEN,*,‡,§ BARBARA A. FIELDING,储 JOHN WAHREN,¶ LEANNE HODSON,储 JULIA PERTTILÄ,‡ TUULIKKI SEPPÄNEN–LAAKSO,# TAPANI SUORTTI,# JOHANNA AROLA,** ROLF HULTCRANTZ,‡‡ SANDRA CASTILLO,# VESA M. OLKKONEN,‡ KEITH N. FRAYN,储 MATEJ OREŠICˇ,# and HANNELE YKI–JÄRVINEN* *Department of Medicine, Division of Diabetes, University of Helsinki, Helsinki, Finland; ‡Minerva Medical Research Institute, Helsinki, Finland; §Diabetes Prevention Unit, National Institute for Health and Welfare, Helsinki, Finland; 储Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, Oxford, United Kingdom; ¶Department of Surgical Sciences, Division of Clinical Physiology, Karolinska Hospital, Stockholm, Sweden; #VTT Technical Research Centre of Finland, Espoo, Finland; **Department of Pathology, University of Helsinki and HUSLAB, Helsinki, Finland; and ‡‡Department of Gastroenterology and Hepatology, Karolinska University Hospital, Stockholm, Sweden

BACKGROUND & AIMS: Animal studies suggest that endocannabinoids could contribute to the development of nonalcoholic fatty liver disease (NAFLD). In addition, NAFLD has been shown to be associated with multiple changes in lipid concentrations in liver biopsies. There are no data on splanchnic free fatty acid (FFA), glycerol, ketone body, endocannabinoid, and lipid fluxes in vivo in subjects with NAFLD. METHODS: We performed hepatic venous catheterization studies in combination with [2H2]palmitate infusion in the fasting state and during a low-dose insulin infusion in 9 subjects with various degrees of hepatic steatosis as determined using liver biopsy. Splanchnic balance of endocannabinoids and individual lipids was determined using ultra performance liquid chromatography coupled to mass spectrometry. RESULTS: Concentrations of the endocannabinoid 2-arachidonoylglycerol were higher in arterialized (91 ⫾ 33 ␮g/L basally) than in hepatic venous (51 ⫾ 19 ␮g/L; P ⬍ .05) plasma. Fasting arterial (r ⫽ 0.72; P ⫽ .031) and hepatic venous (r ⫽ 0.70; P ⫽ .037) concentrations of 2-arachidonoylglycerol were related positively to liver fat content. Analysis of fluxes of 85 different triglycerides showed that the fatty liver overproduces saturated triglycerides. In the plasma FFA fraction in the basal state, the relative amounts of palmitoleate and linoleate were lower and those of stearate and oleate were higher in the hepatic vein than in the artery. Absolute concentrations of all nontriglyceride lipids were comparable in arterialized venous plasma and the hepatic vein both in the basal and insulin-stimulated states. CONCLUSIONS: The human fatty liver takes up 2-arachidonoylglycerol and overproduces triacylglycerols containing saturated fatty acids, which might reflect increased de novo lipogenesis.

Keywords: Hepatic Steatosis; Hepatic Vein; Endocannabinoids; Lipolysis.

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ubjects who are abdominally obese and have the metabolic syndrome or type 2 diabetes, frequently have excess fat (⬎5.5%1) in the liver that is not a result of excess alcohol consumption or other known causes of steatosis (nonalcoholic fatty liver disease [NAFLD]).2–4 Regarding the origin of fatty acids in hepatic triglycerides, in the fasting state in normal subjects, up to 30% of plasma free fatty acids (FFAs), derived from lipolysis of adipose tissue,5 are taken up by the liver.6 A majority of FFAs are re-esterified into triglycerides (TGs) in the liver, and secreted in very low density lipoprotein particles.7,8 The liver also oxidizes fatty acids into carbon dioxide and ketone bodies.6 In human beings, the liver is the main site of hepatic de novo synthesis of fatty acids from acetyl CoA, a process called de novo lipogenesis.9,10 The latter process produces saturated fatty acids such as 16:0 and 18:0.9,10 De novo lipogenesis is increased in subjects with NAFLD compared with those without NAFLD.11,12 We recently showed that TGs containing saturated fatty acids are enriched in livers of subjects with NAFLD.13 We also have shown very low density lipoprotein particles to be relatively enriched with saturated fatty acids in insulin-resistant subjects.14 Whether the fatty liver indeed overproduces excess saturated fatty acids, however, is unknown. Endocannabinoids are endogenously derived arachidonic acid-chain– containing lipids that bind to endocannabinoid receptors types 1 and 2 (CB1 and CB2) and regulate energy balance, glucose, and lipid metabolism.15 The 2 most imAbbreviations used in this paper: 3-OHB, 3-hydroxybutyrate; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CB1, endocannabinoid receptor type 1; CB2, endocannabinoid receptor type 2; FFA, free fatty acid; ICG, indocyanine green; MS, mass spectrometry; NAFLD, nonalcoholic fatty liver disease; TG, triacylglycerol; UPLC, ultra performance liquid chromatography. © 2010 by the AGA Institute 0016-5085/$36.00 doi:10.1053/j.gastro.2010.06.064

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See related article, Suzuki A et al, on page 1062 in CGH.

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portant endocannabinoids are anandamide16 and 2-arachidonoylglycerol.17 Studies in mice have shown that lack of CB1 receptors protects against the development of obesity and a fatty liver.18 Furthermore, liver-specific deletion of CB1 results in similar high-fat diet–induced weight gain as in wild-type mice, but protects against hepatic steatosis, insulin resistance, and obesity-induced metabolic disturbances.19 CB1 activation in the liver increases the activity of the lipogenic transcription factor sterol regulatory element binding protein 1c and its target enzymes fatty acid synthase and acetyl coenzyme-A carboxylase-1, and de novo fatty acid synthesis.18 In wild-type mice fed a high-fat diet, hepatic levels of anandamide are increased compared with lean controls and CB1⫺/⫺ mice.18 It is unknown whether levels of endogenous endocannabinoids are associated with liver fat content in human beings, and whether the liver contributes to increased concentrations of circulating anandamide and 2-arachidonoyl glycerol reported in obese subjects.20 In the present study, we determined splanchnic fluxes of FFA, glycerol, ketone bodies, lipids, and endocannabinoids in vivo in subjects with a various degree of hepatic steatosis, verified by liver biopsy. The study subjects underwent hepatic venous catheterization studies in combination with infusion of [2H2]palmitate in the fasting state and during a low-dose insulin infusion. Splanchnic balance of individual lipids and endocannabinoids was determined using ultra performance liquid chromatography coupled to mass spectrometry (UPLC/MS).

Materials and Methods Subjects Characteristics of study subjects are shown in Table 1. All subjects were recruited among subjects referred to a gastro-

Table 1. Clinical Characteristics of the Study Subjects Mean ⫾ SEM n (women) Age, y Body composition Weight, kg BMI, kg/m2 Waist, cm Hip, cm Laboratory parameters fS-Triglycerides, mmol/L fS-HDL cholesterol, mmol/L fS-LDL cholesterol, mmol/L Plasma-albumin, g/L Serum-ALT, U/L Serum-AST, U/L S-␥-glutamyl transpeptidase, U/L Liver histology Liver fat, % Grades, 0–3 Stages, 0–4

9 (1) 54 ⫾ 5 93 ⫾ 4 29.6 ⫾ 1.1 105 ⫾ 3 100 ⫾ 2 2.6 ⫾ 1.0 1.21 ⫾ 0.11 3.4 ⫾ 0.3 39 ⫾ 1 96 ⫾ 16 62 ⫾ 12 172 ⫾ 66 52 ⫾ 26 1.2 ⫾ 0.4 1.8 ⫾ 0.4

BMI, body mass index; fS, fasting serum; HDL, highdensity lipoprotein; LDL, low-density lipoprotein; SEM, standard error of the mean.

enterologist because of increased liver function tests based on the following inclusion criteria: (1) age 18 to 75 years; (2) no known acute or chronic disease except for obesity based on history, physical examination, and standard laboratory tests (blood counts, serum creatinine, thyroid-stimulating hormone, and electrolyte concentrations) and electrocardiography; and (3) alcohol abuse. The following causes of liver disease were excluded: chronic hepatitis B or C, thyroid dysfunction, autoimmune hepatitis (smooth muscle and antinuclear antibodies), primary biliary cirrhosis (antimitochondrial antibodies), primary sclerosing cholangitis, use of hepatotoxic medications or herbal products, or use of medications known to be associated with steatohepatitis. Three subjects were receiving medications for hypertension. The nature and potential risks of the study were explained to all subjects before obtaining their written informed consent. The study protocol was approved by the ethics committee of the Karolinska Hospital. For ethical and technical reasons, it was not possible to perform a similar study in healthy volunteers.

Design The subjects were studied after an overnight fast. Three intravenous cannulas were inserted. The first was inserted in an antecubital vein for infusions of saline (first 90 min), glucose, insulin, and potassium-[2,22H ]palmitate. The second was inserted retrograde in a 2 contralateral arm in a heated dorsal hand vein. This hand was kept in a heated chamber (60°C) to obtain arterialized venous blood. The third catheter (Cournand) was inserted percutaneously under local anesthesia into a femoral vein and advanced under fluoroscopic control to a right-sided hepatic vein as previously described.21,22 Hepatic blood flow was measured using a constant intravenous infusion of indocyanine green (ICG).23 After 90 minutes of saline infusion, insulin was infused in a primed continuous (0.5 mU/kg/min) fashion as previously described.24,25 Plasma glucose was maintained at 5 mmol/L (90 mg/dL) until 240 minutes using a variable rate of infusion of 20% glucose. Potassium-[2,2-2H2]palmitate bound to human albumin was infused at a rate of 0.05 ␮mol/kg/min starting at 30 minutes to trace FFA and TG metabolism in vivo.

Splanchnic Blood Flow The ICG infusion23 was started at 30 minutes and continued at a rate of 0.04 ␮g/kg/min until 240 minutes, when the hepatic vein catheter was removed. Hepatic blood flow was measured at 90, 100, 110, 120, 200, 220, and 240 minutes. The concentration of ICG in arterialized and hepatic venous plasma was measured using high-pressure liquid chromatography as previously described.23 Splanchnic plasma flow was determined by dividing the rate of ICG infusion by the difference in concentration of ICG in arterialized and hepatic venous plasma. Estimated splanchnic blood flow was calculated by dividing splanchnic plasma flow by (1 - hematocrit).

Blood Sampling Before the saline infusion, blood samples were taken for measurement of fasting plasma glucose, TGs, low-density lipoprotein and high-density lipoprotein cholesterol, serum free insulin, liver enzyme (alanine aminotransferase [ALT], aspartate aminotransferase [AST], ␥-glutamyl transpeptidase), and albumin concentrations. During the saline and insulin infusions, arterialized and hepatic vein blood samples were taken at 10- to 60minute intervals to determine plasma glucose, insulin, FFA, glycerol, 3-hydroxybutyrate (3-OHB), lipid, and endocannabinoid concentrations.

Liver Histology The fat content of the liver biopsy specimens (% of hepatocytes with macrovesicular steatosis) was determined by an experienced liver pathologist (J.A.) in a blinded fashion. The percentage of macrovesicular steatosis was used as the liver fat percentage. NAFLD was determined as the liver fat percentage greater than 10%. Grading of the inflammatory changes and staging of the fibrotic changes was performed as described by Brunt et al.26

Lipidomic Analysis Plasma samples (10 ␮L) were spiked with a standard mixture consisting of 10 lipids (0.2 ␮g/sample) and mixed with 10 ␮L of 0.9% sodium chloride in Eppendorf tubes. Lipids were extracted with 100 ␮L of chloroform/ methanol (2:1) by vortexing for 2 minutes. After 1 hour standing the tubes were centrifuged at 10,000 rpm for 3 minutes and 60 ␮L of the lower organic phase was separated into a vial insert and mixed with a standard mixture containing 3 labeled lipids (0.1 ␮g/sample). The lipid extracts were run on a Waters Q-Tof Premier mass spectrometer (Waters Inc, Milford, MA) combined with an Acquity UPLC (Waters Inc) by using a solvent system including (1) water with 1% 1 mol/L NH4Ac and 0.1% HCOOH and (2) LC/MS grade acetonitrile/isopropanol (5:2) with 1% 1 mol/L NH4Ac, 0.1% HCOOH. The gradient from 65% A/35% B to 100% B lasted for 6 minutes, and the total run time including a 5-minute re-equilibration step was 18 minutes. An Acquity UPLC bridged ethylsiloxane-silica hybrid C18 1 ⫻ 50 mm column with 1.7-␮m particles was used at 50°C and at a flow rate of 0.200 mL/min. The lipid profiling was performed using electrospray ionization ⫹ mode, and the data were collected at a mass range of mass-to-charge ratio 300 –1200. Lipidomics data were processed using MZmine 2 software (available: http://mamine.sourceforge.net), which is an open-source project for metabolomics data processing, with the main focus on LC/MS data. The first step in the data processing was deconvolution of the peaks for each sample. MZmine 2 has several deconvolution algorithms to recognize the chromatographic peaks. The algorithm “local minimum search” was used in the present study. This peak recognition method searches for local

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minima in the chromatogram and considers a peak the region between 2 of these local minima. The next step was alignment of the peaks of all samples and filling the missing peaks. MZmine 2 software provides 2 different alignment methods, of which the “join aligner” was used in the present study. This method aligns the peaks in different samples through a match score. This score is calculated based on the mass and retention time of each peak and ranges of tolerance stipulated in the parameters of the program. The gap filling is performed by returning to raw data and checking again for the presence of corresponding peaks.27 Finally, identification of the peaks was performed using an internal LC/MS lipid library, and the data were normalized based on 5 internal standards.

Endocannabinoid Analyses A total of 450 ␮L acetonitrile, containing 200 mg/L butyl hydroxytoluene, was added to a sirocco plate (Waters Inc). Then 10 ␮L of internal standard solutions 1000 mg/L anandamide-d8 and 20 ␮L 5000 mg/L 2-arachidonoyl glycerol– d8 in acetonitrile were added. A 50-␮L serum sample was added and the sirocco plate was vortexed for 1 minute, after which time the samples were filtered with suction. Filtrate was kept at ⫺18°C for 1 hour and then dried with Fractovap (Carlo Erba, Milan, Italy). The dried samples were dissolved in 30 ␮L ethanol. The instrumentation consisted of a UPLC instrument connected to a micro ultra mass spectrometer. The data acquisition and treatment were performed by Masslynx 4.1 software (all from Waters Inc). An 8-␮L injection was performed to the bridged ethylsiloxanesilica hybrid C18 column (1 ⫻ 50 mm with 1.7-␮m particles) at 60°C. The eluents were 10 mmol/L formic acid in water (eluent A) and 10 mmol/L formic acid in methanol (eluent B). Linear gradient from 70% to 100% B was run during 10 minutes at a flow rate of 100 ␮L/min. The 2-arachidonoyl glycerol was detected with electrospray ionization⫺single reaction monitoring mode by monitoring mass-to-charge ratio 379 ¡287 (cone voltage, 22 V; collision energy, 15 eV). Anandamide was detected with electrospray ionization ⫹ single reaction monitoring mode by monitoring mass-to-charge ratio 348¡62 (cone voltage, 20 V; collision energy, 20 eV).

Hepatic Gene Expression One-half of the liver biopsy was output into RNAlater solution (Ambion, Foster City, CA) and stored in ⫺80°C until messenger RNA extraction. Liver tissue (1–13 mg) was homogenized in RNeasy Lysis Buffer, and total RNA was isolated and purified after DNase treatment using the RNeasy Micro Mini Kit (Qiagen, Hilden, Germany). RNA concentrations and the quality of RNA were measured using the NanoDrop microvolume spectrophotometer (Thermo Scientific, Waltham, MA). Average yields of total RNA were 2–10 ␮g/10 mg liver tissue weight. Liver biopsies from 2 patients did not yield a sufficient amount of RNA. Total RNA (1000 ng) was

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reverse-transcribed using the SuperScript VILO complementary DNA synthesis kit (Invitrogen, Carlsbad, CA). Each sample was amplified in duplicate for the CB1 and CB2 genes and the housekeeping marker 36B4 on a 7000 Sequence Detection System (Applied Biosystems, Carlsbad, CA) by using an SYBR-Green kit (Applied Biosystems). The relative quantification for the gene of interest was corrected to the 36B4 messenger RNA values. The following primers were used: CB1: 5=-AAGACCCTGGTCCTGATCCT-3= (sense) and 5=-CGCAGGTCCTTACTCCTCAG-3= (antisense); CB2: 5=-ATCATGTGGGTCCTCTCAGC-3= (sense) and 5=-GATTCCGGAAAAGAGGAAGG-3= (antisense); 36B4: 5=- CATGCTCAACATCTCCCCCTTCTCC-3= (sense) and 5=-GGGAAGGTGTAATCCGTCTCCACAG-3= (antisense).

Isotopic Enrichment Isotopic enrichment analyses were performed at 75, 80, 85, and 90 minutes basally, and at 180, 190, 200, and 210 minutes during the insulin infusion. Because of tech-

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nical reasons, we could not determine isotopic enrichment during the insulin infusion in 2 subjects. To determine specific fatty acid composition and isotopic enrichment, total lipids were extracted from plasma. Fatty acid methyl esters were prepared from FFA and TG fractions as previously described.28 Fatty acid compositions (mol %) in these fractions were determined by gas chromatography.29 Palmitate concentrations were calculated by multiplying the proportion of palmitate by the corresponding enzymatically determined plasma concentration. [2H2]Palmitate enrichments in the fatty acid methyl esters derivatives of plasma FFA and TG were determined by gas chromatography–MS as previously described.30

Analytic Procedures Plasma TG, glycerol, FFA, and 3-hydroxybutyrate concentrations were determined enzymatically using commercially available kits (Randox Laboratories Ltd, Crumin, UK; Alpha Laboratories Ltd, Eastleigh, Hampshire, UK) and

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Figure 1. Effect of insulin on whole- body and splanchnic FFA and 3-OHB production, and glycerol release. *P ⬍ .05; **P ⬍ .01.

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an ILab 600 Clinical Chemistry System (Instrumentation Laboratory, Warrington, UK). Plasma albumin concentration was determined using a colorimetric method, and serum ALT, AST, ␥-glutamyl transpeptidase, high-density lipoprotein cholesterol, and TG concentrations were determined with enzymatic methods recommended by International Federation of Clinical Chemistry using the Synchron LX system (Beckman Coulter Inc, Fullerton, CA). The concentrations of low-density lipoprotein cholesterol were calculated using the Friedewald et al31 formula.

Calculations Whole-body FFA rate of appearance was calculated by dividing the palmitate tracer infusion rate by the average plasma palmitate tracer-to-tracee ratio basally and during hyperinsulinemia. Net splanchnic production or release of metabolites was calculated from the difference between arterial and hepatic venous concentrations and multiplied by splanchnic plasma or blood flow as appropriate. Splanchnic lipolysis was calculated from the decrease in enrichment of labeled nonesterified palmitate across the splanchnic bed, essentially as described by Nielsen et al32 and multiplied by splanchnic plasma flow. Isotopic labeling of plasma TG did not reach equilibrium during the pre-insulin infusion period; therefore, isotopic calculations of TG production were not performed.

Statistical Methods The data are shown as means ⫾ standard error of the mean. Correlation analyses were performed using the Spearman nonparametric rank correlation coefficient. The paired t test was used to compare the differences in variables between arterialized and hepatic venous plasma. Calculations were made using GraphPad Prism software version 4.00 for Windows (GraphPad Software, San Diego, CA) and

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SPSS 16.0 for Windows (SPSS, Chicago, IL). A P value of less than .05 was considered statistically significant.

Results Splanchnic Turnover of FFA, 3-OHB, and Glycerol The arterial and hepatic venous concentrations and arterial venous differences of FFA, TG, 3-OHB, and glycerol are shown in Figure 1 and Table 2. There was net uptake of FFA across the splanchnic bed both in the basal state (P ⫽ .003) and during the insulin infusion (P ⫽ .023). Net extraction of FFA was 22% and 16% of FFA in the basal state and during hyperinsulinemia, respectively. Splanchnic FFA extraction decreased during the insulin infusion from 2.69 ⫾ 0.34 to 1.20 ⫾ 0.25 ␮mol/kg · min (P ⫽ .0014), mainly because of the diminished arterial concentrations (Table 2). Splanchnic lipolysis also was inhibited by insulin (Figure 1). Liver fat content did not correlate with splanchnic FFA extraction basally (r ⫽ 0.43; P ⫽ NS) but did so during hyperinsulinemia (r ⫽ 0.75; P ⫽ .05; Figure 2A). There was a net production of 3-OHB across the splanchnic bed in the basal state (P ⫽ .007) and during the insulin infusion (P ⫽ .013). Insulin suppressed splanchnic 3-OHB production by 78% (Figure 1). There was a net uptake of glycerol both in the basal state (P ⬍ .0001) and during the insulin infusion (P ⫽ .008) across the splanchnic bed. Splanchnic glycerol uptake was inhibited by insulin by 38% (Figure 1).

Splanchnic Turnover of Total and Individual TGs Total splanchnic TG concentrations were comparable in the artery and hepatic vein. Splanchnic TG production was 0.31 (range, 0.08 – 0.64) ␮mol/kg/min in the

Table 2. Splanchnic Turnover of FFAs, TGs, 3-OHB, and Glycerol in the Basal State and During Euglycemic Hyperinsulinemia

FFA Arterial FFA, ␮mol/L Hepatic venous FFA, ␮mol/L A-HV FFA difference, ␮mol/L Arterial FFA tracer, ␮mol/L Hepatic venous FFA tracer, ␮mol/L A-HV difference tracer, ␮mol/L TG Total arterial TG, ␮mol/L Total hepatic venous TG, ␮mol/L A-V difference, ␮mol/L 3-OHB Arterial 3-OHB, ␮mol/L Hepatic venous 3-OHB, ␮mol/L A-HV 3-OHB difference, ␮mol/L Glycerol Arterial glycerol, ␮mol/L Hepatic venous glycerol, ␮mol/L A-HV difference, ␮mol/L A-HV, arterial– hepatic venous.

Basal

Insulin

Paired t test

814 ⫾ 117 603 ⫾ 105 178 ⫾ 30 4.21 ⫾ 0.57 2.44 ⫾ 0.40 1.684 ⫾ 0.299

438 ⫾ 79 368 ⫾ 60 70 ⫾ 24 3.31 ⫾ 0.50 2.19 ⫾ 0.30 1.084 ⫾ 0.332

.0008 .004 ⬍.0001 .044 .20 .013

1550 ⫾ 260 1540 ⫾ 250 14.3 ⫾ 25.0

1700 ⫾ 270 1750 ⫾ 250 ⫺47 ⫾ 22

.76 .39 .044

288 ⫾ 114 426 ⫾ 149 ⫺138 ⫾ 38

70 ⫾ 26 98 ⫾ 30 ⫺22 ⫾ 7

.06 .048 .033

45 ⫾ 8 14 ⫾ 2 25 ⫾ 8

.001 .032 .011

89 ⫾ 11 21 ⫾ 5 68 ⫾ 9

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Figure 2. (A) The relationship between FFA extraction during hyperinsulinemia and liver fat content. (B) Correlation coefficients between splanchnic production of each of the 85 individual TGs and liver fat content were calculated. These correlation coefficients (Spearman rho) then were plotted against the number of double bonds in the respective TGs. An inverse relationship was observed, implying that the higher the liver fat, the less TGs, which contain many double bonds that are produced by the splanchnic region.

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basal state and 0.41 (range, 0.27– 0.61) ␮mol/kg/min during the insulin infusion (P ⫽ NS). By using UPLC-MS, we identified 85 different TGs in the artery and hepatic vein. The correlation coefficients between splanchnic production of each individual TG and liver fat content were calculated. These correlation coefficients were plotted against the total number of double bonds in the esterified fatty acids of the respective TGs, and to the total number of FA carbons in the respective TGs. An inverse relationship was observed between the correlation coefficient and the number of double bonds (r ⫽ ⫺0.41; P ⫽ .0001; Figure 2B) but not of carbons in the respective TGs, implying that the higher the liver fat, the more saturated TGs are produced by the splanchnic region.

Fatty Acid Composition of FFAs and TGs Across the Splanchnic Region Fatty acid composition of FFAs differed significantly between the artery and hepatic vein (Table 3, Supplementary Table 1). In the basal state, the relative amounts of 16:1n-7 and 18:2n-6 were lower and those of 18:0 and 18:1n-9 were higher in the hepatic vein than in the artery (Table 3). During the insulin infusion, the

relative amounts of 14:0 and 16:1n-7 were lower and those of 18:0 were higher in the hepatic vein than in the artery. The relative amounts of 16:0 and 18:1n-9 in FFA were similar in the hepatic vein and arterialized venous plasma. Absolute concentrations of different fatty acids decreased across the splanchnic bed (Supplementary Table 1). The 18:1n-9/18:0 ratio was higher in the artery (basal: 4.51 ⫾ 0.25; insulin-stimulated: 3.57 ⫾ 0.46) than in the hepatic vein (basal: 3.93 ⫾ 0.31; insulin-stimulated: 3.22 ⫾ 0.41; P ⫽ .0007 and P ⫽ .019). The 18:1n9/18:0 ratio was higher in the basal state than during the insulin infusion (P ⬍ .05 for both the artery and hepatic vein). Fatty acid composition of TG was comparable across the splanchnic bed both in the basal state and during the insulin infusion (Table 3, Supplementary Table 1).

Splanchnic Balance of Endocannabinoids Arterial and hepatic venous concentrations of anandamide were comparable in the basal state (0.30 ⫾ 0.03 and 0.19 ⫾ 0.05 ␮g/L) and during the insulin infusion (0.46 ⫾ 0.16 and 0.22 ⫾ 0.02 ␮g/L; Figure 3). Concentrations of 2-arachidonoylglycerol were higher in

Table 3. Fatty Acid Composition (%) of FFAs and TGs Across the Splanchnic Bed in the Basal State and During Euglycemic Hyperinsulinemia FFA

TG

Basal Arterial

Hepatic venous

14:0 1.66 ⫾ 0.26 1.44 ⫾ 0.23 16:0 25.82 ⫾ 0.69 25.24 ⫾ 0.1.05 16:1n-7 4.73 ⫾ 0.64 3.63 ⫾ 0.64b 18:0 9.92 ⫾ 0.55 12.01 ⫾ 0.88c 18:1n-9 43.65 ⫾ 0.39 45.10 ⫾ 0.78a 18:2n-6 12.12 ⫾ 0.78 10.62 ⫾ 0.66c

Insulin Arterial 2.16 ⫾ 0.43 25.35 ⫾ 1.09 4.69 ⫾ 0.80 11.56 ⫾ 1.13 42.19 ⫾ 1.01 12.01 ⫾ 0.77

Hepatic venous

Basal Arterial

1.79 ⫾ 0.37a 2.32 ⫾ 0.32 25.68 ⫾ 1.13 31.33 ⫾ 1.21 4.11 ⫾ 0.72a 4.83 ⫾ 0.55 12.66 ⫾ 1.07a 4.51 ⫾ 0.25 42.63 ⫾ 0.86 43.62 ⫾ 1.02 11.45 ⫾ 0.96 12.15 ⫾ 1.30

Insulin

Hepatic venous

Arterial

Hepatic venous

2.37 ⫾ 0.32 31.40 ⫾ 1.11 4.82 ⫾ 0.59 4.62 ⫾ 0.37 43.54 ⫾ 1.11 12.17 ⫾ 1.35

2.37 ⫾ 0.31 31.70 ⫾ 1.07 5.16 ⫾ 0.57 4.69 ⫾ 0.31 43.4 ⫾ 1.10 11.44 ⫾ 1.15

2.38 ⫾ 0.32 31.72 ⫾ 1.01 5.19 ⫾ 0.56 4.61 ⫾ 0.31 43.43 ⫾ 1.11 11.44 ⫾ 1.16

NOTE. Data are shown as mean ⫾ standard error of the mean. aP ⬍ .05; bP ⬍ .001; cP ⬍ .01 for differences between artery and hepatic vein (paired t test).

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Figure 3. Arterial and hepatic venous concentrations of (A) anandamide and (B) 2-arachidonoylglycerol (2-AG). The relationships between liver fat and (C) arterial and (D) hepatic venous concentrations of 2-AG. *P ⬍ .05.

the artery (91 ⫾ 33 and 92 ⫾ 30 ␮g/L basally and during the insulin infusion) than in the hepatic vein (51 ⫾ 19 and 46 ⫾ 13 ␮g/L basally and during the insulin infusion; Figure 3). Fasting arterial and hepatic venous concentrations of 2-arachidonoylglycerol were related positively to liver fat content (Figure 3). The net splanchnic uptake of 2-arachidonoylglycerol (mean ⫾ standard error of the mean, 18.2 ⫾ 7.1 ␮g/min) was not related to liver fat content (r ⫽ 0.23; P ⫽ NS). Hepatic expression of the CB1 receptor was related negatively to liver fat content (r ⫽ ⫺0.82; P ⫽ .023), whereas CB2 expression was not correlated significantly with liver fat content (r ⫽ ⫺0.39; P ⫽ NS). Arterial concentrations of 2-arachidonoylglycerol tended to be related negatively to hepatic CB1 expression (r ⫽ ⫺0.65; P ⫽ .11).

Splanchnic Turnover of Non-TG Lipids Absolute concentrations of all non-TG lipids were comparable in arterialized venous plasma and the hepatic vein both in the basal and insulin-stimulated states (Table 4).

Discussion This study examined splanchnic endocannabinoid and splanchnic lipid fluxes using UPLC-MS in human beings. Nine subjects referred to a gastroenterologist be-

cause of chronically increased liver function test results underwent hepatic venous catheterization in combination with infusion of [2H2]palmitate. We found that splanchnic FFA extraction during hyperinsulinemia, measured by tracer extraction, correlated with liver fat content. There was net uptake of 2-arachidonoylglycerol by the splanchnic region in human beings. Gene expression of hepatic CB1 receptor was correlated negatively with liver fat content. Increased liver fat also was found to be related to splanchnic overproduction of TGs containing saturated fatty acids. In healthy lean men, splanchnic uptake of plasma FFAs after an overnight fast has been estimated to be approximately 250 ␮mol/min.33 Fasting splanchnic FFA turnover rates are similar in normal healthy subjects and in subjects with hypertriglyceridemia.34 Splanchnic FFA turnover rates of the present study, measured either in a net sense or by tracer extraction, in subjects with NAFLD are comparable with those previously reported.33–36 This is consistent with the findings of large studies showing that, in the fasting state, serum FFA concentrations do not correlate with liver fat content,37 whereas there is a strong positive association between liver fat and FFA concentrations during euglycemic hyperinsulinemia.38,39 FFA derived from adipose tissue lipolysis are the major source of hepatic fatty acids in the livers of subjects with

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Table 4. Absolute Concentrations of Non-TG Lipids Across Splanchnic Region Basal

PC, ␮mol/L PC(e), ␮mol/L PE, ␮mol/L PE(e), ␮mol/L Lyso PC, ␮mol/L PS, ␮mol/L Sphingomyelins, ␮mol/L Ceramides, ␮mol/L Diacylglycerols, ␮mol/L

Insulin

Arterial

Hepatic venous

Arterial

Hepatic venous

1770 ⫾ 220 19.6 ⫾ 2.0 323 ⫾ 48 148 ⫾ 17 133 ⫾ 15 17.5 ⫾ 2.8 176 ⫾ 18 26.5 ⫾ 2.4 0.83 ⫾ 0.07

1770 ⫾ 120 18.9 ⫾ 1.3 327 ⫾ 15 141 ⫾ 10 131 ⫾ 7 18.8 ⫾ 2.2 166 ⫾ 11 26.5 ⫾ 1.8 0.77 ⫾ 0.07

1970 ⫾ 200 22.7 ⫾ 1.5 376 ⫾ 29 168 ⫾ 12 151 ⫾ 7 29.9 ⫾ 3.2 205 ⫾ 8 26.2 ⫾ 2.2 0.88 ⫾ 0.09

1830 ⫾ 150 18.5 ⫾ 1.8 337 ⫾ 22 145 ⫾ 14 148 ⫾ 11 18.8 ⫾ 2.6 169 ⫾ 10 26.0 ⫾ 2.5 0.74 ⫾ 0.10

NOTE. Data are shown as mean ⫾ standard error of the mean. PC, phosphatidylcholine; PC(e), ether-linked phosphatidylcholine; PE, phosphatidylethanolamine; PE(e), ether-linked phosphatidylethanolamine; Lyso PC, lysophosphatidylcholine; PS, phosphatidylserine.

CLINICAL–LIVER, PANCREAS, AND BILIARY TRACT

NAFLD.11 The present finding that hepatic FFA extraction is associated positively with liver fat content during hyperinsulinemia but not basally is consistent with these data and that insulin resistance of lipolysis contributes to hepatic fat accumulation.38,39 The net splanchnic arterial-hepatic venous difference of different FFAs is positive,40 as also found in the present study (Supplementary Table 1). However, splanchnic handling of different FFAs varies. The splanchnic uptake rate of oleic acid is 70% higher than that of stearic acid.41 On the other hand, splanchnic fractional uptake of arachidonic acid is 60% higher than that of oleic acid.42 Splanchnic fractional uptake of shorter-chain fatty acids is relatively high.40 We found the proportions of palmitoleic and linoleic acids to decrease and stearic and oleic acids to be higher in the fasting state in the hepatic vein as compared with the artery, based on net balance measurements. This is consistent with the data that splanchnic fractional uptakes of palmitoleic and linoleic acids are higher as compared with those of stearic and oleic acids.40 In contrast to FFA, fatty acid composition of TGs did not change across the splanchnic region (Table 3), which in turn allowed us to calculate splanchnic turnover rates of individual TGs applying stable isotope data. We previously have shown that TGs containing saturated fatty acids are enriched in the liver tissue of subjects with NAFLD.13 Consequently, there is a depletion of TGs containing long polyunsaturated fatty acids in the livers of subjects with NAFLD as compared with those without NAFLD.13 One hepatic vein catheterization study showed that in subjects with alcoholic fatty liver, the stimulation of splanchnic TG release by administering an anabolic steroid, norethandrolone, results in relatively greater release of saturated fatty acids,43 which most likely is attributed to accelerated de novo lipogenesis.9,10,43 Here we show that nonalcoholic fatty liver overproduces in vivo TGs containing saturated fatty acids. In subjects with NAFLD, this most likely is attributed to increased de novo lipogenesis.11

Recent studies in animals suggest that endocannabinoids may play an important role in the development of NAFLD. Hepatic steatosis can be reduced dramatically in genetically obese Zucker rats by CB1-receptor blockage.44 Recently, it has been shown that liver-specific deletion of CB1 protects against hepatic steatosis and related metabolic disturbances despite high-fat diet–induced obesity.19 In human beings, daily cannabis use increases the risk of hepatic steatosis in subjects with chronic hepatitis C infection independently of other confounding factors.45 In obese subjects (mean body mass index, 38.3 kg/m2), circulating concentrations of anandamide and 2-arachidonoylglycerol are 35% and 52% higher than in lean subjects (mean body mass index, 23.5 kg/m2).20 In the present study, in which all subjects were overweight (body mass index, ⱖ25 kg/m2 in all), we show that there is a net uptake of 2-arachidonoylglycerol by the splanchnic region. In addition, both arterial and hepatic venous concentrations of 2-arachidonoylglycerol were correlated positively with liver fat content (Figure 3). This is consistent with the only data available in human beings showing that circulating concentrations of 2-arachidonoylglycerol but not of anandamide are related positively to visceral obesity, serum TG level, and measurements of insulin sensitivity, including fasting insulin concentrations and glucose disposal, measured using the euglycemic hyperinsulinemic clamp technique, and correlated inversely with high-density lipoprotein cholesterol.46 All these parameters have been reported to be related to NAFLD.2 This study measured both endocannabinoid concentrations and endocannabinoid-receptor gene expression in the human liver in NAFLD. We found that hepatic expression of the CB1 receptor is significantly negatively correlated with liver fat content and almost significantly to arterial 2-arachidonoylglycerol concentrations. This resembles previously reported data in obese subjects showing that CB1-receptor gene expression is decreased in subcutaneous adipose tissue, whereas serum endocannabinoid concentrations are increased.20 The presence of

CB1 receptor in the human liver has been documented previously.47,48 Our data could be interpreted to suggest that circulating 2-arachidonoylglycerol enters the liver and acts at cannabinoid receptors. Although the origin of circulating endocannabinoids has not been determined, experimental data suggest that the mechanisms behind the chronic activation of the endocannabinoid system in obesity could include increased activity of endocannabinoid-synthesizing enzymes49 and increased dietary supply of fatty acid,18 which serve as endocannabinoid precursors. The observed positive correlation between plasma 2-arachidonoylglycerol concentrations and liver fat content and the negative correlation between hepatic CB1 expression and liver fat content suggests a functional role of endocannabinoids in NAFLD. Osei-Hyiaman et al18 found that in mice, activation of the endocannabinoid system in the liver leads to a 2-fold increase in hepatic fatty acid synthesis, measured using heavy water. The stimulation of CB1 induces activation of sterol regulatory element binding protein 1c,18 a key transcriptional activator of lipogenic genes.50 When treated with a synthetic CB1 agonist, the rate of de novo lipogenesis is lower in liver-specific CB1 knockout than in wild-type mice.19 Recently, hepatic gene expression of sterol regulatory element binding protein 1c and a number of lipogenic genes have been shown to be up-regulated in the livers of subjects with NAFLD compared with control subjects.51 In the present study, we found both higher circulating 2-arachidonoylglycerol concentrations and accelerated splanchnic production of TGs containing saturated fatty acids to associate with increased liver fat content in human beings. These data raise the possibility that the endocannabinoid system may contribute to the development of human NAFLD by affecting hepatic lipid metabolism. We did not find differences in absolute concentrations of non-TG lipids, such as various phospholipids or ceramides, in arterialized and hepatic venous plasma. In the present study, we did not use stable isotope tracers to study the splanchnic metabolism of these lipids, which are present in circulation in relatively small concentrations. Given the potentially important biological functions of these minor lipid species,52–54 there is a need to study their biology and metabolism in vivo in more detail. In conclusion, we found that there is a net uptake of 2-arachidonoylglycerol by the splanchnic region in human beings. Moreover, both increased concentrations of circulating 2-arachidonoylglycerol and splanchnic production of TGs containing saturated fatty acids are related positively to liver fat content. These data are consistent with increased de novo lipogenesis in the human fatty liver11 and suggest that endocannabinoids may regulate liver fat in NAFLD.

Supplementary Material Note: To access the supplementary material accompanying this article, visit the online version of

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Gastroenterology at www.gastrojournal.org, and at doi: 10.1053/j.gastro.2010.06.064. References 1. Szczepaniak LS, Nurenberg P, Leonard D, et al. Magnetic resonance spectroscopy to measure hepatic triglyceride content: prevalence of hepatic steatosis in the general population. Am J Physiol Endocrinol Metab 2005;288:E462–E468. 2. Kotronen A, Westerbacka J, Bergholm R, et al. Liver fat in the metabolic syndrome. J Clin Endocrinol Metab 2007;92:3490 – 3497. 3. Kotronen A, Juurinen L, Hakkarainen A, et al. Liver fat is increased in type 2 diabetic patients and underestimated by serum alanine aminotransferase compared with equally obese nondiabetic subjects. Diabetes Care 2008;31:165–169. 4. Neuschwander-Tetri BA, Caldwell SH. Nonalcoholic steatohepatitis: summary of an AASLD Single Topic Conference. Hepatology 2003;37:1202–1219. 5. Barrows BR, Parks EJ. Contributions of different fatty acid sources to very low-density lipoprotein-triacylglycerol in the fasted and fed states. J Clin Endocrinol Metab 2006;91:1446 –1452. 6. Sidossis LS, Mittendorfer B, Chinkes D, et al. Effect of hyperglycemia-hyperinsulinemia on whole body and regional fatty acid metabolism. Am J Physiol 1999;276:E427–E434. 7. Diraison F, Beylot M. Role of human liver lipogenesis and reesterification in triglycerides secretion and in FFA reesterification. Am J Physiol 1998;274:E321–E327. 8. Parks EJ, Krauss RM, Christiansen MP, et al. Effects of a low-fat, high-carbohydrate diet on VLDL-triglyceride assembly, production, and clearance. J Clin Invest 1999;104:1087–1096. 9. Aarsland A, Wolfe RR. Hepatic secretion of VLDL fatty acids during stimulated lipogenesis in men. J Lipid Res 1998;39: 1280 –1286. 10. Korchak HM. Regulation of hepatic lipogenesis. Tufts Folia Med 1962;8:134 –143. 11. Donnelly KL, Smith CI, Schwarzenberg SJ, et al. Sources of fatty acids stored in liver and secreted via lipoproteins in patients with nonalcoholic fatty liver disease. J Clin Invest 2005;115:1343– 1351. 12. Timlin MT, Parks EJ. Temporal pattern of de novo lipogenesis in the postprandial state in healthy men. Am J Clin Nutr 2005;81: 35– 42. 13. Kotronen A, Seppanen-Laakso T, Westerbacka J, et al. Hepatic stearoyl-CoA desaturase (SCD)-1 activity and diacylglycerol but not ceramide concentrations are increased in the nonalcoholic human fatty liver. Diabetes 2009;58:203–208. 14. Kotronen A, Velagapudi VR, Yetukuri L, et al. Serum saturated fatty acids containing triacylglycerols are better markers of insulin resistance than total serum triacylglycerol concentrations. Diabetologia 2009;52:684 – 690. 15. Di Marzo V. The endocannabinoid system in obesity and type 2 diabetes. Diabetologia 2008;51:1356 –1367. 16. Devane WA, Hanus L, Breuer A, et al. Isolation and structure of a brain constituent that binds to the cannabinoid receptor. Science 1992;258:1946 –1949. 17. Mechoulam R, Ben-Shabat S, Hanus L, et al. Identification of an endogenous 2-monoglyceride, present in canine gut, that binds to cannabinoid receptors. Biochem Pharmacol 1995;50:83–90. 18. Osei-Hyiaman D, DePetrillo M, Pacher P, et al. Endocannabinoid activation at hepatic CB1 receptors stimulates fatty acid synthesis and contributes to diet-induced obesity. J Clin Invest 2005; 115:1298 –1305. 19. Osei-Hyiaman D, Liu J, Zhou L, et al. Hepatic CB1 receptor is required for development of diet-induced steatosis, dyslipidemia, and insulin and leptin resistance in mice. J Clin Invest 2008;118: 3160 –3169.

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20. Engeli S, Bohnke J, Feldpausch M, et al. Activation of the peripheral endocannabinoid system in human obesity. Diabetes 2005; 54:2838 –2843. 21. Fernqvist-Forbes E, Ekberg K, Lindgren BF, et al. Splanchnic exchange of insulin-like growth factor binding protein-1 (IGFBP-1), IGF-I and acid-labile subunit (ALS) during normo- and hyper-insulinaemia in healthy subjects. Clin Endocrinol (Oxf) 1999;51:327– 332. 22. Brundin T, Aksnes AK, Wahren J. Whole body and splanchnic metabolic and circulatory effects of glucose during beta-adrenergic receptor inhibition. Am J Physiol 1997;272:E678 –E687. 23. Bradley SE, Ingelfinger FJ, Bradley GP, et al. The estimation of hepatic blood flow in man. J Clin Invest 1945;24:890 – 897. 24. Ryysy L, Hakkinen AM, Goto T, et al. Hepatic fat content and insulin action on free fatty acids and glucose metabolism rather than insulin absorption are associated with insulin requirements during insulin therapy in type 2 diabetic patients. Diabetes 2000; 49:749 –758. 25. DeFronzo RA, Tobin JD, Andres R. Glucose clamp technique: a method for quantifying insulin secretion and resistance. Am J Physiol 1979;237:E214 –E223. 26. Brunt EM, Janney CG, Di Bisceglie AM, et al. Nonalcoholic steatohepatitis: a proposal for grading and staging the histological lesions. Am J Gastroenterol 1999;94:2467–2474. 27. Katajamaa M, Miettinen J, Oresic M. MZmine: toolbox for processing and visualization of mass spectrometry based molecular profile data. Bioinformatics 2006;22:634 – 636. 28. Heath RB, Karpe F, Milne RW, et al. Selective partitioning of dietary fatty acids into the VLDL TG pool in the early postprandial period. J Lipid Res 2003;44:2065–2072. 29. Evans K, Burdge GC, Wootton SA, et al. Regulation of dietary fatty acid entrapment in subcutaneous adipose tissue and skeletal muscle. Diabetes 2002;51:2684 –2690. 30. Bickerton AS, Roberts R, Fielding BA, et al. Preferential uptake of dietary fatty acids in adipose tissue and muscle in the postprandial period. Diabetes 2007;56:168 –176. 31. Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin Chem 1972;18: 499 –502. 32. Nielsen S, Guo Z, Johnson CM, et al. Splanchnic lipolysis in human obesity. J Clin Invest 2004;113:1582–1588. 33. Boberg J, Carlson LA, Freyschuss U. Determination of splanchnic secretion rate of plasma triglycerides and of total and splanchnic turnover of plasma free fatty acids in man. Eur J Clin Invest 1972;2:123–132. 34. Boberg J, Carlson LA, Freyschuss U, et al. Splanchnic secretion rates of plasma triglycerides and total and splanchnic turnover of plasma free fatty acids in men with normo- and hypertriglyceridaemia. Eur J Clin Invest 1972;2:454 – 466. 35. Eaton RP, Berman M, Steinberg D. Kinetic studies of plasma free fatty acid and triglyceride metabolism in man. J Clin Invest 1969; 48:1560 –1579. 36. Havel RJ, Kane JP, Balasse EO, et al. Splanchnic metabolism of free fatty acids and production of triglycerides of very low density lipoproteins in normotriglyceridemic and hypertriglyceridemic humans. J Clin Invest 1970;49:2017–2035. 37. Seppala-Lindroos A, Vehkavaara S, Hakkinen AM, et al. Fat accumulation in the liver is associated with defects in insulin suppression of glucose production and serum free fatty acids independent of obesity in normal men. J Clin Endocrinol Metab 2002;87:3023–3028. 38. Kotronen A, Juurinen L, Tiikkainen M, et al. Increased liver fat, impaired insulin clearance, and hepatic and adipose tissue insulin resistance in type 2 diabetes. Gastroenterology 2008;135: 122–130.

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39. Korenblat KM, Fabbrini E, Mohammed BS, et al. Liver, muscle, and adipose tissue insulin action is directly related to intrahepatic triglyceride content in obese subjects. Gastroenterology 2008;134:1369 –1375. 40. Hagenfeldt L, Wahren J, Pernow B, et al. Uptake of individual free fatty acids by skeletal muscle and liver in man. J Clin Invest 1972;51:2324 –2330. 41. Hagenfeldt L, Wahren J. Turnover of plasma free stearic and oleic acids in resting and exercising human subjects. Metabolism 1975;24:1299 –1304. 42. Hagenfeldt L, Wahren J. Turnover of plasma-free arachidonic and oleic acids in resting and exercising human subjects. Metabolism 1975;24:799 – 806. 43. Mendenhall CL. Augmented release of hepatic triglycerides with anabolic steroids in patients with fatty liver. Am J Dig Dis 1974; 19:122–126. 44. Gary-Bobo M, Elachouri G, Gallas JF, et al. Rimonabant reduces obesity-associated hepatic steatosis and features of metabolic syndrome in obese Zucker fa/fa rats. Hepatology 2007;46:122– 129. 45. Hezode C, Zafrani ES, Roudot-Thoraval F, et al. Daily cannabis use: a novel risk factor of steatosis severity in patients with chronic hepatitis C. Gastroenterology 2008;134:432– 439. 46. Bluher M, Engeli S, Kloting N, et al. Dysregulation of the peripheral and adipose tissue endocannabinoid system in human abdominal obesity. Diabetes 2006;55:3053–3060. 47. Teixeira-Clerc F, Julien B, Grenard P, et al. CB1 cannabinoid receptor antagonism: a new strategy for the treatment of liver fibrosis. Nat Med 2006;12:671– 676. 48. Xu X, Liu Y, Huang S, et al. Overexpression of cannabinoid receptors CB1 and CB2 correlates with improved prognosis of patients with hepatocellular carcinoma. Cancer Genet Cytogenet 2006;171:31–38. 49. Di Marzo V, Goparaju SK, Wang L, et al. Leptin-regulated endocannabinoids are involved in maintaining food intake. Nature 2001;410:822– 825. 50. Miyazaki M, Dobrzyn A, Man WC, et al. Stearoyl-CoA desaturase 1 gene expression is necessary for fructose-mediated induction of lipogenic gene expression by sterol regulatory element-binding protein-1c-dependent and -independent mechanisms. J Biol Chem 2004;279:25164 –25171. 51. Higuchi N, Kato M, Shundo Y, et al. Liver X receptor in cooperation with SREBP-1c is a major lipid synthesis regulator in nonalcoholic fatty liver disease. Hepatol Res 2008;38:1122–1129. 52. Summers SA. Ceramides in insulin resistance and lipotoxicity. Prog Lipid Res 2006;45:42–72. 53. Zoeller RA, Grazia TJ, LaCamera P, et al. Increasing plasmalogen levels protects human endothelial cells during hypoxia. Am J Physiol Heart Circ Physiol 2002;283:H671–H679. 54. Kim JK, Fillmore JJ, Sunshine MJ, et al. PKC-theta knockout mice are protected from fat-induced insulin resistance. J Clin Invest 2004;114:823– 827.

Received February 10, 2010. Accepted June 24, 2010. Reprint requests Address requests for reprints to: Anna Kotronen, MB, PhD, Department of Medicine, Division of Diabetes, University of Helsinki, PO Box 700, Room C425B, FIN-00029 HUCH, Helsinki, Finland. e-mail: anna.kotronen@helsinki.fi; fax: (358) 9-471 71896. Acknowledgments The authors gratefully acknowledge Eva-Lena Forsberg, Alice Skogholm, Alison Ayres, Monika Jurkiewicz, Agneta Reinholdsson, Mia Urjansson, Katja Sohlo, and Laxman Yetukuri for excellent technical assistance, and the volunteers for their help.

Conflicts of interest The authors disclose no conflicts. Funding This study was supported by research grants from the Academy of Finland, the Sigrid Juselius Foundation, the Finnish Diabetes Research

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Foundation, Orion Foundation, Jalmari and Rauha Ahokas Foundation, Biomedicum Helsinki Foundation, and the Novo Nordisk Foundation. This work is part of the project “Hepatic and adipose tissue and functions in the metabolic syndrome” (www.hepadip.org), which is supported by the European Commission as an Integrated Project under the 6th Framework Programme (contract LSHM-CT-2005-018734).

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Supplementary Table 1. Absolute Concentrations (␮mol/L) of Fatty Acids in FFAs and TGs Across the Splanchnic Region in the Basal State and During Euglycemic Hyperinsulinemia FFA

TG

Basal

14:0 16:0 16:1n-7 18:0 18:1n-9 18:2n-6

Insulin

Basal

Insulin

Arterial

Hepatic venous

Arterial

Hepatic venous

Arterial

Hepatic venous

Arterial

Hepatic venous

16 ⫾ 4 213 ⫾ 32 44 ⫾ 10 77 ⫾ 9 356 ⫾ 52 92 ⫾ 10

11 ⫾ 164 ⫾ 30a 28 ⫾ 8c 71 ⫾ 9b 287 ⫾ 47c 64 ⫾ 9c

10 ⫾ 2 111 ⫾ 21 23 ⫾ 6 45 ⫾ 6 189 ⫾ 37 50 ⫾ 8

6⫾ 96 ⫾ 17 17 ⫾ 4b 43 ⫾ 6 160 ⫾ 28b 39 ⫾ 5b

41 ⫾ 11 507 ⫾ 100 81 ⫾ 19 74 ⫾ 16 666 ⫾ 105 168 ⫾ 18

42 ⫾ 12 502 ⫾ 98 80 ⫾ 19 76 ⫾ 17 657 ⫾ 100 166 ⫾ 17

44 ⫾ 11 555 ⫾ 103 93 ⫾ 20 84 ⫾ 19 727 ⫾ 109 179 ⫾ 21

45 ⫾ 11 570 ⫾ 102 96 ⫾ 20 85 ⫾ 18 747 ⫾ 107 184 ⫾ 20

4a

2a

NOTE. Data are shown as mean ⫾ standard error of the mean. aP ⬍ .01 bP ⱕ .05 for differences between the artery and hepatic vein (paired t test). cP ⬍ .001.