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Contribution of hepatic de novo lipogenesis and reesterification of plasma non esterified fatty acids to plasma triglyceride synthesis during non-alcoholic fatty liver disease
ORIGINAL
A R T I C L E
Contribution of hepatic de novo lipogenesis and reesterification of plasma non esterified fatty acids to plasma triglyceride synthesis during non-alcoholic fatty liver disease F Diraison1, Ph Moulin2, M Beylot1,
3
S UMMARY
R Eu S U M Eu
Background: Non-alcoholic fatty liver disease (NAFLD) is frequently observed in insulin-resistant subjects and can lead to liver fibrosis and cirrhosis. The abnormalities of lipid metabolism behind this development of excess hepatic TG stores are poorly understood. Methods: To clarify these mechanisms we measured triglyceride secretion rate and the contributions of hepatic lipogenesis and reesterification of non-esterified fatty acids (NEFA) to this secretion in healthy subjects and in patients with clear evidence of NAFLD. All subjects were studied in the post-absorptive state. Hepatic lipogenesis was measured with deuterated water. NEFA turnover rate, triglyceride secretion rate and the contribution of NEFA reesterification to this secretion were determined with [1-13C] palmitate infusion. Results: NAFLD patients had higher NEFA concentrations (p < 0.05) but normal NEFA turnover rates (5.23 ± 0.80 vs 5.91 ± 0.97 µmol.kg–1.min–1 in control subjects, ns). Despite a trend for higher plasma triglyceride levels in patients (p < 0.10), triglyceride turnover rates were not increased (0.11 ± 0.01 µmol.kg–1.min–1 in patients vs 0.14 ± 0.01 in controls, ns). However the contribution of hepatic lipogenesis to triglyceride secretion was largely increased in patients (14.9 ± 2.7 vs 4.6 ± 1.1% p < 0.01) while that of NEFA reesterification was reduced (25.1 ± 2.9 vs 52.8 ± 6.2% p < 0.01). Conclusion: Enhanced lipogenesis appears as a major abnormality of hepatic fatty metabolism in subjects with NAFLD. Therapeutic measures aimed at decreasing hepatic lipogenesis would therefore be the most appropriate in order to reduce hepatic TG synthesis and content in such patients.
Contribution de la lipogenèse hépatique de novo et de la réestérification des acides gras plasmatiques non estérifiés à la synthèse des triglycérides au cours de la stéatose hépatique non alcoolique
Key-words: Stable isotopes z Liver z Steatosis z Triglyceride z Insulin-resistance. Diraison F, Moulin Ph, Beylot M. Contribution of hepatic de novo lipogenesis and reesterification of plasma non esterified fatty acids to plasma triglyceride synthesis during non-alcoholic fatty liver disease Diabetes Metab 2003,29,478-85
1 2 3
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INSERM U 499, Faculté RTH Laennec, Lyon, France Department of Endocrinology, Hôpital Louis Pradel, Lyon, France Centre de Recherche en Nutrition Humaine, Hôpital E. Herriot, Lyon, France.
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Objectif : La stéatose hépatique non-alcoolique (NAFLD) est fréquente chez les sujets insulino-résistants et peut évoluer vers la fibrose et la cirrhose. Les anomalies du métabolisme lipidique responsables de cette stéatose sont mal comprises. Méthodes : Pour clarifier ces anomalies nous avons mesurer le débit de secrétion de triglycérides et les contributions de la lipogenèse hépatique et de la réestérification hépatique des acides gras non estérifiés (NEFA) à cette secrétion chez des sujets témoins et des sujets avec NAFLD. Tous les sujets ont été étudiés à l’état post-absorptif. La lipogènese hépatique a été mesurée à l’aide d’eau deutérée. Le débit de renouvellement des NEFA, le débit de secrétion des triglycérides et la contribution de la réestérification des NEFA à cette sécrétion ont été mesurés à l’aide de [1-13C] palmitate. Résultats : Les sujets avec stéatose avaient des concentrations de NEFA élévées (p < 0,05) mais un débit de renouvellement normal (5,23 ± 0,80 vs 5,91 ± 0,97 µmol.kg–1.min–1 chez les témoins, ns). Malgré des concentrations de triglycérides légèrement élevées (p < 0,10) leur débit de sécrétion de triglycérides était normal (0,11 ± 0,01 µmol.kg–1.min–1 vs 0,14 ± 0,01 chez les témoins, ns). Cependant la contribution de la lipogenèse à la sécrétion de triglycérides était très supérieure chez les sujets stéatosiques (14,9 ± 2,7 vs 4,6 ± 1,1 % p < 0,01) alors que celle de la réestérification des NEFA était diminuée (25,1 ± 2,9 vs 52,8 ± 6,2 % p < 0,01). Conclusion : Une augmentation de la lipogenèse apparait comme une anomalie majeure du métabolisme hépatique au cours de la stéatose. Des mesures thérapeutiques visant à normaliser cette lipogenèse pouraient donc permettre de réduire la stéatose hépatique non alcoolique. Mots-clés : Isotopes stables z Foie z Stéatose z Triglycérides z Insulino-résistance.
Address correspondence and reprint requests to: M Beylot. INSERM U499, Faculté RTH Laennec, rue G. Paradin, 69008, Lyon, France. [email protected] Received: February 13th, 2003; revised: June 2nd, 2003
Hepatic steatosis and tg metabolism
N
on-alcoholic fatty liver disease (NAFLD) is a condition characterized by excessive deposition of triglyceride (TG) in hepatocytes of subjects without a history of excessive alcohol consumption. The term NAFLD incorporates the full range of this condition, from steatosis alone to steatosis with inflammation (non-alcoholic hepatosteatitis, NASH) and possibly steatosis with necrosis and fibrosis ending up in cirrhosis [1, 2]. NAFLD is frequently observed in obese and in type 2 diabetic patients [3] but may occur also in insulino-resistant subjects without obesity or impaired glucose tolerance [4, 5]. It is considered as a part of the metabolic syndrome although it remains unclear whether it is a consequence of or a contributor to insulin-resistance. The abnormalities of lipid metabolism leading to NAFLD are poorly understood. The amount of TG present in the liver depends on one hand on hepatic TG synthesis and on the other on the balance between storage and secretion of TG by the liver. The mechanisms controlling these metabolic pathways are only partially known [6, 7], although it is clear that genetic [8] as well as metabolic and hormonal factors [9-11] are implicated. In particular, the contribution of the various sources of fatty acids for liver TG synthesis in subjects with NAFLD has not been determined. These fatty acids can be provided by hepatic lipogenesis (de novo lipogenesis, DNL), the uptake of plasma non esterified fatty acids (NEFA) by liver (reesterification) or the degradation of lipoproteins taken up by liver [7]. In addition, previously stored TG can be hydrolyzed [12] but this does not represent a primary source of fatty acids since these TG were previously synthesized with fatty acids provided by other sources. In normal subjects it is considered that DNL is a minor pathway and that the main pathway for liver TG synthesis is reesterification of NEFA [7, 13, 14]. It has been suggested that this reesterification pathway plays an important role in NAFLD since these subjects have a moderate increase in post-absorptive NEFA levels [4, 5] and lipolytic flux (as appreciated by glycerol turnover rate) and some resistance of lipolysis to the action of insulin [4, 5]. These abnormalities were observed independently of the presence of obesity or diabetes and suggest that NEFA delivery to the liver was increased. On the other hand, DNL is enhanced in insulinresistant state such as obesity [15] and type 2 diabetes [16] and could therefore contribute to the development of NAFLD. Determining the metabolic abnormalities responsible of NAFLD would help to choose the appropriate therapeutic target(s). Therefore we determined simultaneously by using stable isotopes tracers methodology TG turnover rate and the respective contribution of liver DNL and reesterification of NEFA to TG secretion in control subjects and in a group of patients with NAFLD.
Subjects and methods
Materials Tracers (all 99% mole per cent excess) were from Eurisotop (Saint Aubin, France) (2H2O, NaH13CO2) and Mass Trace (Woburn, MA, USA) (1-13C palmitate). Chemical and reactifs were from Sigma (St Louis MI, USA), Boehringer (Mannheim, Germany) or Pierce (Rockford, Illinois, USA).
Subjects Informed written consent was obtained from six healthy volunteers (control subjects, C, two women, four men, aged 22-48 years, body mass index 20-25, mean 22) and five patients with NAFLD (four women, one man, aged 29 to 64 years, body mass index 22-41, mean 29, p < 0.05 vs control subjects) without any history of alcohol consumption. All NAFLD subjects had clear evidence of large increase in TG liver stores as shown by physical examination, hepatic echography and biological data (elevation of plasma levels of gamma-glutamyl transferase, aspartate and alanine aminotransferases above the upper limit of normal values, (normal values: respectively 10 to 65, 10 to 45 and 10 to 65 U/L). One patient had type 2 diabetes and the others were insulinresistant with elevated post-absorptive concentrations of insulin associated with a moderate increase in plasma glucose (Tab I) and impairment of oral glucose tolerance despite a markedly increased insulin response. No control subject had a personal or familial history of diabetes or obesity or was taking any medication; all had normal plasma glucose, lipids, gamma-glutamyl transferase, aspartate and alanine aminotransferases levels. All subjects consumed a weight maintaining diet with at least 200g carbohydrate and abstained from alcohol consumption or heavy physical activity during the week before the study. Women were studied in the follicular period of menstrual cycle. The last meal was ingested between 19: 00 and 20: 00 PM the day before the tests.
Table I
Circulating concentrations of metabolites and hormones in post-absorptive control subjects and insulin-resistant patients (NAFLD).
Glucose (mmol/liter) NEFA (µmol/liter) TG (mmol/liter) Cholesterol (mmol/liter) Insulin (pmol/liter) Glucagon (ng/liter)
Control subjects n=6
NAFLD subjects n=5
4.5 ± 0.2 426 ± 44 0.85 ± 0.07 5.41 ± 0.21 47 ± 5 105 ± 16
6.6 ± 0.8 * 608 ± 52 * 1.04 ± 0.11 5.90 ± 0.16 116 ± 30 * 142 ± 17
* p < 0.05 vs control subjects. Diabetes Metab 2003,29,478-85 • © 2003 Masson, all rights reserved
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Protocols The protocol of the study was approved by the Ethical Committee of Lyon and by the Institut National de la Santé et de la Recherche Médicale, and the study was conducted according to the Hurriet law. All tests were performed in the Centre de Recherche en Nutrition Humaine de Lyon. In the evening before the test the subject drank a loading dose of deuterated water (3 g/kg body water, one-half after the evening meal and one-half at 10: 00 PM). Then until the end of the study, they drank only water enriched with 2H2O (4.5 g 2H2O/l of drinking water). All tests were initiated in the post-absorptive state, after an overnight fast. At 07: 30 AM indwelling catheters were placed in a forearm vein for tracer infusion and in a dorsal vein of the opposite hand, which was kept at 55°C, to obtain arterialized blood. After an initial blood sampling and collection of expired gas in the initial state, a bolus of NaH13CO2 (1.0 µmole/kg) was injected and a primed-continuous infusion of [1-13C] palmitate (0.03 µmoles.kg–1.min–1 after a bolus of 0.3 µmoles/kg) was initiated and continued for 4 h. Blood samples were collected at 60, 120, 180, 200, 220, and 240 min and during the six hours (240 to 600 min) after the interruption of tracer infusion. Expired gas samples were collected every hour, except for the period 180-300 min, when samples were collected every 15 minutes. Respiratory gas exchanges were measured from 120 to 240 min (Deltatrac, Datex, Helsinki, Finland). Urine samples were collected from 0 to 240 min for determination of nitrogen excretion.
Analytic procedures Metabolites were assayed with enzymatic methods [17, 18] on neutralized perchloric extracts of plasma (glucose) or on plasma (NEFA, TG). Plasma insulin [19] and glucagon [20] concentrations were determined by radioimmunoassay. After addition of heptadecanoic acid as internal standard for the determination of plasma palmitate concentration, plasma lipids were extracted by the method of Folch et al. [21]. TG and NEFA were separated by thin-layer chromatography and scraped off the silica plates [22]. The methylated (NEFA) and transmethylated (TG) derivatives of palmitate were prepared according to Morrison and Smith [23]. Total enrichment (i.e, 13C and deuterium) of the palmitate of NEFA and of TG was measured by gas chromatography-mass spectrometry, monitoring ions of m/z 270-273 [22]. Special care was taken to obtain comparable ion peak areas between standard and biological samples adjusting the volume injected or diluting the sample when necessary. Enrichment in 13C was selectively determined by gas chromatography-combustion-isotope ratio mass spectrometry [24]; this method allows accurate determination of 13C enrichment in the presence of very low enrichment level. Deuterium enrichment was calculated by subtracting 13C enrichment from the total (13C and deuterium) enrichment. Palmitate concentrations were calculated 480
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from the area ratio of the peaks corresponding to palmitate and to heptadocanoate. Standard curves containing known concentration ratios of these two fatty acids were run with the samples. Deuterium enrichment in plasma water and 13 C enrichment in expired CO2 were measured as previously described [25, 26].
Calculations Plasma palmitate turnover rate was calculated from its C enrichment measured during the 180 to 240 min period with steady state equation: Palmitate Rt = F/IE where F is the tracer infusion rate, Rt is turnover rate and IE the enrichment expressed in molar ratio (tracer/tracee). Plasma NEFA Rt was calculated from palmitate Rt and the ratio of NEFA to palmitate concentration as NEFARt = PalmitateRt* (NEFA concentration/palmitate concentration). TG kinetics were calculated from the decay of 13C enrichment in TG-palmitate after the end of the infusion of [1-13C] palmitate [14]. This approach is comparable to the one used with radioactive labeled tracers [27]. A single exponential was fitted to the data: IE = IE0e-kt, where IE is the isotopic enrichment and k is the fractional turnover rate (FRt). The half-life time of the plasma TG pool was then calculated as t1/2 = 0.693/FRt. The absolute TGRt was calculated as TGRt = FRt*M, where M is the plasma pool of TG obtained by multiplying the TG concentration by the plasma volume estimated to 45 ml/kg in subjects with normal body weight and 37 ml/kg in subjects with a BMI above 30 [28]. The fractional contribution of hepatic reesterification (FReest) of NEFA to the plasma TG pool was calculated from the 13C enrichment of plasma palmitate and the increase of 13C enrichment in TG-palmitate during the infusion of labeled palmitate: FReest=[(IEt2-IEt1)/(t2-t1)]/ IEpal, where t1 and t2 are the times when samples are taken, IEt are the corresponding IE of TG-palmitate, and IEpal is the 13C enrichment at plateau of plasma palmitate. The comparison of FReest with FRt gives the percent contribution of reesterification to TG turnover rate: Reest % = FReest/FRt. The absolute contribution of reesterification, Reest to the TG secretion is then Reest = Reest %.TG Rt. Three times Reest gives the rate of reesterification of plasma NEFA by the liver (Reest hep). The fractional contribution of lipogenesis to TGpalmitate (DNL%) was calculated from the deuterium enrichments in the palmitate of plasma TG and in plasma water, as previously described [22]. In short the deuterium enrichments that would have been obtained if endogenous synthesis were the only source of plasma TG-palmitate were calculated from plasma water enrichment; the comparison of the actual enrichments observed with these theatrical values gives the contribution of endogenous synthesis to the circulating pool of TG-palmitate. Absolute de novo lipogenesis is then obtained as DNL=DNL%*TGRt*3, where the 13
Hepatic steatosis and tg metabolism
factor 3 acknowledges the fact that there are three fatty acids per molecule of TG. Plasma palmitate oxidation was calculated as PalOx = F13CO2/(IE pal*fc) where F13CO2 is the excretion rate of labeled CO2 produced by palmitate oxidation and fc is the correction factor for incomplete label recovery in expired gas. PalOx was calculated using either the bicarbonate recovery factor (0.80) or the acetate factor measured by Sidossis et al. [29] that takes in account label exchange in Krebs cycle. Total NEFA oxidation was calculated from PalOx and the palmitate over total NEFA concentrations ratio. Total lipid oxidation rate was calculated from respiratory gas exchange by use of stoichiometric equations [30]. Total lipid oxidation was converted into fatty acid oxidation by assuming an average molecular weight of 860g [30] and multiplying by three since there are three fatty acids for one TG. Extracellular, whole body reesterification rate (Reexc) was calculated by subtracting from NEFA Rt oxidation rates obtained from either respiratory gas exchange or 13CO2 excretion rate. Subtracting REhep from Reexc gives the contribution of extrahepatic tissues to plasma NEFA reesterification. All results are shown as means ± SE. Comparisons between control and insulin-resistant subjects were performed with the Mann-Whitney test.
Results
Metabolites and hormones concentrations The concentrations in both groups of subjects of insulin, glucagon and metabolites during the 120 to 240 min period of the test are shown in Table I. NAFLD subjects had higher levels of glucose and insulin, as expected, and also of NEFA
(p<0.05 for all), than controls. In both groups NEFA levels were stable during this period of the study. The trend for higher TG concentrations in NAFLD did not reach significance (p < 0.20). NEFA rose then progressively during the following six hours to reach respectively 703 ± 62 and 692 ± 33 µmol/l at the end of the tests in normal and NAFLD subjects respectively. Plasma glucose levels decreased in NAFLD patients progressively to 4.55 ± 0.36 mmol/l at the end of the study whereas they remained unchanged in control subjects. Plasma TG levels remained stable throughout the study.
Plasma NEFA turnover and oxidation rates The evolution of the 13C enrichment in the palmitate of plasma TG and NEFA is shown in Figure 1. Stable levels of both enrichment and concentration were obtained in NEFA-palmitate and therefore kinetic parameters for NEFA were calculated using equation for steady-state conditions. Enrichment decreased then abruptly in NEFApalmitate when the infusion of tracer was stopped. NEFA Rt were similar in both groups when expressed relative to total body weight (5.23 ± 0.80 and 5.91 ± 0.97 (moles/kg/min in NAFLD and C subjects respectively, ns) or as absolute values per minute (404 ± 56 and 352 ± 56 (moles/min respectively, ns). Plateau levels of labeled carbon enrichment in the CO2 of expired gas were also obtained (data not shown). NEFA oxidation rates (NEFAox) calculated using either the bicarbonate factor or the acetate factor are compared in Table II with total lipid oxidation (Lox) determined from respiratory gas exchange data. This Lox was corrected for the measured hepatic lipogenesis to convert net Lox to total Lox rate. This correction was negligible in control subjects and remained small in NAFLD patients despite their higher
PALMI TATE EI (MPE)
2,4
1,8
1,2
0,6
0,0 0
120
240
360
TIME (MINUTES)
480
600
Figure 1 Evolution of the 13C enrichment of palmitate in the plasma NEFA of control (*) and NAFLD (Φ) subjects and in the plasma TG of control [7] and NAFLD [4] subjects, during the infusion of labelled palmitate (time 0 to 240 minutes) and after its interruption (time 240 to 600 minutes).
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Table II
Plasma NEFA (NEFAox) and total lipid (Lipid ox) oxidation rates. Control subjects n=6
NAFLD patients n=5
3.12 ± 0.45
2.52 ± 0.56
Lipid ox µmol/kg/min NEFA ox: µmol/kg/min “non-NEFA” ox µmol/kg/min
Bicarbonate acetate Bicarbonate acetate 1.84 ± 0.43 2.62 ± 0.61 1.61 ± 0.31 2.31 ± 0.42 Bicarbonate acetate Bicarbonate acetate 1.29 ± 0.30 0.53 ± 0.38 0.90 ± 0.36 0.24 ± 0.30
NEFA oxidation rates were calculated using either the bicarbonate or the acetate factor.
liver lipogenic activity (see next paragraph). Whatever the way lipid oxidation rates were calculated, there was no significant difference between control and NAFLD subjects. Use of the acetate factor raised the contribution of NEFA oxidation to NEFA Rt from 30.7 ± 2.9% to 43.9 ± 4.1% in controls and from 30.6 ± 4.1% to 43.7 ± 5.8% in NAFLD patients. The contribution of NEFA oxidation to total lipid oxidation was raised respectively from 59% to 85% and from 68% to 100%. Therefore the “non-NEFA” oxidation rate (considered to be the oxidation of fatty acids from tissues or plasma TG) was low (0.53 µmoles/kg/min) in controls and negligible in NAFLD patients.
Plasma TG kinetics and contributions of NEFA reesterification and lipogenesis to TG synthesis The kinetics of plasma TG and the respective contributions of lipogenesis and plasma NEFA reesterification to TG secretion rate are shown in Table III. The contributions of plasma NEFA reesterification were calculated from the increase of 13C enrichment in TG-palmitate during the 4 hour infusion of labeled palmitate. In both groups this rise was linear with time (Fig 1) and, calculated from the intercept of the regression line with the time axis, there was a
Table III
Kinetic parameters of plasma TG and contribution of hepatic reesterification of plasma NEFA and lipogenesis to TG synthesis.
FRt h–1 Half-life h Rt µmol.kg–1.min–1 Freest h–1 Reesterification % of Rt Lipogenesis % of Rt
Control subjects n=6
NAFLD subjects n=5
0.21 ± 0.02 3.54 ± 0.32 0.14 ± 0.01 0.112 ± 0.014 52.8 ± 6.2 4.6 ± 1.1
0.18 ± 0.01 3.80 ± 0.19 0.11 ± 0.01 0.046 ± 0.007 ** 25.1 ± 2.9 ** 14.9 ± 2.7 **
** p < 0.01 vs control subjects. FRt: fractional turnover rate. Freest: fractional contribution of plasma NEFA reesterification to plasma TG pool.
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similar delay (about 30 minutes) between initiation of the tracer infusion and the appearance of 13C in TG-palmitate. Despite comparable 13C enrichment in plasma NEFApalmitate the slope of the rise of 13C enrichment in TGpalmitate was much lower in NAFLD than in controls and thus the fractional contribution of reesterification (FReest) to the TG circulating pool was decreased in NAFLD (0.046 ± 0.007/h vs 0.112 ± 0.014/h p < 0.01). There was also a 30 minutes delay between the end of labeled palmitate infusion and the beginning of the decline of 13C enrichment in plasma TG-palmitate. The half-life and FRt of plasma TG were similar in the two groups as well as the absolute Rt, expressed relative to body weight (Tab III) or as µmol/min (9.61 ± 0.96 and 8.65 ± 0.62 in control and NAFLD subjects respectively, ns). Comparison of FReest to FRt shows that reesterification contributed about 50% to TGRt in controls and only 25% (p < 0.01 vs controls) in NAFLD. Deuterium enrichments in plasma water were stable throughout the tests in both groups and were respectively 0.34 ± 0.01 and 0.45 ± 0.04% in control and NAFLD subjects. The calculated contribution of lipogenesis to TG secretion was three time higher in the NAFLD than in the control subjects (14.9 ± 2.7 vs 4.6 ± 1.1% respectively p < 0.05) (Tab III) when measured during the 120-240 minutes period. This contribution decreased to 2.1 ± 0.6% at the end of the test in control subjects (p < 0.05 vs initial value) but remained to 15.9 ± 2.8% in NAFLD. These contributions of lipogenesis during the 120-240 min period represented absolute synthetic rates of fatty acids of 0.021 ± 0.005 and 0.056 ± 0.018 µmol.kg–1.min–1 in control and NAFLD subjects respectively (p < 0.05). Addition of the contributions of lipogenesis and plasma NEFA reesterification shows that about 45% and 60% of the TG secretion rates were not accounted for by these metabolic pathways in control and NAFLD subjects respectively. The contribution of hepatic reesterification to plasma NEFA utilization were calculated to be 0.22 ± 0.02 µmol.kg–1.min–1 in controls and 0.09 ± 0.02 µmol.kg–1.min–1 in NAFLD (p < 0.05 vs controls) and appeared in both groups as a minor contributor to the whole body utilization of NEFA. Thus, liver reesterification of NEFA appears as a minor contributor to total NEFA reesterification.
Discussion We investigated in the present report several aspects of plasma NEFA and TG metabolism in control subjects and in patients with NAFLD. We found no difference in TG kinetics but there was large differences in the source of fatty acids used for the synthesis of TG secreted by the liver: the contribution of DNL was markedly increased in NAFLD while that of plasma NEFA reesterification was decreased. In addition, DNL remained in NAFLD elevated during the prolongation of the fasting period imposed by the experimental protocol.
Hepatic steatosis and tg metabolism
We used for calculating plasma TG kinetic parameters a non-compartmental analysis of the decay curve of 13C enrichment in the palmitate of plasma TG. This approach has been criticized since, contrary to compartmental modeling analysis, it does not allow to resolve the possible tracer recycling through hepatic lipids pool, i.e. release in plasma TG, after stopping the infusion of labelled palmitate, of labelled TG stored during the infusion of the tracer [31]. This release would slow down the decay curve of enrichment in plasma TG and could result in underestimates of TG Rt, although comparable results were found using the compartmental and non-compartmental approach by Lemieux et al. [32]. Compartmental analysis requests to follow the decay curve during a long period (12 hours at least) and this requires initial enrichment levels much higher than the one obtained in the present study. This would have needed a high infusion rate of labelled palmitate which was precluded by ethical consideration (high infusion rate of human albumin) and would have anyway pertubated the pool of plasma palmitate. Lastly, the discrepancy between TG kinetic parameters determined by using compartmental or non-compartmental analysis is marked for TG turnover rates above 0.3 pool/ hour [31], value above those found in the present study. Thus, although we cannot exclude some underestimation of the TG Rt values we found, there is no evidence for an increased TG Rt in the group of NAFLD we studied. Actually this could suggest that these subjects have a preferential orientation of hepatic TG metabolism toward storage rather than secretion. Raised insulin levels could play a role in this orientation. In vitro studies showed that, whereas glucose stimulates both TG synthesis and secretion, insulin stimulates synthesis but decreases secretion resulting in accumulation of stored TG [33]. Short term studies in humans showed also an inhibitory action of insulin on TG secretion, even when the insulin-induced decrease in plasma NEFA concentration was prevented [13]. In addition prolonged hyperinsulinemia could decrease the expression and activity of the microsomal triglyceride transfert protein (MTP) [34] which is necessary for the secretion of TG [35]. It is also possible that subjects prone to the development of NAFLD have genetic variants of MTP [8] resulting in decreased expression and activity of this protein. DNL was markedly increased in NAFLD patients and did not decrease in the fasting state contrary to control subjects. Comparable stimulations of lipogenesis were previously reported in insulin-resistant and obese subjects [15, 36]. Therefore this abnormality is not specific of patients with steatosis but the present results strongly support a role for enhanced DNL in the develoment of NAFLD. This enhanced DNL results probably of the raised insulin levels found in NAFLD subjects, given the known effects of insulin on the expression and activity of the lipogenic pathway in liver [37]. These effects of insulin are probably mediated through the transcription factor SREBP-1c since it plays an
important role in controlling the expression of lipogenic genes in livers and both its mRNA and the nuclear protein levels are increased in mouse models of hepatic steatosis [38]. The decreased contribution of NEFA reesterification appears surprising since plasma NEFA are a major source of fatty acids for liver TG synthesis [7, 13, 14] and NEFA concentrations were increased in NAFLD subjects. Moreover insulin diverts hepatic fatty acids metabolism away from oxidation and toward reesterification [39] It should be kept in mind that we used for the calculation of the contribution of NEFA reesterification to TG secretion the 13C enrichment of NEFA-palmitate in arterial, not portal venous, plasma. There is evidence that the decrease in NEFApalmitate enrichment between arterial and portal venous plasma is low, around 10%, in normal subjects [40, 41] and that therefore the underestimation in the contribution of reesterification is probably low in these subjects. It is possible that the direct release of NEFA from intra-abdominal adipose tissue into the portal vein was higher in NAFLD subjects. This would result in a more important dilution of 13C NEFA-palmitate enrichment in portal venous plasma and thus in a greater underestimate of the contribution of reesterification to TG secretion. Actually this is indirect evidence that this dilution, and therefore the underestimation of NEFA reesterification, could be only around 20% in obese subjects [40]. This lack of stimulation of hepatic reesterification of NEFA in patients with NAFLD could be an aspect of insulin-resistance. Actually there is evidence that hepatic oxidation of NEFA is increased in NAFLD disease [5], and could contribute to the evolution to non-alcoholic steatohepatitis. Further studies of obese and/or insulin-resistant subjects without hepatic steatosis would help to deteremine if this modification of liver fatty acid metabolism is specific or not of subjects with NAFLD. It is also noteworthy that we found at the whole body level no modification of the repartition of NEFA utilization between oxidation and reesterification in NAFLD subjects although it has been clearly shown in control subjects that insulin diverts muscle NEFA metabolism away from oxidation and to reesterification [42]. Lipogenesis and NEFA reesterification accounted only for 55% and 40% of the TG secreted in control and NAFLD subjects respectively. Although reesterification may have been somewhat underestimated, particularly in NAFLD, these data agree with in vitro studies showing that 50-60% of the TG secreted by hepatocytes are provided by sources other than DNL and NEFA uptake [11, 12]. Other possible sources are the degradation of lipoproteins taken up by liver and of TG previously stored in cytosol (recycling) [6, 7]. The higher percentage of TG secretion not accounted for by DNL and NEFA reesterification in NAFLD could be consistent with a more important contribution of the increased cytosolic TG stores. These TG stores must be hydrolyzed by a specific lipase [43]. The fatty acids released are then used for the synthesis of new TG which can be secreted or stored Diabetes Metab 2003,29,478-85 • © 2003 Masson, all rights reserved
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again [12]. Therefore it is possible that in both groups of subjects a part of the TG synthesized with fatty acids provided by DNL and NEFA uptake was directed towards storage in order to replace stored TG which were hydrolyzed. This process would result in a dilution by the fatty acids released from the stored TG of both 13C and deuterium enrichment in the TG secreted by the liver during the period of tracer administration. We sampled only, for obvious ethical reasons, the circulating TG pool and we may therefore have underestimated the true rates of DNL and NEFA reesterification. If we assume that these metabolic pathways contributed also to the maintain of TG stores, i.e. if we normalize their total contribution to TG secretion to 100%, we obtain the upper limits for the estimates of these metabolic rates. This would give in control subjects lipogenic and reesterification rates of fatty acids of 0.038 and 0.40 µmol.kg–1.min–1 instead of 0.021 and 0.22 respectively. In NAFLD the corresponding lipogenic and reesterification rates would be 0.14 and 0.22 µmol.kg–1.min–1 instead of 0.056 and 0.09. This does not modify the differences between the two groups, i.e. increased lipogenic and decreased NEFA reesterification rate in NAFLD. In conclusion we found that a group of subjects with NAFLD had an increased contribution of liver lipogenesis to liver TG synthesis while that of plasma NEFA reesterification appeared decreased. These results support the idea that nutritional or pharmacological interventions aimed at decreasing liver lipogenesis, such as non-digestible carbohydrates [44] or fibrates [16], would be the most appropriate for reducing hepatic TG synthesis and content in these patients.
8.
Acknowledgments − We thank all the subjects who volunteered for this study, J Peyrat for her help during the realization of the tests. This work was supported in part by grants from the Fondation de France, the ALFEDIAM and Association Française de Diabétiques and by the European Economic Community (Contrat Nutrigen FAIR CT97-3011).
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Bernard S, Touzet S, Personne I, et al. Association between microsomal triglyceride transfer protein gene polymorphism and the biological features of liver steatosis in patients with type II diabetes. Diabetologia, 2000, 43, 995-9. Duerden JM, Gibbons GF. Secretion and storage of newly synthesized hepatic triacylglycerol fatty acids in vivo in different nutritional states and in diabetes. Biochem J, 1998, 255, 929-35. Francone OL, Griffaton G, Kalopissis A-D. Effect of a high-fat diet on the incorporation of stored triacylglycerol into hepatic VLDL. Am J Physiol, 1992, 263, E615-23. Gibbons GF, Burnham FJ. Effect of nutritional state on the utilization of fatty acids for hepatic triacylglycerol synthesis and secretion as verylow-density lipoprotein. Biochem J, 1991, 275, 87-92. Wiggins D, Gibbons GF. The lipolysis/esterification cycle of hepatic triacylglycerol. Biochem J, 1992, 284, 457-62. Lewis G, Uffelman K, Szeto L, Weller B, Steiner G. Interaction between free fatty acids and insulin in the acute control of VLDL production in humans. J Clin Invest, 1995, 96, 135-40. Diraison F, Beylot M. Role of human liver lipogenesis and reesterification in triglycerides secretion and in FFA reesterification. Am J Physiol, 1998, 274, E321-7. Diraison F, Dusserre E, Vidal H, Sothier M, Beylot M. Increased hepatic lipogenesis but decreased expression of lipogenic gene in adipose tissue in human obesity. Am J Physiol, 2002, 282, E46-51. Forcheron F, Cachefo A, Thevenon S, Pinteur C, Beylot M. Mechanisms of the triglyceride and cholesterol-lowering effect of fenofibrate in hyperlipidemic type 2 diabetic patients. Diabetes, 2002, 51, 3486-91. Beylot M, Riou F, Bienvenu F, Mornex R. Increased ketonaemia in hyperthyroidism; evidence for a beta-adrenergic mechanism. Diabetologia, 1980, 19, 505-10. Beylot M, Martin C, Laville M, Riou JP, Cohen R, Mornex R. Lipolytic and ketogenic flux in hyperthyroidism. J Clin Endocrinol Metab, 1991, 73, 42-9. Hales CM, Randle PJ. Immunoassay of insulin with insulin antibody precipitate. Biochem J, 1963, 56, 136-42. Harris V, Faloona G, Unger R. Glucagon. In: Jaffe BM, and H.R. Behrman, ed. Methods of radioimmunoassay. New York: Academic Press, 1979, 643-71. Folch J, Lees M, Sloane Stanley GH. A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem, 1957, 226, 497-509. Diraison F, Pachiaudi C, Beylot M. Measuring lipogenesis and cholesterol synthesis in humans with deuterated water: use of simple gas chromatography mass spectrometry techniques. J Mass Spectrom, 1997, 32, 81-6. Morrison WR, Smith L. Preparation of fatty acid methyl esters and dimethylacetals from lipids with boron fluoride-methanol. J Lipid Res, 1964, 5, 600-8. Binnert C, Laville M, Pachiaudi C, Rigalleau V, Beylot M. Use of gas chromatography isotope ratio mass spectrometry to study triglycerides metabolism in humans. Lipids, 1995, 30, 869-73. Yang D, Diraison F, Beylot M, et al. Assay of low deuterium enrichment of water by isotopic exchange with [U-13C] acetone and gas chromatography mass spectrometry. Anal Biochem, 1998, 258, 315-21. Guilly RE, Billion-Rey E, Pachiaudi C, et al. On-line purification and carbon 13 isotopic analysis of carbon dioxide in breath: evaluation of on-line gas chromatography isotope ratio mass spectrometry. Anal Chim Acta, 1992, 259, 193-202. Taskinen MR, Beltz WF, Harper I, et al. Effects of non-insulindependent diabetes on VLDL triglycerides and apolipoprotein B metabolism. Studies before and after sulfonyurea therapy. Diabetes, 1986, 35, 1268-77.
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28. Howard B, Reitman J, Vasquez B, Zezch L. Veryl low density lipoprtein triglyceride metabolism in non-insulin-dependent diabetes mellitus. Relationship to plasma isulin and free fatty acids. Diabetes, 1983, 33, 271-6. 29. Sidossis LS, Coggan AR, Gastaldelli A, Wolfe RR. A new correction factor for use in tracer estimations of plasma fatty acid oxidation. Am J Physiol, 1995, 269, E649-56. 30. Frayn KN. Calculation of substrate oxidation rates in vivo from gaseous exhange. J Appl Physio, 1983, 55, 628-34. 31. Patterson B, Mittendorfer B, Elias N, Satyanarayana R, Klein S. Use of stable isotopically labeled tracers to measure very low density lipoprotein-triglyceride turnover. J Lipid Res, 2002, 43, 223-33. 32. Lemieux S, Patterson B, Carpentier A, Lewis G, Steiner G. A stable isotope method using a [2-H5] glycerol bolus to measure very low density lipoprotein triglyceride kinetics in humans. J Lipid Res, 1999, 40, 2111-7. 33. Durrington PN, Newton RS, Weinstein DB, Steinberg D. Effects of insulin and glucose on very low density lipoprotein secretion by cultured rat hepatocytes. J Clin Invest, 1982, 70, 63-73. 34. Lin MCM, Gordon D, Wettereau JR. Microsomal triglyceride transfer protein (MTP) regulation in HepG2 cells: insulin negatively regulates MTP gene expression. J Lipid Res, 1995, 36, 1073-81. 35. Wettereau JR, Aggerbeck LP, Bouma ME, et al. Abscence of microsomal triglyceride transfer protein in individuals with abetalipoproteinemia. Science, 1992, 258, 999-1001. 36. Faix D, Neese R, Kletke C, et al. Quantification of menstrual and diurnal periodocities in rates of cholesterol and fat synthesis in humans. J Lipid Res, 1993, 34, 2063-75.
37. Girard J, Perdereau P, Foufelle F, Prip-Buus C, Ferre P. Regulation of lipogenic enzyme genes expression by nutrients and hormones. FASEB J, 1994, 8, 36-42. 38. Shimomura I, Bashmakov Y, Horton J. Increased levels of nuclear SREBP-1c associated with fatty livers in two mouse models of diabetes mellitus. J Biol Chem, 1999, 274, 30028-32. 39. Mac Garry JD, Foster D. Regulation of hepatic fatty acids oxidation and ketone body production. Ann Rev Biochem, 1980, 49, 395-411. 40. Havel RJ, Kane JP, Balasse EO, Segel N, Basso LV. 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, 2017-35. 41. Boberg J, Carlson LA, Freyschuss U. Determination of splanchnic secretion rate of plasma triglyceride and of total and splanchnic turnover of plasma free fatty acid in man. European J Clin Invest, 1972, 2, 12330. 42. Rasmussen B, Holmbäck U, Volpi E, Morio-Liondore B, PaddonJones D, Wolfe R. Malonyl coenzyme A and the regulation of functional carnitine palmitoyltransferase-1 activity and fat oxidation in human skeletal muscle. J Clin Invest, 2002, 110, 1687-93. 43. Lehner R, Cui Z, Vance CE. Subcellular localization, developmental expression and characterization of a liver triacylglycerol lipase. Biochem J, 1999, 338, 761-8. 44. Daubioul C, Taper H, De Wispelaere L, Delzenne N. Dietary oligofructose lessens hepatic steatosis, but does not prevent hypertriglyceridemia in obese zucker rats. J Nutr, 2000, 130, 1314-9.
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A R T I C L E
Contribution of hepatic de novo lipogenesis and reesterification of plasma non esterified fatty acids to plasma triglyceride synthesis during non-alcoholic fatty liver disease F Diraison1, Ph Moulin2, M Beylot1,
3
S UMMARY
R Eu S U M Eu
Background: Non-alcoholic fatty liver disease (NAFLD) is frequently observed in insulin-resistant subjects and can lead to liver fibrosis and cirrhosis. The abnormalities of lipid metabolism behind this development of excess hepatic TG stores are poorly understood. Methods: To clarify these mechanisms we measured triglyceride secretion rate and the contributions of hepatic lipogenesis and reesterification of non-esterified fatty acids (NEFA) to this secretion in healthy subjects and in patients with clear evidence of NAFLD. All subjects were studied in the post-absorptive state. Hepatic lipogenesis was measured with deuterated water. NEFA turnover rate, triglyceride secretion rate and the contribution of NEFA reesterification to this secretion were determined with [1-13C] palmitate infusion. Results: NAFLD patients had higher NEFA concentrations (p < 0.05) but normal NEFA turnover rates (5.23 ± 0.80 vs 5.91 ± 0.97 µmol.kg–1.min–1 in control subjects, ns). Despite a trend for higher plasma triglyceride levels in patients (p < 0.10), triglyceride turnover rates were not increased (0.11 ± 0.01 µmol.kg–1.min–1 in patients vs 0.14 ± 0.01 in controls, ns). However the contribution of hepatic lipogenesis to triglyceride secretion was largely increased in patients (14.9 ± 2.7 vs 4.6 ± 1.1% p < 0.01) while that of NEFA reesterification was reduced (25.1 ± 2.9 vs 52.8 ± 6.2% p < 0.01). Conclusion: Enhanced lipogenesis appears as a major abnormality of hepatic fatty metabolism in subjects with NAFLD. Therapeutic measures aimed at decreasing hepatic lipogenesis would therefore be the most appropriate in order to reduce hepatic TG synthesis and content in such patients.
Contribution de la lipogenèse hépatique de novo et de la réestérification des acides gras plasmatiques non estérifiés à la synthèse des triglycérides au cours de la stéatose hépatique non alcoolique
Key-words: Stable isotopes z Liver z Steatosis z Triglyceride z Insulin-resistance. Diraison F, Moulin Ph, Beylot M. Contribution of hepatic de novo lipogenesis and reesterification of plasma non esterified fatty acids to plasma triglyceride synthesis during non-alcoholic fatty liver disease Diabetes Metab 2003,29,478-85
1 2 3
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INSERM U 499, Faculté RTH Laennec, Lyon, France Department of Endocrinology, Hôpital Louis Pradel, Lyon, France Centre de Recherche en Nutrition Humaine, Hôpital E. Herriot, Lyon, France.
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Objectif : La stéatose hépatique non-alcoolique (NAFLD) est fréquente chez les sujets insulino-résistants et peut évoluer vers la fibrose et la cirrhose. Les anomalies du métabolisme lipidique responsables de cette stéatose sont mal comprises. Méthodes : Pour clarifier ces anomalies nous avons mesurer le débit de secrétion de triglycérides et les contributions de la lipogenèse hépatique et de la réestérification hépatique des acides gras non estérifiés (NEFA) à cette secrétion chez des sujets témoins et des sujets avec NAFLD. Tous les sujets ont été étudiés à l’état post-absorptif. La lipogènese hépatique a été mesurée à l’aide d’eau deutérée. Le débit de renouvellement des NEFA, le débit de secrétion des triglycérides et la contribution de la réestérification des NEFA à cette sécrétion ont été mesurés à l’aide de [1-13C] palmitate. Résultats : Les sujets avec stéatose avaient des concentrations de NEFA élévées (p < 0,05) mais un débit de renouvellement normal (5,23 ± 0,80 vs 5,91 ± 0,97 µmol.kg–1.min–1 chez les témoins, ns). Malgré des concentrations de triglycérides légèrement élevées (p < 0,10) leur débit de sécrétion de triglycérides était normal (0,11 ± 0,01 µmol.kg–1.min–1 vs 0,14 ± 0,01 chez les témoins, ns). Cependant la contribution de la lipogenèse à la sécrétion de triglycérides était très supérieure chez les sujets stéatosiques (14,9 ± 2,7 vs 4,6 ± 1,1 % p < 0,01) alors que celle de la réestérification des NEFA était diminuée (25,1 ± 2,9 vs 52,8 ± 6,2 % p < 0,01). Conclusion : Une augmentation de la lipogenèse apparait comme une anomalie majeure du métabolisme hépatique au cours de la stéatose. Des mesures thérapeutiques visant à normaliser cette lipogenèse pouraient donc permettre de réduire la stéatose hépatique non alcoolique. Mots-clés : Isotopes stables z Foie z Stéatose z Triglycérides z Insulino-résistance.
Address correspondence and reprint requests to: M Beylot. INSERM U499, Faculté RTH Laennec, rue G. Paradin, 69008, Lyon, France. [email protected] Received: February 13th, 2003; revised: June 2nd, 2003
Hepatic steatosis and tg metabolism
N
on-alcoholic fatty liver disease (NAFLD) is a condition characterized by excessive deposition of triglyceride (TG) in hepatocytes of subjects without a history of excessive alcohol consumption. The term NAFLD incorporates the full range of this condition, from steatosis alone to steatosis with inflammation (non-alcoholic hepatosteatitis, NASH) and possibly steatosis with necrosis and fibrosis ending up in cirrhosis [1, 2]. NAFLD is frequently observed in obese and in type 2 diabetic patients [3] but may occur also in insulino-resistant subjects without obesity or impaired glucose tolerance [4, 5]. It is considered as a part of the metabolic syndrome although it remains unclear whether it is a consequence of or a contributor to insulin-resistance. The abnormalities of lipid metabolism leading to NAFLD are poorly understood. The amount of TG present in the liver depends on one hand on hepatic TG synthesis and on the other on the balance between storage and secretion of TG by the liver. The mechanisms controlling these metabolic pathways are only partially known [6, 7], although it is clear that genetic [8] as well as metabolic and hormonal factors [9-11] are implicated. In particular, the contribution of the various sources of fatty acids for liver TG synthesis in subjects with NAFLD has not been determined. These fatty acids can be provided by hepatic lipogenesis (de novo lipogenesis, DNL), the uptake of plasma non esterified fatty acids (NEFA) by liver (reesterification) or the degradation of lipoproteins taken up by liver [7]. In addition, previously stored TG can be hydrolyzed [12] but this does not represent a primary source of fatty acids since these TG were previously synthesized with fatty acids provided by other sources. In normal subjects it is considered that DNL is a minor pathway and that the main pathway for liver TG synthesis is reesterification of NEFA [7, 13, 14]. It has been suggested that this reesterification pathway plays an important role in NAFLD since these subjects have a moderate increase in post-absorptive NEFA levels [4, 5] and lipolytic flux (as appreciated by glycerol turnover rate) and some resistance of lipolysis to the action of insulin [4, 5]. These abnormalities were observed independently of the presence of obesity or diabetes and suggest that NEFA delivery to the liver was increased. On the other hand, DNL is enhanced in insulinresistant state such as obesity [15] and type 2 diabetes [16] and could therefore contribute to the development of NAFLD. Determining the metabolic abnormalities responsible of NAFLD would help to choose the appropriate therapeutic target(s). Therefore we determined simultaneously by using stable isotopes tracers methodology TG turnover rate and the respective contribution of liver DNL and reesterification of NEFA to TG secretion in control subjects and in a group of patients with NAFLD.
Subjects and methods
Materials Tracers (all 99% mole per cent excess) were from Eurisotop (Saint Aubin, France) (2H2O, NaH13CO2) and Mass Trace (Woburn, MA, USA) (1-13C palmitate). Chemical and reactifs were from Sigma (St Louis MI, USA), Boehringer (Mannheim, Germany) or Pierce (Rockford, Illinois, USA).
Subjects Informed written consent was obtained from six healthy volunteers (control subjects, C, two women, four men, aged 22-48 years, body mass index 20-25, mean 22) and five patients with NAFLD (four women, one man, aged 29 to 64 years, body mass index 22-41, mean 29, p < 0.05 vs control subjects) without any history of alcohol consumption. All NAFLD subjects had clear evidence of large increase in TG liver stores as shown by physical examination, hepatic echography and biological data (elevation of plasma levels of gamma-glutamyl transferase, aspartate and alanine aminotransferases above the upper limit of normal values, (normal values: respectively 10 to 65, 10 to 45 and 10 to 65 U/L). One patient had type 2 diabetes and the others were insulinresistant with elevated post-absorptive concentrations of insulin associated with a moderate increase in plasma glucose (Tab I) and impairment of oral glucose tolerance despite a markedly increased insulin response. No control subject had a personal or familial history of diabetes or obesity or was taking any medication; all had normal plasma glucose, lipids, gamma-glutamyl transferase, aspartate and alanine aminotransferases levels. All subjects consumed a weight maintaining diet with at least 200g carbohydrate and abstained from alcohol consumption or heavy physical activity during the week before the study. Women were studied in the follicular period of menstrual cycle. The last meal was ingested between 19: 00 and 20: 00 PM the day before the tests.
Table I
Circulating concentrations of metabolites and hormones in post-absorptive control subjects and insulin-resistant patients (NAFLD).
Glucose (mmol/liter) NEFA (µmol/liter) TG (mmol/liter) Cholesterol (mmol/liter) Insulin (pmol/liter) Glucagon (ng/liter)
Control subjects n=6
NAFLD subjects n=5
4.5 ± 0.2 426 ± 44 0.85 ± 0.07 5.41 ± 0.21 47 ± 5 105 ± 16
6.6 ± 0.8 * 608 ± 52 * 1.04 ± 0.11 5.90 ± 0.16 116 ± 30 * 142 ± 17
* p < 0.05 vs control subjects. Diabetes Metab 2003,29,478-85 • © 2003 Masson, all rights reserved
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Protocols The protocol of the study was approved by the Ethical Committee of Lyon and by the Institut National de la Santé et de la Recherche Médicale, and the study was conducted according to the Hurriet law. All tests were performed in the Centre de Recherche en Nutrition Humaine de Lyon. In the evening before the test the subject drank a loading dose of deuterated water (3 g/kg body water, one-half after the evening meal and one-half at 10: 00 PM). Then until the end of the study, they drank only water enriched with 2H2O (4.5 g 2H2O/l of drinking water). All tests were initiated in the post-absorptive state, after an overnight fast. At 07: 30 AM indwelling catheters were placed in a forearm vein for tracer infusion and in a dorsal vein of the opposite hand, which was kept at 55°C, to obtain arterialized blood. After an initial blood sampling and collection of expired gas in the initial state, a bolus of NaH13CO2 (1.0 µmole/kg) was injected and a primed-continuous infusion of [1-13C] palmitate (0.03 µmoles.kg–1.min–1 after a bolus of 0.3 µmoles/kg) was initiated and continued for 4 h. Blood samples were collected at 60, 120, 180, 200, 220, and 240 min and during the six hours (240 to 600 min) after the interruption of tracer infusion. Expired gas samples were collected every hour, except for the period 180-300 min, when samples were collected every 15 minutes. Respiratory gas exchanges were measured from 120 to 240 min (Deltatrac, Datex, Helsinki, Finland). Urine samples were collected from 0 to 240 min for determination of nitrogen excretion.
Analytic procedures Metabolites were assayed with enzymatic methods [17, 18] on neutralized perchloric extracts of plasma (glucose) or on plasma (NEFA, TG). Plasma insulin [19] and glucagon [20] concentrations were determined by radioimmunoassay. After addition of heptadecanoic acid as internal standard for the determination of plasma palmitate concentration, plasma lipids were extracted by the method of Folch et al. [21]. TG and NEFA were separated by thin-layer chromatography and scraped off the silica plates [22]. The methylated (NEFA) and transmethylated (TG) derivatives of palmitate were prepared according to Morrison and Smith [23]. Total enrichment (i.e, 13C and deuterium) of the palmitate of NEFA and of TG was measured by gas chromatography-mass spectrometry, monitoring ions of m/z 270-273 [22]. Special care was taken to obtain comparable ion peak areas between standard and biological samples adjusting the volume injected or diluting the sample when necessary. Enrichment in 13C was selectively determined by gas chromatography-combustion-isotope ratio mass spectrometry [24]; this method allows accurate determination of 13C enrichment in the presence of very low enrichment level. Deuterium enrichment was calculated by subtracting 13C enrichment from the total (13C and deuterium) enrichment. Palmitate concentrations were calculated 480
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from the area ratio of the peaks corresponding to palmitate and to heptadocanoate. Standard curves containing known concentration ratios of these two fatty acids were run with the samples. Deuterium enrichment in plasma water and 13 C enrichment in expired CO2 were measured as previously described [25, 26].
Calculations Plasma palmitate turnover rate was calculated from its C enrichment measured during the 180 to 240 min period with steady state equation: Palmitate Rt = F/IE where F is the tracer infusion rate, Rt is turnover rate and IE the enrichment expressed in molar ratio (tracer/tracee). Plasma NEFA Rt was calculated from palmitate Rt and the ratio of NEFA to palmitate concentration as NEFARt = PalmitateRt* (NEFA concentration/palmitate concentration). TG kinetics were calculated from the decay of 13C enrichment in TG-palmitate after the end of the infusion of [1-13C] palmitate [14]. This approach is comparable to the one used with radioactive labeled tracers [27]. A single exponential was fitted to the data: IE = IE0e-kt, where IE is the isotopic enrichment and k is the fractional turnover rate (FRt). The half-life time of the plasma TG pool was then calculated as t1/2 = 0.693/FRt. The absolute TGRt was calculated as TGRt = FRt*M, where M is the plasma pool of TG obtained by multiplying the TG concentration by the plasma volume estimated to 45 ml/kg in subjects with normal body weight and 37 ml/kg in subjects with a BMI above 30 [28]. The fractional contribution of hepatic reesterification (FReest) of NEFA to the plasma TG pool was calculated from the 13C enrichment of plasma palmitate and the increase of 13C enrichment in TG-palmitate during the infusion of labeled palmitate: FReest=[(IEt2-IEt1)/(t2-t1)]/ IEpal, where t1 and t2 are the times when samples are taken, IEt are the corresponding IE of TG-palmitate, and IEpal is the 13C enrichment at plateau of plasma palmitate. The comparison of FReest with FRt gives the percent contribution of reesterification to TG turnover rate: Reest % = FReest/FRt. The absolute contribution of reesterification, Reest to the TG secretion is then Reest = Reest %.TG Rt. Three times Reest gives the rate of reesterification of plasma NEFA by the liver (Reest hep). The fractional contribution of lipogenesis to TGpalmitate (DNL%) was calculated from the deuterium enrichments in the palmitate of plasma TG and in plasma water, as previously described [22]. In short the deuterium enrichments that would have been obtained if endogenous synthesis were the only source of plasma TG-palmitate were calculated from plasma water enrichment; the comparison of the actual enrichments observed with these theatrical values gives the contribution of endogenous synthesis to the circulating pool of TG-palmitate. Absolute de novo lipogenesis is then obtained as DNL=DNL%*TGRt*3, where the 13
Hepatic steatosis and tg metabolism
factor 3 acknowledges the fact that there are three fatty acids per molecule of TG. Plasma palmitate oxidation was calculated as PalOx = F13CO2/(IE pal*fc) where F13CO2 is the excretion rate of labeled CO2 produced by palmitate oxidation and fc is the correction factor for incomplete label recovery in expired gas. PalOx was calculated using either the bicarbonate recovery factor (0.80) or the acetate factor measured by Sidossis et al. [29] that takes in account label exchange in Krebs cycle. Total NEFA oxidation was calculated from PalOx and the palmitate over total NEFA concentrations ratio. Total lipid oxidation rate was calculated from respiratory gas exchange by use of stoichiometric equations [30]. Total lipid oxidation was converted into fatty acid oxidation by assuming an average molecular weight of 860g [30] and multiplying by three since there are three fatty acids for one TG. Extracellular, whole body reesterification rate (Reexc) was calculated by subtracting from NEFA Rt oxidation rates obtained from either respiratory gas exchange or 13CO2 excretion rate. Subtracting REhep from Reexc gives the contribution of extrahepatic tissues to plasma NEFA reesterification. All results are shown as means ± SE. Comparisons between control and insulin-resistant subjects were performed with the Mann-Whitney test.
Results
Metabolites and hormones concentrations The concentrations in both groups of subjects of insulin, glucagon and metabolites during the 120 to 240 min period of the test are shown in Table I. NAFLD subjects had higher levels of glucose and insulin, as expected, and also of NEFA
(p<0.05 for all), than controls. In both groups NEFA levels were stable during this period of the study. The trend for higher TG concentrations in NAFLD did not reach significance (p < 0.20). NEFA rose then progressively during the following six hours to reach respectively 703 ± 62 and 692 ± 33 µmol/l at the end of the tests in normal and NAFLD subjects respectively. Plasma glucose levels decreased in NAFLD patients progressively to 4.55 ± 0.36 mmol/l at the end of the study whereas they remained unchanged in control subjects. Plasma TG levels remained stable throughout the study.
Plasma NEFA turnover and oxidation rates The evolution of the 13C enrichment in the palmitate of plasma TG and NEFA is shown in Figure 1. Stable levels of both enrichment and concentration were obtained in NEFA-palmitate and therefore kinetic parameters for NEFA were calculated using equation for steady-state conditions. Enrichment decreased then abruptly in NEFApalmitate when the infusion of tracer was stopped. NEFA Rt were similar in both groups when expressed relative to total body weight (5.23 ± 0.80 and 5.91 ± 0.97 (moles/kg/min in NAFLD and C subjects respectively, ns) or as absolute values per minute (404 ± 56 and 352 ± 56 (moles/min respectively, ns). Plateau levels of labeled carbon enrichment in the CO2 of expired gas were also obtained (data not shown). NEFA oxidation rates (NEFAox) calculated using either the bicarbonate factor or the acetate factor are compared in Table II with total lipid oxidation (Lox) determined from respiratory gas exchange data. This Lox was corrected for the measured hepatic lipogenesis to convert net Lox to total Lox rate. This correction was negligible in control subjects and remained small in NAFLD patients despite their higher
PALMI TATE EI (MPE)
2,4
1,8
1,2
0,6
0,0 0
120
240
360
TIME (MINUTES)
480
600
Figure 1 Evolution of the 13C enrichment of palmitate in the plasma NEFA of control (*) and NAFLD (Φ) subjects and in the plasma TG of control [7] and NAFLD [4] subjects, during the infusion of labelled palmitate (time 0 to 240 minutes) and after its interruption (time 240 to 600 minutes).
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Table II
Plasma NEFA (NEFAox) and total lipid (Lipid ox) oxidation rates. Control subjects n=6
NAFLD patients n=5
3.12 ± 0.45
2.52 ± 0.56
Lipid ox µmol/kg/min NEFA ox: µmol/kg/min “non-NEFA” ox µmol/kg/min
Bicarbonate acetate Bicarbonate acetate 1.84 ± 0.43 2.62 ± 0.61 1.61 ± 0.31 2.31 ± 0.42 Bicarbonate acetate Bicarbonate acetate 1.29 ± 0.30 0.53 ± 0.38 0.90 ± 0.36 0.24 ± 0.30
NEFA oxidation rates were calculated using either the bicarbonate or the acetate factor.
liver lipogenic activity (see next paragraph). Whatever the way lipid oxidation rates were calculated, there was no significant difference between control and NAFLD subjects. Use of the acetate factor raised the contribution of NEFA oxidation to NEFA Rt from 30.7 ± 2.9% to 43.9 ± 4.1% in controls and from 30.6 ± 4.1% to 43.7 ± 5.8% in NAFLD patients. The contribution of NEFA oxidation to total lipid oxidation was raised respectively from 59% to 85% and from 68% to 100%. Therefore the “non-NEFA” oxidation rate (considered to be the oxidation of fatty acids from tissues or plasma TG) was low (0.53 µmoles/kg/min) in controls and negligible in NAFLD patients.
Plasma TG kinetics and contributions of NEFA reesterification and lipogenesis to TG synthesis The kinetics of plasma TG and the respective contributions of lipogenesis and plasma NEFA reesterification to TG secretion rate are shown in Table III. The contributions of plasma NEFA reesterification were calculated from the increase of 13C enrichment in TG-palmitate during the 4 hour infusion of labeled palmitate. In both groups this rise was linear with time (Fig 1) and, calculated from the intercept of the regression line with the time axis, there was a
Table III
Kinetic parameters of plasma TG and contribution of hepatic reesterification of plasma NEFA and lipogenesis to TG synthesis.
FRt h–1 Half-life h Rt µmol.kg–1.min–1 Freest h–1 Reesterification % of Rt Lipogenesis % of Rt
Control subjects n=6
NAFLD subjects n=5
0.21 ± 0.02 3.54 ± 0.32 0.14 ± 0.01 0.112 ± 0.014 52.8 ± 6.2 4.6 ± 1.1
0.18 ± 0.01 3.80 ± 0.19 0.11 ± 0.01 0.046 ± 0.007 ** 25.1 ± 2.9 ** 14.9 ± 2.7 **
** p < 0.01 vs control subjects. FRt: fractional turnover rate. Freest: fractional contribution of plasma NEFA reesterification to plasma TG pool.
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similar delay (about 30 minutes) between initiation of the tracer infusion and the appearance of 13C in TG-palmitate. Despite comparable 13C enrichment in plasma NEFApalmitate the slope of the rise of 13C enrichment in TGpalmitate was much lower in NAFLD than in controls and thus the fractional contribution of reesterification (FReest) to the TG circulating pool was decreased in NAFLD (0.046 ± 0.007/h vs 0.112 ± 0.014/h p < 0.01). There was also a 30 minutes delay between the end of labeled palmitate infusion and the beginning of the decline of 13C enrichment in plasma TG-palmitate. The half-life and FRt of plasma TG were similar in the two groups as well as the absolute Rt, expressed relative to body weight (Tab III) or as µmol/min (9.61 ± 0.96 and 8.65 ± 0.62 in control and NAFLD subjects respectively, ns). Comparison of FReest to FRt shows that reesterification contributed about 50% to TGRt in controls and only 25% (p < 0.01 vs controls) in NAFLD. Deuterium enrichments in plasma water were stable throughout the tests in both groups and were respectively 0.34 ± 0.01 and 0.45 ± 0.04% in control and NAFLD subjects. The calculated contribution of lipogenesis to TG secretion was three time higher in the NAFLD than in the control subjects (14.9 ± 2.7 vs 4.6 ± 1.1% respectively p < 0.05) (Tab III) when measured during the 120-240 minutes period. This contribution decreased to 2.1 ± 0.6% at the end of the test in control subjects (p < 0.05 vs initial value) but remained to 15.9 ± 2.8% in NAFLD. These contributions of lipogenesis during the 120-240 min period represented absolute synthetic rates of fatty acids of 0.021 ± 0.005 and 0.056 ± 0.018 µmol.kg–1.min–1 in control and NAFLD subjects respectively (p < 0.05). Addition of the contributions of lipogenesis and plasma NEFA reesterification shows that about 45% and 60% of the TG secretion rates were not accounted for by these metabolic pathways in control and NAFLD subjects respectively. The contribution of hepatic reesterification to plasma NEFA utilization were calculated to be 0.22 ± 0.02 µmol.kg–1.min–1 in controls and 0.09 ± 0.02 µmol.kg–1.min–1 in NAFLD (p < 0.05 vs controls) and appeared in both groups as a minor contributor to the whole body utilization of NEFA. Thus, liver reesterification of NEFA appears as a minor contributor to total NEFA reesterification.
Discussion We investigated in the present report several aspects of plasma NEFA and TG metabolism in control subjects and in patients with NAFLD. We found no difference in TG kinetics but there was large differences in the source of fatty acids used for the synthesis of TG secreted by the liver: the contribution of DNL was markedly increased in NAFLD while that of plasma NEFA reesterification was decreased. In addition, DNL remained in NAFLD elevated during the prolongation of the fasting period imposed by the experimental protocol.
Hepatic steatosis and tg metabolism
We used for calculating plasma TG kinetic parameters a non-compartmental analysis of the decay curve of 13C enrichment in the palmitate of plasma TG. This approach has been criticized since, contrary to compartmental modeling analysis, it does not allow to resolve the possible tracer recycling through hepatic lipids pool, i.e. release in plasma TG, after stopping the infusion of labelled palmitate, of labelled TG stored during the infusion of the tracer [31]. This release would slow down the decay curve of enrichment in plasma TG and could result in underestimates of TG Rt, although comparable results were found using the compartmental and non-compartmental approach by Lemieux et al. [32]. Compartmental analysis requests to follow the decay curve during a long period (12 hours at least) and this requires initial enrichment levels much higher than the one obtained in the present study. This would have needed a high infusion rate of labelled palmitate which was precluded by ethical consideration (high infusion rate of human albumin) and would have anyway pertubated the pool of plasma palmitate. Lastly, the discrepancy between TG kinetic parameters determined by using compartmental or non-compartmental analysis is marked for TG turnover rates above 0.3 pool/ hour [31], value above those found in the present study. Thus, although we cannot exclude some underestimation of the TG Rt values we found, there is no evidence for an increased TG Rt in the group of NAFLD we studied. Actually this could suggest that these subjects have a preferential orientation of hepatic TG metabolism toward storage rather than secretion. Raised insulin levels could play a role in this orientation. In vitro studies showed that, whereas glucose stimulates both TG synthesis and secretion, insulin stimulates synthesis but decreases secretion resulting in accumulation of stored TG [33]. Short term studies in humans showed also an inhibitory action of insulin on TG secretion, even when the insulin-induced decrease in plasma NEFA concentration was prevented [13]. In addition prolonged hyperinsulinemia could decrease the expression and activity of the microsomal triglyceride transfert protein (MTP) [34] which is necessary for the secretion of TG [35]. It is also possible that subjects prone to the development of NAFLD have genetic variants of MTP [8] resulting in decreased expression and activity of this protein. DNL was markedly increased in NAFLD patients and did not decrease in the fasting state contrary to control subjects. Comparable stimulations of lipogenesis were previously reported in insulin-resistant and obese subjects [15, 36]. Therefore this abnormality is not specific of patients with steatosis but the present results strongly support a role for enhanced DNL in the develoment of NAFLD. This enhanced DNL results probably of the raised insulin levels found in NAFLD subjects, given the known effects of insulin on the expression and activity of the lipogenic pathway in liver [37]. These effects of insulin are probably mediated through the transcription factor SREBP-1c since it plays an
important role in controlling the expression of lipogenic genes in livers and both its mRNA and the nuclear protein levels are increased in mouse models of hepatic steatosis [38]. The decreased contribution of NEFA reesterification appears surprising since plasma NEFA are a major source of fatty acids for liver TG synthesis [7, 13, 14] and NEFA concentrations were increased in NAFLD subjects. Moreover insulin diverts hepatic fatty acids metabolism away from oxidation and toward reesterification [39] It should be kept in mind that we used for the calculation of the contribution of NEFA reesterification to TG secretion the 13C enrichment of NEFA-palmitate in arterial, not portal venous, plasma. There is evidence that the decrease in NEFApalmitate enrichment between arterial and portal venous plasma is low, around 10%, in normal subjects [40, 41] and that therefore the underestimation in the contribution of reesterification is probably low in these subjects. It is possible that the direct release of NEFA from intra-abdominal adipose tissue into the portal vein was higher in NAFLD subjects. This would result in a more important dilution of 13C NEFA-palmitate enrichment in portal venous plasma and thus in a greater underestimate of the contribution of reesterification to TG secretion. Actually this is indirect evidence that this dilution, and therefore the underestimation of NEFA reesterification, could be only around 20% in obese subjects [40]. This lack of stimulation of hepatic reesterification of NEFA in patients with NAFLD could be an aspect of insulin-resistance. Actually there is evidence that hepatic oxidation of NEFA is increased in NAFLD disease [5], and could contribute to the evolution to non-alcoholic steatohepatitis. Further studies of obese and/or insulin-resistant subjects without hepatic steatosis would help to deteremine if this modification of liver fatty acid metabolism is specific or not of subjects with NAFLD. It is also noteworthy that we found at the whole body level no modification of the repartition of NEFA utilization between oxidation and reesterification in NAFLD subjects although it has been clearly shown in control subjects that insulin diverts muscle NEFA metabolism away from oxidation and to reesterification [42]. Lipogenesis and NEFA reesterification accounted only for 55% and 40% of the TG secreted in control and NAFLD subjects respectively. Although reesterification may have been somewhat underestimated, particularly in NAFLD, these data agree with in vitro studies showing that 50-60% of the TG secreted by hepatocytes are provided by sources other than DNL and NEFA uptake [11, 12]. Other possible sources are the degradation of lipoproteins taken up by liver and of TG previously stored in cytosol (recycling) [6, 7]. The higher percentage of TG secretion not accounted for by DNL and NEFA reesterification in NAFLD could be consistent with a more important contribution of the increased cytosolic TG stores. These TG stores must be hydrolyzed by a specific lipase [43]. The fatty acids released are then used for the synthesis of new TG which can be secreted or stored Diabetes Metab 2003,29,478-85 • © 2003 Masson, all rights reserved
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again [12]. Therefore it is possible that in both groups of subjects a part of the TG synthesized with fatty acids provided by DNL and NEFA uptake was directed towards storage in order to replace stored TG which were hydrolyzed. This process would result in a dilution by the fatty acids released from the stored TG of both 13C and deuterium enrichment in the TG secreted by the liver during the period of tracer administration. We sampled only, for obvious ethical reasons, the circulating TG pool and we may therefore have underestimated the true rates of DNL and NEFA reesterification. If we assume that these metabolic pathways contributed also to the maintain of TG stores, i.e. if we normalize their total contribution to TG secretion to 100%, we obtain the upper limits for the estimates of these metabolic rates. This would give in control subjects lipogenic and reesterification rates of fatty acids of 0.038 and 0.40 µmol.kg–1.min–1 instead of 0.021 and 0.22 respectively. In NAFLD the corresponding lipogenic and reesterification rates would be 0.14 and 0.22 µmol.kg–1.min–1 instead of 0.056 and 0.09. This does not modify the differences between the two groups, i.e. increased lipogenic and decreased NEFA reesterification rate in NAFLD. In conclusion we found that a group of subjects with NAFLD had an increased contribution of liver lipogenesis to liver TG synthesis while that of plasma NEFA reesterification appeared decreased. These results support the idea that nutritional or pharmacological interventions aimed at decreasing liver lipogenesis, such as non-digestible carbohydrates [44] or fibrates [16], would be the most appropriate for reducing hepatic TG synthesis and content in these patients.
8.
Acknowledgments − We thank all the subjects who volunteered for this study, J Peyrat for her help during the realization of the tests. This work was supported in part by grants from the Fondation de France, the ALFEDIAM and Association Française de Diabétiques and by the European Economic Community (Contrat Nutrigen FAIR CT97-3011).
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