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O.4 Insulin infusion restores surgery-induced reductionof insulin-stimulated glucose uptake without increasing substrate oxidation or translocation of Glut4
ORAL PRESENTATIONS 1st S e s s i o n - I N T E R M E D I A R Y M E T A B O L I S M , L I P O P R O T E I N M E T A B O L I S M , G E N E R E G U L A T I O N
O.1 Excessive muscle proteolysis during one-leg exercise is exclusively attended by increased de novo synthesis of glutamine, not of alanine A. J. M. Wagenmakers, G. van Ha// and B. Sa/tin ~
Department of Human Biology, University of Limburg, Maastricht, The Netherlands and I Copenhagen Muscle Research Centre, Copenhagen, Denmark. During prolonged one-leg knee extensor kicking exercise, the workload (W) per kg muscle is 2- to 3-fold higher than during two-legged cycling. The aim of this study was to investigate whether exercise under these extreme conditions leads to a high proteolytic rate in skeletal muscle and to increased de novo synthesis of alanine and glutamine. Six subjects performed 90 min exercise at 60% of the one leg Wrnax. Net amino acid production was estimated at rest and in the 10-90 min exercise period from the leg exchange (femoral arterio-venous plasma difference x plasma flow measured by thermodilution), and changes in the muscle amino acid concentration (percutaneous biopsies) between 10 and 90 rain of exercise. Exercise-induced changes were assumed to originate from the 3 kg of active muscle. Mean values were used of the leg exchange after 10, 30, 60 and 90min of exercise. During exercise the net production (in #mol.kg 1 dry muscle.min 1) of the sum of six amino acids that are not metabolized (threonine, methionine, phenylalanine, lysine, glycine and tyrosine) increased from 4.8 _+0.9 to 53 _+ 14 (m + SEM, P < 0.05). Using the net production rates and the relative occurrence of amino acids in human muscle protein, alanine production by net proteolysis was calculated at 1.4 (rest) and 15 (exercise). Values observed were 6.3 _+1.3 (rest) and 8 _+23 (exercise). Glutamine net production was calculated at 1.0 (rest) and 10.5 (exercise) and measured at 7.3 + 1.0 and 75 _+42. When the observed production rate exceeds the calculated rate then de novo synthesis must occur from other amino acids. We conclude that net proteolysis (synthesis-degradation) is increased 11-fold during one-leg exercise. The acceleration of proteolysis is attended by a massive increase of de novo glutamine synthesis (from other amino acids), and not of de hove alanine synthesis.
0.2 Measurement of glutamine metabolism in gastrointestinal cancer patients B. A. C. van Acker, tt(. ~ E. Hulsewe, A. J. M. Wagenmakers ~, N. E. P. Deutz, B. K. van Kreel 2, R B. Soeters and M. F. von Meyenfeldt Depts of Surgery, 1Human Biology and 2Clinical Chemistry, University Hospital Maastricht, The Netherlands.
0.3 Troglitazone inhibits hepatic fatty acid oxidation and esterification, and gluconeogenesis J. P. Fulgencio 1, C. Kohl 2, J. Girard 2 and J. P. Pegorier 2
l l-F~ H Mender, Cr6teil, France and 2CEREMOD, Meudon Bellevue, France. Insulin resistance in obesity and diabetes is associated with an active hepatic gluconeogenesis (GNG) supported by an increased hepatic fatty acid oxidation. Troglitazone (Tro) decreases hepatic GNG and circulating VLDL-triglycerides, and improves hepatic insulin resistance. This work was designed to investigate the mechanisms involved in the effects of Tro in isolated hepatocytes from 24-h starved rats, incubated for 1 h in the absence (control, C) or in the presence of 1 mM Tro. Fatty acid oxidation and esterification were estimated with 0.3 mM [1J4C]-oleate. GNG was measured in the presence of alanine or dihydroxyacetene (DHA), each at 10 mM. The Km and Vmaxof acyI-CoA synthetase (ACS) were determined in mitochondria and microsomes isolated from 1-h incubated C or Tro hepatocytes. Oleate oxidation was respectively 88 _+ 6 and 30 + 2 nmol/h/106 cells in C and Tro hepatocytes (P < 0.05). The production of ~-hydroxybutyrate was nearly abolished leading to a very oxidized mitochondrial redox state, as estimated by the fall in the ~hydroxybutyrate/acetoacetate ratio. Tro inhibited GNG, either in the presence of alanine (81 _+8%) or DHA (40 _+6%). As DHA bypasses the gluconeogenic steps controlled by fatty acid oxidation, these results suggest that Tro could partly inhibit GNG secondarily to the decreased availability in acetyI-CoA, ATP and reduced equivalents (NADH). Triglyceride synthesis was 10.5 -+ 1.0 and 1.4 _+0.1 nmol/h/106 cells in C and "Fro hepatocytes respectively (P < 0.05), whereas phospholipid synthesis was unaffected. AcyI-CoA synthesis is an obligatory step prior to fatty acid metabolism. The mitochondrial and microsomal Vm~×of ACS was respectively decreased by 58 + 6% and 61 _+15% by Tro (P < 0.05), whereas its Km was unaffected. In conclusion, Tro inhibits hepatic mitochondrial and microsomal ACS, leading to a decrease in triglyceride synthesis and fatty acid oxidation. The inhibition of fatty acid oxidation is partly responsible for the decrease in hepatic GNG.
0.4 Insulin infusion restores surgery-induced reduction of insulin-stimulated glucose uptake without increasing substrate oxidation or translocation of Glut4 A. Thorell, J. Nygren, S. Nair1, M. Hirshman 2, E. S. Horton 2, L.
Aim: To determine the plasma appearance rate (Ra) of glutamine in
Goodyear 2 and O. Ljungqvist Dept of Surgery, Karolinska Hospital, Stockholm, IDept of Endocrinology, Mayo Clinic, Minnesota, USA, 2joslin r)iabetes Center, Harvard Medical School, Boston, USA.
patients with gastrointestinal malignancies. Methods: Ten preoperative metabolically stable patients with colon or gastric cancer were given a 6-h (n = 5) or 11-h (n= 5) primed intravenous infusion of L-[5-15N]-glutamine (0.67 ~tmol/kg/h, 1-h prime) in the postabsorptive state. Arterial blood samples and percutaneous muscle biopsies were taken and analysed for 15N glutamine enrichment by GC-combustion-IRMS. The plasma Ra of glutamine was calculated via the standard isotope dilution expression for dilution of the infused ~SN glutamine in plasma: Ra = i[EI/Ep-1]. Results: The 15N glutamine enrichment in plasma gradually increased in each patient during both 6-h and 11-h tracer infusion periods, indicating that isotopic steady state had not been achieved. Consequently, the plasma Ra of glutamine decreased during the course of the study from 240 _+14 ~mol/kg/h (t = 2 h; n = 10, mean + SEM) to 212 _+13 llmol/kg/h (t = 6h; n= 10, P< 0.01 vs t= 2h) and 185 + 141tmol/kg/h (t= 11 h; n= 5, P < 0.05 vs t= 6 h). The 15N glutamine enrichment in muscle as percentage of the plasma enrichment, increased from 6.9 _+0.6% (t = 2 h, n = 5) to 11.6 _+2.0% (t = 4 h, n = 4), 15.3 _+1.2% (t = 6 h, n = 10) and 24.5 _+3.0% (t =11h, n=5). Conclusion: The results of the present study show that the 15N glutamine tracer enters the large glutamine pool in muscle. As equilibration between the plasma and muscle pool is slow, it takes probably more than 24h before steady state conditions are established. Previous suggestions that the plasma Ra of glutamine can be calculated via the standard isotope dilution expression between 2 and 4 h of tracer infusion, in our hands leads to a considerable overestimation of glutamine Ra.
Background:The underlying molecular mechanisms that cause surgeryand trauma-induced whole body insulin resistance and decreased rates of skeletal muscle glucose uptake are not known. In skeletal muscle, glucose transport is rate-limiting for glucose disposal. In healthy human subjects, insulin-stimulated glucose transport is due, at least in part, to the translocation of the Glut4 glucose transporter proteins from inside the cell to the surface (plasma) membranes. Methods: Six otherwise healthy patients (5M/1F, age 59 (3) years, BMI 26.5 (1.0)), scheduled for total hip replacement, underwent :2 euglycaemic (5 mmol), hyperinsulinaemic (60 #U/ml) clamps; one a week prior to surgery (pre-op) and one immediately post-op. Before and at the end of each clamp (=120 rain), body glucose uptake (Rd), glucose and fat oxidation and non-oxidative glucose uptake (NOGU) were measured. Biopsies of vastus lateralis muscle (=1 g) were obtained and used to purify plasma membranes for the measurement of Glut4 translocation. Immediately after the post-op clamp, in an attempt to restore preoperative insulin-stimulated glucose uptake, the patients were given sufficient amounts of insulin to match preoperative glucose infusion rate (feedback clamp), and a last sampling period was performed. Results: (Wilcoxon's signed rank test, mean (SEM)). For the pre-op clamp, insulin infusion increased Rd to 3.41 (0.4) mg/kg/min, increased Glut4 translocation to 128 (8) arbitrary units, increased glucose oxidation to 2.06 (0.35) mg/kg/min and decreased fat oxidation to 0.5,1 (0.13) mg/kg/min (all P < 0.05 vs basal). In the first phase of the post-op clamp, Rd was 2.27 (0.21) mg/kg/min (P < 0.05 vs pre-op) while translocatien of Glut4 and substrate oxidation were not different. During feedback clamp
(serum insulin 92 + 21 #U/ml), Rd was normalized to pre-op levels (3.15 (0.52) mg/kg/min) and an increase of NOGU occurred without further alterations of translocations of Glut4 or substrate oxidation. Conclusions: Immediate postoperatively, insulin resistance develops, but can be reversed by insulin infusion. Alterations in the degree of translocation of Glut4 can not explain these events. Postoperatively, insulin infusion primarily seems to stimulate the non-oxidative pathway intracellularly.
0.5 Assessment of perioperative glycerol metabolism by stable isotope tracer technique Th. Schricker, U. Pfeiffer, A. Berroth, M. Schreiber, W. Geisser,
A. Goertz and M. Georgieff Clinic of Anesthesiology, UIm, Germany.
Introduction: The objective of this study was to investigate metabolic changes during and after lower abdominal surgery with specific regard to glycerol metabolism. Methods: With the approval of the ethics committee, 7 otherwise healthy patients receiving abdominal hysterectomy (47 _+8 years, 162 4- 4 cm, 64 4- 10kg) were studied. Glycerol turnover (GTO) - a measure of whole body lipolysis - and hepatic glucose production (HGP) were quantified by stable isotope tracer technique ([1,1,2,3,3-2Hs]-glycerol, [6,6-2H2]-glucose), before and 3 h after surgery. Metabolic substrates (glycerol, nonesterified fatty acids (NEFA), ~-hydroxybutyrate (BOH), glucose) and hormones (adrenaline (A), noradrenaline (NA), cortisol (C), insulin, glucagon) were determined pre-, intra- and postoperatively. Values are means 4- SD. Results: Hysterectomy induced an increase of GTO from 3.56 4- 1.28 to 6.46 4- 2.44 pmol/kg/min (P < 0.05). This increment was inversely related to the patients' age (r = 0.872, P < 0.05). HGP and glucose postoperatively rose from 9.8 _ 1.6 to 12.8 _+1.5#mol/kg/min (P < 0.05) and from 4.6 4-0.9 to 6.2 4-0.9 mmol/I (P < 0.05), respectively. NEFA and BOH also significantly increased post surgery (NEFA: 506 _+ 172 vs. 766 _+ 116t~mol/I, BOH: 174 4-107 vs. 655 _+276 p.mol/I, P< 0.05), while glycerol revealed no pedoperative change. Cortisol (5 _+3 vs. 37 4- 14 }~g/dl, P < 0.05) and catecholamines (A: 25 4- 7 vs. 145 _+96 pg/mt, NA: 183 _+70 vs. 631 _+299 pg/ml, P < 0.05) increased after the operation, but glucagon and insulin were not altered. Conclusions: The postoperative enhancement of lipolysis was not detectable from plasma glycerol concentrations, which remained unchanged. In addition hysterectomy caused postoperative hyperglycemia due to stimulated H G P - common features of the metabolic response to surgical trauma. The results gave further evidence that observations based on static metabolite levels provide only limited insight into the underlying dynamic biochemical events, especially under perioperative conditions. 0.6 Does the presence of fish oil (FO) in emulsion particles affect the elimination of MCT/LCT emulsion? Y. A. Carpentier, V. S. Siderova, M. Richefle, Ft. J. Decke/baum, D.
Eggerickx and F. Sultan~ Universit# Libre de Bruxelles, Brussels, Belgium and ~Braun M6dical, Paris, France. Triglycerides (TG) containing long-chain n-3 polyunsaturated fatty acids (LCPFA) are poorly hydrolyzed by lipoprotein lipase (LPL); further, when FO emulsions are mixed with soy TG emulsions, they can have a suppressive effect on total lipolysis. The purpose of the present study was to determine whether including 10% FOTG in particles containing 50% MCT and 40% LCT would affect TG clearance and intravascular metabolism during infusion in man, using the TG clamp model. Eight healthy normolipidaemic subjects (mean age 23 _+3 years) were infused during 4 consecutive days with MCT/LCT alone and, 4 weeks later, with MCT/LCT + FO (MLF). Glucose and amino acids were also infused to minimize the contribution of endogenous fat stores. TG level was clamped at 3 mmol/L for 5h by adjusting the infusion rate. TG elimination is expressed by the mean rate (mg TG/kg b.w./h _+SD) during the 2 last h of infusion:
D1
D2
MCT/LCT 162_+35 178_+53 MLF 205 -+39*** 207 _+52* ***P< 0.01, **P< 0.025, *P< 0.05.
D3
D4
174_+57 199 _+48**
164_+62 204 -+40***
The ability to clear infused TG remained fairly constant over each 4-day period in each subject (CV = 11.6% with MCT/LCT, 7.3% with MLF), but large interindividual differences were observed (98-203 mgTG/kg/h with
MCT/LCT, 134-278mgTG/kg/h with MLF). Infusion of both emulsions resulted in a similar increase of plasma free fatty acids (1.0 4- 0.1 mmol/L with MCT/LCT vs. 1.2 + 0.1 mmol/L with MLF, NS). In vitro hydrolysis of both emulsions with LPL showed very similar rates of FFA release, both with respect to time and to varying enzyme concentrations (data not shown). Conclusions: 1. The presence of 10% FO in MCT/LCT particles does not reduce, but rather enhances elimination of infused TG. 2. The difference may not be related to a higher rate of hydrolysis, but to a more efficient uptake of remnant particles.
0.7 Lipoprotein lipase activity and mass in plasma following administration of exogenous fat in healthy subjects A. ThSrne, J. NordenstrSm and T. Olivecrona*
Department of Surget34, Huddinge University Hospital and *Department of Medical Biochemistry and Biophysics, Ume& University, Sweden. Lipoprotein lipase (LPL) plays a pivotal role in the handling of fat emulsions, e.g. unloading plasma triglycerides at the endothelial surface in peripheral tissues. Previous studies have shown that after an overnight fast a majority of circulating LPL is catalytically inactive. Heparin administration is followed by an increase in both LPL activity and mass, which is assumed to reflect the available amount of active LPL Administration of exogenous fat is followed by a rise in LPL activity but whether the LPL mass is influenced is unknown. The purpose of the study was to examine the influence of exogenous fat administration, as a hypertriglyceddaemic (HTG) clamp, on LPL activity and mass in health subjects. Methods: A priming dose of a fat emulsion (Intratipid®, 20%, Pharmacia AB), 13 mgTG/kg BW x min, was given in order to reach a TG concentration of 4 mmol/I. The infusion rate was thereafter adjusted to maintain TG at 4 mmol/I during 180 rain. Twenty healthy men with an average age of 52 years (19-78 years) and body mass index (BMI) of 24 (19-27) participated in the study. All studies were performed after an overnight fast (10 h) and blood samples were collected in the basal fasting stage and then throughout the study period for analysis of LPL activity and mass. Results: In the basal state the average (_+SEM) LPL activity and mass were 0.44 _+0.07mU/ml and 194 _+ 13ng/ml and the calculated specific activity was 0.0025 _+0.0004mU/ng. During infusion of fat at an average rate of 0.202 _+0.021 mmol/min, the LPL activity increased (P < 0.001) 5fold to 2.10 + 0.29mU/ml, while the LPL mass remained virtually unchanged (183 -+ 14 ng/ml, n.s.). The corresponding calculated specific activity increased (P < 0.001 ) 5-fold to 0.0130 4- 0.0020 mU/ng. Assuming the specific activity of active plasma LPL to be 0.30 mU/ng, the calculated proportion of active LPL was 0.84 + 0.14% after an overnight fast and, during lipid infusion, it increased (P< 0.001) to 4.34 -+0.68% and the amount of inactive LPL protein tended to decrease (P=0.05) from 192 + 13 to 177 4-14 ng/ml. In conclusion, administration of exogenous fat as a HTG clamp in healthy man is accompanied by augmented plasma levels of active LPL and a tendency towards decline in catalytically inactive LPL protein. An enhanced release from the endothelium reflects the former and the latter could conceivably be due to an accelerated catabolism through binding to emulsion particles which in turn are rapidly cleared from plasma.
0.8 Role of nuclear transcription factors in regulation of albumin synthesis in protein-deprived rat liver M. Yano, T. Tsujinaka, C. Ebisui, T. Morimoto, M. Kishibuchi, S. Morita, M. Taniguchi, H. Shiozaki and M. Monden Department of Surgery II, Osaka University Medical School, Osaka, Japan.
A. Ogawa,
Serum albumin level and albumin mRNA level in the liver are known to decrease in rats when fed with a protein-free diet, although its precise mechanism is unknown. The albumin gene has six cis-acting elements in the proximal promoter region. Among these elements, B and D site are important for strong and tissue specific expression of the albumin gene. Liver-specific transcription factors, CCAAT/enhancer binding protein ~ (C/EBPc 0, C/EBP[3 and D site binding protein (DBP) bind to D site and hepatocyte nuclear factor-1 (HNF-1) binds to B site. This study was designed to elucidate the transcriptional regulation of the albumin gene and the role of nuclear transcription factors under protein deprivation. Male Donryu rats were fed with a protein-free diet (PF) or a control diet (CON) for 10 days and were killed to obtain the liver and blood. Hepatic
O.1 Excessive muscle proteolysis during one-leg exercise is exclusively attended by increased de novo synthesis of glutamine, not of alanine A. J. M. Wagenmakers, G. van Ha// and B. Sa/tin ~
Department of Human Biology, University of Limburg, Maastricht, The Netherlands and I Copenhagen Muscle Research Centre, Copenhagen, Denmark. During prolonged one-leg knee extensor kicking exercise, the workload (W) per kg muscle is 2- to 3-fold higher than during two-legged cycling. The aim of this study was to investigate whether exercise under these extreme conditions leads to a high proteolytic rate in skeletal muscle and to increased de novo synthesis of alanine and glutamine. Six subjects performed 90 min exercise at 60% of the one leg Wrnax. Net amino acid production was estimated at rest and in the 10-90 min exercise period from the leg exchange (femoral arterio-venous plasma difference x plasma flow measured by thermodilution), and changes in the muscle amino acid concentration (percutaneous biopsies) between 10 and 90 rain of exercise. Exercise-induced changes were assumed to originate from the 3 kg of active muscle. Mean values were used of the leg exchange after 10, 30, 60 and 90min of exercise. During exercise the net production (in #mol.kg 1 dry muscle.min 1) of the sum of six amino acids that are not metabolized (threonine, methionine, phenylalanine, lysine, glycine and tyrosine) increased from 4.8 _+0.9 to 53 _+ 14 (m + SEM, P < 0.05). Using the net production rates and the relative occurrence of amino acids in human muscle protein, alanine production by net proteolysis was calculated at 1.4 (rest) and 15 (exercise). Values observed were 6.3 _+1.3 (rest) and 8 _+23 (exercise). Glutamine net production was calculated at 1.0 (rest) and 10.5 (exercise) and measured at 7.3 + 1.0 and 75 _+42. When the observed production rate exceeds the calculated rate then de novo synthesis must occur from other amino acids. We conclude that net proteolysis (synthesis-degradation) is increased 11-fold during one-leg exercise. The acceleration of proteolysis is attended by a massive increase of de novo glutamine synthesis (from other amino acids), and not of de hove alanine synthesis.
0.2 Measurement of glutamine metabolism in gastrointestinal cancer patients B. A. C. van Acker, tt(. ~ E. Hulsewe, A. J. M. Wagenmakers ~, N. E. P. Deutz, B. K. van Kreel 2, R B. Soeters and M. F. von Meyenfeldt Depts of Surgery, 1Human Biology and 2Clinical Chemistry, University Hospital Maastricht, The Netherlands.
0.3 Troglitazone inhibits hepatic fatty acid oxidation and esterification, and gluconeogenesis J. P. Fulgencio 1, C. Kohl 2, J. Girard 2 and J. P. Pegorier 2
l l-F~ H Mender, Cr6teil, France and 2CEREMOD, Meudon Bellevue, France. Insulin resistance in obesity and diabetes is associated with an active hepatic gluconeogenesis (GNG) supported by an increased hepatic fatty acid oxidation. Troglitazone (Tro) decreases hepatic GNG and circulating VLDL-triglycerides, and improves hepatic insulin resistance. This work was designed to investigate the mechanisms involved in the effects of Tro in isolated hepatocytes from 24-h starved rats, incubated for 1 h in the absence (control, C) or in the presence of 1 mM Tro. Fatty acid oxidation and esterification were estimated with 0.3 mM [1J4C]-oleate. GNG was measured in the presence of alanine or dihydroxyacetene (DHA), each at 10 mM. The Km and Vmaxof acyI-CoA synthetase (ACS) were determined in mitochondria and microsomes isolated from 1-h incubated C or Tro hepatocytes. Oleate oxidation was respectively 88 _+ 6 and 30 + 2 nmol/h/106 cells in C and Tro hepatocytes (P < 0.05). The production of ~-hydroxybutyrate was nearly abolished leading to a very oxidized mitochondrial redox state, as estimated by the fall in the ~hydroxybutyrate/acetoacetate ratio. Tro inhibited GNG, either in the presence of alanine (81 _+8%) or DHA (40 _+6%). As DHA bypasses the gluconeogenic steps controlled by fatty acid oxidation, these results suggest that Tro could partly inhibit GNG secondarily to the decreased availability in acetyI-CoA, ATP and reduced equivalents (NADH). Triglyceride synthesis was 10.5 -+ 1.0 and 1.4 _+0.1 nmol/h/106 cells in C and "Fro hepatocytes respectively (P < 0.05), whereas phospholipid synthesis was unaffected. AcyI-CoA synthesis is an obligatory step prior to fatty acid metabolism. The mitochondrial and microsomal Vm~×of ACS was respectively decreased by 58 + 6% and 61 _+15% by Tro (P < 0.05), whereas its Km was unaffected. In conclusion, Tro inhibits hepatic mitochondrial and microsomal ACS, leading to a decrease in triglyceride synthesis and fatty acid oxidation. The inhibition of fatty acid oxidation is partly responsible for the decrease in hepatic GNG.
0.4 Insulin infusion restores surgery-induced reduction of insulin-stimulated glucose uptake without increasing substrate oxidation or translocation of Glut4 A. Thorell, J. Nygren, S. Nair1, M. Hirshman 2, E. S. Horton 2, L.
Aim: To determine the plasma appearance rate (Ra) of glutamine in
Goodyear 2 and O. Ljungqvist Dept of Surgery, Karolinska Hospital, Stockholm, IDept of Endocrinology, Mayo Clinic, Minnesota, USA, 2joslin r)iabetes Center, Harvard Medical School, Boston, USA.
patients with gastrointestinal malignancies. Methods: Ten preoperative metabolically stable patients with colon or gastric cancer were given a 6-h (n = 5) or 11-h (n= 5) primed intravenous infusion of L-[5-15N]-glutamine (0.67 ~tmol/kg/h, 1-h prime) in the postabsorptive state. Arterial blood samples and percutaneous muscle biopsies were taken and analysed for 15N glutamine enrichment by GC-combustion-IRMS. The plasma Ra of glutamine was calculated via the standard isotope dilution expression for dilution of the infused ~SN glutamine in plasma: Ra = i[EI/Ep-1]. Results: The 15N glutamine enrichment in plasma gradually increased in each patient during both 6-h and 11-h tracer infusion periods, indicating that isotopic steady state had not been achieved. Consequently, the plasma Ra of glutamine decreased during the course of the study from 240 _+14 ~mol/kg/h (t = 2 h; n = 10, mean + SEM) to 212 _+13 llmol/kg/h (t = 6h; n= 10, P< 0.01 vs t= 2h) and 185 + 141tmol/kg/h (t= 11 h; n= 5, P < 0.05 vs t= 6 h). The 15N glutamine enrichment in muscle as percentage of the plasma enrichment, increased from 6.9 _+0.6% (t = 2 h, n = 5) to 11.6 _+2.0% (t = 4 h, n = 4), 15.3 _+1.2% (t = 6 h, n = 10) and 24.5 _+3.0% (t =11h, n=5). Conclusion: The results of the present study show that the 15N glutamine tracer enters the large glutamine pool in muscle. As equilibration between the plasma and muscle pool is slow, it takes probably more than 24h before steady state conditions are established. Previous suggestions that the plasma Ra of glutamine can be calculated via the standard isotope dilution expression between 2 and 4 h of tracer infusion, in our hands leads to a considerable overestimation of glutamine Ra.
Background:The underlying molecular mechanisms that cause surgeryand trauma-induced whole body insulin resistance and decreased rates of skeletal muscle glucose uptake are not known. In skeletal muscle, glucose transport is rate-limiting for glucose disposal. In healthy human subjects, insulin-stimulated glucose transport is due, at least in part, to the translocation of the Glut4 glucose transporter proteins from inside the cell to the surface (plasma) membranes. Methods: Six otherwise healthy patients (5M/1F, age 59 (3) years, BMI 26.5 (1.0)), scheduled for total hip replacement, underwent :2 euglycaemic (5 mmol), hyperinsulinaemic (60 #U/ml) clamps; one a week prior to surgery (pre-op) and one immediately post-op. Before and at the end of each clamp (=120 rain), body glucose uptake (Rd), glucose and fat oxidation and non-oxidative glucose uptake (NOGU) were measured. Biopsies of vastus lateralis muscle (=1 g) were obtained and used to purify plasma membranes for the measurement of Glut4 translocation. Immediately after the post-op clamp, in an attempt to restore preoperative insulin-stimulated glucose uptake, the patients were given sufficient amounts of insulin to match preoperative glucose infusion rate (feedback clamp), and a last sampling period was performed. Results: (Wilcoxon's signed rank test, mean (SEM)). For the pre-op clamp, insulin infusion increased Rd to 3.41 (0.4) mg/kg/min, increased Glut4 translocation to 128 (8) arbitrary units, increased glucose oxidation to 2.06 (0.35) mg/kg/min and decreased fat oxidation to 0.5,1 (0.13) mg/kg/min (all P < 0.05 vs basal). In the first phase of the post-op clamp, Rd was 2.27 (0.21) mg/kg/min (P < 0.05 vs pre-op) while translocatien of Glut4 and substrate oxidation were not different. During feedback clamp
(serum insulin 92 + 21 #U/ml), Rd was normalized to pre-op levels (3.15 (0.52) mg/kg/min) and an increase of NOGU occurred without further alterations of translocations of Glut4 or substrate oxidation. Conclusions: Immediate postoperatively, insulin resistance develops, but can be reversed by insulin infusion. Alterations in the degree of translocation of Glut4 can not explain these events. Postoperatively, insulin infusion primarily seems to stimulate the non-oxidative pathway intracellularly.
0.5 Assessment of perioperative glycerol metabolism by stable isotope tracer technique Th. Schricker, U. Pfeiffer, A. Berroth, M. Schreiber, W. Geisser,
A. Goertz and M. Georgieff Clinic of Anesthesiology, UIm, Germany.
Introduction: The objective of this study was to investigate metabolic changes during and after lower abdominal surgery with specific regard to glycerol metabolism. Methods: With the approval of the ethics committee, 7 otherwise healthy patients receiving abdominal hysterectomy (47 _+8 years, 162 4- 4 cm, 64 4- 10kg) were studied. Glycerol turnover (GTO) - a measure of whole body lipolysis - and hepatic glucose production (HGP) were quantified by stable isotope tracer technique ([1,1,2,3,3-2Hs]-glycerol, [6,6-2H2]-glucose), before and 3 h after surgery. Metabolic substrates (glycerol, nonesterified fatty acids (NEFA), ~-hydroxybutyrate (BOH), glucose) and hormones (adrenaline (A), noradrenaline (NA), cortisol (C), insulin, glucagon) were determined pre-, intra- and postoperatively. Values are means 4- SD. Results: Hysterectomy induced an increase of GTO from 3.56 4- 1.28 to 6.46 4- 2.44 pmol/kg/min (P < 0.05). This increment was inversely related to the patients' age (r = 0.872, P < 0.05). HGP and glucose postoperatively rose from 9.8 _ 1.6 to 12.8 _+1.5#mol/kg/min (P < 0.05) and from 4.6 4-0.9 to 6.2 4-0.9 mmol/I (P < 0.05), respectively. NEFA and BOH also significantly increased post surgery (NEFA: 506 _+ 172 vs. 766 _+ 116t~mol/I, BOH: 174 4-107 vs. 655 _+276 p.mol/I, P< 0.05), while glycerol revealed no pedoperative change. Cortisol (5 _+3 vs. 37 4- 14 }~g/dl, P < 0.05) and catecholamines (A: 25 4- 7 vs. 145 _+96 pg/mt, NA: 183 _+70 vs. 631 _+299 pg/ml, P < 0.05) increased after the operation, but glucagon and insulin were not altered. Conclusions: The postoperative enhancement of lipolysis was not detectable from plasma glycerol concentrations, which remained unchanged. In addition hysterectomy caused postoperative hyperglycemia due to stimulated H G P - common features of the metabolic response to surgical trauma. The results gave further evidence that observations based on static metabolite levels provide only limited insight into the underlying dynamic biochemical events, especially under perioperative conditions. 0.6 Does the presence of fish oil (FO) in emulsion particles affect the elimination of MCT/LCT emulsion? Y. A. Carpentier, V. S. Siderova, M. Richefle, Ft. J. Decke/baum, D.
Eggerickx and F. Sultan~ Universit# Libre de Bruxelles, Brussels, Belgium and ~Braun M6dical, Paris, France. Triglycerides (TG) containing long-chain n-3 polyunsaturated fatty acids (LCPFA) are poorly hydrolyzed by lipoprotein lipase (LPL); further, when FO emulsions are mixed with soy TG emulsions, they can have a suppressive effect on total lipolysis. The purpose of the present study was to determine whether including 10% FOTG in particles containing 50% MCT and 40% LCT would affect TG clearance and intravascular metabolism during infusion in man, using the TG clamp model. Eight healthy normolipidaemic subjects (mean age 23 _+3 years) were infused during 4 consecutive days with MCT/LCT alone and, 4 weeks later, with MCT/LCT + FO (MLF). Glucose and amino acids were also infused to minimize the contribution of endogenous fat stores. TG level was clamped at 3 mmol/L for 5h by adjusting the infusion rate. TG elimination is expressed by the mean rate (mg TG/kg b.w./h _+SD) during the 2 last h of infusion:
D1
D2
MCT/LCT 162_+35 178_+53 MLF 205 -+39*** 207 _+52* ***P< 0.01, **P< 0.025, *P< 0.05.
D3
D4
174_+57 199 _+48**
164_+62 204 -+40***
The ability to clear infused TG remained fairly constant over each 4-day period in each subject (CV = 11.6% with MCT/LCT, 7.3% with MLF), but large interindividual differences were observed (98-203 mgTG/kg/h with
MCT/LCT, 134-278mgTG/kg/h with MLF). Infusion of both emulsions resulted in a similar increase of plasma free fatty acids (1.0 4- 0.1 mmol/L with MCT/LCT vs. 1.2 + 0.1 mmol/L with MLF, NS). In vitro hydrolysis of both emulsions with LPL showed very similar rates of FFA release, both with respect to time and to varying enzyme concentrations (data not shown). Conclusions: 1. The presence of 10% FO in MCT/LCT particles does not reduce, but rather enhances elimination of infused TG. 2. The difference may not be related to a higher rate of hydrolysis, but to a more efficient uptake of remnant particles.
0.7 Lipoprotein lipase activity and mass in plasma following administration of exogenous fat in healthy subjects A. ThSrne, J. NordenstrSm and T. Olivecrona*
Department of Surget34, Huddinge University Hospital and *Department of Medical Biochemistry and Biophysics, Ume& University, Sweden. Lipoprotein lipase (LPL) plays a pivotal role in the handling of fat emulsions, e.g. unloading plasma triglycerides at the endothelial surface in peripheral tissues. Previous studies have shown that after an overnight fast a majority of circulating LPL is catalytically inactive. Heparin administration is followed by an increase in both LPL activity and mass, which is assumed to reflect the available amount of active LPL Administration of exogenous fat is followed by a rise in LPL activity but whether the LPL mass is influenced is unknown. The purpose of the study was to examine the influence of exogenous fat administration, as a hypertriglyceddaemic (HTG) clamp, on LPL activity and mass in health subjects. Methods: A priming dose of a fat emulsion (Intratipid®, 20%, Pharmacia AB), 13 mgTG/kg BW x min, was given in order to reach a TG concentration of 4 mmol/I. The infusion rate was thereafter adjusted to maintain TG at 4 mmol/I during 180 rain. Twenty healthy men with an average age of 52 years (19-78 years) and body mass index (BMI) of 24 (19-27) participated in the study. All studies were performed after an overnight fast (10 h) and blood samples were collected in the basal fasting stage and then throughout the study period for analysis of LPL activity and mass. Results: In the basal state the average (_+SEM) LPL activity and mass were 0.44 _+0.07mU/ml and 194 _+ 13ng/ml and the calculated specific activity was 0.0025 _+0.0004mU/ng. During infusion of fat at an average rate of 0.202 _+0.021 mmol/min, the LPL activity increased (P < 0.001) 5fold to 2.10 + 0.29mU/ml, while the LPL mass remained virtually unchanged (183 -+ 14 ng/ml, n.s.). The corresponding calculated specific activity increased (P < 0.001 ) 5-fold to 0.0130 4- 0.0020 mU/ng. Assuming the specific activity of active plasma LPL to be 0.30 mU/ng, the calculated proportion of active LPL was 0.84 + 0.14% after an overnight fast and, during lipid infusion, it increased (P< 0.001) to 4.34 -+0.68% and the amount of inactive LPL protein tended to decrease (P=0.05) from 192 + 13 to 177 4-14 ng/ml. In conclusion, administration of exogenous fat as a HTG clamp in healthy man is accompanied by augmented plasma levels of active LPL and a tendency towards decline in catalytically inactive LPL protein. An enhanced release from the endothelium reflects the former and the latter could conceivably be due to an accelerated catabolism through binding to emulsion particles which in turn are rapidly cleared from plasma.
0.8 Role of nuclear transcription factors in regulation of albumin synthesis in protein-deprived rat liver M. Yano, T. Tsujinaka, C. Ebisui, T. Morimoto, M. Kishibuchi, S. Morita, M. Taniguchi, H. Shiozaki and M. Monden Department of Surgery II, Osaka University Medical School, Osaka, Japan.
A. Ogawa,
Serum albumin level and albumin mRNA level in the liver are known to decrease in rats when fed with a protein-free diet, although its precise mechanism is unknown. The albumin gene has six cis-acting elements in the proximal promoter region. Among these elements, B and D site are important for strong and tissue specific expression of the albumin gene. Liver-specific transcription factors, CCAAT/enhancer binding protein ~ (C/EBPc 0, C/EBP[3 and D site binding protein (DBP) bind to D site and hepatocyte nuclear factor-1 (HNF-1) binds to B site. This study was designed to elucidate the transcriptional regulation of the albumin gene and the role of nuclear transcription factors under protein deprivation. Male Donryu rats were fed with a protein-free diet (PF) or a control diet (CON) for 10 days and were killed to obtain the liver and blood. Hepatic