Applied Physiology
Eur J Appl Physiol (1990) 60:293-299
European Journal of
and Occupational Physiology © Springer-Verlag 1990
Evidence that hyperglycaemia per se does not inhibit hepatic glucose production in man Manfred J. Miiller 1, Kevin J. Acheson 2"3, Albert G. Burger 4, and Eric Jequier 2 1 Medizinische Hochschule Hannover, Gastroenterologie und Hepatologie, Konstanty-Gutschow-Strasse 8, D-3000 Hannover 61, West Germany 2 Universit6 de Lausanne, Institute de Physiologie, CH-1005 Lausanne, Switzerland 3 Nestle' Research Center, Nestlec Ltd., Vers-Chez-Les-Blanc, CH-1005 Lausanne, Switzerland 4 H6pital Cantonal de G~neve, Division Endocrinology, CH-1211 Geneva, Switzerland Accepted September 4, 1989
Summary. The effect of hyperglycaemia on hepatic glucose production (Ra) was investigated in nine healthy men using sequential clamp protocols during somatostatin infusion and euglycaemia (0-150 min), at plasma glucose levels of 165 mg-d1-1 (9.2mM, 150-270 min) and during insulin infusion (1.0 mU. k g - l . m i n - 1 , 2 7 0 360 min) in study 1 or during hypo-insulinaemia and plasma glucose levels of 220 mg.d1-1 (12.2 raM; 270390 min) in study 2. Somatostatin decreased Ra and glucose disposal rate (Rd) but increased plasma free fatty acids (FFA) and lipid oxidation during euglycaemia. Increasing plasma glucose to 165 mg.d1-1 (9.2 mM) and hypo-insulinaemia increased Rd, but no suppressive effects o n Ra, plasma FFA and lipid oxidation were observed. By contrast hyperinsulinaemia (study 1), as well as a further increase in plasma glucose (study 2), both decreased R~. However, more pronounced hyperglycaemia increased insulin secretion despite somatostatin resulting in a fall in plasma FFA and lipid oxidation. Our data questions the accepted dogma that hyperglycaemia inhibits Ra independently of insulin action.
Key words: Hyperglycaemia - Hepatic glucose production - Insulin action - Glucose turnover - Glucose-free fatty acid interaction
Introduction The effect of glucose on hepatic glucose production (Ra) has been investigated for nearly 50 years. Soskin et al. (1938) demonstrated a reciprocal relationship between Ra and plasma glucose concentration; infusing glucose into dogs decreased Ra and enhanced glucose disposal (Rd) in proportion to the degree of hyperglycaemia. The concept of glucose autoregulation of Ra has been supported by more recent findings. The inhibitory effect of glucose on Ra has been reported to be
Offprint requests to: M. J. Mfiller
independent of the hormonal changes induced by glucose, as hyperglycaemia was effective in the absence of changes in plasma insulin and glucagon (Sacca et al. 1978; Shulman et al. 1978; Liljenquist et al. 1979; DeFronzo et al. 1983; Bell et al. 1986a; Wolfe et al. 1986; Adkins et al. 1987; Mfiller et al. 1988b). This idea is based mainly on studies where glucose has been infused together with somatostatin with or without basal amounts of insulin a n d / o r glucagon. It is well known that somatostatin induces a sustained decrease in plasma insulin within 3 to 5 min, but the biological effect of insulin on hepatic Ra decays with an apparent half-life between 40 to 50 min in healthy man (Baron et al. 1985 ; Prager et al. 1986). From this finding it becomes evident that the de-activation of the action of cellular insulin lags behind the disappearance of the hormone from plasma. Thus, the conclusion that glucose by itself inhibits Ra may be erroneous, as part of the insulin action was still present during the studies cited above. It follows that it should be possible to demonstrate the effect of hyperglycaemia per se in the absence of any insulin action, e.g. after prolonged somatostatin infusion. We, therefore, performed two sequential clamp protocols in healthy subjects during prolonged hypo-insulinaemia and subsequent hyperglycaemia with and without hyperinsulinaemia. Our data provide evidence that the possible effect of hyerglycaemia on Ra depends on the presence of insulin, thus, raising questions about accepted dogma.
Methods Subjects. Experiments were performed on nine healthy male subjects (23.5 years, SD 4.4, 71.1 kg body mass, SD 6.3, 179.7 cm, SD 10.1, 1.86 mz, SD 0.10, 22.1 kg-m -2 body mass index, SD 2.6. No subject had a family history of diabetes mellitus and none was taking any medication before and during the experiments. The protocol was reviewed and accepted by the Ethical Committee of the Faculty of Medicine, University of Lausanne, and carefully explained to each volunteer, who gave written consent before being acceped to participate in the study.
294
Experimentalprotocol. Each subject was requested to keep a log of his diet and physical activities and was instructed to eat a diet rich in carbohydrates (at least 250 g per day) for 3 days before the study. The volunteers spent the night before the test at the Institute. After an overnight fast, the subject was wakened at 6.30 a.m. and after voiding urine he was transferred to the room in which the test was to be performed. One venous catheter (Venflon 2, Viggo, AB, Helsingborg, Sweden) was placed in an antecubital vein for the infusion of test substances while another (19-gauge Butterfly, Abbott Ireland Ltd, Sligo, Rep. of Ireland) was inserted retrogradely into a wrist vein for blood sampling and was kept patent with saline. The hand was then placed in a heated box (60 ° 70 ° C) to achieve arterialization of venous blood. All studies were performed in a recumbent position. At - 1 2 0 min (33H)-glucose was given as a primed (20~tCi) constant infusion (0.20 ~tCi.min -1) as described previously (Mfiller et al. 1984). Respiratory exchange measurements were begun at - 6 0 min and were continued until the end of the experiments. Two experimental protocols were performed:
Study. Six subjects underwent our original study 1. After a baseline period of 1 h a sequential euglycaemic clamp protocol was started at hypo-insulinaemia for 150 minutes, then plasma glucose was increased to a target blood glucose concentration of about 165 mg-d1-1 at ongoing hypo-insulinaemia (150-270 min). Between 270 and 360 min insulin was infused at 1.0 mU- k g - l - m i n - ~ .
berg, The Netherlands). The total 3H label was counted in the supernatant and 3H20 was calculated by substracting (33H)-glucose counts from total counts. To determine the doses of infused tracer accurately, five separate 1:100 dilutions of each injectate were run in parallel through the Somogyi procedure and counted together with the plasma samples. The tritiated water content of the infusate was below 1% of the total activity and calculations were corrected for the "true" (33H)-glucose infusion rates. All measurements were performed in duplicate and each sample was counted for 15 rain. Plasma insulin (sensitivity: 1 mU.1-1) and C-peptide were measured by radie-immunoassays, plasma lactate by fluorometry and plasma FFA by colorimetry on the dole extract ( N E F A C, Wako Chem., Neuss, FRG) (Mfiller et al. 1984; Christin et al. 1986; Mfiller et al. 1988a). Urinary nitrogen was analysed after digestion on a Technicon auto-analyser (Technicon Instruments Corp., Tarrytown, NY, USA).
Data analyses. For data presentation the mean values obtained during the last 30 min of each experimental period were considered (i.e. basal state: minus 30-0 min; bypo-insulinaemia and euglycaemia: 120-150 min; hypo-insulinaemia and hyperglycaemia: 240-270 min; hyperinsulinaemia and hyperglycaemia: 330360 min). Glucose kinetics were calculated using a two-compartment model and assuming a total glucose distribution volume of 200 ml.kg body mass -1 (Ferrannini et al. 1986). The amount of
somatostatin
Study 2. Two subjects who underwent study 1 and three other volunteers participated in study 2, which followed the protocol of study 1 up to 270 min, then plasma glucose was further increased to about 225 mg. dl-1 at ongoing insulin deficiency. Hypoinsulinaemia was brought about by somatostatin infusion (Stilamin, Serono, SA, Aubonne, Switzerland; diluted in 90 ml of normal saline to which 4 ml of the subject's own blood had been added) which was infused at 500 lxg-h-1 from 0 to 360 min and euglycaemia was maintained by an exogenous glucose infusion (20% D-glucose) up to 150 min. Measurement of the glucose concentration in the infusate revealed values between 19.6% and 20.5%. Plasma glucose concentration was raised to 165 or 225 rag. d l - I by a priming dose of glucose, which was given in a logarithmically decreasing manner to fill up the glucose space. This hypo-insulinaemic-hyperglycaemic state was maintained up to 270 (study 1) or 390 min (study 2) by varying the glucose infusion rate. In study 1 insulin (diluted in 0.9% saline to which 4 ml of the subject's own blood had been added) was given over a period of 10 min in a logarithmically falling manner (comp. Christin et al. 1986) and then infused at a constant rate of 1.0 m U - k g - I . m i n - i , for a further 80 min. Measurement of insulin concentration in the infusate resulted in an actual insulin infusion rate of 0.91 m U . k g -1 .min -1. Continuous respiratory exchange measurements were performed for the duration of the test using a ventilated hood, open circuit, indirect calorimeter (Christin et al. 1986; Mfiller et al. 1988a). Blood samples were taken every 5 min for plasma glucose determination. In addition four blood samples were taken during the last 30 rain of each experimental period, which were analysed for labelled glucose, hormones (insulin, C-peptide) and metabolires (blood urea nitrogen, plasma lactate and free fatty acids (FFA). Urine was collected at the beginning and end of the test and analysed for glucose and nitrogen.
]insulin glucose (mg • d1-1)
t
~.:;;:~.~
2oo~ 150 4
glucose infused (mg • kg -1.10 min -1)
30 10
i
endogenous R a (mg. kg -1. rain -1)
aI I
,
:)
0 Rd (mg • kg -1. min -1)
', I
i
6
!
I
0
i
t
I
MCRg -1 (ml. Rg • min-1)
J I
-30 0
60
I
7
120 180 240 300 360 time (min)
Analyses. Plasma glucose was analysed in a Beckman II glucose
mean and SD
analyser (Beckman Instruments, Inc. Fullerton, CA, USA). For measurements of radio-active glucose, 0.5 ml plasma was deproteinized (Somogyi's method) and 0.8 ml of the protein free supernatant were evaporated to dryness in a vacuum at - 7 0 ° C for 12 h to eliminate 3H20 (MOiler et al. 1983). The residue was redissolved in 1 ml H20 and radio-activity was measured after the addition of 10 ml of liquid scintillant (Lumagel, Lumac, 3M, Schaes-
Fig. 1. Kinetic alterations in plasma glucose, the amount of glucose infused to maintain euglycaemia or hyperglycaemia, endogenous glucose production (R~), glucose disposal (Rd) and the metabolic clearance rate of glucose (MCRg) during the different periods of the experimental protocol. Data are given as means and SD, n = 6
295 glucon metabolized (M) was calculated from the glucose infusion rate after correction for changes in the glucose space (Bergman et al. 1985). During the clamp, the rate of endogenous R~ was calculated from the difference between isotopically determined total R~ and the amount of glucose infused. Substrate oxidation rates were calculated according to previously reported methods (Christin et al. 1986). The following constants were used: 6.25 g of protein to produce 1 g of urinary nitrogen, 966.3 ml O2 was consumed to oxidize 1 g of protein, which produced 773.9 ml CO2. The respiratory quotient for complete lipid oxidation is 0.707 and 1.000 for complete glucose oxidation. The 02 consumed per g substrate oxidized was 2.019 1.g -1 fat and 0.8291 1.g -1 glycogen and 0.746 1.g- ~ glucose. The constants for glycogen were used in the basal state. During glucose infusion, the constants of glucose were used. Since it is possible that during hypo-insulinaemia and euglycaemia a considerable amount of glucose oxidized is derived from intracellular glycogen, our data were recalculated using the constant for glycogen up to 150 min of our protocol. Glucose oxidation was calculated from the respiratory exchange data, which were continuously recorded, integrated every 5 min and then averaged for the last 30 min of each period. Non-oxidative Re was calculated by substracting glucose oxidation from total Rd. Protein oxidation was calculated from the urinary N-excretion after correction for changes in the urea pool (Christin et al. 1986). Statistical analyses were performed using analysis of variance (ANOVA). All data are given as means and SD.
Results
Study 1 Infusing s o m a t o s t a t i n r e d u c e d arterial insulin a n d Cpeptide (Table 3). C o n c o m i t a n t l y e n d o g e n o u s Ra, Ra, glucose o x i d a t i o n as well as the rate o f non-oxidative glucose m e t a b o l i s m all decreased (Fig. 1, Tables 1, 2), but lipid o x i d a t i o n a n d p l a s m a F F A b o t h increased (Tables 2, 3). To m a i n t a i n e u g l y c a e m i a glucose h a d to be infused u p to 150 min (Fig. 1). The coefficient o f
variation (CV) o f p l a s m a glucose was 0.5%, SD 0.6% (basal period) a n d increased to 2.4%, S D 1.2% during period 1 ( e u g l y c a e m i a + hypo-insulinaemia). D u r i n g the course o f somatostatin infusion glucose infusion was r e d u c e d due to the recovery in e n d o g e n o u s Ra, which p r e c e e d e d the r e b o u n d in Ra (Fig. 1). Calculating means b e t w e e n 120 and 150 min s h o w e d that endogenous Ra a n d Ra were each r e d u c e d to a b o u t 74% (Fig. 1, Tables 1, 2). N o significant changes were observed for p l a s m a lactate (Table 3). D u r i n g period 2 (hyperglycaemia + h y p o - i n s u l i n a e m i a ) glucose infusion was increased to reach h y p e r g l y c a e m i a (target glucose 165 m g . d 1 - 1 , 9 . 1 7 m M ) , but was subsequently r e d u c e d once the glucose space h a d been filled (Fig. 1). Plasma glucose was c l a m p e d at 168 m g - d 1 - 1 (9.33 m M ) with a CV o f 1.9%, S D 1.0%. D u r i n g this period e n d o g e n o u s R~, Rd a n d n o n - o x i d a t i v e glucose m e t a b o l i s m all increased, whereas metabolic clearance rate for glucose (MCRg), glucose a n d lipid oxidation as well as p l a s m a F F A r e m a i n e d u n c h a n g e d (Fig. 1, Tables 2, 3). D u r i n g p e r i o d 3 ( h y p e r g l y c a e m i a + hyperinsulinaemia) insulin was infused at u n c h a n g e d p l a s m a glucose c o n c e n t r a t i o n s (CV: 2.8%, SD 1.5%). A l t h o u g h steady-state conditions were not r e a c h e d within 90 min insulin infusion increased Ra, MCRg, glucose oxidation, the rate o f n o n o x i d a t i v e glucose m e t a b o l i s m a n d p l a s m a lactate (Fig. 1, Tables 2, 3) but decreased Ra, lipid oxidation a n d p l a s m a F F A (Fig. 1, Tables 1, 2,
3). Study 2 I n order to p e r f o r m an a p p r o p r i a t e control study study 2 was d o n e following our original p r o t o c o l o f study 1
Table 1. Alterations in plasma glucose, rate of glucose appearance (Ra), endogenous glucose production (endog Ra) and glucose utilization (Rd) during the basal state, euglycaemia plus hypo-insulinaemia (period 1), hyperglycaemia plus hypoinsulinaemia (period 2) and hyperglycaemia plus hyperinsulinaemia (study 1, period 3) or more pronounced hyperglycaemia plus hypoinsulinaemia (study 2, period 3). Data are means and SD; significance: * P
Eur J Appl Physiol (1990) 60:293-299
European Journal of
and Occupational Physiology © Springer-Verlag 1990
Evidence that hyperglycaemia per se does not inhibit hepatic glucose production in man Manfred J. Miiller 1, Kevin J. Acheson 2"3, Albert G. Burger 4, and Eric Jequier 2 1 Medizinische Hochschule Hannover, Gastroenterologie und Hepatologie, Konstanty-Gutschow-Strasse 8, D-3000 Hannover 61, West Germany 2 Universit6 de Lausanne, Institute de Physiologie, CH-1005 Lausanne, Switzerland 3 Nestle' Research Center, Nestlec Ltd., Vers-Chez-Les-Blanc, CH-1005 Lausanne, Switzerland 4 H6pital Cantonal de G~neve, Division Endocrinology, CH-1211 Geneva, Switzerland Accepted September 4, 1989
Summary. The effect of hyperglycaemia on hepatic glucose production (Ra) was investigated in nine healthy men using sequential clamp protocols during somatostatin infusion and euglycaemia (0-150 min), at plasma glucose levels of 165 mg-d1-1 (9.2mM, 150-270 min) and during insulin infusion (1.0 mU. k g - l . m i n - 1 , 2 7 0 360 min) in study 1 or during hypo-insulinaemia and plasma glucose levels of 220 mg.d1-1 (12.2 raM; 270390 min) in study 2. Somatostatin decreased Ra and glucose disposal rate (Rd) but increased plasma free fatty acids (FFA) and lipid oxidation during euglycaemia. Increasing plasma glucose to 165 mg.d1-1 (9.2 mM) and hypo-insulinaemia increased Rd, but no suppressive effects o n Ra, plasma FFA and lipid oxidation were observed. By contrast hyperinsulinaemia (study 1), as well as a further increase in plasma glucose (study 2), both decreased R~. However, more pronounced hyperglycaemia increased insulin secretion despite somatostatin resulting in a fall in plasma FFA and lipid oxidation. Our data questions the accepted dogma that hyperglycaemia inhibits Ra independently of insulin action.
Key words: Hyperglycaemia - Hepatic glucose production - Insulin action - Glucose turnover - Glucose-free fatty acid interaction
Introduction The effect of glucose on hepatic glucose production (Ra) has been investigated for nearly 50 years. Soskin et al. (1938) demonstrated a reciprocal relationship between Ra and plasma glucose concentration; infusing glucose into dogs decreased Ra and enhanced glucose disposal (Rd) in proportion to the degree of hyperglycaemia. The concept of glucose autoregulation of Ra has been supported by more recent findings. The inhibitory effect of glucose on Ra has been reported to be
Offprint requests to: M. J. Mfiller
independent of the hormonal changes induced by glucose, as hyperglycaemia was effective in the absence of changes in plasma insulin and glucagon (Sacca et al. 1978; Shulman et al. 1978; Liljenquist et al. 1979; DeFronzo et al. 1983; Bell et al. 1986a; Wolfe et al. 1986; Adkins et al. 1987; Mfiller et al. 1988b). This idea is based mainly on studies where glucose has been infused together with somatostatin with or without basal amounts of insulin a n d / o r glucagon. It is well known that somatostatin induces a sustained decrease in plasma insulin within 3 to 5 min, but the biological effect of insulin on hepatic Ra decays with an apparent half-life between 40 to 50 min in healthy man (Baron et al. 1985 ; Prager et al. 1986). From this finding it becomes evident that the de-activation of the action of cellular insulin lags behind the disappearance of the hormone from plasma. Thus, the conclusion that glucose by itself inhibits Ra may be erroneous, as part of the insulin action was still present during the studies cited above. It follows that it should be possible to demonstrate the effect of hyperglycaemia per se in the absence of any insulin action, e.g. after prolonged somatostatin infusion. We, therefore, performed two sequential clamp protocols in healthy subjects during prolonged hypo-insulinaemia and subsequent hyperglycaemia with and without hyperinsulinaemia. Our data provide evidence that the possible effect of hyerglycaemia on Ra depends on the presence of insulin, thus, raising questions about accepted dogma.
Methods Subjects. Experiments were performed on nine healthy male subjects (23.5 years, SD 4.4, 71.1 kg body mass, SD 6.3, 179.7 cm, SD 10.1, 1.86 mz, SD 0.10, 22.1 kg-m -2 body mass index, SD 2.6. No subject had a family history of diabetes mellitus and none was taking any medication before and during the experiments. The protocol was reviewed and accepted by the Ethical Committee of the Faculty of Medicine, University of Lausanne, and carefully explained to each volunteer, who gave written consent before being acceped to participate in the study.
294
Experimentalprotocol. Each subject was requested to keep a log of his diet and physical activities and was instructed to eat a diet rich in carbohydrates (at least 250 g per day) for 3 days before the study. The volunteers spent the night before the test at the Institute. After an overnight fast, the subject was wakened at 6.30 a.m. and after voiding urine he was transferred to the room in which the test was to be performed. One venous catheter (Venflon 2, Viggo, AB, Helsingborg, Sweden) was placed in an antecubital vein for the infusion of test substances while another (19-gauge Butterfly, Abbott Ireland Ltd, Sligo, Rep. of Ireland) was inserted retrogradely into a wrist vein for blood sampling and was kept patent with saline. The hand was then placed in a heated box (60 ° 70 ° C) to achieve arterialization of venous blood. All studies were performed in a recumbent position. At - 1 2 0 min (33H)-glucose was given as a primed (20~tCi) constant infusion (0.20 ~tCi.min -1) as described previously (Mfiller et al. 1984). Respiratory exchange measurements were begun at - 6 0 min and were continued until the end of the experiments. Two experimental protocols were performed:
Study. Six subjects underwent our original study 1. After a baseline period of 1 h a sequential euglycaemic clamp protocol was started at hypo-insulinaemia for 150 minutes, then plasma glucose was increased to a target blood glucose concentration of about 165 mg-d1-1 at ongoing hypo-insulinaemia (150-270 min). Between 270 and 360 min insulin was infused at 1.0 mU- k g - l - m i n - ~ .
berg, The Netherlands). The total 3H label was counted in the supernatant and 3H20 was calculated by substracting (33H)-glucose counts from total counts. To determine the doses of infused tracer accurately, five separate 1:100 dilutions of each injectate were run in parallel through the Somogyi procedure and counted together with the plasma samples. The tritiated water content of the infusate was below 1% of the total activity and calculations were corrected for the "true" (33H)-glucose infusion rates. All measurements were performed in duplicate and each sample was counted for 15 rain. Plasma insulin (sensitivity: 1 mU.1-1) and C-peptide were measured by radie-immunoassays, plasma lactate by fluorometry and plasma FFA by colorimetry on the dole extract ( N E F A C, Wako Chem., Neuss, FRG) (Mfiller et al. 1984; Christin et al. 1986; Mfiller et al. 1988a). Urinary nitrogen was analysed after digestion on a Technicon auto-analyser (Technicon Instruments Corp., Tarrytown, NY, USA).
Data analyses. For data presentation the mean values obtained during the last 30 min of each experimental period were considered (i.e. basal state: minus 30-0 min; bypo-insulinaemia and euglycaemia: 120-150 min; hypo-insulinaemia and hyperglycaemia: 240-270 min; hyperinsulinaemia and hyperglycaemia: 330360 min). Glucose kinetics were calculated using a two-compartment model and assuming a total glucose distribution volume of 200 ml.kg body mass -1 (Ferrannini et al. 1986). The amount of
somatostatin
Study 2. Two subjects who underwent study 1 and three other volunteers participated in study 2, which followed the protocol of study 1 up to 270 min, then plasma glucose was further increased to about 225 mg. dl-1 at ongoing insulin deficiency. Hypoinsulinaemia was brought about by somatostatin infusion (Stilamin, Serono, SA, Aubonne, Switzerland; diluted in 90 ml of normal saline to which 4 ml of the subject's own blood had been added) which was infused at 500 lxg-h-1 from 0 to 360 min and euglycaemia was maintained by an exogenous glucose infusion (20% D-glucose) up to 150 min. Measurement of the glucose concentration in the infusate revealed values between 19.6% and 20.5%. Plasma glucose concentration was raised to 165 or 225 rag. d l - I by a priming dose of glucose, which was given in a logarithmically decreasing manner to fill up the glucose space. This hypo-insulinaemic-hyperglycaemic state was maintained up to 270 (study 1) or 390 min (study 2) by varying the glucose infusion rate. In study 1 insulin (diluted in 0.9% saline to which 4 ml of the subject's own blood had been added) was given over a period of 10 min in a logarithmically falling manner (comp. Christin et al. 1986) and then infused at a constant rate of 1.0 m U - k g - I . m i n - i , for a further 80 min. Measurement of insulin concentration in the infusate resulted in an actual insulin infusion rate of 0.91 m U . k g -1 .min -1. Continuous respiratory exchange measurements were performed for the duration of the test using a ventilated hood, open circuit, indirect calorimeter (Christin et al. 1986; Mfiller et al. 1988a). Blood samples were taken every 5 min for plasma glucose determination. In addition four blood samples were taken during the last 30 rain of each experimental period, which were analysed for labelled glucose, hormones (insulin, C-peptide) and metabolires (blood urea nitrogen, plasma lactate and free fatty acids (FFA). Urine was collected at the beginning and end of the test and analysed for glucose and nitrogen.
]insulin glucose (mg • d1-1)
t
~.:;;:~.~
2oo~ 150 4
glucose infused (mg • kg -1.10 min -1)
30 10
i
endogenous R a (mg. kg -1. rain -1)
aI I
,
:)
0 Rd (mg • kg -1. min -1)
', I
i
6
!
I
0
i
t
I
MCRg -1 (ml. Rg • min-1)
J I
-30 0
60
I
7
120 180 240 300 360 time (min)
Analyses. Plasma glucose was analysed in a Beckman II glucose
mean and SD
analyser (Beckman Instruments, Inc. Fullerton, CA, USA). For measurements of radio-active glucose, 0.5 ml plasma was deproteinized (Somogyi's method) and 0.8 ml of the protein free supernatant were evaporated to dryness in a vacuum at - 7 0 ° C for 12 h to eliminate 3H20 (MOiler et al. 1983). The residue was redissolved in 1 ml H20 and radio-activity was measured after the addition of 10 ml of liquid scintillant (Lumagel, Lumac, 3M, Schaes-
Fig. 1. Kinetic alterations in plasma glucose, the amount of glucose infused to maintain euglycaemia or hyperglycaemia, endogenous glucose production (R~), glucose disposal (Rd) and the metabolic clearance rate of glucose (MCRg) during the different periods of the experimental protocol. Data are given as means and SD, n = 6
295 glucon metabolized (M) was calculated from the glucose infusion rate after correction for changes in the glucose space (Bergman et al. 1985). During the clamp, the rate of endogenous R~ was calculated from the difference between isotopically determined total R~ and the amount of glucose infused. Substrate oxidation rates were calculated according to previously reported methods (Christin et al. 1986). The following constants were used: 6.25 g of protein to produce 1 g of urinary nitrogen, 966.3 ml O2 was consumed to oxidize 1 g of protein, which produced 773.9 ml CO2. The respiratory quotient for complete lipid oxidation is 0.707 and 1.000 for complete glucose oxidation. The 02 consumed per g substrate oxidized was 2.019 1.g -1 fat and 0.8291 1.g -1 glycogen and 0.746 1.g- ~ glucose. The constants for glycogen were used in the basal state. During glucose infusion, the constants of glucose were used. Since it is possible that during hypo-insulinaemia and euglycaemia a considerable amount of glucose oxidized is derived from intracellular glycogen, our data were recalculated using the constant for glycogen up to 150 min of our protocol. Glucose oxidation was calculated from the respiratory exchange data, which were continuously recorded, integrated every 5 min and then averaged for the last 30 min of each period. Non-oxidative Re was calculated by substracting glucose oxidation from total Rd. Protein oxidation was calculated from the urinary N-excretion after correction for changes in the urea pool (Christin et al. 1986). Statistical analyses were performed using analysis of variance (ANOVA). All data are given as means and SD.
Results
Study 1 Infusing s o m a t o s t a t i n r e d u c e d arterial insulin a n d Cpeptide (Table 3). C o n c o m i t a n t l y e n d o g e n o u s Ra, Ra, glucose o x i d a t i o n as well as the rate o f non-oxidative glucose m e t a b o l i s m all decreased (Fig. 1, Tables 1, 2), but lipid o x i d a t i o n a n d p l a s m a F F A b o t h increased (Tables 2, 3). To m a i n t a i n e u g l y c a e m i a glucose h a d to be infused u p to 150 min (Fig. 1). The coefficient o f
variation (CV) o f p l a s m a glucose was 0.5%, SD 0.6% (basal period) a n d increased to 2.4%, S D 1.2% during period 1 ( e u g l y c a e m i a + hypo-insulinaemia). D u r i n g the course o f somatostatin infusion glucose infusion was r e d u c e d due to the recovery in e n d o g e n o u s Ra, which p r e c e e d e d the r e b o u n d in Ra (Fig. 1). Calculating means b e t w e e n 120 and 150 min s h o w e d that endogenous Ra a n d Ra were each r e d u c e d to a b o u t 74% (Fig. 1, Tables 1, 2). N o significant changes were observed for p l a s m a lactate (Table 3). D u r i n g period 2 (hyperglycaemia + h y p o - i n s u l i n a e m i a ) glucose infusion was increased to reach h y p e r g l y c a e m i a (target glucose 165 m g . d 1 - 1 , 9 . 1 7 m M ) , but was subsequently r e d u c e d once the glucose space h a d been filled (Fig. 1). Plasma glucose was c l a m p e d at 168 m g - d 1 - 1 (9.33 m M ) with a CV o f 1.9%, S D 1.0%. D u r i n g this period e n d o g e n o u s R~, Rd a n d n o n - o x i d a t i v e glucose m e t a b o l i s m all increased, whereas metabolic clearance rate for glucose (MCRg), glucose a n d lipid oxidation as well as p l a s m a F F A r e m a i n e d u n c h a n g e d (Fig. 1, Tables 2, 3). D u r i n g p e r i o d 3 ( h y p e r g l y c a e m i a + hyperinsulinaemia) insulin was infused at u n c h a n g e d p l a s m a glucose c o n c e n t r a t i o n s (CV: 2.8%, SD 1.5%). A l t h o u g h steady-state conditions were not r e a c h e d within 90 min insulin infusion increased Ra, MCRg, glucose oxidation, the rate o f n o n o x i d a t i v e glucose m e t a b o l i s m a n d p l a s m a lactate (Fig. 1, Tables 2, 3) but decreased Ra, lipid oxidation a n d p l a s m a F F A (Fig. 1, Tables 1, 2,
3). Study 2 I n order to p e r f o r m an a p p r o p r i a t e control study study 2 was d o n e following our original p r o t o c o l o f study 1
Table 1. Alterations in plasma glucose, rate of glucose appearance (Ra), endogenous glucose production (endog Ra) and glucose utilization (Rd) during the basal state, euglycaemia plus hypo-insulinaemia (period 1), hyperglycaemia plus hypoinsulinaemia (period 2) and hyperglycaemia plus hyperinsulinaemia (study 1, period 3) or more pronounced hyperglycaemia plus hypoinsulinaemia (study 2, period 3). Data are means and SD; significance: * P
