AJCN 19th International Congress of Nutrition
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American Journal of Clinical Nutrition, Vol. 69, No. 6, 1174-1182, June 1999
© 1999 American Society for Clinical Nutrition

Original Research Communications

Euglycemic hyperinsulinemic clamp to assess posthepatic glucose appearance after carbohydrate loading. 1. Validation in pigs1,2,3

Vincent Lang, Pierre Vaugelade, Françoise Bernard, Béatrice Darcy-Vrillon, Catherine Alamowitch, Gérard Slama, Pierre-Henri Duée and Francis RJ Bornet


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Precise knowledge of the rate of glucose absorption after meal feeding requires invasive methods in humans.

Objective: This study aimed to validate in an animal model a technique combining the euglycemic hyperinsulinemic clamp and oral carbohydrate loading (OC-Clamp) as a noninvasive procedure to quantify the posthepatic appearance of glucose after oral carbohydrate loading.

Design: Twenty-one pigs were fitted with arterial, jugular, portal, and duodenal catheters and a portal blood flow probe. At glucose clamp steady state, duodenal glucose (0.9 g/kg; DG-Clamp) and oral carbohydrate (140 g corn or mung bean starch as part of a mixed meal; OC-Clamp) were administered while the glucose infusion was progressively reduced to compensate for the incremental posthepatic appearance of glucose. [3-3H]glucose was used to assess the glucose turnover rate.

Results: Hepatic glucose production was totally suppressed by insulin infusion, and the whole-body glucose turnover rate remained stable during glucose absorption. The incremental portal appearance of glucose after the DG load was not altered by hyperinsulinemia, and the cumulative posthepatic appearance of glucose was 63 ± 3% ( x ± SEM) of the DG load. The net hepatic portal appearance of glucose remained constant during absorption (34 ± 3% of the load). After the OC load, the respective portal appearance rates of glucose were significantly different between carbohydrate sources; however, the rates paralleled those of the posthepatic appearance of glucose. Again, net hepatic glucose uptake expressed as portal appearance was similar for both carbohydrates.

Conclusions: The results validate the OC-Clamp method to monitor the posthepatic appearance of glucose after carbohydrate ingestion and to discriminate between different carbohydrate sources. The results suggest that the technique be used in humans.

Key Words: Glucose • insulin • intestinal absorption • intes-tinal metabolism • portal vein catheter technique • carbohydrate load • euglycemic clamp • endogenous glucose production • glucose turnover rate • pigs


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
After an overnight fast, blood glucose homeostasis is maintained by a precise balance between the rate of hepatic and renal glucose production and the amount of glucose taken up by peripheral tissues. After glucose ingestion, hepatic glucose production is depressed and splanchnic and peripheral glucose uptake are highly stimulated through the combined action of transient hyperglycemia and hyperinsulinemia (1–4). Therefore, exogenous glucose is efficiently metabolized, stored as glycogen, or both, and the blood glucose concentration rapidly returns to preprandial values.

The amount of glucose available for peripheral disposal represents the part of the exogenous glucose not extracted by the splanchnic bed together with the residual glucose production. Thus, when the control of glucose homeostasis is pathologically altered, the rate of glucose appearance into the systemic circulation may be impaired (4, 5). As a consequence, nutritional management may be beneficial in these circumstances (6, 7).

Such a nutritional strategy requires precise knowledge of the kinetics of glucose absorption after a meal. Because the direct measurement of the rate of glucose appearance in the portal circulation is not ethically feasible in humans, several indirect methods have been developed. However, these methods provide only a qualitative determination of the rate of intestinal glucose absorption after a carbohydrate load. Indeed, intubation techniques and breath-hydrogen tests indicate the amount of carbohydrates not absorbed after a meal (8, 9). Similarly, determination of the glycemic index from postprandial plasma glucose concentrations allows only a qualitative comparison between different carbohydrate sources (10, 11). Lastly, isotopic methods indicate the rate of systemic appearance of exogenous glucose that may be unrelated to the kinetics of glucose absorption after a meal because hepatic glucose production can remain operative (2, 3, 12, 13).

The aim of the present study was to validate in pigs the euglycemic hyperinsulinemic clamp technique as a method to quantify the posthepatic appearance of glucose after glucose loading, assuming that a certain level of hyperinsulinemia can totally suppress hepatic glucose production. This method was applied recently in humans to quantify splanchnic glucose uptake after a glucose load (14, 15). Moreover, preliminary data indicated its potential to provide information on the posthepatic appearance of glucose when intestinal glucose absorption is active (16). The pig represents a good animal model in digestive physiology for chronic portal and hepatic catheterization (17). Thus, a concomitant comparison between the rate of portal appearance of glucose and determination of the rate of posthepatic appearance of glucose could be performed after duodenal glucose (DG) or oral carbohydrate (OC) loads.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and surgical procedure
Experiments were performed on 21 female pigs (mean live weight at the time of surgery: 58 ± 2 kg) of the Large White breed. Two weeks before surgery, animals were kept in restraining cages and fed twice daily 800 g of a semisynthetic diet (casein, 178 g/kg; purified cornstarch, 516 g/kg; sucrose, 65 g/kg; soybean oil, 150 g/kg; purified cellulose, 50 g/kg; mineral and vitamin supplements, 41 g/kg; UFAC, Limours, France) mixed with 1.6 L water. While the pigs were under general anesthesia induced by halothane (Fluothan; Laboratoires Belamont, Paris), 3 polyvinyl chloride catheters (1.27 mm internal diameter, 2.29 mm outside diameter; Tygon Norton, Cleveland) were implanted in their portal and jugular veins and right carotid arteries; portal blood flow probes (Transonic probes; Transonic Systems, Ithaca, NY) were also placed according to procedures described previously (18) for continuous recording of portal blood flow. Arterial hepatic blood flow probes and hepatic vein catheters were also implanted in 5 of the 21 animals as described previously (19). For the experiments with DG infusion, a permanent fistulation of the duodenum was performed. Experiments began 8–10 d after the surgical procedure, when the animals had resumed presurgical feed intakes. All aspects of the protocol conformed to the International guidelines for biomedical research involving animals.

Experimental design
Validation with duodenal glucose
Eight animals were randomly submitted to 3 protocols: 1) a DG load combined with a euglycemic hyperinsulinemic clamp (DG-Clamp), 2) a glucose-free water load into the duodenum combined with a euglycemic hyperinsulinemic clamp, and 3) a DG load. There was a lag period of >=3–4 d between each experimental protocol. Five additional animals were submitted to the DG-Clamp combined with the direct measurement of net hepatic glucose balance.

DG-Clamp
. After the pigs were deprived of food for 24 h, a primed (40 mU/kg for 1 min), continuous (2 mU•kg-1•min-1) 240-min infusion of porcine insulin (Organon, Saint-Denis, France) into the jugular vein was begun to suppress hepatic glucose production. The arterial blood glucose concentration was maintained at its basal value by a computer-adjusted infusion of a 30% glucose solution (BRAUN Medical, Boulogne, France) into the jugular vein. To monitor the glucose clamp, 20 µL arterial blood was collected at 2–5-min intervals for glucose determination (One-Touch II; Lifescan, Roissy, France). Steady state concentrations of arterial plasma glucose and insulin were obtained 90 min after the glucose clamp was started. After a 30-min plateau period, a DG load (0.9 g D-glucose/kg in 500 mL water) was administered over 60 min according to a 3-step linear degressive rate: 50%, 33%, and 17% of the glucose load for 3 successive 20-min periods. The arterial blood glucose concentration was maintained at the prior concentration for 2 h by adjusting the rate of glucose infusion to offset the increment in the posthepatic appearance of glucose. The glucose turnover rate was also measured by using a primed [2220 kBq (60 µCi) for 1 min], continuous infusion of [3-3H] glucose (Dupont de Nemours, Les Ulis, France) into the jugular vein for 1 h before the insulin infusion began (basal glucose turnover rate) and during the clamp procedure (euglycemic hyperinsulinemic glucose turnover rate). The specific activity of blood glucose was kept constant by progressively increasing the rate of tracer infusion from 22.2 kBq/min (0.6 µCi/min) to 62.9 kBq/min (1.7 µCi/min) after the insulin infusion began.

Control clamp
. As a control, the clamp procedure was also performed in combination with a glucose-free water load (500 mL) into the duodenum according to the protocol described for the DG-Clamp, except that there was no infusion of the glucose tracer.

DG load
. After the pigs were deprived of food for 24 h, a 60-min DG load (0.9 g D-glucose/kg in 500 mL water) was administered according to the 3-step linear degressive rate described above.

Validation with oral carbohydrates
OC-Clamp
. Eight animals were submitted to 2 glucose clamp procedures as described above for the DG-Clamp. The DG load was replaced with an OC load as part of a 0.2-kg mixed meal. The meal consisted of either 700 g cornstarch or mung bean starch per kilogram mixed with other macronutrients (casein, 160 g/kg; soybean oil, 99 g/kg; and minerals and vitamins, 41 g/kg). Thus, both test meals contained 140 g starch and were ingested within 5 min. Indeed, cornstarch is known to be digested rapidly (20), whereas mung bean starch is digested slowly on the basis of in vivo and in vitro assessments (21). There was no tracer infusion.

Arterial and portal blood samples were drawn at 10- to 15-min intervals throughout these different protocols to determine blood glucose and plasma insulin concentrations. Blood was collected regularly to determine lactate, glutamine, and alanine concentrations during the DG and DG-Clamp procedures, and hepatic vein blood was collected during the DG-Clamp procedure to assess glucose concentration. As indicated by blood hematocrit values, the cumulative blood volume removed was <10% of the total at the end of the experiment.

Analytic methods
To determine the specific activity of glucose, 100-µL blood samples were deproteinized in Ba(OH)2ZnSO4 as described previously (22) and immediately centrifuged (16000 x g for 5 min at 4°C). A specific enzymatic method was used to determine blood glucose from the supernate (23). An aliquot (500 µL) was evaporated to dryness to eliminate tritiated water and then the [3-3H]D-glucose content was determined with a liquid scintillation counter (LKB-Pharmacia, Saint-Quentin-en-Yvelines, France).

To determine the substrate concentration, 1 mL blood was collected and immediately deproteinized in 2 mL ice-cold 6% (wt:vol) perchloric acid. Arterial and portal blood glucose, lactate, glutamine, and alanine concentrations were determined in the neutralized perchloric acid filtrates by using standard enzymatic methods (23). Blood samples (1 mL) were collected simultaneously into heparin- and EDTA-containing tubes, and plasma was obtained after centrifugation at 16000 x g for 5 min at 4°C. Plasma insulin was determined by radioimmunoassay (Kit ERIA Pasteur; Marnes La Coquette, France). All enzymes and coenzymes used for enzymatic assays were from Boehringer (Meylan, France).

Calculations and statistical analysis
The rate of net portal appearance of glucose and the intestinal balances of lactate, glutamine, and alanine were calculated as the difference between portal and arterial concentrations multiplied by the flow rate in the portal vein as described previously (18). The total rate of glucose appearance into the glucose pool was calculated by using standard equations for a continuous infusion of tracer during steady state or non–steady state conditions (24). The rate of hepatic glucose production was calculated as the difference between the rate of appearance of glucose and the rate of exogenous glucose infusion.

The rate of net posthepatic appearance of glucose was calculated by subtracting the rate of exogenous glucose infusion from the mean steady state rate of exogenous glucose infusion obtained before glucose or carbohydrate administration. The integrated posthepatic appearance of glucose over the time required for complete glucose absorption was simultaneously compared with the corresponding portal appearance of glucose. The net hepatic glucose balance was calculated as the difference between hepatic glucose output (calculated from hepatic vein glycemia and total hepatic blood flow) and input (sum of arterial hepatic and portal vein glucose fluxes as calculated from arterial and portal glycemia and respective blood flow).

The results are expressed as means ± SEMs. Statistical analysis consisted of repeated-measures analysis of variance (with Scheffe's test for post hoc analysis), Student's t test, and a two-factor analysis of variance for blood metabolite values (STATVIEW, version 1.03; Abacus Concepts, Berkeley, CA). A P value <0.05 indicated significant differences.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Validation with duodenal glucose
Blood flow measurement
During experiments of validation with DG, portal blood flow remained constant. Basal portal blood flow was 33.0 ± 1.3 mL• kg-1· min-1 (n = 8). Neither insulin infusion (33.9 ± 1.4 mL•kg-1· min-1) nor DG infusion (DG load: 34.1 ± 1.4 mL• kg-1• min-1; DG-Clamp: 35.4 ± 1.2 mL•kg-1•min-1) significantly modified portal blood flow (n = 8). Arterial hepatic blood flow during the DG-Clamp experiments was 17.2 ± 1.8% of total hepatic blood flow (n = 5), at the glucose clamp steady state and during glucose absorption.

Blood glucose and plasma insulin concentrations
Before the insulin infusion, arterial and portal blood glucose concentrations were 3.4 ± 0.1 and 3.3 ± 0.1 mmol/L, respectively (n = 13). During the glucose clamp steady state, the arterial glucose concentration was maintained at 3.8 ± 0.1 mmol/L (Figure 1AGo) and portal (n = 13) and hepatic (n = 5) vein glucose concentrations were 3.4 ± 0.1 and 3.1 ± 0.1 mmol/L, respectively. During absorption of the DG load (n = 8), the arterial glucose concentration remained constant (4.0 ± 0.1 mmol/L) and the portal glucose concentration increased significantly beginning 10 min after infusion and rose to peak values (7.3 ± 0.4 mmol/L) 30 min after the DG infusion began.



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  		FIGURE 1. Mean (±SEM) arterial carotid (squares) and portal vein (circles) blood glucose and plasma insulin concentrations during duodenal glucose (DG) infusion in pigs (n = 8) under euglycemic hyperinsulinemic clamp (DG-Clamp; open symbols) and nonclamp (DG load; closed symbols) conditions.

 
Basal arterial and portal plasma insulin were 8 ± 4 mU/L and 15 ± 6 mU/L (n = 8), respectively. During the DG-Clamp procedure (n = 8), insulin infusion increased plasma insulin concentrations to steady state arterial (116 ± 3 mU/L) and portal (101 ± 6 mU/L) concentrations (Figure 1AGo). The arterial insulin concentration was stable during the absorption period; however, the portal insulin concentration increased significantly 10 min after the DG infusion began, but thereafter returned to steady state (P > 0.05). The time course of arterial and portal blood glucose and plasma insulin concentrations (n = 8) after DG infusion were as expected, increasing rapidly immediately after infusion, reaching peak values at 30 min and returning to basal values by 75 min (Figure 1BGo).

Rate of glucose appearance
The basal total rate of glucose appearance determined from the specific activity of blood [3-3H]glucose, which was stable, was 21.7 ± 1.2 µmol·kg-1·min-1 (n = 8). During the insulin infusion (DG-Clamp), the specific activity of blood glucose remained constant, even during the period of glucose absorption (data not shown). The rate of glucose appearance increased notably during insulin infusion (106.7 ± 2.8 µmol•kg-1•min-1) and remained constant during the DG load (Figure 2Go, inset).



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  		FIGURE 2. Mean (±SEM) rates of glucose infusion in pigs (n = 8) submitted to euglycemic hyperinsulinemic clamp conditions plus duodenal glucose infusion (0.9 g/kg; thick black line) or an infusion of water (500 mL) alone (thin black line). The glucose and water infusions began at time 0. The steady state rate of glucose infusion was similar after both the duodenal glucose and water infusions. The overall rate of glucose appearance determined from the specific activity of [3-3H]glucose was constant during the duodenal glucose infusion under clamp conditions (inset).

 
During the DG-Clamp, the rate of exogenous glucose infusion rapidly increased to reach a steady state value (110.0 ± 5.5 µmol•kg-1•min-1; n = 8) within 30–60 min before DG infusion. Comparison of both the total rate of glucose appearance and the rate of exogenous glucose infusion indicated that residual glucose production was totally suppressed before the DG infusion began. Indeed, the calculated hepatic glucose production (-3.3 ± 2.6 µmol•kg-1•min-1) was not significantly different from zero (P > 0.05). Thereafter, the rate of exogenous glucose infusion significantly decreased beginning 10 min after the DG infusion began to a nadir of 51.8 ± 11.5 µmol•kg-1•min-1 obtained at 35 min. (Figure 2Go). The rate of exogenous glucose infusion returned to the prior steady state value at {approx}72 ± 3 min (P > 0.05). When water was administered alone without glucose, the rate of exogenous glucose infusion did not differ from the steady state value (Figure 2Go).

Net portal and posthepatic glucose appearance
As shown in Figure 3Go, portal glucose kinetics were similar during both experimental conditions (DG load and DG-Clamp). The amounts of glucose absorbed relative to the glucose load were 93 ± 4% (DG) and 95 ± 7% (DG-Clamp). The net posthepatic appearance of glucose, calculated from the decrease in the rate of exogenous glucose infusion, was 63 ± 3% of the glucose load. Furthermore, the time at which glucose absorption was achieved, calculated as the time when the portal glucose concentration did not differ from the arterial glucose concentration, was also similar: 65 ± 5 min (DG) and 71 ± 5 min (DG-Clamp).



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  		FIGURE 3. Mean (±SEM) net rate of portal and posthepatic glucose appearance in pigs (n = 8) after duodenal glucose infusion (0.9 g/kg) determined from the arterio-portal difference either under nonclamp (•) or euglycemic hyperinsulinemic clamp ({circ}) conditions. (Because the lines overlap, the open circles are hidden in some cases.) The net posthepatic appearance of glucose ({triangleup}) was calculated as the decrease in the rate of glucose infusion during the clamp procedure (see Figure 2Go).

 
Net hepatic glucose balance
When the DG-Clamp was combined with hepatic vein catheterization, the rates of hepatic glucose input and output calculated at steady state were 122.5 ± 7.7 and 109.0 ± 11.0 µmol•kg-1•min-1, respectively (n = 5). The net hepatic glucose balance was –13.5 ± 5.2 µmol•kg-1•min-1 (n = 5), suggesting hepatic glucose uptake. During glucose absorption, the rates of both hepatic glucose input and output increased significantly beginning 10 min after the glucose infusion began, peaked at 30 min (240.0 ± 18.7 and 182.3 ± 20.8 µmol•kg-1•min-1, respectively), and then returned to values not significantly different from steady state values (117.6 ± 17.8 and 105.1 ± 9.8 µmol•kg-1•min-1, respectively) at 75 min. Cumulative hepatic glucose input and output during glucose absorption were 14.48 ± 0.71 and 11.63 ± 0.70 mmol/kg, respectively (n = 5). Thus, net hepatic glucose uptake was 2.85 ± 0.61 mmol (n = 5), or 19.7 ± 3.8% of the hepatic glucose input.

For a comparison of the kinetics of the posthepatic appearance of glucose calculated from the decrease in the rate of exogenous glucose infusion, hepatic glucose input and output were also calculated after subtraction of corresponding steady state rates. Therefore, cumulative hepatic glucose input and output after DG administration (ie, the integration of incremental values over steady state rates) were 5.29 ± 0.63 and 3.46 ± 0.25 mmol/kg, respectively (n = 5), 75 min postinfusion (Figure 4Go), corresponding to a net hepatic glucose uptake of 34 ± 3% of the hepatic glucose input (n = 5). Furthermore, net hepatic glucose uptake was constant as early as 20 min after the DG infusion began (Figure 4Go, inset). When calculated from the decrease in rate of exogenous glucose infusion (see above), the net hepatic glucose uptake [ie, (portal appearance - posthepatic appearance)/portal appearance] was identical (34 ± 3%, n = 8).



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  		FIGURE 4. Mean (±SEM) cumulative rate of net hepatic glucose input and output in pigs (n = 5) after duodenal glucose infusion (0.9 g/kg). The respective amounts of glucose corresponding to the increased values over steady state were determined under euglycemic hyperinsulinemic clamp conditions plus hepatic vein catheterization and hepatic blood flow was assessed continuously. The net hepatic glucose uptake (input - output) was constant beginning 20 min postinfusion (inset).

 
Validation with oral carbohydrates
OC-Clamp experiments were performed with corn and mung bean starches as the carbohydrate sources because mung bean starch has a low glycemic index and cornstarch has a high glycemic index (25). During the OC-Clamp, portal blood flow increased significantly beginning 15 min after the meal was fed. It increased from 33.1 ± 1.5 mL•kg-1•min-1 before the meal to a peak of 38.2 ± 1.1 mL•kg-1•min-1 60 min after the meal began, regardless of the type of carbohydrate ingested (n = 8).

During the OC-Clamp experiments with both carbohydrates, arterial blood glucose was 3.2 ± 0.1 mmol/L at steady state and remained constant during the glucose absorption period (3.3 ± 0.4 mmol/L, n = 8). Similarly, the arterial plasma insulin concentration was clamped at 86 ± 2 mU/L. At steady state, the rate of exogenous glucose infusion was 106 ± 5 µmol•kg-1•min-1 (n = 8).

During glucose absorption, the rate of exogenous glucose infusion decreased to compensate for the increment in the posthepatic appearance of glucose. The rate of exogenous glucose infusion decreased significantly beginning 20 min after meal feeding. Then, it plateaued from 35 to 70 min after the cornstarch meal (56.7 ± 7.7 µmol•kg-1•min-1) and from 35 to 55 min after the mung bean starch meal (77.8 ± 10.5 µmol•kg-1•min-1). As shown in Figure 5Go, the kinetics of the posthepatic appearance of glucose were significantly different between the 2 carbohydrate meals. As expected, the portal appearance of glucose after the 2 carbohydrate meals was significantly different (P < 0.05), but paralleled the respective time courses of the posthepatic appearance of glucose (Figure 5Go). The net portal appearance of glucose during the 2 h of absorption was 257 ± 14 and 90 ± 21 mmol for the corn and mung bean starches, respectively. Moreover, net hepatic glucose uptake was similar for both carbohydrates (cornstarch: 21 ± 3%; mung bean starch: 20 ± 3%; NS).



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  		FIGURE 5. Mean (±SEM) net posthepatic and portal appearance of glucose in pigs (n = 8) after a 2-h euglycemic hyperinsulinemic clamp procedure combined with an oral carbohydrate load consisting of either cornstarch (•) or mung bean starch ({circ}) as part of a mixed meal.

 
Blood arterial metabolite and intestinal metabolism
Insulin infusion profoundly changed intestinal glucose uptake determined before DG infusion. Indeed, intestinal glucose uptake significantly increased from 2.6 ± 0.4 to 13.9 ± 2.5 µmol•kg-1•min-1 (n = 8), whereas arterial glucose remained constant. The arterial glutamine concentration significantly decreased during the hyperinsulinemic clamp, whereas intestinal glutamine uptake decreased slightly (Table 1Go). However, the DG load stimulated intestinal glutamine uptake both in basal and hyperinsulinemic conditions.


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  		TABLE 1. Blood arterial concentration and intestinal balance of glutamine, alanine, and lactate after duodenal glucose (DG) infusion in the basal state and during euglycemic hyperinsulinemic clamp conditions1
 
Arterial alanine concentrations decreased significantly after insulin infusion; however, intestinal alanine production did not change significantly after either insulin or glucose infusion (Table 1Go). The arterial blood lactate concentration was significantly enhanced either by insulin infusion or the glucose load, and this stimulating effect seemed to be additive (Table 1Go). However glucose administration only altered intestinal lactate balance. Indeed, the glucose load significantly reversed the intestinal lactate balance from uptake to release when glucose was absorbed (Table 1Go).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The objective of the present study was to validate the glucose clamp technique as a noninvasive method to quantify the kinetics of the posthepatic appearance of glucose after a carbohydrate load. Because direct determination of the portal appearance of glucose was required in parallel with the euglycemic hyperinsulinemic clamp procedure, the pig was used as the animal model because its size makes it suitable for chronic catheterization. In addition, the kinetics of glucose absorption after an oral or DG load and the parameters of glucose metabolism in basal or postprandial conditions are quite similar in humans and pigs, as shown in the present study and others (2–4, 26–28).

The validation consisted of 2 steps: 1) a well-controlled glucose load was infused directly in the pigs' duodenum, and 2) 1 of 2 sources of carbohydrate (cornstarch or mung bean starch) with different glycemic indexes were fed as part of a mixed meal.

After DG infusion, the overall rate of glucose appearance corresponded to both exogenous glucose absorption and residual endogenous glucose production. With the glucose clamp procedure, mild hyperinsulinemia was established to totally suppress endogenous glucose production. The plasma insulin concentration was kept to a physiologic level ({approx}100 mU/L), close to the peak value obtained during a DG load under nonclamp conditions. In addition, because the glucose turnover rate and endogenous glucose production are often underestimated during insulin administration (29, 30), tracer glucose was infused at a rate allowing a constant specific activity of blood glucose (31). As expected, hyperinsulinemia totally suppressed the rate of endogenous glucose production and markedly increased the rates of both glucose infusion and glucose turnover. Moreover, the 5-fold increase in the glucose infusion rate was high enough to allow its subsequent reduction to compensate for glucose absorption (14, 15, 32). Because the overall glucose turnover rate and glycemia were kept constant during the time of glucose absorption, the rate of exogenous glucose infusion determined at steady state before the DG load can be used to estimate the rate of posthepatic appearance of glucose. Moreover, the present study proved that the rate of portal appearance of glucose was not altered significantly by the clamp procedure and that it paralleled the posthepatic appearance of glucose during glucose absorption. In turn, the time when the rate of exogenous glucose infusion again reaches steady state can be considered the time of complete glucose absorption.

The present study showed that hepatic glucose uptake during mild hyperinsulinemia was limited before glucose loading (11% of the hepatic glucose input), but increased during the time of glucose absorption. Indeed, net hepatic glucose uptake determined by 2 distinct procedures was 34% of the hepatic glucose input calculated after subtraction of the steady state rate (also equivalent to the incremental portal glucose appearance), or 26 µmol•kg-1•min-1, which is within the range previously found in other species (33).

Previous experiments indicated that hyperinsulinemia increases net hepatic uptake only when there is a negative arterial-portal glucose gradient (33–36). Thus, enhanced portal glycemia or a portal signal could explain the hepatic response. Net hepatic glucose uptake was confirmed by an increase in the hepatic glycogen content from 0.2 ± 0.1 to 3.2 ± 0.1 mmol/kg body wt during the period of absorption (n = 4; F Bernard, P Vaugelade, unpublished observations, 1997).

Present data also indicate that net hepatic glucose uptake, expressed as the rate of glucose absorbed, remains constant as early as 20 min after the beginning of glucose absorption, allowing a quantitative comparison of the net posthepatic appearance of glucose from various carbohydrate sources. In the basal state as well as during hyperinsulinemia, the rate of intestinal glucose utilization was 12–13% of overall glucose utilization. This confirms that, in pigs, the gut represents a site of high glucose utilization, as documented previously (1, 26–28). During glucose absorption, it was not possible to determine the amount of glucose utilization by intestinal tissues on the basis of differences between arterial and portal concentrations. However, net intestinal glucose utilization can be as high as 5% of the glucose load during the time of absorption ({approx}70 min after infusion), assuming that exogenous glucose not found in the portal vein is metabolized by the gut. Thus, net glucose utilization was 3.6 µmol•kg-1•min-1, close to the determination from intestinal lactate output (4.6 µmol • kg-1•min-1). This suggests that intestinal glucose utilization during a DG-Clamp decreases during DG administration, as found previously in isolated enterocytes (28). Besides glucose, intestinal tissues use glutamine and release alanine, as reported previously (37–39). Moreover, glutamine uptake is related to arterial glutamine concentrations and is enhanced during glucose absorption, confirming prior observations (40).

To extend the validation of the method, experiments were carried out with 2 sources of carbohydrates with different glycemic indexes (25) as a part of mixed meal. The findings of the present study support the use of the euglycemic hyperinsulinemic clamp procedure for determining the posthepatic appearance of glucose after meal feeding. Indeed, the relations between portal appearance and posthepatic appearance of glucose were similar after the rapidly digested cornstarch and the slowly digested mung bean starch meals. Furthermore, the net hepatic glucose uptake, expressed as the net portal appearance of glucose, was similar with both carbohydrate meals. However, net hepatic glucose uptake after a mixed meal was lower than that after the DG load, which can be explained by the change in the ratio of insulin to glucagon that occurs when carbohydrate is fed together with protein (41).

In summary, the present data prove that the posthepatic appearance of glucose after a carbohydrate load can be assessed with a euglycemic hyperinsulinemic clamp procedure, assuming that hepatic glucose production is totally suppressed and that overall glucose utilization is kept constant. Because net hepatic glucose uptake remains proportional to the rate of glucose absorbed (ie, constant extraction) and because intestinal glucose utilization represents only a small part of splanchnic glucose uptake, the results of the present study support the extension of this method to use in human studies. However, the present study suggests that comparison of various carbohydrates requires well-controlled nutritional conditions.


   ACKNOWLEDGMENTS
 
We thank Malcolm Watford for his helpful discussion.


   FOOTNOTES
 
1 From the Institut National de la Recherche Agronomique, Jouy-en-Josas, France; INSERM U 341, Service de Diabétologie, Hôpital Hôtel-Dieu, Paris; and éridania Béghin-Say, Nutrition and Health Service, Vilvoorde, Belgium.

2 Supported in part by a grant from the Ministère chargé de la Recherche (Aliment Demain 94 G 0267, Paris) and by Nutrition et Santé SA (Revel, France).

3 Reprints not available. Address correspondence to P-H Duée, Institut National de la Recherche Agronomique, LNSA, 78352 Jouy-en-Josas Cedex, France. E-mail: phd{at}jouy.inra.fr.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Abumrad NN, Cherrington AD, Williams PE, Lacy WW, Rabin D. Absorption and disposition of a glucose load in the conscious dog. Am J Physiol 1982;242:E398–406.[Abstract/Free Full Text]
  2. Ferrannini E, Björkman O, Reichard GA, et al. The disposal of an oral glucose load in healthy subjects. A quantitative study. Diabetes 1985;34:580–8.[Abstract]
  3. Jackson RA, Roshania RD, Hawa MI, Sim BM, DiSilvio L. Impact of glucose ingestion on hepatic and peripheral glucose metabolism in man: an analysis based on simultaneous use of the forearm and double isotope techniques. J Clin Endocrinol Metab 1986;63:541–9.[Abstract]
  4. Consoli A. Pathways of glucose metabolism following ingestion of an oral glucose load. Diabetes Nutr Metab 1994;7:97–107.
  5. De Fronzo RA. Pathogenesis of type 2 (non-insulin dependent) diabetes mellitus: a balanced overview. Diabetologia 1992;35:389–97.[Medline]
  6. Slama G, Jean-Joseph P, Goicola I, et al. Sucrose taken during mixed meal has no additional hyperglycaemic action over isocaloric amounts of starch in well-controlled diabetics. Lancet 1984;2:122–6.[Medline]
  7. Bornet F, Haardt MJ, Costagliola D, Blayo A, Slama G. Sucrose or honey at breakfast have no additional acute hyperglycaemic effect over an isoglucidic amount of bread in Type 2 diabetic patients. Diabetologia 1984;28:213–7.
  8. Stephen AM, Haddad AC, Phillips SF. Passage of carbohydrate into the colon. Direct measurements in humans. Gastroenterology 1983;85:589–95.[Medline]
  9. Levitt MD, Hirsh P, Fetzer CA, Sheahan M, Levine AS. H2 excretion after ingestion of complex carbohydrates. Gastroenterology 1987;92:383–9.[Medline]
  10. Jenkins DJA, Wolever TMS, Taylor RH, et al. Glycemic index of foods: a physiological basis for carbohydrate exchange. Am J Clin Nutr 1981;34:362–6.[Abstract/Free Full Text]
  11. Bornet FR, Costagliola D, Rizkalla SW, et al. Insulinemic and glycemic indexes of six starch-rich foods taken alone and in a mixed meal by type 2 diabetics. Am J Clin Nutr 1987;45:588–95.[Abstract/Free Full Text]
  12. Lacroix M, Mosora F, Pontus M, Lefebvre P, Luyckx A, Lopez-Habib G. Glucose naturally labelled with 13C: use for metabolic studies in man. Science 1973;181:445–6.[Abstract/Free Full Text]
  13. Normand S, Pachiaudi C, Khalfallah Y, Guilluy R, Mornex R, Riou JP. 13C appearance in plasma glucose and breath CO2 during feeding with naturally 13C-enriched starchy food in normal humans. Am J Clin Nutr 1992;55:430–5.[Abstract/Free Full Text]
  14. Ludvik B, Nolan J, Roberts A, et al. A noninvasive method to measure splanchnic glucose uptake after oral glucose administration. J Clin Invest 1995;95:2232–8.
  15. Ludvik B, Nolan J, Roberts A, et al. Evidence for decreased splanchnic glucose uptake after oral glucose administration in non-insulin-dependent diabetes mellitus. J Clin Invest 1997;100:2354–61.[Medline]
  16. Lang V, Vaugelade P, Bernard F, et al. Experimental validation in pig of a noninvasive method for quantifying glucose absorption kinetics. Diabetes 1995;44(suppl):93A (abstr).
  17. Moughan PJ, Rowan AM. The pig as a model animal for human nutrition research. Proc Nutr Soc N Z 1989;14:116–23.
  18. Rérat A, Chayvialle JA, Kandé J, Vaissade P, Vaugelade P, Bourrier T. Metabolic and hormonal effects of test meals with various protein contents in pigs. Can J Physiol Pharmacol 1985;63:1547–59.[Medline]
  19. Simoes Nunes C, Rérat A, Galibois I, Vaugelade P, Vaissade P. Hepatic and gut balances of glucose, amino-nitrogen, ammonia and urea in the pig after ingestion of casein or rapeseed proteins. Nutr Rep Int 1989;40:901–7.
  20. Rerat A, Vaissade P, Vaugelade P. Absorption kinetics of some carbohydrates in conscious pigs. Br J Nutr 1984;51:517–29.[Medline]
  21. Kabir M, Rizkalla S, Champ M, et al. Dietary amylose-amylopectin starch content affects glucose and lipid metabolism in adipocytes of normal and diabetic rats. J Nutr 1998;128:35–43.[Abstract/Free Full Text]
  22. Somogyi M. Determination of blood sugar. J Biol Chem 1945;160: 69–73.[Free Full Text]
  23. Bergmeyer HU. Methods of enzymatic analysis. New York: Academic Press, 1974.
  24. Steele R. Influence of glucose loading and of insulin on hepatic glucose output. Proc N Y Acad Sci 1959;82:420–30.
  25. Bornet F, Fontvieille A, Rizkalla S, et al. Insulin and glycemic responses in healthy humans to native starches processed in different ways: correlation with in vitro {alpha}-amylase hydrolysis. Am J Clin Nutr 1989;50:315–23.[Abstract/Free Full Text]
  26. Björkman O, Eriksson LS, Nyberg B, Wahren J. Gut exchange of glucose and lactate in basal state and after oral glucose ingestion in post operative patients. Diabetes 1990;39:747–51.[Abstract]
  27. Müller MJ, Paschen U, Seitz HJ. Glucose production measured by tracer and balance data in conscious miniature pig. Am J Physiol 1983;244:E236–44.[Abstract/Free Full Text]
  28. Vaugelade P, Posho L, Darcy-Vrillon B, Bernard F, Morel MT, Duée PH. Intestinal oxygen uptake and glucose metabolism during nutrient absorption in the pig. Proc Soc Exp Biol Med 1994;207:309–16.[Abstract]
  29. Argoud GM, Schade DS, Eaton RP. Underestimation of hepatic glucose production by radioactive and stable tracers. Am J Physiol 1987;252:E606–15.[Abstract/Free Full Text]
  30. Neely RDG, Rooney DP, Atkinson AB, et al. Underestimation of glucose turnover determined using [6-3H] glucose tracer in non-steady state. Diabetologia 1990;33:681–7.[Medline]
  31. Finegood DT, Bergman RN, Vranic M. Estimation of endogenous glucose production during hyperinsulinaemic-euglycaemic glucose clamps. Diabetes 1987;36:914–24.[Abstract]
  32. Jacot E, De Fronzo RA, Jéquier E, Maeder E, Felber JP. The effect of hyperglycemia, hyperinsulinemia, and route of glucose administration on glucose oxidation and glucose storage. Metabolism 1982;31:922–30.[Medline]
  33. Pagliassotti MJ, Cherrington AD. Regulation of net hepatic glucose uptake in vivo. Annu Rev Physiol 1992;54:847–60.[Medline]
  34. Shulman GI, Rossetti L. Influence of the route of glucose administration on hepatic glycogen repletion. Am J Physiol 1989;257:E681–5.[Abstract/Free Full Text]
  35. Adkins BA, Myers SR, Hendrick GK, Stevenson RW, Williams PE, Cherrington AD. Importance of the route of intravenous glucose delivery to hepatic glucose balance in the conscious dog. J Clin Invest 1987;79:557–65.
  36. De Fronzo RA, Ferrannini E, Hendler R, Wahren J, Felig P. Influence of hyperinsulinemia, hyperglycemia, and the route of glucose administration on splanchnic glucose exchange. Proc Natl Acad Sci U S A 1978;75:5173–7.[Abstract/Free Full Text]
  37. Windmueller HG. Enterohepatic aspects of glutamine metabolism. In: Mora J, Palacios R, eds. Glutamine: metabolism, enzymology, and regulation. New York: Academic Press, 1980:235–57.
  38. Watford M. Glutamine metabolism in rat small intestine: synthesis of three-carbon products in isolated enterocytes. Biochim Biophys Acta 1994;1200:73–8.[Medline]
  39. Duée PH, Darcy-Vrillon B, Blachier F, Morel MT. Fuel selection in intestinal cells. Proc Nutr Soc 1995;54:83–94.[Medline]
  40. Weber FL, Veach G, Friedman DW. Stimulation of ammonia production from glutamine by intraluminal glucose in small intestine of dogs. Am J Physiol 1982;242:G552–7.[Abstract/Free Full Text]
  41. Pagliassotti MJ, Horton, TJ. Hormonal and neural regulation of hepatic glucose uptake. In: Pagliassotti MJ, Davis SN, Cherrington AD, eds. The role of the liver in maintaining glucose homeostasis. Austin, TX: Landes RG Co, 1994:45–70.
Received for publication August 20, 1998. Accepted for publication December 17, 1998.




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