Hepatic glucose disposition during concomitant portal glucose and amino acid infusions in the dog

Mary Courtney Moore1, Paul J. Flakoll2, Po-Shiuan Hsieh1, Michael J. Pagliassotti1, Doss W. Neal2, Michael T. Monohan1, Carol Venable1, and Alan D. Cherrington1,2

1 Department of Molecular Physiology and Biophysics and 2 Diabetes Research and Training Center, Vanderbilt University, Nashville, Tennessee 37232-0615

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The effect of concomitant intraportal infusion of glucose and gluconeogenic amino acids (AA) on net hepatic glucose uptake (NHGU) and glycogen synthesis was examined in 42-h-fasted dogs. After a basal period, there was a 240-min experimental period during which somatostatin was infused continuously into a peripheral vein and insulin and glucagon (at 3-fold basal and basal rates, respectively) and glucose (18.3 µmol · kg-1 · min-1) were infused intraportally. One group (PoAA, n = 7) received an AA mixture intraportally at 7.6 µmol · kg-1 · min-1, whereas the other group (NoAA, n = 6) did not receive AA. Arterial blood glucose concentrations and hepatic glucose loads were the same in the two groups. NHGU averaged 4.8 ± 2.0 (PoAA) and 9.4 ± 2.0 (NoAA) µmol · kg-1 · min-1 (P < 0.05), and tracer-determined hepatic glucose uptake was 4.6 ± 1.6 (PoAA) and 10.0 ± 1.7 (NoAA) µmol · kg-1 · min-1 (P < 0.05). AA data for PoAA and NoAA, respectively, were as follows: arterial blood concentrations, 1,578 ± 133 vs. 1,147 ± 86 µM (P < 0.01); hepatic loads, 56 ± 3 vs. 32 ± 4 µmol · kg-1 · min-1 (P < 0.01); and net hepatic uptakes, 14.1 ± 1.4 vs. 5.6 ± 0.4 µmol · kg-1 · min-1 (P < 0.01). The rate of net hepatic glycogen synthesis was 7.5 ± 1.9 (PoAA) vs. 10.7 ± 2.3 (NoAA) µmol · kg-1 · min-1 (P = 0.1). In a net sense, intraportal gluconeogenic amino acid delivery directed glucose carbon away from the liver. Despite this, net hepatic carbon uptake was equivalent in the presence and absence of amino acid infusion.

liver; hyperglycemia; liver nerves; glycogen; mixed meal

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

NET HEPATIC GLUCOSE UPTAKE (NHGU) in the conscious dog is two- to threefold greater in response to intraportal glucose infusion than in response to peripheral glucose infusion, even when the hormone and glucose levels are made identical (3, 26). The enhancement of NHGU in response to intraportal glucose delivery is accompanied by an enhancement of hepatic glycogen synthesis (26).

We have postulated that a signal (termed portal signal) generated during portal glucose infusion (or absorption of enterally administered glucose) results in enhanced NHGU (3). Neurophysiological evidence is consistent with this postulate. For example, the afferent firing rate in the hepatic branch of the vagus nerve of the guinea pig is inversely proportional to the portal vein glucose concentration (23). Also, injection of glucose into the portal vein increases the efferent firing rate in the vagal pancreatic nerve, whereas injection of the same amount of glucose into the jugular vein does not (23). Moreover, functional connections have been identified between glucose-sensitive afferent neurons in the portal vein and glucose-sensitive neurons in the central nervous system (2).

During the postprandial period after intragastric administration of a mixed meal containing protein, glucose, and lipid, NHGU in conscious dogs was ~80% less than would have been predicted based on the increases in the insulin concentration (>9-fold basal) and the hepatic glucose load (~2-fold basal) (20). The rise in glucagon (~20 pg/ml) during the postprandial period may have contributed to the unexpectedly low NHGU (20). During intraportal glucose (hepatic glucose load 2-fold basal) and insulin (4-fold basal) infusion, a 20 pg/ml increase in glucagon reduced NHGU ~50% compared with that observed in the presence of basal glucagon (16). It is therefore unlikely that the increase in glucagon which occurred in response to the mixed meal was sufficient by itself to account for the marked blunting of NHGU. Another possible explanation for the very small NHGU observed after consumption of a mixed meal is the potential interaction between macronutrients when they are delivered simultaneously.

There exists a potential for complex interactions, and possibly competition, between signals created in response to intraportal delivery of glucose and other substrates (in particular amino acids) contained in mixed meal feedings. It has been shown, for example, that intraportal or intraperitoneal injection of solutions of single amino acids or mixtures of amino acids alters the afferent discharge rate of the hepatic branch of the vagus nerve (24, 25). It is therefore possible that net hepatic uptake of substrates may differ when they are administered singly and when they are administered in combination. We hypothesized that intraportal delivery of glycogenic substrates other than glucose (e.g., gluconeogenic amino acids) might suppress NHGU during intraportal glucose delivery. The aim of the present study was to quantify NHGU and hepatic glycogen synthesis during concomitant intraportal delivery of glucose and a mixture of gluconeogenic amino acids under conditions in which the insulin and glucagon levels were fixed (3-fold basal and basal, respectively) using somatostatin and intraportal hormone replacement.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Animals, diets, and experimental preparation. Studies were carried out on conscious 42-h-fasted adult dogs of either sex with a mean weight of 22 ± 1 kg. Housing and diet have previously been described (20). The protocol was approved by the Vanderbilt University Medical Center Animal Care Committee, and animals were housed according to American Association for the Accreditation of Laboratory Animal Care International guidelines. Approximately 16 days before study, all dogs underwent a laparotomy under general anesthesia, and silicone rubber catheters (Dow Corning, Midland, MI) were inserted in the portal and left common hepatic veins, a splenic and a jejunal vein, and the femoral artery (26). Ultrasonic flow probes (Transonic Systems, Ithaca, NY) were positioned around the portal vein and hepatic artery, and their proximal ends were placed in subcutaneous pockets.

Approximately 2 days before study, blood was drawn from each animal. A dog was studied only if it met established criteria: leukocyte count <18,000/mm3, hematocrit >35%, consumption of all of the daily food ration, and normal stools. On the morning of the study, the proximal ends of the flow probes and surgically implanted catheters were exteriorized, the catheters were cleared, the dog was placed in a Pavlov harness, and intravenous access was established in three peripheral veins.

Experimental design. At -120 min, a primed (40 µCi), continuous (0.4 µCi/min) peripheral infusion of D-[3-3H]glucose and a continuous peripheral infusion of indocyanine green (ICG; 4 µg · kg-1 · min-1; Becton Dickinson, Cockeysville, MD) dye were begun. The latter provided confirmation of hepatic vein catheter placement and a second measurement of hepatic blood flow. After 80 min (-120 to -40) of dye equilibration, there was a 40-min (-40 to 0) control or basal period followed by a 240-min (0 to 240) experimental period. At time 0, constant infusions of several solutions were begun, and these infusions continued for the next 240 min (experimental period). Somatostatin (0.8 µg · kg-1 · min-1; Bachem, Torrance, CA) was infused to suppress endogenous insulin and glucagon secretion. Insulin (7.2 pmol · kg-1 · min-1; 3-fold basal) and glucagon (both hormones obtained from Eli Lilly, Indianapolis, IN) were delivered into the portal circulation via the jejunal and splenic infusion catheters. In two dogs in each group, the rate of glucagon infusion was 0.65 ng · kg-1 · min-1. This rate resulted in circulating glucagon levels slightly higher than basal, however, and therefore the infusion rate was lowered to 0.5 ng · kg-1 · min-1 in the remainder of the dogs. Dextrose (20%, 18.3 µmol · kg-1 · min-1; Baxter Healthcare, Deerfield, IL) mixed with p-aminohippuric acid (PAH; delivered at 1.7 µmol · kg-1 · min-1 Sigma, St. Louis, MO) was also infused intraportally. PAH was used to assess mixing of the infused glucose with blood in the portal and hepatic veins as described previously (26). In one group of dogs (PoAA, n = 7), a mixture of gluconeogenic amino acids (L-isomers of glutamine, glutamate, threonine, serine, glycine, and alanine; molar ratio 1.0, 0.2, 0.5, 0.2, 0.4, and 0.4, respectively) was infused intraportally at 7.6 µmol · kg-1 · min-1. The ratios and rate were chosen to mimic the ratios and the absorption rates that we observed previously after delivery of a liquid mixed meal to conscious dogs (20). The amino acid mixture was prepared just before time 0 by dissolving the individual crystalline amino acids (Sigma) in deionized water. The second group (NoAA, n = 6) received intraportal saline rather than amino acids. In addition to the constant infusions, a primed, continuous peripheral infusion of 50% dextrose was begun in both groups at time 0, so that the blood glucose could be quickly clamped at its desired value. Blood samples of 0.2 ml were obtained from the artery every 5 min to permit measurement of the plasma glucose concentration, and the peripheral glucose infusion rate was adjusted on the basis of these measurements to maintain the hepatic glucose load at 1.5-fold basal throughout the experimental period. Larger blood samples (4-9 ml) for data acquisition were obtained from the artery, portal vein, and hepatic vein every 20 min during the basal period and every 15-30 min during the experimental period. The collection, processing, and analysis of blood samples have been described in detail elsewhere (20).

After completion of the experiment, each animal was killed with an overdose of pentobarbital, the liver was removed, and a tissue sample from each liver lobe was freeze clamped within 5 min of pentobarbital administration and stored at -70°C to await analysis.

Processing and analysis of samples. Blood glucose, lactate, alanine, glycerol, and hematocrit; plasma glucose, insulin, and glucagon concentrations; plasma [3H]glucose specific activity; and liver glycogen concentrations were determined as described previously (20). HPLC was used to determine amino acid concentrations on sulfosalicylic acid-deproteinized blood samples (32). Blood glutamine and glutamate concentrations were measured on perchloric acid-deproteinized samples (20). PAH was measured in perchloric acid-deproteinized blood as previously described (26).

Calculations. The thoroughness of mixing of the infused glucose in the portal vein was assessed by comparing recovery of PAH (which was mixed with glucose) in the portal and hepatic veins with the PAH infusion rate (26). Because of the magnitude of the coefficient of variation of the method for assessing PAH balance, samples were considered statistically unmixed (>95% confidence that mixing did not occur) if hepatic or portal vein recovery of PAH was 40% greater or less than the actual amount of PAH infused. An experiment was defined as having poor mixing (and was excluded from data base) if poor mixing was observed at at least three of the eight time points in the experimental period. In PoAA mixing of infused amino acids was considered to have occurred if mixing of glucose occurred in the same animal, since glucose and amino acids were infused through the same catheters. Seventeen dogs were studied; one is not included in the data base because of malfunction of sampling catheters, and three were excluded because of poor mixing. In the 13 animals included in the data base, mixing failed to occur at <10% of the time points. Because mixing errors, when they occurred, were random, individual data points were not excluded if the experiment as a whole was included. Good mixing was evident in both groups. The ratio of recovered to infused PAH in the portal and hepatic veins was 0.9 ± 0.1 and 0.9 ± 0.1, respectively (with a ratio of 1.0 representing ideal mixing).

Hepatic blood flow (HBF) was calculated by two methods, ultrasonic flow probes and dye extraction (20). The results obtained with ultrasonic flow probes and dye were not significantly different. Because the flow probes make it possible to determine the relative proportions of the hepatic blood flow provided by the hepatic artery and the portal vein, calculations reported in here utilize HBF obtained from the flow probes when flow probes were available. One or the other of the flow probes did not function in two dogs in PoAA and three in NoAA. In these animals, ICG-derived flows were used, and the portal vein was assumed to provide 80% of hepatic blood flow during the basal period and 74% during the experimental period. These assumptions were based on the findings in the present studies in the animals having functional flow probes and were also consistent with our findings in previous studies conducted in the presence and absence of somatostatin (e.g., Refs. 21, 26).

The rate of substrate delivery to the liver, or hepatic substrate load, was calculated by a direct (d) method as
load<SUB>in(d)</SUB> = ([S]<SUB>A</SUB> × ABF) + ([S]<SUB>P</SUB> × PBF)
where [S] is the substrate concentration, A and P refer to artery and portal vein, respectively, and ABF and PBF refer to blood flow through the hepatic artery and portal vein, respectively. To avoid any potential errors arising from either incomplete mixing of glucose during intraportal infusion or lack of precise measurements of the distribution of hepatic blood flow, hepatic glucose load was also calculated by an indirect (i) method
load<SUB>in(i)</SUB> = (G<SUB>A</SUB> × HBF) + GIR<SUB>Po</SUB> − GUG
where G is the blood glucose concentration, GIRPo is the intraportal glucose infusion rate, and GUG is the uptake of glucose by the gastrointestinal tract, calculated based on the previously described relationship between the arterial blood glucose concentration and GUG (26).

The load of a substrate exiting the liver was calculated as
load<SUB>out</SUB> = [S]<SUB>H</SUB> × HBF
where H represents the hepatic vein.

Direct and indirect methods were used in calculation of net hepatic balance (NHB). The direct calculation was as follows: NHBd = loadout - loadin(d). The indirect calculation was as follows: NHBi = loadout - loadin(i). A negative value indicates net uptake. Both equations were used in calculation of net hepatic glucose and amino acid balance, but only the direct calculation was employed for other substrates. The results for NHB of glucose and amino acids did not differ regardless of the method used in calculation. The results given in this report utilize the indirect calculation, because this method is less likely to be affected by any inadequate mixing of infused substrates in the portal vein.

Net fractional substrate extraction by the liver was calculated directly and indirectly as the ratio of NHB to loadin.

Endogenous rate of appearance (Endo Ra) was calculated using a two-compartment model (18) with canine parameters (10), deducting the rate of exogenous (portal and peripheral) glucose infusion. The rate of hepatic glucose uptake was calculated as the balance of D-[3-3H]glucose across the liver, using the same formula as for NHB but substituting hepatic plasma flow measurements. The results were divided by the weighted inflowing glucose specific activity (expressed as dpm/µmol glucose). The weighted specific activity was the sum of the arterial and portal glucose specific activities, weighted for the proportion of flow provided by each vessel. These calculations assume that 3H measurements were obtained before dilution of the tracer glucose resulting from the addition of unlabeled glucose to the plasma as it passes through the liver. Nevertheless, the calculation should be accurate even if the assumption is not correct, since there was little or no change in glucose specific activity across the liver.

The trapezoidal rule was used to determine the area under the curve (AUC). Both positive and negative excursions (i.e., net balance) were included in the calculations for net hepatic disposition of substrates.

Net hepatic glycogen synthesis was calculated as the difference between poststudy glycogen concentrations in the glucose-infused dogs and basal concentrations in 11 dogs killed after a 42-h fast (corresponding to time 0 in experimental animals) (21). The glycogen concentrations for each dog represent the mean of the values for the seven liver lobes, weighted for the percentage of liver mass accounted for by each lobe (21). The contribution of the direct pathway of glycogen synthesis (glucoseright-arrowglucose 6-phosphateright-arrowglucose 1-phosphateright-arrowUDP-glucoseright-arrowglycogen) was also assessed by dividing the number of 3H dpm in the liver by the average inflowing [3-3H]glucose specific activity.

Data are presented as means ± SE. SYSTAT (Evanston, IL) was used for statistical analysis. Time-course data were analyzed with repeated-measures ANOVA with post hoc analysis by univariate F tests. Independent-sample t-tests were used for analysis of glycogen data and comparison of AUC. Results were considered statistically significant at P < 0.05.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Plasma insulin and glucagon concentrations. Plasma insulin concentrations in the two groups did not differ at any time (Fig. 1). Arterial plasma insulin concentrations during the experimental period (158 ± 12 pM in PoAA and 146 ± 17 pM in NoAA) were ~3- to 3.5-fold basal, mimicking concentrations in the postprandial state. Arterial plasma glucagon levels remained at basal throughout the study in both groups and did not differ between groups at any time.


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Fig. 1.   Arterial plasma insulin and glucagon concentrations in 42-h-fasted dogs during basal sampling period (-40 to 0 min) and experimental period (0-240 min). During experimental period, all dogs received a peripheral infusion of somatostatin and intraportal infusions of insulin (4-fold basal), glucagon (basal), and glucose (18.3 µmol · kg-1 · min-1). PoAA group (n = 7) received an intraportal infusion of gluconeogenic amino acids (7.6 µmol · kg-1 · min-1); NoAA group (n = 6) received no amino acid infusion. There are no significant differences between groups.

Hepatic blood flow, blood glucose metabolism, and glucose infusion rates. Total HBF did not differ among the groups at any time (mean: 28 ± 2 and 25 ± 3 ml · kg-1 · min-1 in PoAA and NoAA, respectively), nor did it change significantly between the basal and experimental periods in either group (data not shown).

Arterial blood glucose concentrations in the groups were similar throughout the studies (basal concentrations: 4.2 ± 0.1 and 4.2 ± 0.2 mM in PoAA and NoAA, respectively; experimental period concentrations: 6.2 ± 0.2 and 6.5 ± 0.4 mM in PoAA and NoAA, respectively; Fig. 2). The mean hepatic glucose loads were 130 ± 12 (PoAA) and 133 ± 18 (NoAA) µmol · kg-1 · min-1 during the basal period and 205 ± 18 (PoAA) and 203 ± 29 (NoAA) µmol · kg-1 · min-1 during the experimental period (NS; Fig. 2). The peripheral glucose infusion rates required to maintain this increase in the hepatic glucose load were ~40% less in PoAA (Table 1), but the variance among animals resulted in a P value of 0.3. 


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Fig. 2.   Arterial blood glucose concentrations and hepatic glucose load. See legend to Fig. 1 for description of study design. There are no differences between groups.

                              
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Table 1.   Peripheral glucose infusion rates in dogs receiving intraportal glucose infusion with or without concomitant intraportal infusion of gluconeogenic amino acids

Net hepatic glucose output was similar in the two groups during the basal period (9.8 ± 2.0 and 10.1 ± 1.7 µmol · kg-1 · min-1 in PoAA and NoAA, respectively; Fig. 3). During the experimental period, NHGU (µmol · kg-1 · min-1) averaged 4.8 ± 2.0 in PoAA and 9.4 ± 2.0 in NoAA (P < 0.05). (As previously stated in MATERIALS AND METHODS, these results were obtained using the indirect calculation. The direct calculation yielded a difference of 5.5 ± 2.1 µmol · kg-1 · min-1 in NHGU between the groups during the experimental period, which was not different from the results with the indirect calculation.) The net hepatic fractional extraction of glucose (Table 2) during the experimental period in PoAA was approximately one-half of that in NoAA (2.6 ± 0.7% vs. 4.8 ± 0.9%; P < 0.05).


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Fig. 3.   Net hepatic balance of glucose and gluconeogenic amino acids. See legend to Fig. 1 for description of study design. Net hepatic glucose uptake was significantly greater (P < 0.05) in NoAA, and net hepatic uptake of gluconeogenic amino acids was greater (P < 0.01) in PoAA.

                              
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Table 2.   Net hepatic fractional extraction of glucose in dogs receiving intraportal glucose infusion with or without a concomitant intraportal infusion of gluconeogenic amino acids

Endo Ra decreased similarly in both groups during the experimental period to rates that were no different from zero (Table 3). The mean tracer-determined hepatic glucose uptake rates during the experimental period were 4.8 ± 1.4 and 10.4 ± 2.0 µmol · kg-1 · min-1 in PoAA and NoAA, respectively (Table 3).

                              
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Table 3.   Net hepatic glucose balance, endogenous Ra, and tracer-determined hepatic glucose uptake in dogs receiving a gluconeogenic amino acid mixture or no amino acids

Gluconeogenic amino acid metabolism. During the basal period, parameters related to gluconeogenic amino acid metabolism (arterial and portal blood concentrations, hepatic load, net hepatic uptake, and net hepatic fractional extraction; Table 4, Figs. 3 and 4) were similar in the two groups. During the experimental period, however, arterial and portal blood amino acids concentrations were higher in PoAA than NoAA (mean values for total of gluconeogenic amino acids in artery: 1,573 ± 133 vs. 1,147 ± 86 µM, P < 0.05; in portal vein: 2,055 ± 175 vs. 1,137 ± 85, P < 0.05). Similarly, the hepatic load of gluconeogenic amino acids (Fig. 4) was higher (P < 0.01) in PoAA at every time point during the experimental period (mean values were 56 ± 3 and 32 ± 4 µmol · kg-1 · min-1 in PoAA and NoAA, respectively). As would be expected with the greater hepatic load of amino acids in PoAA, the rate of net hepatic amino acid uptake was greater in PoAA than in NoAA throughout the experimental period (P < 0.01; mean rates for total gluconeogenic amino acid uptake were 14 ± 2 and 6 ± 1 µmol · kg-1 · min-1 in PoAA and NoAA, respectively; Fig. 3). In both groups, the net hepatic fractional extraction of alanine, glutamate, glutamine, and serine increased significantly (P < 0.05) compared with baseline, and there was a trend toward an increase in net hepatic fractional extraction of glycine (Table 4). The experimental period values for net hepatic fractional extraction of threonine were similar in the two groups, but the change from baseline reached statistical significance only in PoAA.

                              
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Table 4.   Arterial and portal blood concentrations, net hepatic uptake, and net hepatic fractional extraction of individual gluconeogenic amino acids in dogs receiving intraportal glucose infusion with or without a concomitant intraportal infusion of gluconeogenic amino acids


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Fig. 4.   Arterial and portal vein concentrations and hepatic load of gluconeogenic amino acids (serine, threonine, glutamine, glutamate, glycine, and alanine). See legend to Fig. 1 for description of study design. Values were significantly greater in PoAA than NoAA during experimental period (arterial and portal concentrations, P < 0.05; hepatic load, P < 0.01).

Lactate metabolism. There were no differences between groups in arterial blood lactate concentrations or net hepatic lactate balance. Both groups exhibited net hepatic lactate uptake (~7 µmol · kg-1 · min-1) during the basal period, shifted to net hepatic lactate output within 30 min of the start of the experimental period, and returned to net hepatic lactate uptake before the end of the study (data not shown).

Net hepatic glycogen synthesis. The mean rates of net hepatic glycogen synthesis were 7.5 ± 1.9 and 10.7 ± 2.3 µmol glucosyl residues · kg-1 · min-1 in PoAA and NoAA, respectively (P = 0.1). The hepatic 3H content (expressed per kg body wt) and the inflowing plasma [3H]glucose specific activities were 939,076 ± 220,971 dpm and 1,165 ± 220 dpm/µmol in PoAA and 992,130 ± 407,759 dpm and 612 ± 233 dpm/µmol in NoAA. Glycogen synthesis via the direct pathway, measured by deposition of tritiated glycogen, averaged 3.4 ± 0.4 and 6.8 ± 0.5 µmol glucosyl residues · kg-1 · min-1 in PoAA and NoAA, respectively (P < 0.01).

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

NHGU was reduced 50% in the group receiving intraportal amino acid infusion (4.8 ± 2.0 vs. 9.4 ± 2.0 µmol · kg-1 · min-1, P < 0.05), and tracer-determined hepatic glucose uptake was reduced by a similar amount. Normally glucagon increases in the postprandial period after intake of glucose and amino acids or a mixed meal (20) as a result of the stimulatory effect of amino acids on glucagon secretion. If we had increased circulating glucagon levels, rather than maintaining basal glucagonemia, it could be anticipated that NHGU would have been reduced even further (16), thus increasing the impact of amino acids on glucose handling by the liver.

The amino acid infusion maintained relatively stable circulating levels of gluconeogenic amino acids in PoAA, but in NoAA the amino acid concentrations declined significantly in response to hyperinsulinemia, hyperglycemia, and the portal signal. The fall in amino acid concentrations was associated with an increase in their net hepatic fractional extraction (Table 4). Levels of gluconeogenic amino acids have been observed to decline ~20%-50% from basal values in the presence of physiological hyperinsulinemia (peripheral insulin concentrations of 120-240 pM) in the rat, dog, and human (12, 17, 19, 28). Although insulin is an upregulator of hepatic system A activity, it does not affect other amino acid transport systems such as ASC, N, or L (11). Thus, other than a small effect on alanine, insulin has little capacity to alter net hepatic fractional extraction of amino acids. In the presence of a euglycemic, hyperinsulinemic (~120 pM) clamp, the fractional extraction of gluconeogenic amino acids did not change compared with basal conditions (28). Therefore the increase in fractional extraction of the amino acids in the current report is unlikely to be due merely to hyperinsulinemia. On the other hand, hyperglycemia by itself is also unable to alter the fractional extraction of amino acids. During a hyperglycemic (~10.5 mM), euinsulinemic clamp in conscious dogs, the fractional extraction of the gluconeogenic amino acids remained stable (29). It therefore appears that the combination of hyperinsulinemia and hyperglycemia or the presence of the portal signal must have been responsible for the enhanced net fractional extraction of the amino acids by the liver.

There are several possible reasons for the inhibition of NHGU in the PoAA group, including substrate competition, a relative insulin resistance, and neural signals induced by amino acid infusion (14, 25). The possibility of substrate competition cannot be adequately assessed from the current studies. If the glycogen content is indeed lower in PoAA, then a mechanism other than or in addition to must have been operating. As to the possibility of insulin resistance, investigators using the hyperinsulinemic-euglycemic clamp technique in the human have reported that amino acids caused insulin resistance both by reducing whole body glucose utilization and by interfering with suppression of hepatic glucose production by insulin, primarily by enhancing gluconeogenesis (1, 31). However, glucagon was not controlled, and glucagon levels were consistently higher during amino acid delivery than in control studies without amino acids (1, 31). A selective increase in glucagon in subjects receiving an amino acid load was found to enhance endogenous glucose production and inhibit amino acid-induced protein synthesis (8). When glucagon was maintained at basal concentrations in human subjects receiving infusions of somatostatin, 10-fold basal insulin, a balanced amino acid mixture, and glucose to maintain euglycemia, there was no reduction in glucose disposal, and endogenous glucose production was fully suppressed (6). Moreover, use of tracer-determined glucose oxidation (to overcome inherent limitations of indirect calorimetry in measuring substrate oxidation rates during amino acid administration) demonstrated no reduction of glucose oxidation during amino acid infusion in humans (30). In the current study, the peripheral glucose infusion rate required to maintain the hepatic glucose load at 150% basal did not differ between groups, although it tended to be lower in the group receiving amino acids. Thus we have no clear evidence that amino acid infusion resulted in insulin resistance.

Finally, amino acids might have inhibited NHGU by eliciting a neural signal that conflicted with or modulated the "portal signal" created by intraportal glucose infusion. Neural sensors for many amino acids have been identified in the portal region (24, 25, 27). Intraportal injection of several amino acids, including glycine and threonine, results in dose-dependent depression of the afferent firing rate in the hepatic branch of the vagus nerve (25). In contrast, intraportal injection of other amino acids, including alanine and serine, increases the afferent hepatic branch firing rate (25). The efferent firing rates in the pancreatic branches of the vagal and splanchnic nerves change inversely and directly, respectively, with the afferent firing rate in the hepatic vagal branch, suggesting that sensors in the hepatoportal system reflexively regulate the neuroendocrine response to amino acid-containing feedings (23). Neural signals resulting from intraportal substrate delivery also appear to affect the liver directly. In the absence of hepatic nerves, NHGU during intraportal glucose delivery in conscious dogs is blunted compared with the response in the presence of hepatic nerves, even with insulin and glucagon concentrations fixed at similar levels in the hepatic-innervated and -denervated groups (4). In addition to the hepatoportal region, the amino acids could also have been sensed in other sites. For example, they might have affected the central nervous system directly. Also, the concentrations of the gluconeogenic amino acids were higher in the arterial circulation in PoAA than in NoAA, and thus we cannot rule out a peripheral effect of the amino acids. Further studies are underway in our laboratory to explore this question more thoroughly. However, functional evidence suggests that the portal delivery route may have a unique role to play in hepatic amino acid metabolism. Intraportally delivered leucine stimulated hepatic fibrinogen synthesis in conscious dogs, whereas peripherally infused leucine had no such effect (5).

It is not clear whether it was one or two amino acids, or the combination of the six amino acids, that were responsible for the results obtained. It is noteworthy that fractional extraction of glutamine and glutamate increased during intraportal infusion of amino acids (Table 4). Glutamate is recognized as a neurotransmitter in many sites (7), and there is evidence that glutamine can modulate both the intestinal absorption and hepatic uptake of glucose. Delivery of glutamine into the intestinal lumen, but not into the superior mesenteric artery, was found to increase hepatic glucose uptake in a combined liver-intestine perfusion preparation in which both the hepatic and intestinal nerve plexuses remained intact but not in an isolated (and denervated) liver perfusion system (13). This suggests that enterohepatic nerves, humoral factors, or absorbed substrates may act singly or jointly to regulate hepatic glucose uptake during enteral administration of glutamine. NHGU was decreased, rather than increased, during intraportal amino acid infusion in the current studies, indicating that enteric control mechanisms had been bypassed, a mixture of amino acids may produce different effects than glutamine given alone, or the effect of glutamine on NHGU may be dosage dependent, as shown in vitro (13).

Net hepatic glycogen synthesis was reduced ~30% in the animals receiving the amino acid infusion relative to those not receiving amino acids, although this reduction did not reach statistical significance (P = 0.1). This is consistent with our previous findings that net hepatic glycogen synthesis is strongly correlated with NHGU in dogs receiving glucose infusion (peripherally or portally), intraportal insulin (basal or 4-fold basal), and basal glucagon (26). The failure to achieve statistical significance was probably a result of a type II statistical error; we calculated the n required for this study based on the variance and expected difference in NHGU, the primary variable of interest. Net hepatic uptake of glycogenic substrates (sum of glucose, gluconeogenic amino acids, and glycerol) was the same in the two groups (Fig. 5); thus glycogen accumulation in PoAA was not impaired by any reduction in net substrate supply to the liver. In each group, NHGU was sufficient to account for all glycogen deposition via the direct pathway, as well as net hepatic release of lactate during the experimental period. Net hepatic glycogen synthesis and lactate production accounted for 88% of the net hepatic carbon uptake by the NoAA group. Much of the carbon remaining unaccounted for (~1.5 µmol glucose equivalents · kg-1 · min-1) was undoubtedly oxidized, with a small amount being utilized for lipogenesis (20). In the PoAA group, only 70% of net hepatic substrate uptake could be accounted for by net hepatic glycogen synthesis and lactate release. We have previously observed that the postprandial hepatic oxidation rate averaged ~1.5 µmol glucose equivalents · kg-1 · min-1 and that very little de novo lipogenesis occurred in the liver in a group of dogs receiving an intragastric mixed meal in which glucose provided all the carbohydrate (20). If hepatic oxidation and lipogenic rates are assumed to be the same in PoAA and mixed-meal-fed dogs, ~2 µmol glucose equivalents · kg-1 · min-1 were available for purposes other than glycogen formation, lactate production, and oxidation in the livers of PoAA. There is no evidence of enhanced gluconeogenesis in the PoAA group; Endo Ra fell to a similar, low rate in both groups (Table 3). Administration of amino acids may have stimulated hepatic protein synthesis and/or reduced hepatic protein breakdown. Humans receiving a meal containing glucose, lipid, and amino acids were observed to have significantly enhanced synthesis of albumin compared with subjects receiving an isocaloric meal containing only glucose and lipid (9, 33). The combination of amino acid delivery and hyperinsulinemia enhanced albumin synthesis more than hyperinsulinemia alone (33). Selective hypothreoninemia or hypoisoleucinemia for 4 h impaired whole body protein synthesis but not hepatic protein synthesis (17). Thus, even though the amino acid mixture administered in these studies was lacking in most of the essential amino acids, an enhancement of intrahepatic protein synthesis may have occurred, with intrahepatic proteolysis, or possibly proteolysis in other tissues, providing the additional amino acids needed. On the other hand, a specific group of regulatory amino acids, including glutamine, has been observed to inhibit hepatic protein degradation (22), and thus a decrease in the rate of autophagy may explain the greater hepatic amino acid retention in the group receiving amino acids. The mechanism or mechanisms responsible for determining the fate of carbon entering the liver remain to be elucidated, but hepatic nerves present one possibility for directing carbon away from one biosynthetic pathway (e.g., glycogenosis) and into another (e.g., protein synthesis).


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Fig. 5.   Summary of net hepatic uptake of glycogenic precursors, glucose, gluconeogenic amino acids, and glycerol, in dogs receiving intraportal glucose infusion with (PoAA, n = 7) or without (NoAA, n = 6) concomitant intraportal gluconeogenic amino acid infusion. Total net hepatic glycogenic precursor uptake did not differ between groups.

In a net sense, intraportal infusion of gluconeogenic amino acids concurrent with intraportal glucose infusion directs glucose carbon away from the liver and toward other tissues but causes a reciprocal increase in gluconeogenic amino acid uptake by the liver. Thus net hepatic uptake of glycogenic carbon is equivalent in animals receiving intraportal glucose with or without concomitant intraportal infusion of amino acids. Despite this, the animals receiving amino acids deposited ~30% less hepatic glycogen (P = 0.1), probably because of increased requirements for carbon to be used for hepatic protein synthesis or because of a decrease in intrahepatic proteolysis. These data are consistent with the existence of a generalized energy receptor (24), or a coordinated system of receptors for individual substrates, which is responsible for determining the contribution of the liver to substrate disposition.

    ACKNOWLEDGEMENTS

The authors appreciate the assistance of Jon Hastings, Wanda Snead, and Pam Venson.

    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants R-01-DK-43706 and DK-40936 and Diabetes Research and Training Center Grant DK-20593.

A preliminary report of this work was presented at the American Diabetes Association Annual Meeting, Atlanta, GA, June 1995, and published in abstract form (Diabetes 44, Suppl. 1: 90A, 1995).

Present address of M. J. Pagliassotti: Sect. of Pediatric Nutrition, Dept. of Pediatrics, University of Colorado Health Sciences Center, Denver, CO 80262.

Address for reprint requests: M. C. Moore, 702 Light Hall, Dept. of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN 37232-0615.

Received 14 July 1997; accepted in final form 23 January 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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