A negative arterial-portal venous glucose gradient increases net hepatic glucose uptake in euglycemic dogs

Pietro Galassetti, Chang An Chu, Doss W. Neal, George W. Reed, David H. Wasserman, and Alan D. Cherrington

Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37232-0615


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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We investigated whether a negative arterial-portal venous (a-pv) glucose gradient, or "portal signal," can increase net hepatic glucose uptake (NHGU) and decrease muscle glucose uptake at euglycemia as it does at hyperglycemia. Twenty 42-h fasted dogs were studied during a basal and two 120-min euglycemic periods (period I and period II). Glucagon was maintained at basal levels, and insulin was raised 3-fold (3×Ins, n = 10) or 15-fold (15×Ins, n = 10). During period I, dogs received glucose only peripherally. During period II, one-half of the dogs continued the peripheral infusion; the other one-half received glucose intraportally (4 mg · kg-1 · min-1 and reduced peripheral glucose infusion). A negative a-pv glucose gradient was present during intraportal glucose infusion. All 3×Ins and 15×Ins dogs had similar NHGU in period I. In period II, it was 2.1 ± 0.3 (3×Ins) and 2.5 (15×Ins) mg · kg-1 · min-1 greater in the presence than in the absence of the portal signal (P < 0.001). The net glucose fractional extraction data paralleled NHGU. In 3×Ins, but not in 15×Ins, whole body nonhepatic glucose uptake was lower in the presence of the portal signal than in its absence. In conclusion, in hyperinsulinemic, but not hyperglycemic conditions, the portal signal is effective in activating NHGU. The inhibition of nonhepatic glucose uptake, on the other hand, is minimal under euglycemic as opposed to hyperglycemic conditions.

liver; skeletal muscle


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE LIVER is a central organ in the regulation of glucose homeostasis. In the postabsorptive state (when all nutrients from the last meal have been absorbed from the intestine), the liver is a net producer of glucose. If adequate amounts of glucose are administered at this point, the liver switches from net glucose production to net glucose uptake. Peak rates of net hepatic glucose uptake (NHGU) can reach 5-8 mg · kg-1 · min-1 in both humans and animals (1, 5, 23). Hyperglycemia, hyperinsulinemia, and a negative arterial-portal venous (a-pv) glucose gradient ("portal signal") are known to positively affect the rate of NHGU. Only when these three factors are simultaneously present can peak rates of NHGU be reached (19, 23, 24). Circulating insulin increased NHGU in a dose-dependent manner in the presence of a fixed hyperglycemia (20). At all insulin levels tested (20), NHGU was greater in the presence than in the absence of the portal signal. If arterial insulin was maintained constant, both at basal (3, 24) or at fourfold basal levels (19), NHGU increased as the glucose levels were progressively increased, and again NHGU was greater in the presence of the portal signal than in its absence (19).

The modulation of net glucose uptake by the portal signal also extends to extrahepatic tissues. A decrease in net glucose uptake by nonhepatic tissues in the presence of the portal signal was indirectly demonstrated in several previous studies (2, 24). This reduction was of the same magnitude as the increase in NHGU, thus suggesting that the portal signal controlled the whole body distribution of glucose. Galassetti et al. (8) recently demonstrated that the reduction in extrahepatic glucose uptake caused by the portal signal was primarily the result of a decrease in glucose uptake by skeletal muscle.

Although all previous studies investigating the portal signal were conducted under hyperglycemic conditions, indirect evidence indicates that its effects may be present even under euglycemic conditions. Several papers (6, 7, 9) showed that the sympathoadrenal response to hypoglycemia was blunted when systemic hypoglycemia was allowed to occur, but the hepatoportal region was kept euglycemic by portal glucose infusion. In the process of maintaining hepatic euglycemia, the authors created a negative a-pv glucose gradient. Therefore, the decrease in the sympathoadrenal response may well have been due to the effects of the portal signal. If the portal signal can be activated under euglycemic conditions, it should increase NHGU in response to insulin and possibly exert its inhibitory effect on glucose uptake by nonhepatic tissues even in the absence of hyperglycemia.

The aim of the present study, therefore, was to determine whether the enhancement of NHGU and the inhibition of muscle glucose uptake caused by the portal signal under hyperglycemic conditions are also evident under euglycemic conditions. This aim was addressed with arteriovenous balance techniques in the chronically catheterized conscious dog.


    MATERIALS AND METHODS
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MATERIALS AND METHODS
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Animal care and surgical procedures. Studies were performed on 20 42-h fasted conscious mongrel dogs of either sex, averaging 22.8 ± 1.2 kg in weight. All animals were maintained on a daily diet of meat and chow (34% protein, 14.5% fat, 46% carbohydrate, and 5.5% fiber based on dry wt). The animals were housed in a facility meeting the American Association for Accreditation of Laboratory Animal Care guidelines, and the protocol was approved by the Vanderbilt University Animal Care Subcommittee.

Seventeen days before each study, dogs underwent a laparotomy under general anesthesia (0.8% isoflurane). Sampling catheters were inserted into the right common hepatic vein, the hepatic portal vein, a common iliac vein, and an external iliac artery (5, 25). Catheters were also placed in a splenic and a jejunal vein for intraportal infusion of insulin, glucagon, glucose, and p-aminohippurate (PAH). Doppler flow probes (Instrumentation Development Laboratory, Baylor College of Medicine, Houston, TX) were placed around the portal vein, the hepatic artery, and an external iliac artery as previously described (8).

Dogs were studied only if 48 h before a study they had a leukocyte count <18,000/mm3, a hematocrit >35%, a good appetite, and normal stools. On the day of study, 16-gauge venous catheters were inserted into limb veins for infusion of indocyanine green (ICG), glucose, PAH, and somatostatin (Bachem, Torrance, CA).

Experimental design. Each experiment consisted of a total of 380 min, divided into a 110-min equilibration period (from -140 to -30 min), a 30-min basal period (from -30 to 0 min), and two 120-min euglycemic-hyperinsulinemic periods (period I, 0-120 min; period II, 120-240 min; Fig. 1). In all experiments, a constant infusion of ICG (0.1 mg · m-2 · min-1) was initiated at -140 min and maintained throughout the study. At 0 min, a constant infusion of somatostatin (0.8 µg · kg-1 · min-1) was begun to suppress endogenous insulin and glucagon secretion, and a constant intraportal infusion of glucagon (0.5 ng · kg-1 · min-1) was established to maintain the plasma glucagon concentration at basal values.


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Fig. 1.   Experimental design. For each insulin level, in one-half of animals euglycemia was maintained through period I (0-120 min) and period II (120-240 min) via peripheral glucose infusion only; in other one-half of animals euglycemia was maintained via peripheral glucose infusion in period I, and via intraportal glucose infusion in period II. ICG, indocyanine green; 3×Ins, insulin raised 3-fold; 15×Ins, insulin raised 15-fold; Po, intraportal; Pe, peripheral.

At t = 0 min, an intraportal insulin infusion was also started. In one-half of the dogs, the portal insulin infusion rate was 1 mU · kg-1 · min-1, which obtained arterial insulin levels 3-fold basal (3×Ins, n = 10), and in the other one-half, the infusion rate was 3.5 mU · kg-1 · min-1, which obtained arterial insulin levels 15-fold basal (15×Ins, n = 10). Among the 3×Ins animals, euglycemia was maintained in one-half of the dogs (n = 5) via a glucose infusion in a peripheral vein throughout the study. This group of dogs will be referred to as 3×Ins-Pe. Euglycemia was maintained in the remaining 3×Ins dogs (n = 5) via a glucose infusion in a peripheral vein during period I and via constant intraportal glucose infusion (4 mg · kg-1 · min-1) supplemented by glucose infusion into a peripheral vein to maintain euglycemia during period II. This group of dogs will be referred to as 3×Ins-Po. Similarly, in 15×Ins animals, euglycemia was maintained in one-half of the dogs (n = 5) via a glucose infusion in a peripheral vein throughout the study. This group of dogs will be referred to as 15×Ins-Pe. Euglycemia was maintained in the remaining 15×Ins dogs (n = 5) via a glucose infusion in a peripheral vein during period I and via constant intraportal glucose infusion (4 mg · kg-1 · min-1) supplemented by glucose infusion into a peripheral vein to maintain euglycemia during period II. This group of dogs will be referred to as 15×Ins-Po.

External iliac artery, portal vein, hepatic vein, and common iliac vein blood samples were taken every 15 min during the baseline period and every 10 min during the last 30 min of periods I and II. The euglycemic clamp was monitored by means of small (0.4 ml) arterial blood samples drawn every 5 min and analyzed for glucose within 90 s. The peripheral glucose infusion rate was adjusted as required. The total volume of blood withdrawn did not exceed 20% of the total blood volume of the animal, and two volumes of normal saline were infused for each volume of blood withdrawn. The methods for collecting and processing blood samples have been described previously (17).

Analytical procedures. Plasma glucose concentrations were determined on all samples with the glucose-oxidase method (12) on a Beckman glucose analyzer (Beckman Instruments, Fullerton, CA). Blood concentrations of glucose were obtained in triplicate on perchloric acid extracts with a Technicon Autoanalyzer according to the method of Lloyd et al. (14). Blood lactate, alanine, glycerol (perchloric acid extracts), and plasma free fatty acid (FFA) concentrations were determined in triplicate by enzymatic methods, with a Monarch 2000 centrifugal analyzer (Instrumentation Laboratory, Lexington, MA) as previously described (14). PAH concentrations were determined according to the method of Brun (4). The immunoreactive glucagon was determined with a modified version of the method of Morgan and Lazarow (18), with an interassay coefficient of variation (CV) of 10%. Immunoreactive insulin was measured as previously described (18), with an interassay CV of 4%.

Calculations and data analysis. Hindlimb blood flow was estimated with a Doppler flow probe (Transonic Systems, Ithaca, NY; Refs. 10, 11). Hepatic blood flow was estimated with both ICG (13) and Doppler flow probes. These two independent techniques yielded similar values. Doppler-determined hepatic blood flow was used in data calculation, whereas the ICG technique was used as a backup for the Doppler measurements. The distribution of hepatic blood flow was 81% portal vein and 19% hepatic artery in the baseline period, 74% portal vein and 26% hepatic artery in period I, and 73% portal vein and 27% hepatic artery in period II; these data are consistent with previous observations (2, 3, 19, 26).

To assess the mixing of intraportally infused glucose in the blood, the PAH recovery technique was utilized (19, 24). Among the 10 animals (5 from each group) that underwent intraportal glucose infusion in period II, poor mixing, according to the above method, was obtained in one of the four time points of the intraportal period in two animals and in two of the four time points of the intraportal period in two more animals. In the 10 animals, the ratio of PAH recovery in the portal vein to the intraportal PAH infusion rate was 0.91 ± 0.03 and the ratio of PAH recovery in the hepatic vein to the PAH infusion rate was 0.89 ± 0.03 (a ratio of 1.0 would represent perfect mixing). The infusate failed to mix with the blood only 15% of the time (6 out of 40 measurements). More importantly, when mixing errors did occur, they were random; therefore, all time points from these 10 animals were included in the database.

The hepatic substrate load was calculated directly as
load<SUB>in(D)</SUB> = [(C<SUB>A</SUB> × ABF) + (C<SUB>p</SUB> × PBF)]
where CA and CP represent the substrate concentrations in arterial and portal venous blood and ABF and PBF represent the hepatic artery and the portal vein blood flows. In glucose balance calculations, plasma glucose values were converted to whole blood values with a correction factor (CF) obtained by calculating the ratio of the whole blood glucose value to the plasma glucose value at each time point throughout the study. The mean CF was 0.73 ± 0.01 in the artery, iliac vein, and hepatic vein throughout the whole study. The CF in the portal vein was 0.73 ± 0.01 throughout the whole study for the 10 dogs that underwent peripheral glucose infusion only, 0.73 ± 0.01 during baseline and period I, and 0.72 ± 0.01 during period II in the dogs that had glucose infused intraportally. To circumvent any potential errors arising from incomplete mixing of glucose during intraportal glucose infusion, a second, indirect method of calculating the hepatic glucose load was used. It utilized the formula
load<SUB>in(I)</SUB> = [(G<SUB>A</SUB> × HBF) + GIR<SUB>p</SUB>] − GUG
where GA is arterial blood glucose concentration, GIRP is portal glucose infusion rate, GUG is gastrointestinal tract glucose uptake, and HBF is the total hepatic blood flow. GUG was measured during peripheral glucose infusion as the product of the a-pv glucose difference and the portal vein blood flow and was considered to be the same during intraportal and peripheral glucose infusions, if the arterial glucose concentration was identical between periods I and II. Small differences in arterial glucose concentrations between periods I and II were compensated for according to the previously demonstrated correlation between GUG and arterial blood glucose concentration (19).

The load of substrates exiting the liver was calculated as
load<SUB>out</SUB> = C<SUB>H</SUB> × HBF
where CH is the concentration of substrate in the hepatic vein.

Net hepatic glucose balance (NHGB) was calculated by two separate methods as described previously (19, 26). The first method, which will be referred to as the direct calculation, used the equation
NHGB<SUB>D</SUB> = load<SUB>out</SUB> − load<SUB>in(D)</SUB>
and the second, referred to as the indirect calculation, used the equation
NHGB<SUB>I</SUB> = load<SUB>out</SUB> − load<SUB>in(I)</SUB>
In these calculations, a positive value indicates net output. In the RESULTS section, the hepatic glucose load and NHGB shown were determined with the indirect calculation. The estimate of NHGB was similar regardless of which method was used in calculations. For other substrates, the direct calculation was used to calculate net hepatic balance.

Net hindlimb substrate balances were calculated as the product of external iliac artery blood flow and the arterial-venous difference in substrate concentration, as measured with samples from the external iliac artery and common iliac vein.

For both the liver and hindlimb, net substrate fractional extraction was calculated as the ratio between net substrate uptake and substrate load. Whole body, net nonhepatic glucose balance was calculated during periods I and II as the difference between the total glucose infusion rate and net hepatic glucose uptake.

Statistical analysis. Statistics were performed with SuperAnova (Abacus Concepts, Berkley, CA) on a MacIntosh PowerPC. Statistical comparisons between groups and over time were made with ANOVA designed to account for repeated measures. Specific time points were examined for significance with contrasts solved by univariate repeated measures. Changes in a given variable that occurred between period I and period II within a group were compared with paired t-tests. Differences were considered significant when P values were <0.05. Data are expressed as means ± SE.


    RESULTS
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ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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Hormonal levels. The arterial plasma insulin concentration was increased by 3-fold over basal through periods I and II in all 3×Ins dogs and by 15-fold over basal in all 15×Ins dogs (Fig. 2). Arterial plasma glucagon was kept at basal levels throughout the study in all four groups of dogs.


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Fig. 2.   Arterial plasma insulin (A) and glucagon (B) in 42-h-fasted conscious dogs during baseline (basal insulin-euglycemia), period I (hyperinsulinemia-euglycemia via peripheral glucose infusion), and period II (hyperinsulinemia-euglycemia via either peripheral or intraportal glucose infusion). Data are group means ± SE; n = 5 in each group. In each dog, value from baseline period is mean of 3 measurements, whereas data from periods I and II are means of 4 measurements each. Samples were taken over the last 30 min of each period. * P < 0.05 vs. baseline.

Glucose levels and a-pv glucose gradient and blood flows. Arterial blood glucose concentrations were similar in all groups at baseline and were maintained at similar baseline levels throughout the study in all animals (Fig. 3; Table 1). The a-pv blood glucose gradient was positive in all animals at baseline and during period I. During period II, it remained positive in the 3×Ins-Pe and 15×Ins-Pe groups, whereas it became markedly negative in 3×Ins-Po (-10 ± 1 mg/dl) and 15×Ins-Po (-10 ± 2 mg/dl, nonsignificant vs. 3×Ins-Po).


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Fig. 3.   Arterial blood glucose (A) and arterial-portal venous glucose gradient (B) in 42-h-fasted conscious dogs during baseline (basal insulin-euglycemia), period I (hyperinsulinemia-euglycemia via peripheral glucose infusion), and period II (hyperinsulinemia-euglycemia via either peripheral or intraportal glucose infusion). Data are group means ± SE; n = 5 in each group. In each dog, value from baseline period is mean of 3 measurements, whereas data from periods I and II are means of 4 measurements each. Samples were taken over the last 30 min of each period. * P < 0.05 vs. baseline. # P < 0.05 vs. period I within same group.


                              
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Table 1.   External iliac artery, hepatic artery, portal vein, and total hepatic blood flows in 42-h-fasted conscious dogs during baseline, period I, and period II

Total hepatic blood flow (Table 1) was similar in all four groups of dogs at baseline and did not change significantly throughout the study. This was true despite the fact that the hepatic artery blood flow was significantly higher in the 3×Ins-Po than in the 3×Ins-Pe dogs in all three study periods. External iliac artery blood flows were slightly lower in the 3×Ins-Po than in the 3×Ins-Pe dogs and in the 15×Ins-Pe than in the 15×Ins-Po dogs, but within each group they remained stable throughout the whole study.

Hepatic glucose metabolism. The hepatic glucose load was similar in all animals at baseline and remained unchanged during period I (Fig. 4). During period II, the hepatic glucose load remained unchanged in the 3×Ins-Pe and 15×Ins-Pe dogs, whereas, as a necessary feature of our experimental design, it rose slightly in the 3×Ins-Po and 15×Ins-Po groups (3×Ins-Pe: 21 ± 1 and 21 ± 1 mg · kg-1 · min-1 in periods I and II, respectively; 3×Ins-Po: 25 ± 1 and 29 ± 1 mg · kg-1 · min-1 in periods I and II, respectively; 15×Ins-Pe: 20 ± 1 and 20 ± 1 mg · kg-1 · min-1 in periods I and II, respectively; 15×Ins-Po: 22 ± 1 and 29 ± 2 mg · kg-1 · min-1 in periods I and II, respectively).


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Fig. 4.   Net hepatic glucose uptake (A), fractional extraction (B), and load (C) in 42-h-fasted conscious dogs during baseline (basal insulin-euglycemia), period I (hyperinsulinemia-euglycemia via peripheral glucose infusion), and period II (hyperinsulinemia-euglycemia via either peripheral or intraportal glucose infusion). Data are group means ± SE; n = 5 in each group. In each dog, value from baseline period is mean of 3 measurements, whereas data from periods I and II are means of 4 measurements each. Samples were taken over the last 30 min of each period. * P < 0.05 vs. baseline. # P < 0.05 vs. period I within same group.

The net hepatic fractional extraction of glucose was close to zero during period I in all 3×Ins dogs. During period II, this variable remained close to zero in the 3×Ins-Pe group and rose significantly to 7.4 ± 0.5% in the 3×Ins-Po group. In all 15×Ins dogs, net hepatic glucose fractional extraction was ~2.0% during period I. During period II, net hepatic glucose fractional extraction remained unchanged in the 15×Ins-Pe dogs, whereas it increased significantly to 10.3 ± 1.8% in the 15×Ins-Po group. It should be noted that the increase in net hepatic glucose fractional extraction between period I and period II was similar in the 3×Ins-Po and 15×Ins-Po groups.

At baseline, all groups of dogs showed net hepatic output of glucose (1.6-1.9 mg · kg-1 · min-1). In all 3×Ins dogs, the net hepatic glucose balance was close to zero during period I. During period II, the net hepatic glucose balance remained close to zero in the 3×Ins-Pe group (Delta  vs. period I = 0.3 ± 0.2 mg · kg-1 · min-1), whereas in the 3×Ins-Po group it increased significantly (Delta  vs. period I = 2.2 ± 0.3 mg · kg-1 · min-1, P < 0.01 vs. 3×Ins-Pe). All 15×Ins dogs displayed a NHGU of ~0.5 mg · kg-1 · min-1 during period I. During period II, NHGU remained unchanged in the 15×Ins-Pe group (Delta  vs. period I = -0.1 ± 0.2 · kg-1 · min-1), whereas in the 15×Ins-Po group it increased significantly (Delta  vs. period I = 2.5 ± 0.6 mg · kg-1 · min-1, P < 0.01 vs. 3×Ins-Pe). It should be noted that the increase in net hepatic glucose uptake between period I and period II was similar in the 3×Ins-Po and 15×Ins-Po groups.

Hindlimb glucose metabolism. Net hindlimb glucose uptake was similar in all groups of dogs at baseline. During period I net hindlimb glucose uptake rose on average by ~70% in 3×Ins dogs and by ~400% in the 15×Ins dogs (Fig. 5). Net hindlimb glucose uptake was not different in period II compared with period I, regardless of group. The behavior of the net hindlimb fractional extraction of glucose closely resembled that of net glucose uptake.


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Fig. 5.   Net hindlimb glucose uptake (A) and fractional extraction (B) in 42-h-fasted conscious dogs during baseline (basal insulin-euglycemia), period I (hyperinsulinemia-euglycemia via peripheral glucose infusion), and period II (hyperinsulinemia-euglycemia via either peripheral or intraportal glucose infusion). Data are group means ± SE; n = 5 in each group. In each dog, value from baseline period is mean of 3 measurements, whereas data from periods I and II are means of 4 measurements each. Samples were taken over the last 30 min of each period. * P < 0.05 vs. baseline.

Total glucose infusion rate and net whole body nonhepatic glucose uptake. The total glucose infusion rate was similar in 3×Ins-Pe and 3×Ins-Po during period I, and in both groups it rose similarly by ~2 mg · kg-1 · min-1 during period II. Likewise the total glucose infusion rate was similar in 15×Ins-Pe and 15×Ins-Po during period I (Fig. 6). During period II it increased by 1.7 ± 0.6 mg · kg-1 · min-1 in 15×Ins-Pe and by 3.3 ± 1.4 mg · kg-1 · min-1 in 15×Ins-Po (nonsignificant).


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Fig. 6.   Total glucose infusion rate (A) and net whole body nonhepatic glucose uptake (B) in 42-h-fasted conscious dogs during baseline (basal insulin-euglycemia), period I (hyperinsulinemia-euglycemia via peripheral glucose infusion), and period II (hyperinsulinemia-euglycemia via either peripheral or intraportal glucose infusion). Data are group means ± SE; n = 5 in each group. In each dog, value from baseline period is mean of 3 measurements, whereas data from periods I and II are means of 4 measurements each. Samples were taken over the last 30 min of each period. * P < 0.05 vs. baseline. # P < 0.05 vs. period I within same group.

Whole body nonhepatic glucose uptake was similar in all subgroups of dogs at baseline (Fig. 6B). Whole body nonhepatic glucose uptake was similar in 3×Ins-Pe and 3×Ins-Po during period I. During period II, the net whole body nonhepatic glucose uptake increased by 1.7 ± 0.5 mg · kg-1 · min-1 in the 3×Ins-Pe group (P < 0.05 vs. period I), whereas it remained unchanged in the 3×Ins-Po group (Delta  vs. period I = 0.1 ± 0.3 mg · kg-1 · min-1; Fig. 6B).

Whole body nonhepatic glucose uptake was also similar in 15×Ins-Pe and 15×Ins-Po during period I. During period II, neither 15×Ins-Pe nor 15×Ins-Po dogs displayed a significant increase in whole body nonhepatic glucose uptake over that seen in period I.

Arterial levels and net hepatic uptake of lactate. Arterial lactate levels and net hepatic lactate uptake were not significantly different in the four groups at baseline (Fig. 7). During period I, although the 3×Ins-Pe group had higher arterial lactate and lower net hepatic lactate uptake than at baseline, no difference in either variable was present between the 3×Ins-Pe and the 3×Ins-Po groups. In the 15×Ins-Pe and 15×Ins-Po groups, lactate levels rose similarly and net hepatic lactate uptake remained at baseline levels during period I. During period II, lactate levels remained unchanged in all dogs; net hepatic lactate uptake was similar in period I and period II in 15×Ins-Pe dogs, whereas it increased significantly in the 15×Ins-Po group.


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Fig. 7.   Arterial lactate (A) and net hepatic lactate uptake (B) in 42-h-fasted conscious dogs during baseline (basal insulin-euglycemia), period I (hyperinsulinemia-euglycemia via peripheral glucose infusion), and period II (hyperinsulinemia-euglycemia via either peripheral or intraportal glucose infusion). Data are group means ± SE; n = 5 in each group. In each dog, value from baseline period is mean of 3 measurements, whereas data from periods I and II are means of 4 measurements each. Samples were taken over the last 30 min of each period. * P < 0.05 vs. baseline. # P < 0.05 vs. period I within same group.

Arterial concentrations and net hepatic uptake of glycerol and FFA. Arterial concentrations of glycerol and FFA were similar in all subgroups of dogs at baseline. Glycerol concentrations decreased similarly by ~50% during period I in all groups and remained unchanged through period II (Table 2). FFA concentrations decreased by 50% in 3×Ins dogs and by >80% in 15×Ins dogs, with no difference due to route of glucose infusion. Net hepatic glycerol and FFA uptakes were lower than basal in all four subgroups during period I but changed little thereafter (period II).

                              
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Table 2.   Glycerol and FFA arterial concentrations and net hepatic balances in 42-h-fasted conscious dogs during baseline, period I, and period II


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the postprandial state, the liver becomes an important site of glucose removal. The amount of glucose taken up by the liver is regulated by circulating glucose and insulin concentrations and by the a-pv glucose gradient (portal signal; Ref. 23). The importance of the latter was only recently identified, and its effectiveness has to date been demonstrated only under hyperglycemic conditions. The present study sought to determine whether the signal created by intraportal glucose infusion could enhance NHGU or decrease muscle glucose uptake in the presence of hyperinsulinemia but in the absence of hyperglycemia. The results show that NHGU and net hepatic fractional extraction of glucose were greater in the presence than in the absence of a negative a-pv glucose gradient, even under euglycemic conditions. This was true with both a 3- and a 15-fold increase in arterial insulin. Decrements in muscle glucose uptake were harder to detect. Our data thus indicate that hyperglycemia is not necessary for the portal signal to regulate the uptake of glucose by the liver; it is less certain whether the same is true with respect to changes in peripheral glucose uptake.

The stimulatory effect of the portal signal was clearly present in both our experimental groups of animals. Interestingly, although one insulin level used in this study was five times higher than the other, NHGU and net hepatic glucose fractional extraction were only 20-40% greater in the presence of higher insulin. These findings are consistent with those of Myers et al. (20) who induced the portal signal under hyperglycemic conditions at various levels of hyperinsulinemia. In this study, the effect of the portal signal on NHGU increased proportionally to insulin only until hyperinsulinemia was about fourfold basal, at which point the effect appeared to have reached saturation. When we previously induced the portal signal under hyperglycemic-hyperinsulinemic (3-fold basal) conditions (8), NHGU was greater (5.8 mg · kg-1 · min-1) than in the present study (2.3 mg · kg-1 · min-1). Of the 5.8 mg · kg-1 · min-1 previously reported, however, only 3.4 mg · kg-1 · min-1 could be ascribed to hyperglycemia alone; the increase in NHGU induced by the portal signal was therefore 2.4 mg · kg-1 · min-1, similar to the increases (2.2 mg · kg-1 · min-1 in 3×Ins and 2.5 mg · kg-1 · min-1 in 15×Ins) observed in the present study. Taken together, these observations indicate that the effect of the portal signal on NHGU is independent of the increases in circulating levels of insulin or glucose.

Our experimental design required that we maintain arterial euglycemia; thus the hepatic glucose load was increased slightly during intraportal glucose infusion. This raises the question of how much of the increase in NHGU was due to the increase in hepatic glucose load. A positive linear correlation exists between hepatic glucose load and NHGU (19). In the presence of a fourfold increase in insulin, an increase in hepatic glucose load of 1 mg · kg-1 · min-1 induced an increase in NHGU of 0.088 mg · kg-1 · min-1. In our experimental setting, the differences in load were 4 and 7 mg · kg-1 · min-1 in the 3×Ins and in the 15×Ins groups, respectively. If we assume that the data of Myers et al. (19) apply to our experiments, the measured differences in load could have accounted for 0.35 and 0.62 mg · kg-1 · min-1 of the increase in NHGU seen during intraportal infusion in the presence of the 3- and 15-fold increases in arterial insulin, respectively. The measured increases in NHGU were much greater, 2.2 and 2.5 mg · kg-1 · min-1 in the presence of the 3- and 15-fold increases in arterial insulin, respectively. Further evidence against a mere hepatic load effect comes from the net hepatic fractional glucose extraction data. This parameter is much more independent of changes in hepatic glucose load (19) than is NHGU. Net hepatic glucose fractional extraction increased during intraportal glucose infusion from 0 to 7% in the 3×Ins group and from 2 to 10% in the 15×Ins group.

The design of the present study did not allow direct assessment of the intrahepatic fate of glucose. It has been previously demonstrated (24) that the increase in NHGU induced by the portal signal is a result of the stimulation of hepatic glucose uptake processes and not a suppression of hepatic glucose production. The rise in hepatic glucose uptake results in increased intracellular concentrations of glucose 6-phosphate and other hexose monophosphates, which in turn stimulate glycogen synthase, leading to significant glycogen deposition. In fact, ~75% of the extra glucose taken up by the liver in response to the portal signal is stored as glycogen, whereas the remainder primarily leaves the liver as lactate. Other metabolic pathways (oxidation, pentose phosphate cycle, conversion to lipids) appear to play minor roles in the intrahepatic fate of glucose. The response of all measured metabolic variables is compatible with the notion that similar intrahepatic events occurred under the euglycemic conditions of the present study.

Under hyperglycemic conditions, the increase in NHGU induced by the portal signal is paralleled by a proportional decrease in net glucose uptake by nonhepatic tissues (2, 8, 24). In the present study, the same observation was made with a threefold increase in insulin under euglycemic conditions. When insulin was raised 15-fold, peripheral (nonhepatic) glucose uptake was not significantly different in the two groups of dogs (i.e., with or without the portal signal), possibly reflecting a saturation of glucose transport mechanisms at supraphysiological arterial insulin levels. The decrease in peripheral (nonhepatic) glucose uptake induced by the portal signal during hyperglycemia occurs primarily in the skeletal muscle (8). In the present study, net glucose uptake by the hindlimb, which is mostly skeletal muscle, was not affected when the route of glucose infusion was shifted from peripheral to portal at either insulin level. The failure of the limb balance data to confirm the whole body, nonhepatic glucose uptake data is probably explained by the fact that under euglycemic conditions the magnitude of the response is small, especially at the lower insulin level, whereas the inherent noise of the arteriovenous glucose balance measurements across the whole limb is large.

Recent studies (3, 15, 16) suggest that the autonomic nervous system probably represents the mechanism by which the portal signal exerts its effects. Both adrenergic and cholinergic nerve terminals have been found within the liver (21, 22), and NHGU appears to result from the balance between inhibitory sympathetic impulses (27) and stimulatory parasympathetic impulses (28). The hepatic branch of the vagus (21) provides a pathway to transfer information about the portal glucose level to the brain. Efferent impulses to the liver alter the sympathetic-parasympathetic balance in favor of greater NHGU. The autonomic nervous system has also been implicated in the extrahepatic effects of the portal signal. Minokoshi et al. (16) observed an increase in glucose uptake by the skeletal muscle in response to stimulation of the ventromedial hypothalamus. Xie and Lautt (29, 30) reported that a selective hepatic parasympathetic blockade induced insulin resistance in the skeletal muscle of hyperinsulinemic cats. The complete elucidation of the mechanisms by which the portal signal exerts its hepatic and extrahepatic effects, however, will require additional work.

Several studies published over the last few years (6, 7, 9) have investigated the hypothesis that portal glucosensors trigger the counterregulatory response to hypoglycemia. The above authors showed that when arterial hypoglycemia is generated, but glucose is infused in the portal vein, a significant part of the sympathoadrenal response to hypoglycemia is suppressed. They concluded that systemic hypoglycemia is sensed less well when intraportal glucose infusion restores euglycemia locally in the hepatic region because the hypothetical hepatic glucosensors are not stimulated. The findings of the present study allow an alternative explanation for their observations. The reported blunting of the sympathoadrenal response always occurred in the presence of a negative a-pv glucose gradient, or portal signal. This raises the possibility that the decreased sympathoadrenal response reflected the addition of a "feeding" signal to a hypoglycemic setting. Portal glucose infusion has been shown to alter the firing rate of the splanchnic nerves. Evidence that the portal signal can stimulate NHGU at close to euglycemic conditions did not previously exist. The present data indicate that such stimulation can occur. This suggests that the reduction in the sympathetic response to hypoglycemia seen during glucose infusion into the portal vein might reflect a feeding effect rather than the detection of hepatic hypoglycemia per se. This is important because a negative arterial-portal glucose gradient would not usually be present during insulin-induced hypoglycemia. This interpretation would support the concept that hypoglycemia sensing per se occurs in the brain.

In summary, hyperinsulinemia (3- or 15-fold basal) was generated in 42-h-fasted conscious dogs, while glucagon was maintained at its basal concentration. Euglycemia was maintained via infusion of glucose either in the portal vein or into a peripheral vein. At each insulin level, NHGU was significantly greater during glucose infusion in the portal vein than in a peripheral vein. Our findings indicate that hyperglycemia is not a necessary cofactor for the activation of the effects of the portal signal on the liver and suggest that the portal signal may have played an important role in previously observed hepatic responses to hypoglycemia.


    ACKNOWLEDGEMENTS

We thankfully acknowledge the excellent technical assistance of Wanda Snead and Pamela Venson from the Vanderbilt University DRTC Hormone Core Laboratory and Brittina Murphy from the Vanderbilt University Animal Care facility.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants R-01-DK-43706, DK-40936, and DK-50277 and the Diabetes Research and Training Center Grant DK-20593. P. Galassetti was supported by the National Institutes of Health Training Grant DK-07061.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: P. Galassetti, Rm. 754, MRB I, Vanderbilt Univ. Medical Center, Nashville, TN 37232-0615 (E-mail : pietro.galassetti{at}vanderbilt.edu).

Received 29 October 1998; accepted in final form 15 March 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Endocrinol Metab 277(1):E126-E134
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