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Am J Physiol Endocrinol Metab 276: E806-E813, 1999;
0193-1849/99 $5.00
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Vol. 276, Issue 4, E806-E813, April 1999

Effect of a selective rise in hepatic artery insulin on hepatic glucose production in the conscious dog

Dana K. Sindelar, Kayano Igawa, Chang A. Chu, Jim H. Balcom, Doss W. Neal, and Alan D. Cherrington

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study we compared the hepatic effects of a selective increase in hepatic sinusoidal insulin brought about by insulin infusion into the hepatic artery with those resulting from insulin infusion into the portal vein. A pancreatic clamp was used to control the endocrine pancreas in conscious overnight-fasted dogs. In the control period, insulin was infused via peripheral vein and the portal vein. After the 40-min basal period, there was a 180-min test period during which the peripheral insulin infusion was stopped and an additional 1.2 pmol · kg-1 · min-1 of insulin was infused into the hepatic artery (HART, n = 5) or the portal vein (PORT, n = 5, data published previously). In the HART group, the calculated hepatic sinusoidal insulin level increased from 99 ± 20 (basal) to 165 ± 21 pmol/l (last 30 min). The calculated hepatic artery insulin concentration rose from 50 ± 8 (basal) to 289 ± 19 pmol/l (last 30 min). However, the overall arterial (50 ± 8 pmol/l) and portal vein insulin levels (118 ± 24 pmol/l) did not change over the course of the experiment. In the PORT group, the calculated hepatic sinusoidal insulin level increased from 94 ± 30 (basal) to 156 ± 33 pmol/l (last 30 min). The portal insulin rose from 108 ± 42 (basal) to 192 ± 42 pmol/l (last 30 min), whereas the overall arterial insulin (54 ± 6 pmol/l) was unaltered during the study. In both groups hepatic sinusoidal glucagon levels remained unchanged, and euglycemia was maintained by peripheral glucose infusion. In the HART group, net hepatic glucose output (NHGO) was suppressed from 9.6 ± 2.1 µmol · kg-1 · min-1 (basal) to 4.6 ± 1.0 µmol · kg-1 · min-1 (15 min) and eventually fell to 3.5 ± 0.8 µmol · kg-1 · min-1 (last 30 min, P < 0.05). In the PORT group, NHGO dropped quickly (P < 0.05) from 10.0 ± 0.9 (basal) to 7.8 ± 1.6 (15 min) and eventually reached 3.1 ± 1.1 µmol · kg-1 · min-1 (last 30 min). Thus NHGO decreases in response to a selective increase in hepatic sinusoidal insulin, regardless of whether it comes about because of hyperinsulinemia in the hepatic artery or portal vein.

glycogenolysis; gluconeogenesis


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ALTHOUGH THE ABILITY of insulin to suppress hepatic glucose production (HGP) has long been recognized, both the site and mechanism by which the hormone acts remain controversial. Recent work showed that the suppression of HGP correlated strongly with changes in the peripheral insulin level (1, 7, 8, 21), giving rise to the suggestion that it is peripheral rather than hepatic sinusoidal insulin that is important in controlling HGP. We recently showed that, although HGP is sensitive to increases in peripheral insulin, it is also sensitive to increases in portal vein insulin per se (27, 28). Other investigators have also shown in humans (14) and diabetic dogs (8) that insulin has a direct action on the liver. In our previous study, the response of the liver to a selective 84 pmol/l increase in portal vein insulin (occurring in the absence of a change in arterial insulin) caused HGP to decline quickly (15 min) and to fall ~50% within 3 h (27). This decline in HGP resulted solely from a decrease in glycogenolysis. Similarly, when portal insulin was selectively decreased by 120 pmol/l, again with no change in arterial insulin (26), net hepatic glucose output (NHGO) increased rapidly by 22 µmol · kg-1 · min-1 and remained elevated for 3 h relative to an equivalently hyperglycemic control group. Again, this change reflected an alteration in glycogenolysis.

A selective 84 pmol/l increase in peripheral insulin (no change in portal vein insulin) also suppressed HGP by ~50%, but it did so much more slowly (60 min) than the increase in portal vein insulin. The fall in HGP due to the selective increase in peripheral insulin depended on a combination of factors, including a decrease in gluconeogenesis and a diversion of glycogenolytically derived carbon to lactate rather than glucose. The latter correlated with a fall in the nonesterified fatty acid (NEFA) level and a rise in net hepatic lacate output (27, 28). When the NEFA level was prevented from falling by infusion of a lipid emulsion, the decline in HGP was blunted and the increase in net hepatic lactate output was prevented (28). Rebrin and co-workers (23, 24) also found that the NEFA level plays a role in mediating the effects of peripheral insulin on HGP. Finally, the remainder of the effects of the selective increase in peripheral insulin were ascribed to a decrease in glycogenolysis due to the slight rise in the hepatic sinusoidal insulin level that resulted from the rise in insulin in the hepatic artery. It has been clearly demonstrated that although both peripheral insulin and portal vein insulin inhibit HGP, they appear to act through distinct mechanisms.

Because the liver exhibits zonal heterogeneity with regard to metabolism (12) and microcirculation (3, 25), insulin delivered to the liver directly by either the portal vein or hepatic artery may not have the same effect on HGP. The earlier studies (1, 7, 8, 21, 23, 24) attributed the indirect effect of insulin on HGP solely to peripheral actions mediated by suppression of NEFA; however, the authors did not account for changes in hepatic sinusoidal insulin brought about by the hepatic artery. We have hypothesized in our earlier studies (27, 28) that part of the suppression in HGP by peripheral insulin may be due to a rise in hepatic sinusoidal insulin brought about by an increase in hepatic artery insulin. Therefore, we wished to determine whether a selective increase in hepatic sinusoidal insulin brought about directly by insulin infusion into the hepatic artery would produce an effect different from that achieved by a rise brought about by portal vein insulin infusion. Our aim was to determine whether the effects of insulin delivered directly to the liver sinusoids depend on the route of delivery (portal vein vs. hepatic artery).


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animal Care and Surgical Procedures

Experiments were conducted on 10 conscious mongrel dogs (18-26 kg) of either sex that had been fed a meat and chow diet [34% protein, 46% carbohydrate, 14.5% fat, and 5.5% fiber based on dry weight; Kal Kan beef dinner (Vernon, CA) and Purina Lab Canine Diet no. 5006] once daily. The surgical facility met the standards published by the American Association for the Accreditation of Laboratory Animal Care, and the protocols were approved by the Vanderbilt University Medical Center Animal Care Committee.

Each dog underwent a laporatomy performed under general anesthesia (15 mg/kg pentothal sodium, presurgery, and 1% isoflurane inhalation anesthetic during surgery) 2 wk before the experiment. By use of previously described sterile techniques (2), Silastic catheters (0.03 in. ID; Dow Corning, Midland, MI) were placed into a splenic and a jejunal vein for intraportal infusions as required. In the hepatic artery group (HART), the gastric artery was ligated ~4 cm from the juncture with the hepatic artery, and a Silastic infusion catheter (0.03 in. ID; Dow Corning) was advanced ~3 cm into the hepatic artery. Catheters (0.04 in. ID) for blood sampling were placed in the left common hepatic vein, the hepatic portal vein, and the femoral artery as described previously (5). All catheters were filled with saline containing heparin (200 U/ml; Abbott Laboratories, North Chicago, IL), and their free ends were knotted before closure of the skin. Doppler flow probes (Instrument Development Laboratories, Baylor College of Medicine, Houston, TX; Transonic Flowprobe, Ithaca, NY) were placed around the hepatic artery and portal vein to determine hepatic blood flow, as previously described (20). The Doppler leads, along with the catheters, were placed in a subcutaneous pocket before closure of the abdominal skin. The positions of the catheter tips were confirmed on autopsy.

Only dogs that had a leukocyte count <18,000/mm3, a hematocrit >35%, normal stools, and had consumed their daily food ration were used for a study. On the day of the experiment, after an 18-h fast, the catheters and flow probe leads were exteriorized under local anesthesia (2% lidocaine; Astra Pharmaceutical, Worcester, MA). The contents of each catheter were aspirated, and the catheters were flushed with saline. The intraportal catheters (splenic and jejunal) were used for the infusion of insulin and glucagon (Eli Lilly, Indianapolis, IN). Angiocaths (Deseret Medical, Becton-Dickinson, Sandy, UT) were inserted percutanously into the left cephalic vein for [3-3H]glucose (New England Nuclear, Boston, MA) plus indocyanine green (Becton-Dickinson, Cockeysville, MD) infusion, and into a saphenous vein for somatostatin (Bachem, Torrance, CA) plus insulin infusion. An angiocath was inserted into the right cephalic vein for peripheral glucose infusion. Each animal was allowed to rest quietly in a Pavlov harness for 30 min before the experiment was begun.

Experimental Procedure

Each experiment consisted of a tracer and dye equilibration period (-140 to -40 min), a basal period (-40 to 0 min), and an experimental period (0 to 180 min). At -140 min, a priming dose of [3-3H]glucose (25 µCi) was given and a continual infusion of [3-3H]glucose (0.21 µCi/min) was begun to allow assessment of HGP. Constant infusions of indocyanine green (0.07 mg/min) and somatostatin (0.8 µg · kg-1 · min-1) were started simultaneously (t = -140 min) via a leg vein to measure hepatic blood flow (HF) and to inhibit the endogenous secretion of insulin and glucagon, respectively. A constant intraportal infusion of glucagon (0.5 ng · kg-1 · min-1) was given to replace endogenous glucagon secretion. A constant infusion of insulin (0.48 pmol · kg-1 · min-1) was given via a peripheral vein, and a variable insulin infusion was given via the portal infusion catheters. The rate of portal insulin infusion was adjusted to maintain preexisting plasma glucose levels. Once the plasma glucose level had been stabilized at a euglycemic value for 30 min, the basal sampling period was begun. At the end of the basal sampling period, the peripheral insulin infusion was stopped, and insulin was infused into either the hepatic artery or portal vein in the manner described in the following protocols.

Protocol I. Hepatic artery insulin group (HART, n = 5). During the basal period, the portal insulin infusion rate averaged 1.6 pmol · kg-1 · min-1. On completion of the basal period, the peripheral insulin infusion (0.48 pmol · kg-1 · min-1) was turned off and insulin was infused into the hepatic artery at 1.2 pmol · kg-1 · min-1. This caused a selective increase in hepatic artery and hepatic sinusoidal insulin, with no change in portal or nonhepatic arterial insulin concentrations. Euglycemia was maintained during the experimental period by use of a variable glucose infusion given through a peripheral vein.

Protocol II. Portal insulin group (PORT, n = 5). The portal insulin infusion during the basal period averaged 0.9 pmol · kg-1 · min-1. On completion of the basal period, the peripheral insulin infusion (0.48 pmol · kg-1 · min-1) was turned off, and the rate of portal insulin infusion was increased by 1.2 pmol · kg-1 · min-1. This resulted in a selective increase in the hepatic sinusoidal and portal vein insulin concentrations without a change in the arterial insulin level. Euglycemia was again maintained during the experimental period by use of a variable glucose infusion given through a peripheral vein. Data from this group have been presented elsewhere (27).

Arterial blood samples were taken every 10 min during the basal period and every 15 min during the experimental period. Portal and hepatic vein blood samples were drawn every 20 min during the basal period, 15 and 30 min after the initiation of the experimental period, and every 30 min thereafter. The arterial plasma glucose level was monitored every 5 min during the experimental period to assess glycemia. The total volume of blood withdrawn did not exceed 20% of the animal's blood volume, and two volumes of saline were given for each volume of blood withdrawn. No significant decreases in hematocrit occurred with this procedure (<5%). The arterial and portal blood samples were collected simultaneously ~30 s before collection of the hepatic venous sample in an attempt to compensate for transit time of glucose through the liver (11) and thus allow the most accurate estimates of net hepatic balance to be obtained.

Analytic Procedures

The handling and immediate processing of blood samples have been previously described (27). Blood samples were processed for the later determination of acetoacetate, beta -hydroxybutyrate, glycerol, and lactate, and the gluconeogenic amino acids alanine, glutamine, glutamate, glycine, serine, and threonine. Plasma samples were obtained for immediate analysis of glucose by the glucose oxidase method in a Beckman glucose analyzer (Beckman Instruments, Fullerton, CA). Plasma samples were also processed for the later determination of [3H]glucose, immunoreactive glucagon and insulin, NEFA, and cortisol. All samples were kept in an ice bath during processing and then were stored at -70°C until they were assayed.

For the determination of plasma [3H]glucose, 1-ml plasma samples were deproteinized with 5 ml of 0.067 N barium hydroxide and 5 ml of 0.067 N zinc sulfate (Sigma Chemical). A 5-ml aliquot of the supernatant was evaporated, the residue was reconstituted in 1 ml of water, and 10 ml of liquid scintillation fluid [EcoLite(+), ICN Biomedicals, Irvine, CA] was added. Tritium in the sample was determined by liquid scintillation counting with a Beckman LS 5000TD. Whole blood metabolite concentrations were determined according to the methods developed by Lloyd et al. (15) for the Technicon Autoanalyzer (Tarrytown, NY) and Monarch 2000 centrifugal analyzer (Lexington, MA). Whole blood glutamine and glutamate concentrations were determined by the methods described in Wasserman et al. (31). Plasma NEFA were determined spectrophotometrically (Wako Chemicals, Richmond, VA). Immunoreactive insulin was measured using a double-antibody procedure (19). Immunoreactive glucagon was measured using a modification of the double-antibody insulin radioimmunnoassay method (19). Insulin and glucagon antibodies and 125I tracers were obtained from Linco Research (St. Louis, MO). Blood gluconeogenic amino acids were determined by HPLC separation (30). Indocyanine green was measured spectrophotometrically at 810 nm to estimate HF according to the method of Leevy et al. (13). Blood acetoacetate concentrations were determined spectrophotometrically (22). Enzymes and coenzymes for metabolic analyses were obtained from Boehringer-Mannheim Biochemicals (Mannheim, Germany) and Sigma Chemical.

Tracer Calculations

Tracer-determined glucose production (TDGP) and glucose utilization were measured using a primed, continual infusion of [3-3H]glucose. Data calculation was carried out using a two-compartment model described by Mari (16) with canine parameters reported by Dobbins et al. (6). Endogenous glucose production was calculated as the difference between TDGP and the exogenous glucose infusion rate.

Arteriovenous Difference Calculations

NHGO and the net hepatic balance of gluconeogenic substrates were calculated using the formula [H - (0.28A + 0.72P)] × HF, where H, A, and P are the substrate concentrations in the hepatic vein, femoral artery, and portal vein blood or plasma, respectively; HF is total hepatic flow of blood or plasma as estimated from indocyanine green; and 0.28 and 0.72 represent the approximate contributions of the hepatic artery and the portal vein, respectively, to total HF during somatostatin infusion (20). With this calculation, a positive value represents net production by the liver, and a negative value represents net hepatic uptake. Plasma glucose values were multiplied by 0.73 to convert them to blood glucose values for the net hepatic balance calculation (18). The data displayed in RESULTS were calculated using indocyanine green-determined blood flows. Doppler flow probes were functional in only 6 of 10 studies, precluding their use in the entire data base. Nevertheless, the Doppler flow data obtained confirmed that the ratio of arterial to portal blood flow used was correct. Also, as demonstrated in previous studies (20), the Doppler and indocyanine green-determined blood flows were not significantly different, and therefore the method of flow determination used had little effect on the net hepatic balance calculation. Hepatic sinusoidal hormone concentrations were calculated using the formula (0.28A + 0.72P), where A and P represent the hormone levels in arterial and portal plasma, respectively. For the calculated increase in hepatic artery insulin concentration in the HART group, the hepatic artery insulin infusion rate was divided by the contribution of the hepatic artery to total hepatic plasma flow. Maximal gluconeogenesis from circulating precursors was calculated by summing the net hepatic uptake rates of all of the gluconeogenic precursors and dividing by two to account for the incorporation of the three-carbon precursor into the six-carbon glucose molecule. This method has been shown to provide an estimate of gluconeogenesis (10) very similar to that obtained by using [U-14C]alanine and by measuring hepatic [14C]phosphoenolpyruvate and UDPG specific activities (9). Lactate and the individual gluconeogenic amino acids were only considered for inclusion in the gluconeogenic precursor uptake calculation (or total amino acid uptake) if net hepatic uptake was evident. The mean lactate and gluconeogenic amino acid data, on the other hand, represent the entire database regardless of the sign of net balance. For calculation of hepatic glucose uptake by the liver, net hepatic [3-3H]glucose uptake (dpm · kg-1 · min-1) was divided by the arterial glucose specific activity (dpm/µmol). The calculation of hepatic glucose uptake assumes that uptake of glucose occurs before production and that the resulting dilution of glucose specific activity across the liver is minimal. Because there is no difference between arterial and portal vein glucose specific activity and a minimal drop (6%) across the liver, the impact of this assumption is negligible.

Statistics

The level of significance was P < 0.05 (2-sided test). The data were analyzed for differences on the basis of group-by-group comparisons and for changes from intragroup baseline values. Statistical comparisons between groups were calculated using two-way analysis of variance, and intragroup difference from baseline was calculated using one-way analysis of variance (Statview, Calabasas, CA). The Scheffé procedure and Fisher's protected least significant difference test for multiple comparisons were used post hoc when significant F ratios were obtained.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of a Selective Increase in Hepatic Artery Insulin

When insulin was infused into the hepatic artery, neither the systemic (nonhepatic arterial) nor portal insulin levels changed (Fig. 1). The calculated hepatic artery insulin concentration rose from 50 ± 8 (basal) to 308 ± 38 (15 min) and 289 ± 19 pmol/l (last 30 min). As a result, the sinusoidal insulin level rose from 99 ± 20 (basal) to 173 ± 27 (15 min) and 165 ± 21 pmol/l (last 30 min). This change in hepatic sinusoidal insulin was reflected in the change seen in hepatic vein insulin (55 ± 16, basal to 113 ± 31 at 15 min, and 106 ± 29 pmol/l for last 30 min). The hepatic sinusoidal glucagon level fell minimally (~7%), whereas the arterial plasma glucose concentration (Table 1) and plasma glucose specific activity (Table 2) remained unaltered. Net hepatic glucose output fell from 9.6 ± 2.1 to 4.6 ± 1.0 by 15 min and to 3.5 ± 0.8 µmol · kg-1 · min-1 by the last 30 min of the study (Fig. 2, P < 0.05). A fall in glucose production was also evident from the TDGP data (Table 2, P < 0.05). As expected, the rise in hepatic sinusoidal insulin did not alter whole body glucose utilization (Table 2, P < 0.05).


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  		Fig. 1.   Arterial and hepatic sinusoidal insulin levels in 18-h-fasted dogs during the basal period (-40 to 0 min) and during selective increases in hepatic artery (HART) or portal vein (PORT) insulin created during the experimental period as described in the text. Data are expressed as group means ± SE.

                              
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  		Table 1.   Plasma glucose and glucagon levels and hepatic blood flow in 18-h-fasted dogs studied during a basal period and during selective increases in hepatic artery or portal vein insulin level


                              
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  		Table 2.   Tracer-determined glucose production and utilization, arterial plasma glucose specific activity, and glucose infusion for 18-h-fasted dogs studied during a basal period and during selective increases in HART or PORT insulin level



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  		Fig. 2.   Change in net hepatic glucose output (NHGO) in 18-h-fasted dogs during the basal period (-40 to 0 min) and during selective increases in HART or PORT insulin created during the experimental period as described in the text. Horizontal dashed line, a control group that had no change in insulin made during the experimental period. Data are expressed as group means ± SE.

Arterial blood lactate levels (Table 2) did not change. Net hepatic lactate production (Fig. 3) fell significantly (P < 0.05) from 1.0 ± 3.2 µmol · kg-1 · min-1 to net hepatic uptake of 3.4 ± 1.7 µmol · kg-1 · min-1 by the end of the study. The arterial blood glycerol level did not change (Table 2), whereas arterial blood gluconeogenic amino acid levels dropped ~20% (see Table 4). Neither net hepatic uptake of glycerol (1.4 ± 0.3, basal vs. 1.2 ± 0.4 µmol · kg-1 · min-1, last 30 min, Table 3, P < 0.05) nor the net hepatic uptake of the gluconeogenic amino acids (5.5 ± 1.1, basal, to 5.2 ± 0.6 µmol · kg-1 · min-1, last 30 min, Table 4) was decreased. Total net hepatic gluconeogenic precursor uptake (including changes in net hepatic lactate uptake when such occurred) did not increase appreciably (9.4 ± 2.0, basal to 10.5 ± 2.1 µmol · kg-1 · min-1, last 30 min). This rise in gluconeogenic precursor uptake, if real, would explain an increase in glucose release of no more than ~0.6 µmol · kg-1 · min-1. Neither the arterial plasma NEFA level (Table 2) nor hepatic NEFA uptake (Fig. 4) changed. As a result, ketone metabolism was not altered (Tables 3 and 5).


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  		Fig. 3.   Changes in net hepatic lactate output in 18-h-fasted dogs during the basal period (-40 to 0 min) and during selective increases in HART or PORT insulin created during the experimental period as described in the text. Data are expressed as group means ± SE.

                              
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  		Table 3.   Whole blood lactate, glycerol, beta -OHB, AcAc, and plasma NEFA concentrations in 18-h-fasted dogs studied during a basal period and during selective increases in HART or PORT insulin levels


                              
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  		Table 4.   Arterial blood concentrations and net hepatic uptake rates of gluconeogenic amino acids in 18-h-fasted dogs studied during a basal period and during selective increases in the HART or PORT insulin levels



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  		Fig. 4.   Net hepatic nonesterified fatty acid (NEFA) uptake in 18-h-fasted dogs during the basal period (-40 to 0 min) and during selective increases in HART and PORT insulin created during the experimental period as described in the text. Data are expressed as group means ± SE.

                              
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  		Table 5.   Net hepatic uptake of glycerol and net hepatic production of beta -OHB and AcAc for 18-h-fasted dogs studied during a basal period and during selective increases in HART or PORT insulin levels

Effects of a Selective Increase in Portal Insulin

In the PORT group, the portal insulin level increased from 108 ± 42 (basal) to 216 ± 48 at 15 min and 192 ± 42 pmol/l during the last 30 min of the experiment, whereas the arterial insulin level (regardless of site) did not change over the course of the experiment (Fig. 1). The calculated hepatic sinusoidal insulin level rose from 94 ± 30 (basal) to 170 ± 37 (15 min) and 156 ± 33 pmol/l (last 30 min). The liver sinusoidal glucagon level fell minimally (7%), and the arterial plasma glucose level and the plasma glucose specific activity remained unaltered (Table 1). Net hepatic glucose output (Fig. 2) dropped quickly from 10.0 ± 0.8 (basal) to 7.7 ± 1.6 µmol · kg-1 · min-1 by 15 min (P < 0.05) and remained suppressed thereafter (3.1 ± 1.2 µmol · kg-1 · min-1, last 30 min). A fall in glucose production was also evident from the tracer data (Table 2). Again the selective rise in liver sinusoidal insulin did not change whole body glucose utilization (Table 2).

Arterial blood lactate levels (Table 3) remained unaltered during the study; however, net hepatic lactate production dropped from 4.0 ± 2.4 (basal) to 0.1 ± 1.0 µmol · kg-1 · min-1 (last 30 min, P < 0.05, Fig. 3). Arterial blood gluconeogenic amino acid (Table 4) and glycerol levels remained unaltered. Neither the net hepatic uptake of gluconeogenic amino acids (5.2 ± 0.7, basal, to 5.8 ± 0.3 µmol · kg-1 · min-1, last 30 min) nor that of glycerol (1.0 ± 0.2 to 1.1 ± 0.1 µmol · kg-1 · min-1, Table 5) fell. Total net hepatic gluconeogenic precursor uptake (including changes in net hepatic lactate uptake when such occurred) increased minimally from 6.5 ± 0.7 (basal) to 7.7 ± 0.7 µmol · kg-1 · min-1 (last 30 min, P < 0.05). This rise in the gluconeogenic precursor uptake rate could explain an increase in glucose release of no more than ~0.6 µmol · kg-1 · min-1. Neither arterial plasma NEFA levels (Table 3) nor net hepatic NEFA uptake (Fig. 4) changed. As a result, ketone metabolism (Tables 3 and 5) was not altered.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

These studies clearly demonstrate the potent inhibitory effect of hepatic sinusoidal insulin on HGP. The increase in the liver sinusoidal insulin concentration of ~66 pmol/l due to the increase in hepatic artery insulin suppressed NHGO from 9.6 ± 2.1 to 4.6 ± 1.0 µmol · kg-1 · min-1 within 15 min and eventually reduced it to 3.5 ± 0.8 µmol · kg-1 · min-1 (Delta 6.1 µmol · kg-1 · min-1). This suppression occurred in the absence of any change in the peripheral or portal vein insulin level, the liver sinusoidal glucagon level, the plasma glucose level, the plasma NEFA level, or net hepatic NEFA uptake.

The increase in hepatic sinusoidal insulin, resulting from the portal insulin infusion, was ~62 pmol/l. This suppressed NHGO from 10.0 ± 0.9 to 7.7 ± 1.6 µmol · kg-1 · min-1 within 15 min and to 3.1 ± 1.1 µmol · kg-1 · min-1 by the last 30 min of the study (Delta 6.9 µmol · kg-1 · min-1). This suppression also occurred in the absence of any change in the peripheral or hepatic artery insulin level, the liver sinusoidal glucagon level, the plasma glucose level, the plasma NEFA level, or net hepatic NEFA uptake. When the drop in NHGO in previous control studies in which the plasma insulin was not increased (Delta 2.1 µmol · kg-1 · min-1; see dotted line in Fig. 2) is taken into account (27), it is evident that ~70% of the decrease in NHGO from baseline observed in both groups was caused by the selective increase in hepatic sinusoidal insulin.

The rapidity of the suppression in NHGO was also not significantly different between the two groups. Although the decline at 15 min appeared greater in the HART group, the difference was not significant. Furthermore, by 30 min NHGO was equally and markedly decreased in both groups. Likewise, in both groups there was a slight increase in net hepatic gluconeogenic precursor uptake, which could at best account for an increase in NHGO of 0.6 µmol · kg-1 · min-1. Because hepatic gluconeogenesis may have risen slightly in both groups, it is clear that an inhibition gluconeogenesis cannot explain the fall in glucose production caused by the increase in hepatic sinusoidal insulin. It is therefore evident that a decrease in glycogenolysis must provide the explanation for the fall. This conclusion is consistent with the rapidity of the liver's response to a selective rise in hepatic sinusoidal insulin and our previous findings.

Although the tracer data confirmed the suppression of HGP by the increase in hepatic sinusoidal insulin, regardless of the route of access to the liver, it indicated a slightly smaller decrease in the HART group. This in part reflects the fact that hepatic glucose uptake (HGU, measured by [3H]glucose balance) increased slightly more in that group (Delta 1.3 µmol · kg-1 · min-1, PORT, and Delta 1.6 µmol · kg-1 · min-1, HART). The change in total hepatic glucose release (HGR = NHGO + HGU) should have been 8.2 µmol · kg-1 · min-1 in the PORT group and 7.7 µmol · kg-1 · min-1 in the HART group. In fact, the decreases in TDGP were 7.1 µmol · kg-1 · min-1 in the PORT group and 4.0 µmol · kg-1 · min-1 in the HART group. Thus, in the PORT group the two estimates were very similar, but in the HART group they were less so. The difference between the TDGP and HGR in the latter group is unlikely to be accounted for by changes in renal glucose production (4, 17, 29), which would not be evident in HGR because the hormonal mileu at the kidneys did not change, so that renal glucose production probably did not change. In all likelihood, the small difference in the two estimates was due to noise in the data obtained with the two methods. If this is the case, then one could argue that the true fall in HGP might best be represented by the average of the two methods and thus might have been slightly less in the HART group. If so, this would fit with the observation that the entry of the hepatic artery occurs somewhere within the first third of the liver sinusoids (32). Thus a slightly smaller response might be predicted when insulin is delivered via the hepatic artery. Consistent with this is our observation that the glucose infusion in the HART group was slightly less than in the PORT group (Delta 2.3 µmol · kg-1 · min-1 over the last 30 min). In addition, because the sinusoidal glucagon level appears to be slightly higher in the PORT group, this could have resulted in a somewhat greater glycogenolytic response and thus a slightly greater inhibition. However, the slight elevation in glucagon levels may be due to differences in the level of cross-reacting material and not to differences in true glucagon. Nevertheless, the present data indicate that a rise in liver sinusoidal insulin decreases glucose release by the liver, regardless of the route by which it enters the hepatic sinusoids.

The response of the liver to the rise in hepatic sinusoidal insulin that occurred in the present study is consistent with our finding when the hepatic sinusoidal insulin level was selectively decreased 120 pmol/l [i.e., no accompanying change in arterial insulin (26)]. When selective hepatic sinusoidal insulin deficiency was brought about, NHGO increased 22 µmol · kg-1 · min-1 within 15 min. It then remained elevated relative to the rate evident in an equivalently hyperglycemic control group by ~20 µmol · kg-1 · min-1 for the remainder of the experiment. Clearly, the rapid and sensitive changes in NHGO that occur in response to increases or decreases in hepatic sinusoidal insulin demonstrate that the direct action of insulin on the liver plays a very important role in the minute-to-minute regulation of hepatic glucose output. Furthermore, the route (hepatic artery or portal vein) by which insulinization of the liver occurs is of little or no consequence to the hepatic action of the hormone.

In summary, the present results indicate that the effects of an increase in hepatic sinusoidal insulin brought about by hepatic artery or portal vein insulin infusion were rapid in onset and very similar. The rapid suppression of glucose production observed after an increase in hepatic artery or portal vein insulin reflects the change in liver glycogenolysis that occurs in response to the alteration in the insulin level in the hepatic sinusoids.


    ACKNOWLEDGEMENTS

We thank Jon Hastings, Pam Venson, Wanda Snead, Paul Flakoll, and Pat Donahue for their excellent technical assistance.


    FOOTNOTES

This research was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grants 2RO1-DK-18243 and 5P60-DK-2059.

Present address for D. K. Sindelar: Puget Sound VA Health Care System, Metabolism (151), 1660 South Columbian Way, Seattle, WA 98108-1597.

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: A. D. Cherrington, Dept. of Molecular Physiology and Biophysics, 702 Light Hall, Vanderbilt Univ. School of Medicine, 21st Ave. South and Garland, Nashville, TN 37232-0615 (E-mail: alan.cherrington{at}mcmail.vanderbilt.edu).

Received 5 June 1998; accepted in final form 22 December 1998.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Endocrinol Metab 276(4):E806-E813
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society



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