AJP - Endo Add DOIs to your references at manuscript stage!
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Endocrinol Metab 294: E768-E777, 2008. First published January 22, 2008; doi:10.1152/ajpendo.00184.2007
0193-1849/08 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
294/4/E768    most recent
00184.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Moore, M. C.
Right arrow Articles by Cherrington, A. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Moore, M. C.
Right arrow Articles by Cherrington, A. D.

Hepatic portal venous delivery of a nitric oxide synthase inhibitor enhances net hepatic glucose uptake

Mary Courtney Moore,1,2 Catherine A. DiCostanzo,1 Marta S. Smith,1 Ben Farmer,1 Tiffany D. Rodewald,1 Doss W. Neal,1 Phillip E. Williams,2,3 and Alan D. Cherrington1,2

1Department of Molecular Physiology and Biophysics, 2Diabetes Research and Training Center, and 3Department of Surgery, Vanderbilt University School of Medicine, Nashville, Tennessee

Submitted 22 March 2007 ; accepted in final form 20 January 2008


   ABSTRACT
TOP
 ABSTRACT
RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Hepatic portal venous infusion of nitric oxide synthase (NOS) inhibitors causes muscle insulin resistance, but the effects on hepatic glucose disposition are unknown. Conscious dogs underwent a hyperinsulinemic (4-fold basal) hyperglycemic (hepatic glucose load 2-fold basal) clamp, with assessment of liver metabolism by arteriovenous difference methods. After 90 min (P1), dogs were divided into two groups: control (receiving intraportal saline infusion; n = 8) and LN [receiving NG-nitro-L-arginine methyl ester (L-NAME), a nonspecific NOS inhibitor; n = 11] intraportally at 0.3 mg·kg–1·min–1 for 90 min (P2). During the final 60 min of study (P3), L-NAME was discontinued, and five LN dogs received the NO donor SIN-1 intraportally at 6 µg·kg–1·min–1 while six received saline (LN/SIN-1 and LN/SAL, respectively). Net hepatic fractional glucose extraction (NHFE) in control dogs was 0.034 ± 0.016, 0.039 ± 0.015, and 0.056 ± 0.019 during P1, P2, and P3, respectively. NHFE in LN was 0.045 ± 0.009 and 0.111 ± 0.007 during P1 and P2, respectively (P < 0.05 vs. control during P2), and 0.087 ± 0.009 and 0.122 ± 0.016 (P < 0.05) during P3 in LN/SIN-1 and LN/SAL, respectively. During P2, arterial glucose was 204 ± 5 vs. 138 ± 11 mg/dl (P < 0.05) in LN vs. control to compensate for L-NAME's effect on blood flow. Therefore, another group (LNlow; n = 4) was studied in the same manner as LN/SAL, except that arterial glucose was clamped at the same concentrations as in control. NHFE in LNlow was 0.052 ± 0.008, 0.093 ± 0.023, and 0.122 ± 0.021 during P1, P2, and P3, respectively (P < 0.05 vs. control during P2 and P3), with no significant difference in glucose infusion rates. Thus, NOS inhibition enhanced NHFE, an effect partially reversed by SIN-1.

NG-nitro-L-arginine methyl ester; 3-morpholynosydnonimine; dog

MODULATION OF LIVER NITRIC OXIDE (NO) levels alters whole body insulin sensitivity in the rat (16, 17, 22, 32). Intraportal administration of the nonspecific NO synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 1.0 mg/kg) reduced whole body insulin sensitivity in rats by 55%, whereas the same dose of L-NAME given via a peripheral vein did not cause a significant decrease in insulin sensitivity (32). The NOS inhibitor N-monomethyl-L-arginine (0.73 mg/kg) also reduced insulin sensitivity when administered intraportally, an effect reversed by intraportal injection of the NO donor 3-morpholynosydnonimine (SIN-1) (32).

The reduction in insulin sensitivity due to hepatic NOS blockade resulted from inhibition of insulin action in skeletal muscle, with no effect on glucose uptake by the liver, gut, or adipose tissue (21). However, the aforementioned studies (16, 17, 32) were carried out under hyperinsulinemic euglycemic conditions, when the skeletal muscle is responsible for much of the glucose disposal and the role of the liver is minimized. Under hyperinsulinemic hyperglycemic conditions, the liver is responsible for a substantial portion of total glucose uptake [~25–33%, depending on the conditions of study (7)]. This raises the question of what effect NO has on net hepatic glucose uptake (NHGU).

The regulation of NHGU is particularly relevant because hepatic extraction of glucose in the postprandial period is impaired in individuals with type 2 diabetes (3), contributing to postprandial hyperglycemia. Furthermore, postprandial hyperglycemia is strongly associated with morbidity and mortality in people with diabetes (5). There is evidence to link hepatic NO levels and the magnitude of NHGU. Infusion of SIN-1 into the hepatic portal circulation of conscious dogs significantly blunted NHGU in the presence of hyperinsulinemia, hyperglycemia, and intraportal glucose delivery (2), conditions that mimic the postprandial state. Therefore, the current study was undertaken to quantify NHGU under hyperglycemic and hyperinsulinemic conditions in the presence of a reduction of endogenous NO brought about by intraportal infusion of L-NAME.


   RESEARCH DESIGN AND METHODS
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals and Surgical Procedures

Studies were carried out on conscious 42-h-fasted adult mongrel dogs of either sex weighing 19–25 kg. Diet and housing were as described previously (28), and the protocol was approved by the Vanderbilt University Institutional Animal Care and Use Committee. Approximately 16 days before study, each dog underwent a laparotomy for placement of ultrasonic flow probes (Transonic Systems, Ithaca, NY) around the portal vein and the hepatic artery as well as insertion of silicone rubber catheters for sampling in a hepatic vein, the portal vein, and a femoral artery and for infusion into a splenic and a jejunal vein, as described in detail elsewhere (28). Criteria for health of the animals prior to study were as described previously (28). On the morning of the study, catheters and flow probe leads were exteriorized from their subcutaneous pockets. Intravenous access was established in three peripheral veins.

Experimental Design

Each experiment consisted of a 90-min equilibration period (–120 to –30 min), a 30-min basal period (–30 to 0 min), and a 240-min experimental period (0–240 min) that was divided into three subperiods (P1, 0–90 min; P2, 90–180 min; and P3, 180–240 min). At –120 min, a continuous infusion of indocyanine green dye (0.08 mg/min; Sigma, St. Louis, MO) was begun in all dogs. At 0 min, a constant, peripheral infusion of somatostatin (0.8 µg·kg–1·min–1; Bachem, Torrance, CA) was begun to suppress endogenous insulin and glucagon secretion. Insulin was infused intraportally via the splenic and jejunal catheters at fourfold basal (1.2 mU·kg–1·min–1; Eli Lilly, Indianapolis, IN), and glucagon was replaced intraportally in basal amounts (0.57 ng·kg–1·min–1, GlucaGen; Bedford Laboratories, Bedford, OH). In addition, a primed, continuous, variable-rate 50% dextrose infusion was begun through a peripheral vein to create hyperglycemia. During P1, all dogs received intraportal saline infusion. In the control group (dogs receiving intraportal saline infusion; n = 8), the intraportal saline infusion continued throughout P2 and P3. In the LN group (dogs receiving L-NAME; n = 11), the dogs received intraportal L-NAME (Cayman Chemical, Ann Arbor, MI) at 0.3 mg·kg–1·min–1 during P2. This infusion rate, which is similar to that previously used in studies in humans (15), was derived from a canine dose-response study (data not shown). L-NAME infusion stopped at the end of P2, and the LN dogs were divided into two subgroups at 180 min. During P3, six dogs received intraportal saline after the cessation of L-NAME infusion (LN/SAL), and five received intraportal SIN-1 infusion (LN/SIN-1; Cayman Chemical) at 6 µg·kg–1·min–1. The large changes in hepatic blood flow brought about by L-NAME infusion raised questions about what effect this might have had on hepatic glucose balance. It was also unclear whether the hepatic glucose load or the concentration of glucose at the liver was most important in stimulating glucose uptake (35). Therefore, to determine whether the results could be due to the effects of the L-NAME infusion rate used or the elevation of the arterial glucose concentration, an additional group [dogs receiving a low rate of L-NAME infusion in P2 (LNlow); n = 4] was treated in the same manner as the LN group, except that the L-NAME infusion rate was 0.075 mg·kg–1·min–1 and the arterial glucose concentration was clamped at the same level as in the control group.

Femoral artery, portal vein, and hepatic vein blood samples were taken every 15–30 min throughout the study as described previously (28). Arterial samples were also taken every 5 min throughout the experimental period to monitor the plasma glucose level and allow appropriate adjustment of the glucose infusion rate. Blood pressure and heart rate were obtained with an arterial transducer (DigiMed; Micro-Med, Louisville, KY).

Processing and Analysis of Samples

Hematocrit, blood lactate, and glycerol and plasma glucose, insulin, glucagon, and cortisol were measured as described previously (26). In the LN/SAL, LNlow, and control groups, hepatic glycogen concentrations were determined by the method of Keppler and Decker (20).

Calculations and Data Analysis

Hepatic blood flow was measured using ultrasonic probes and indocyanine green extraction. The two methods yielded similar results, but the data reported here utilize the ultrasonic-determined flows, because their use did not require an assumption regarding the relative contribution of arterial and portal flow to total hepatic blood flow.

The rate of glucose delivery to the liver or hepatic glucose load, net hepatic substrate balance, net hepatic fractional substrate extraction, net hepatic carbon retention, and hepatic sinusoidal hormone concentrations were calculated as described previously (26). Nonhepatic glucose uptake equaled the glucose infusion rate minus NHGU; the rate was corrected for changes in the size of the glucose pool, using a pool fraction of 0.65 (11) and assuming that the volume of distribution for glucose equaled the volume of the extracellular fluid, or ~22% of the dog's weight (37). Hepatic glucose clearance was calculated as NHGU divided by the weighted (for the proportion of hepatic blood flow contributed by the hepatic artery vs. the portal vein) inflowing arterial and portal venous glucose concentration. Net hepatic carbon retention, an index of hepatic glycogen storage, was calculated as the difference between NHGU and lactate output, assuming that net hepatic uptake of nonlactate gluconeogenic precursors was offset by the rate of hepatic glucose oxidation. We (26) have previously shown this calculated variable to be closely related to the actual rate of glycogen synthesis. For all glucose balance calculations, glucose concentrations were converted from plasma to blood values by using correction factors (ratio of the blood to the plasma concentration) previously established in our laboratory (19, 28). Net hepatic glycogen synthesized over the course of the study was calculated as the glycogen concentration in the terminal liver biopsies minus the concentrations in 42-h-fasted dogs that did not undergo study.

Statistical Analysis

All data are presented as means ± SE. Time course data were analyzed with repeated-measures ANOVA, and Tukey's test was used for post hoc comparisons (SigmaStat, Point Richmond, CA). Statistical significance was accepted at P < 0.05. Where mean data for an experimental period are presented, they are the mean of the sampling points in the last 30 min of that period, when steady-state conditions prevailed.


   RESULTS
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
High-Dose L-NAME With or Without SIN-1 Infusion

Hepatic blood flow and cardiovascular parameters. Hepatic blood flow did not differ between the control and LN groups during the basal period or P1 (Fig. 1). In both groups the portal vein blood flow decreased modestly with the onset of somatostatin infusion, whereas there was a small increase in hepatic artery flow, such that total hepatic flow decreased by ~15%. L-NAME infusion significantly reduced both hepatic artery and portal vein flow such that total hepatic blood flow was only 58% of that in the control group during the last 30 min of P2 (P < 0.05 between groups). In LN/SAL, portal vein flow increased significantly after cessation of L-NAME infusion, but hepatic artery flow did not change. Consequently, total hepatic flow rose but remained significantly suppressed. Infusion of SIN-1 during P3 had no effect on portal vein flow compared with the rate in LN/SAL, but it elevated hepatic artery flow (P < 0.05). The changes in hepatic blood flow were similar regardless of the method of measurement (indocyanine green-determined flow not shown).


Figure 1
View larger version (19K):
[in this window]
[in a new window]

  		Fig. 1. Hepatic artery, portal vein, and total hepatic blood flow from the ultrasonic flow probes. The control group (n = 8) received intraportal saline infusion from 0 to 240 min, whereas the combined LN [dogs receiving intraportal NG-nitro-L-arginine methyl ester (L-NAME) infusion] group received intraportal saline from 0 to 90 min [experimental period 1 (P1)], followed by intraportal infusion of L-NAME from 90 to 180 min (P2). After L-NAME infusion ceased, the LN group was divided into 2 subgroups from 180 to 240 min (P3): LN/SAL (receiving intraportal saline infusion; n = 6) and LN/SIN-1 (receiving intraportal infusion of SIN-1; n = 5). *P < 0.05 vs. control.

 
Blood pressure and heart rate were stable during the basal period and P1 and did not differ between groups (Table 1). However, both systolic and diastolic blood pressure rose modestly in response to L-NAME, and the increase continued to be evident even after L-NAME infusion ceased. Infusion of SIN-1 post-L-NAME reduced systolic pressure to the level seen in the control group but failed to normalize diastolic pressure. Concomitant with the hypertensive changes, the heart rate was significantly reduced both during and after L-NAME infusion. SIN-1 partially reversed the bradycardia.


View this table:
[in this window]
[in a new window]

  		Table 1. Cardiovascular parameters

 
Hormone concentrations. The hepatic sinusoidal insulin concentrations rose ~35% (P < 0.05) in the LN group during P2 vs. P1; they increased further in LN/SAL during P3 but declined in LN/SIN-1 (Table 2). Arterial plasma glucagon concentrations remained basal throughout P1–P3 in all animals. Hepatic sinusoidal glucagon concentrations in the LN group were ~10–20% greater than those in the control group during P2, but this did not reach statistical significance; the concentrations in LN/SAL remained elevated ~14% (not significant) above control levels during P3. Arterial plasma cortisol concentrations did not change significantly from basal in either control or LN and differed between groups only during P1, when experimental conditions were identical in the groups. Arterial plasma norepinephrine did not differ significantly between the control and the LN groups during the basal period, P1, or P2 (Table 2). During P3 it was significantly greater than control values in the LN/SIN-1 group but not the LN/SAL group. Arterial plasma epinephrine concentrations were significantly greater in the LN group than in the control group during P2, and they remained higher in LN/SAL and LN/SIN-1 than in the control group during P3.


View this table:
[in this window]
[in a new window]

  		Table 2. Plasma hormone concentrations in the control, LN/SAL, and LN/SIN-1 groups

 
Glucose metabolism. Basal arterial blood glucose concentrations (~76–80 mg/dl) did not differ between the control and LN groups (Fig. 2). Similarly, the mean values during P1 were 139 ± 10 and 134 ± 4 mg/dl in the control and LN dogs, respectively (not significant). By design, the hepatic glucose load was increased ~60% over basal during the experimental period, with no differences between groups at any time (Fig. 2). Since hepatic blood flow did not change significantly during P1, P2, and P3 in the control group, the arterial blood glucose was also maintained at a constant level throughout the experimental period. However, the marked decline in hepatic blood flow during L-NAME infusion necessitated that sufficient glucose be infused to increase the arterial blood glucose concentrations ~50% (Fig. 2) to maintain a stable load of glucose to the liver. During P3, arterial blood glucose was gradually lowered in the LN subgroups to adjust for decrements in hepatic blood flow. During P1 and P3, the glucose infusion rates did not differ significantly between the control and LN dogs, but the glucose infusion rate during L-NAME infusion was ~45% greater than during the comparable time period in the control dogs.


Figure 2
View larger version (18K):
[in this window]
[in a new window]

  		Fig. 2. Arterial blood glucose, glucose infusion rate, and hepatic glucose load in the control and combined LN groups and in the LN/SAL and LN/SIN-1 subgroups. Study conditions were as described in the legend to Fig. 1. *P < 0.05 vs. control.

 
During the basal period, both the control and LN groups exhibited net hepatic glucose output at 1.5 mg·kg–1·min–1 (Fig. 3). In the presence of hyperinsulinemia and hyperglycemia, both groups shifted to NHGU, with the rates during the last 30 min of P1 averaging 1.3 ± 0.6 and 1.6 ± 0.3 mg·kg–1·min–1 in control and LN, respectively (not significant). Infusion of L-NAME markedly enhanced NHGU, with rates during P2 averaging 1.5 ± 0.5 vs. 3.5 ± 0.2 mg·kg–1·min–1 in control and LN, respectively (P < 0.001). NHGU remained significantly enhanced even after L-NAME infusion ceased (i.e., LN/SAL), but infusion of SIN-1 partially reversed the L-NAME-induced enhancement of NHGU (rates in control, LN/SAL, and LN/SIN-1: 2.2 ± 0.7, 4.7 ± 0.8, and 2.8 ± 0.5 mg·kg–1·min–1, respectively; P < 0.05 for LN/SAL vs. control and LN/SIN-1). During P2, hepatic glucose clearance averaged 1.00 ± 0.32 vs. 1.79 ± 0.11 ml·kg–1·min–1 in the control vs. LN groups, respectively (P < 0.01), and during P3 it was 1.48 ± 0.39 vs. 2.33 ± 0.26 ml·kg–1·min–1 in control vs. LN/SAL, respectively (P < 0.05). The infusion of SIN-1 tended to reverse the L-NAME-induced stimulation of hepatic glucose clearance (1.83 ± 0.19 ml·kg–1·min–1 in LN/SIN-1, P = 0.07 vs. LN/SAL and P = 0.10 vs. control during P3). The rates of NHGU and hepatic glucose clearance obtained with the indocyanine green-derived blood flows did not differ significantly from those with the ultrasonic blood flow data. Net hepatic fractional extraction of glucose mirrored the results for NHGU, with L-NAME infusion increasing the value >2.5-fold. The enhancement continued after cessation of L-NAME and was partially reversed by infusion of SIN-1 (Fig. 3). Nonhepatic glucose uptake in LN was significantly greater than in control during the first 30 min of P2, but it declined so that it was no different from control during the last half of P2.


Figure 3
View larger version (22K):
[in this window]
[in a new window]

  		Fig. 3. Net hepatic glucose balance and fractional extraction and nonhepatic glucose uptake in the control and combined LN groups and in the LN/SAL and LN/SIN-1 subgroups. Study conditions were as described in the legend to Fig. 1. *P < 0.05 vs. control. {dagger}P < 0.05 for LN/SAL vs. LN/SIN-1.

 
The mass of hepatic glycogen synthesized over the course of the study tended to be greater in LN/SAL than in control (1.74 ± 0.40 vs. 1.29 ± 0.37 g/100 g liver, P = 0.20).

Lactate, glycerol, and nonesterified fatty acid metabolism: net hepatic carbon retention. Arterial blood lactate concentrations increased with the onset of hyperglycemic hyperinsulinemia in both groups, and the concentrations did not differ between groups at any time (Table 3). Net hepatic lactate balance shifted from uptake to output during P1 in both groups. Net hepatic carbon retention, an index of glycogen storage, was significantly enhanced by L-NAME infusion (Table 3).


View this table:
[in this window]
[in a new window]

  		Table 3. Lactate and glycerol data and net hepatic carbon retention in the control, LN/SAL, and LN/SIN-1 groups

 
Arterial blood glycerol concentrations declined from basal during hyperinsulinemic hyperglycemia in all animals and did not differ between groups at any time. Similarly, net hepatic uptakes of glycerol fell during P1 in all dogs and did not differ among treatments through P2 (Table 3). Infusion of SIN-1 tended to increase arterial blood glycerol (P = 0.14) and significantly stimulated net hepatic glycerol uptake.

Low-Dose L-NAME Infusion

In LNlow, total hepatic blood flow decreased ~25% during P2 compared with P1 (P < 0.05; Table 4). Neither the systolic nor the diastolic blood pressure increased significantly between P2 and P1, but the heart rate fell {approx}20% (P < 0.05; Table 4).


View this table:
[in this window]
[in a new window]

  		Table 4. Blood flow, cardiovascular parameters, and plasma hormone concentrations in LNlow

 
The arterial insulin concentrations did not differ from those in the control group during any period, but the hepatic sinusoidal concentrations were significantly higher in LNlow than in control throughout P1, P2, and P3 (Table 4). The arterial and hepatic sinusoidal glucagon concentrations in LNlow were not significantly different from those in control throughout P1, P2, and P3.

The arterial blood glucose concentration was clamped at 135 ± 2, 134 ± 2, and 134 ± 3 mg/dl in LNlow during P1, P2, and P3, respectively, not different from the concentrations in the control group (Fig. 4). The glucose infusion rate did not differ significantly between groups, although it tended to be lower ({approx}35%, P = 0.36) in LNlow than in the control group during P3. In P1, the hepatic glucose load was equivalent in the two groups (34 ± 3 and 33 ± 2 mg·kg–1·min–1 in the control and LNlow groups, respectively). Because of the fall in hepatic blood flow, the hepatic glucose load during and after L-NAME infusion was lower (P < 0.05) in LNlow than in control (25 ± 1 vs. 36 ± 4 mg·kg–1·min–1 in P2 and 24 ± 2 vs. 38 ± 4 mg·kg–1·min–1 in P3). NHGU tended to be increased in the LNlow vs. control group during P2 and P3 (61 and 35%, respectively, P = 0.15 and 0.23; Fig. 5) despite the smaller glucose load. Net hepatic fractional extraction of glucose was enhanced (P < 0.05) during and after L-NAME infusion (0.093 ± 0.023 and 0.122 ± 0.021 in LNlow vs. 0.039 ± 0.015 and 0.056 ± 0.019 in control during P2 and P3, respectively).


Figure 4
View larger version (16K):
[in this window]
[in a new window]

  		Fig. 4. Arterial blood glucose, glucose infusion rate, and hepatic glucose load in the control and LNlow (dogs receiving a low rate of L-NAME infusion in P2) groups. The control group is the same one shown in the previous figures. *P < 0.05 vs. control.

 

Figure 5
View larger version (20K):
[in this window]
[in a new window]

  		Fig. 5. 5 Net hepatic glucose uptake and fractional extraction and nonhepatic glucose uptake in the control and LNlow groups. The control group is the same one shown in Figs. 1–3. *P < 0.05 vs. control.

 
Nonhepatic glucose uptake was significantly reduced during P3 but not P2 in LNlow vs. control (Fig. 5). Arterial lactate concentrations, net hepatic lactate balance, and net hepatic carbon retention in the LNlow group did not differ from those in the control group (Table 5). The mass of hepatic glycogen synthesized in the LNlow group tended to be greater than in the control group (1.78 ± 0.43 vs. 1.29 ± 0.37 g/100 g liver, P = 0.16). Arterial blood glycerol and net hepatic glycerol uptake declined in a similar manner to the control group; there was a significant increase in both parameters in LNlow at the final time point.


View this table:
[in this window]
[in a new window]

  		Table 5. Lactate and glycerol data and net hepatic carbon retention in LNlow

 

   DISCUSSION
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The current report is the first to examine the effects of NOS inhibition on glucose metabolism in vivo under controlled hyperinsulinemic hyperglycemic conditions, when substantial NHGU occurs. Intraportal administration of L-NAME at 0.3 mg·kg–1·min–1 significantly increased the uptake and fractional extraction of glucose by the liver. In this investigation we did not use intraportal glucose delivery, which augments NHGU, due to our concern that it could result in our erroneously concluding that suppression of hepatic NOS has no effect on NHGU. Such an error would occur if NOS inhibition is the method by which portal glucose delivery (the "portal signal") stimulates NHGU or if NOS inhibition and the portal signal are unrelated but not additive mechanisms for enhancing NHGU. The effects of L-NAME on NHGU and fractional glucose extraction did not wane during P3, after L-NAME infusion ceased, consistent with the finding (10) that the vasoactive effects of L-NAME in rats persist for as long as 6 h after an injection of the drug.

The blood glucose concentrations during L-NAME infusion at 0.3 mg·kg–1·min–1 were clamped at higher levels than in the control group to maintain a stable hepatic glucose load [a key determinant of NHGU (7)], raising the question of whether the increased hyperglycemia might have been responsible for the enhancement of NHGU. However, even when NHGU was adjusted for the glucose concentrations (i.e., hepatic glucose clearance), a significant augmentation of hepatic glucose disposal was evident in the group receiving the NOS inhibitor. Nevertheless, because of concern that the additional hyperglycemia might have affected the results, an additional group of dogs (LNlow) was studied in which no attempt was made to clamp the hepatic glucose load at the same level as in the control group. Instead, the arterial glucose concentration was clamped at control levels; as L-NAME brought about a fall in hepatic blood flow, the hepatic glucose load declined to basal. Despite of the fall in hepatic glucose load, NHGU tended to be enhanced during and after L-NAME infusion, and net hepatic fractional extraction of glucose was significantly stimulated. The fact that NHGU was not higher during L-NAME infusion is not surprising, given that the liver does not make a major contribution to glucose disposal when the hepatic glucose load is basal or near basal. Galassetti et al. (13) carried out studies in conscious dogs in which glucagon was delivered intraportally at a basal rate, insulin was infused intraportally at either three- or 15-fold basal rates (resulting in arterial plasma insulin concentrations of ~15 and 120 µU/ml, respectively), and the hepatic glucose load was maintained at basal levels by peripheral venous glucose infusion. NHGU during the final 2 h of these 4-h clamp studies averaged only ~0.1 and 0.5 mg·kg–1·min–1 with three- and 15-fold basal insulin, respectively. Thus, even with marked hyperinsulinemia, NHGU was very low. On the other hand, net hepatic fractional extraction of glucose was significantly stimulated. Fractional extraction takes into account the magnitude of the hepatic glucose load, making a difference between groups more readily apparent.

Calculation of NHGU is dependent on the accuracy of hepatic blood flow measurements. With a vasoconstrictive agent such as L-NAME, there is a concern that blood flow might not be measured accurately by the ultrasonic method, which is dependent on the diameter of the vessel. However, Transonic flow probes have previously been reported (42) to function appropriately during vasoconstrictive changes. In addition, the rates of NHGU obtained with the ultrasonic technique did not differ significantly from those obtained with blood flow derived from the dye method, a technique that is not dependent on vessel diameter.

In contrast to our current findings with NOS inhibition during acute hyperglycemia and hyperinsulinemia, in normal dogs that had received total parenteral nutrition (TPN) for 5 days, initiation of an intraportal infusion of the NOS inhibitor NG-nitro-L-arginine (L-NNA, the active metabolite of L-NAME) had no impact on NHGU (6). The delivery rate of the NOS inhibitor in the current study was ~6.6- and 1.6-fold greater in the LN and LNlow groups, respectively, than that in the TPN-infused dogs, and this might explain the difference in the results. Additionally, the TPN-adapted dogs exhibited high rates of NHGU before the L-NNA (with the liver extracting ~45% of a glucose load under clamp conditions) was begun despite the presence of euglycemia (115–120 mg/dl) and only mild hyperinsulinemia (~16 µU/ml) (23). Thus, the hepatic fractional extraction of glucose in the TPN-adapted dogs was approximately four times greater than that observed in the control group in the current study, and it could be that NOS inhibition was ineffective in increasing hepatic glucose uptake over the already elevated (for the prevailing insulin and glycemic levels) rate.

Net hepatic lactate output accounted for only ~30% of the enhancement in NHGU in LN. Thus, there was a significant stimulation of net hepatic carbon retention, consistent with enhancement of glycogen synthesis. Moreover, net hepatic glycogen synthesis in the LNlow group was 38% greater than in the control group (P = 0.16), consistent with the magnitude of stimulation of NHGU and net hepatic carbon retention in the LNlow group. Our findings are in accord with the observation that, in the presence of glucose and amino acids, treatment of isolated hepatocytes with the NO donor S-nitroso-N-acetylpenicillamine reduced glycogen synthesis in a dose-dependent manner, due to inhibition of glycogen synthase phosphatase (36). Additionally, treatment of lean rats with L-NAME for 4 wk resulted in greater insulin sensitivity, increased hepatic glycogen content, and increased insulin-stimulated phosphorylation of protein kinase B/Akt and glycogen synthase kinase-3 in liver, consistent with enhanced hepatic insulin signaling (38). Moreover, during a hyperinsulinemic hyperglycemic clamp concomitant with portal glucose infusion, intraportal infusion of SIN-1 significantly reduced net hepatic carbon retention in the dog (2). Thus, providing increased NO produced the effect opposite from reducing it.

We cannot determine from our data how NOS inhibition augmented net uptake and fractional extraction of glucose. Certainly the fall in hepatic blood flow with L-NAME, and particularly the fall in hepatic artery flow, might have been predicted to blunt hepatic glucose uptake rather than stimulate it. Suppression of hepatic arterial flow results in shunting of blood away from the sinusoids, where most glucose uptake takes place, and toward the hepatic vein (1). Complete occlusion or ligation of the hepatic artery reduces liver glucose uptake in vitro (1) and in vivo (24). Conversely, intraportal delivery of acetylcholine, which increased hepatic artery blood flow more than twofold but did not alter portal vein blood flow, enhanced NHGU 70% under hyperglycemic hyperinsulinemic conditions (35). NO is regarded as a key regulator of the hepatic microcirculation (4), and it has been suggested (34) that the hepatic insulin resistance previously reported in mice with global knockout of endothelial NOS is a result of impaired hepatic perfusion.

Since the changes in hepatic blood flow do not explain the enhancement of NHGU, other explanations must be sought. The change in hepatic sinusoidal hormone concentrations resulting from the reduction of hepatic blood flow during L-NAME infusion could have impacted both positively (insulin) and negatively (glucagon) on NHGU, but the magnitude of the impacts is difficult to quantify. In our previous work (27), a doubling of the sinusoidal insulin concentration was sufficient to increase NHGU and fractional extraction ~1–1.5 mg·kg–1·min–1 and two- to threefold, respectively. Thus the 35% increase in sinusoidal insulin that we observed in LN/SAL is unlikely to have accounted for a large proportion of the enhancement in liver glucose uptake. Moreover, the increase in sinusoidal insulin in LNlow was much more modest (10 and 12% in P2 and P3, respectively, compared with P1) and would have little impact on NHGU and fractional extraction. Also, our previous data (27) were obtained in the presence of basal glucagon concentrations. In the current studies, glucagon tended to increase concomitantly with insulin, and this would have offset insulin's stimulatory effect on liver glucose uptake (12, 18, 33). Additionally, epinephrine concentrations were elevated in the LN group vs. control, and this would have stimulated hepatic glucose output (8), thereby reducing the net uptake and fractional extraction of glucose by the liver. Another possible explanation for our data is that NOS inhibition limits the activity of AMP-activated protein kinase (AMPK). One pathway for activation of AMPK is triggered by peroxynitrite, which can be generated by the spontaneous reaction of NO with superoxide anion (43). Consistent with this possibility, intraportal infusion of the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside induced acute hepatic insulin resistance in the conscious dog (29).

Nonhepatic glucose disposal was difficult to evaluate in the high-dose LN groups because the dogs were exposed to very different peripheral glucose loads during P1–P3. Nonhepatic glucose uptake in the LNlow group reached a relative plateau during P2 and P3, but it steadily increased in the control group over the course of the experiment. During P3, the divergence in response between groups reached statistical significance. The reduction in nonhepatic glucose uptake in association with an enhancement of liver glucose disposal is in agreement with our previous findings (14, 25, 28) of a reciprocal relationship between the liver and nonhepatic tissues in disposition of glucose under a variety of experimental conditions. The glucose infusion rate, in absolute terms, was ~3.5 mg·kg–1·min–1 less in LNlow than in the control group during P3, but because of the variability of the measurement, it did not reach statistical significance. Therefore, it is not clear whether the L-NAME infusion reduced whole body glucose disposal or merely redirected glucose toward the liver and away from the peripheral tissues. In lean, standard-diet-fed mice with or without L-NAME added to their drinking water for 12 wk, there were no differences in glucose tolerance, insulin tolerance, or insulin signaling in skeletal muscle between groups (40). This suggests that L-NAME treatment in our studies simply directed glucose toward the liver.

Blood pressure and heart rate during the basal period and P1 were within the ranges previously reported by our group (9) and others (39) in dogs similar in size to those in the current investigation. The hypertensive and bradycardic effects of L-NAME were consistent with those observed in humans, dogs, and rats (30, 31, 41) and appeared to be dose dependent. Despite the observed cardiovascular changes, the arterial concentrations of norepinephrine in the combined LN group did not differ significantly from those in the control group during P2, suggesting little change in sympathetic output. Only SIN-1 infusion was associated with a consistent and significant rise in catecholamine concentrations.

In conclusion, acute intraportal infusion of L-NAME stimulated NHGU under hyperinsulinemic and hyperglycemic conditions. The increase in NHGU was accompanied by an increase in hepatic carbon retention (glycogen storage) and may reflect an enhancement of hepatic insulin signaling in the presence of L-NAME (38).


   GRANTS
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
These studies were supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant RO1-DK-43706 and Diabetes Research and Training Center Grant SP-60-AM20593.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. C. Moore, 702 Light Hall, Dept. of Molecular Physiology & Biophysics, Vanderbilt University School of Medicine, Nashville, TN 37232-0615 (e-mail: genie.moore{at}vanderbilt.edu)

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. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Alexander B, Rogers C, Naftalin R. Hepatic arterial perfusion decreases intrahepatic shunting and maintains glucose uptake in the rat liver. Pflugers Arch 444: 291–298, 2002.[CrossRef][Web of Science][Medline]
  2. An Z, DiCostanzo CA, Moore MC, Edgerton DS, Dardevet DP, Neal DW, Cherrington AD. Effects of the nitric oxide donor SIN-1 on net hepatic glucose uptake in the conscious dog. Am J Physiol Endocrinol Metab 294: E300–E306, 2008.[Abstract/Free Full Text]
  3. Basu A, Basu R, Shah P, Vella A, Johnson CM, Jensen M, Nair KS, Schwenk WF, Rizza RA. Type 2 diabetes impairs splanchnic uptake of glucose but does not alter intestinal glucose absorption during enteral glucose feeding: additional evidence for a defect in hepatic glucokinase activity. Diabetes 50: 1351–1362, 2001.[Abstract/Free Full Text]
  4. Bauer C, Walcher F, Kalweit U, Larsen R, Marzi I. Role of nitric oxide in the regulation of the hepatic microcirculation in vivo. J Hepatol 27: 1089–1095, 1997.[CrossRef][Web of Science][Medline]
  5. Ceriello A, Davidson J, Hanefeld M, Leiter L, Monnier L, Owens D, Tajima N, Tuomilehto J. Postprandial hyperglycaemia and cardiovascular complications of diabetes: an update. Nutr Metab Cardiovasc Dis 16: 453–456, 2006.[CrossRef][Web of Science][Medline]
  6. Chen SS, Donmoyer CM, Ejiofor J, McCay J, Archuletta R, Lacy DB, McGuinness OP. Impact of intraportal N(omega)-nitro-L-arginine infusion on hepatic glucose metabolism in total parenteral nutrition-adapted dogs: interaction with infection. Metabolism 51: 274–283, 2002.[CrossRef][Web of Science][Medline]
  7. Cherrington AD. Banting Lecture 1997. Control of glucose uptake and release by the liver in vivo. Diabetes 48: 1198–1214, 1999.[Web of Science][Medline]
  8. Chu CA, Sindelar DK, Neal DW, Allen EJ, Donahue EP, Cherrington AD. Comparison of the direct and indirect effects of epinephrine on hepatic glucose production. J Clin Invest 99: 1044–1056, 1997.[Web of Science][Medline]
  9. Chu CA, Sindelar DK, Neal DW, Cherrington AD. Hepatic and gut clearance of catecholamines in the conscious dog. Metabolism 48: 259–263, 1999.[CrossRef][Web of Science][Medline]
  10. Conner EM, Aiko S, Fernandez M, Battarbee HD, Gray L, Grisham MB. Duration of the hemodynamic effects of N(G)-nitro-L-arginine methyl ester in vivo. Nitric Oxide 4: 85–93, 2000.[CrossRef][Web of Science][Medline]
  11. Cowan JS, Hetenyi G Jr. Glucoregulatory responses in normal and diabetic dogs recorded by a new tracer method. Metabolism 20: 360–372, 1971.[CrossRef][Web of Science][Medline]
  12. Dinneen S, Alzaid A, Turk D, Rizza R. Failure of glucagon suppression contributes to postprandial hyperglycaemia in IDDM. Diabetologia 38: 337–343, 1995.[Web of Science][Medline]
  13. Galassetti P, Chu CA, Neal DW, Reed GW, Wasserman DH, Cherrington AD. A negative arterial-portal venous glucose gradient increases net hepatic glucose uptake in euglycemic dogs. Am J Physiol Endocrinol Metab 277: E126–E134, 1999.[Abstract/Free Full Text]
  14. Galassetti P, Shiota M, Zinker BA, Wasserman DH, Cherrington AD. A negative arterial-portal vein glucose gradient decreases skeletal muscle glucose uptake in the conscious dog. Am J Physiol Endocrinol Metab 275: E101–E111, 1998.[Abstract/Free Full Text]
  15. Gentilcore D, Visvanathan R, Russo A, Chaikomin R, Stevens JE, Wishart JM, Tonkin A, Horowitz M, Jones KL. Role of nitric oxide mechanisms in gastric emptying of, and the blood pressure and glycemic responses to, oral glucose in healthy older subjects. Am J Physiol Gastrointest Liver Physiol 288: G1227–G1232, 2005.[Abstract/Free Full Text]
  16. Guarino MP, Afonso RA, Raimundo N, Raposo JF, Macedo MP. Hepatic glutathione and nitric oxide are critical for hepatic insulin-sensitizing substance action. Am J Physiol Gastrointest Liver Physiol 284: G588–G594, 2003.[Abstract/Free Full Text]
  17. Guarino MP, Correia NC, Lautt WW, Macedo MP. Insulin sensitivity is mediated by the activation of the ACh/NO/cGMP pathway in rat liver. Am J Physiol Gastrointest Liver Physiol 287: G527–G532, 2004.[Abstract/Free Full Text]
  18. Holste LC, Connolly CC, Moore MC, Neal DW, Cherrington AD. Physiological changes in circulating glucagon alter hepatic glucose disposition during portal glucose delivery. Am J Physiol Endocrinol Metab 273: E488–E496, 1997.[Abstract/Free Full Text]
  19. Hsieh PS, Moore MC, Neal DW, Emshwiller M, Cherrington AD. Rapid reversal of the effects of the portal signal under hyperinsulinemic conditions in the conscious dog. Am J Physiol Endocrinol Metab 276: E930–E937, 1999.[Abstract/Free Full Text]
  20. Keppler D, Decker K. Glycogen: determination with amyloglucosidase. In: Methods in Enzymatic Analysis, edited by Bergmeyer HU. New York: Verlag Chemie Weinheim, Academic, 1974, p. 1127–1131.
  21. Lautt WW. Practice and principles of pharmacodynamic determination of HISS-dependent and HISS-independent insulin action: methods to quantitate mechanisms of insulin resistance. Med Res Rev 23: 1–14, 2003.[CrossRef][Web of Science][Medline]
  22. Lautt WW, Macedo MP, Sadri P, Legare DJ, Reid MA, Guarino MP. Pharmaceutical reversal of insulin resistance. Proc West Pharmacol Soc 47: 30–32, 2004.[Medline]
  23. McGuinness OP, Donmoyer C, Ejiofor J, McElligott S, Lacy DB. Hepatic and muscle glucose metabolism during total parenteral nutrition: impact of infection. Am J Physiol Endocrinol Metab 275: E763–E769, 1998.[Abstract/Free Full Text]
  24. Moore MC, Burish MJ, Farmer B, Neal DW, Pan C, Cherrington AD. Chronic hepatic artery ligation does not prevent liver from differentiating portal vs. peripheral glucose delivery. Am J Physiol Endocrinol Metab 285: E845–E853, 2003.[Abstract/Free Full Text]
  25. Moore MC, Hsieh PS, Neal DW, Cherrington AD. Nonhepatic response to portal glucose delivery in conscious dogs. Am J Physiol Endocrinol Metab 279: E1271–E1277, 2000.[Abstract/Free Full Text]
  26. Moore MC, Satake S, Lautz M, Soleimanpour SA, Neal DW, Smith M, Cherrington AD. Nonesterified fatty acids and hepatic glucose metabolism in the conscious dog. Diabetes 53: 32–40, 2004.[Abstract/Free Full Text]
  27. Myers SR, McGuinness OP, Neal DW, Cherrington AD. Intraportal glucose delivery alters the relationship between net hepatic glucose uptake and the insulin concentration. J Clin Invest 87: 930–939, 1991.[Web of Science][Medline]
  28. Pagliassotti MJ, Holste LC, Moore MC, Neal DW, Cherrington AD. Comparison of the time courses of insulin and the portal signal on hepatic glucose and glycogen metabolism in the dog. J Clin Invest 97: 81–91, 1996.[Web of Science][Medline]
  29. Pencek RR, Shearer J, Camacho RC, James FD, Lacy DB, Fueger PT, Donahue EP, Snead W, Wasserman DH. 5-Aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside causes acute hepatic insulin resistance in vivo. Diabetes 54: 355–360, 2005.[Abstract/Free Full Text]
  30. Picker O, Scheeren TW, Arndt JO. Nitric oxide synthases in vagal neurons are crucial for the regulation of heart rate in awake dogs. Basic Res Cardiol 96: 395–404, 2001.[CrossRef][Web of Science][Medline]
  31. Rossoni G, Manfredi B, De Gennaro Colonna V, Berti M, Guazzi M, Berti F. Sildenafil reduces L-NAME-induced severe hypertension and worsening of myocardial ischaemia-reperfusion damage in the rat. Br J Pharmacol 150: 567–576, 2007.[CrossRef][Web of Science][Medline]
  32. Sadri P, Lautt WW. Blockade of hepatic nitric oxide synthase causes insulin resistance. Am J Physiol Gastrointest Liver Physiol 277: G101–G108, 1999.[Abstract/Free Full Text]
  33. Shah P, Vella A, Basu A, Basu R, Schwenk WF, Rizza RA. Lack of suppression of glucagon contributes to postprandial hyperglycemia in subjects with type 2 diabetes mellitus. J Clin Endocrinol Metab 85: 4053–4059, 2000.[Abstract/Free Full Text]
  34. Shankar RR, Wu Y, Shen HQ, Zhu JS, Baron AD. Mice with gene disruption of both endothelial and neuronal nitric oxide synthase exhibit insulin resistance. Diabetes 49: 684–687, 2000.[Abstract]
  35. Shiota M, Jackson P, Galassetti P, Scott M, Neal DW, Cherrington AD. Combined intraportal infusion of acetylcholine and adrenergic blockers augments net hepatic glucose uptake. Am J Physiol Endocrinol Metab 278: E544–E552, 2000.[Abstract/Free Full Text]
  36. Sprangers F, Sauerwein HP, Romijn JA, van Woerkom GM, Meijer AJ. Nitric oxide inhibits glycogen synthesis in isolated rat hepatocytes. Biochem J 330: 1045–1049, 1998.[Web of Science][Medline]
  37. Steele R, Wall JS, DeBodo RC, Altszuler N. Measurement of size and turnover rate of body glucose pool by the isotope dilution method. Am J Physiol 187: 15–24, 1956.[Abstract/Free Full Text]
  38. Tarsitano CA, Paffaro VA Jr, Pauli JR, da Silva GH, Saad MJ, Salgado I, da Cruz-Höfling MA, Hyslop S. Hepatic morphological alterations, glycogen content and cytochrome P450 activities in rats treated chronically with N(omega)-nitro-L-arginine methyl ester (L-NAME). Cell Tissue Res 329: 45–58, 2007.[CrossRef][Web of Science][Medline]
  39. Thrasher TN, Shifflett C. Effect of carotid or aortic baroreceptor denervation on arterial pressure during hemorrhage in conscious dogs. Am J Physiol Regul Integr Comp Physiol 280: R1642–R1649, 2001.[Abstract/Free Full Text]
  40. Tsuchiya K, Sakai H, Suzuki N, Iwashima F, Yoshimoto T, Shichiri M, Hirata Y. Chronic blockade of nitric oxide synthesis reduces adiposity and improves insulin resistance in high fat-induced obese mice. Endocrinology 148: 4548–4556, 2007.[Abstract/Free Full Text]
  41. van der Linde NA, Boomsma F, van den Meiracker AH. Potentiation of L-NAME-induced systemic and renal vasoconstrictor responses by alpha1-adrenoceptor antagonism. J Hypertens 23: 1017–1024, 2005.[Web of Science][Medline]
  42. Walkowska A, Badzynska B, Kompanowska-Jezierska E, Johns EJ, Sadowski J. Effects of renal nerve stimulation on intrarenal blood flow in rats with intact or inactivated NO synthases. Acta Physiol Scand 183: 99–105, 2005.[CrossRef][Web of Science][Medline]
  43. Zou MH, Hou XY, Shi CM, Kirkpatick S, Liu F, Goldman MH, Cohen RA. Activation of 5'-AMP-activated kinase is mediated through c-Src and phosphoinositide 3-kinase activity during hypoxia-reoxygenation of bovine aortic endothelial cells. Role of peroxynitrite. J Biol Chem 278: 34003–34010, 2003.[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
294/4/E768    most recent
00184.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Moore, M. C.
Right arrow Articles by Cherrington, A. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Moore, M. C.
Right arrow Articles by Cherrington, A. D.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2008 by the American Physiological Society.