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Am J Physiol Endocrinol Metab 294: E300-E306, 2008. First published November 20, 2007; doi:10.1152/ajpendo.00380.2007
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Effects of the nitric oxide donor SIN-1 on net hepatic glucose uptake in the conscious dog

Zhibo An,* Catherine A. DiCostanzo,* Mary C. Moore, Dale S. Edgerton, Dominique P. Dardevet, Doss W. Neal, and Alan D. Cherrington

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

Submitted 18 June 2007 ; accepted in final form 19 November 2007


   ABSTRACT
TOP
 ABSTRACT
RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
To determine the role of nitric oxide in regulating net hepatic glucose uptake (NHGU) in vivo, studies were performed on three groups of 42-h-fasted conscious dogs using a nitric oxide donor [3-morpholinosydnonimine (SIN-1)]. The experimental period was divided into period 1 (0–90 min) and period 2 (P2; 90–240 min). At 0 min, somatostatin was infused peripherally, and insulin (4-fold basal) and glucagon (basal) were given intraportally. Glucose was delivered intraportally (22.2 µmol·kg–1·min–1) and peripherally (as needed) to increase the hepatic glucose load twofold basal. At 90 min, an infusion of SIN-1 (4 µg·kg–1·min–1) was started in a peripheral vein (PeSin-1, n = 10) or the portal vein (PoSin-1, n = 12) while the control group received saline (SAL, n = 8). Both peripheral and portal infusion of SIN-1, unlike saline, significantly reduced systolic and diastolic blood pressure. Heart rate rose in PeSin-1 and PoSin-1 (96 ± 5 to 120 ± 10 and 88 ± 6 to 107 ± 5 beats/min, respectively, P < 0.05) but did not change in response to saline. NHGU during P2 was 31.0 ± 2.4 and 29.9 ± 2.0 µmol·kg–1·min–1 in SAL and PeSin-1, respectively but was 23.7 ± 1.7 in PoSin-1 (P < 0.05). Net hepatic carbon retention during P2 was significantly lower in PoSin-1 than SAL or PeSin-1 (21.4 ± 1.2 vs. 27.1 ± 1.5 and 26.1 ± 1.0 µmol·kg–1·min–1). Nonhepatic glucose uptake did not change in response to saline or SIN-1 infusion. In conclusion, portal but not peripheral infusion of the nitric oxide donor SIN-1 inhibited NHGU.

3-morpholinosydnominine; nitric oxide; net hepatic glucose uptake; hyperglycemia

THE LIVER IS A PIVOTAL ORGAN in disposal of ingested glucose and, therefore, in limiting postprandial hyperglycemia. It has been shown that the response to infusion of glucose directly in the hepatic portal vein mimics the response to oral glucose delivery in the conscious dog (5). In comparing the effects of peripheral vs. portal venous glucose delivery on net hepatic glucose uptake (NHGU) during hyperglycemic clamps, we found that NHGU was considerably greater in the presence of intraportal glucose delivery, even when the hepatic glucose loads were well matched and insulin and glucagon levels were equivalent between groups (1, 2, 26, 28). This led us to suggest that a "portal glucose signal" is at least as important as insulin in determining NHGU after an oral glucose load. In addition, the portal glucose signal reduces glucose uptake by nonhepatic tissues, primarily muscle, at the same time as it increases NHGU, thereby ensuring appropriate distribution of glucose to muscle and liver (2). To date, it remains unclear how these coordinated responses of muscle and liver come about.

Nitric oxide (NO) is a potent biological mediator produced in a variety of tissues during the catabolism of L-arginine to L-citrulline under the control of nitric oxide synthase (NOS). The latter exists in three isoforms including endothelial (eNOS), neuronal (nNOS) and inducible (iNOS) forms. Emerging evidence supports a role for endogenous NO in the regulation of glucose, fatty acid, and amino acid metabolism in mammals (19). Physiological levels of NO can stimulate glucose uptake and oxidation in skeletal muscle and adipocytes (30). Moreover, chronic administration of NG-nitro-L-arginine methyl ester (L-NAME), a NOS inhibitor, in drinking water decreased glucose tolerance in rats (4). eNOS knockout mice have hyperinsulinemia and impaired insulin-stimulated glucose uptake relative to control mice (8). These findings suggest that NO could play an important regulatory role in glucose metabolism.

All three isoforms of NOS have been reported to be expressed in liver (20). In vitro studies have shown that NO can have direct effects on isolated hepatocytes, which results in an inhibition of gluconeogenesis, a reduction in glycogen synthesis, and an increase in glucose output (14, 36). Lautt (17) and others have investigated the role of hepatic NO in regulating whole body glucose metabolism. They (17) hypothesized the existence of a novel neurohumoral mechanism by which hepatic parasympathetic nerves, through permissive release of a putative insulin-sensitizing substance [hepatic insulin-sensitizing substance (HISS)] from the liver, regulate peripheral glucose disposal. According to their theory, insulin stimulates hepatic parasympathetic nerve firing and thereby promotes the release of NO, which then increases the release of HISS from the liver. This, in turn, stimulates peripheral glucose uptake. Sadri and Lautt (31) showed that portal but not systemic administration of an NO donor or antagonist alters glucose metabolism in vivo. Consistent with this, Sprangers et al. (35) found that systemic administration of a NOS inhibitor, N{omega}-monomethyl-L-arginine, in humans did not alter hepatic glucose production. Together, these in vivo data support the concept that the liver is an important NO target, and its hepatic effects may influence nonhepatic glucose uptake. All of the published studies relating to HISS (12, 13, 17, 24, 31, 32) have, however, been carried out under euglycemic conditions, when the role of the liver in glucose disposition is minimal and the role of muscle is dominant. As a result of that and the difficulty in directly measuring NHGU in the human or the rodent, virtually nothing is known about the effect of NO on hepatic glucose uptake in vivo.

As noted above, it has been shown that muscle and hepatic glucose uptake are altered reciprocally in response to portal glucose delivery (2, 15, 29). Thus it is possible that hepatic NO, or some downstream signal that it generates, has a stimulatory effect on glucose uptake by muscle and an inhibitory effect on glucose uptake by the liver. If that were to be the case, it raises the possibility that downregulation of hepatic NO action may be responsible for the effect of portal glucose delivery on muscle and liver glucose uptake. We hypothesized, therefore, that portal infusion of the NO donor 3-morpholinosydnonimine (SIN-1) would reduce NHGU and increase peripheral glucose uptake.


   RESEARCH DESIGN AND METHODS
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals and surgical procedures. Studies were carried out on healthy, conscious 42-h-fasted mongrel dogs (22.7 ± 0.5 kg). A fast of this duration was chosen because it produces a metabolic state resembling that in the overnight-fasted human and results in liver glycogen levels that are at a stable minimum. The animals were fed a standard diet one time per day, water was provided ad libitum, and they were housed in a facility that met American Association for Accreditation of Laboratory Animal Care guidelines. The protocol was approved by the Vanderbilt University Medical Center Animal Care Committee.

Approximately 16 days before study, each dog underwent a laparotomy, and silicone rubber catheters for sampling were inserted in the hepatic vein, the portal vein, and a femoral artery. Catheters for intraportal infusion were placed in a splenic and a jejunal vein, whereas ultrasonic flow probes (Transonic Systems, Ithaca, NY) were placed around the portal vein and the hepatic artery as described elsewhere (1).

On the morning of the study, catheters and flow probe leads were exteriorized as described previously (1). Angiocaths (Deseret Medical, Becton-Dickinson, Sandy, UT) were inserted in the cephalic and saphenous veins. Each dog was allowed to stand quietly in a Pavlov harness throughout the experiment and was studied only one time.

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), which was divided into a 90-min period (P1) followed by a 150-min period (P2). In all experiments, a constant infusion of indocyanine green dye (0.076 mg/min; Sigma Immunochemicals, St. Louis, MO) was initiated at –120 min via the left cephalic vein, and p-aminohippuric acid (PAH; 0.4 mg·kg–1·min–1; Sigma) was infused via the left saphenous vein from –120 to 0 min. At 0 min, a constant infusion of somatostatin (0.8 µg·kg–1·min–1; Bachem, Torrance, CA) was begun via the left saphenous vein to suppress endogenous insulin and glucagon secretion, and basal glucagon (0.57 ng·kg–1·min–1; Glucagen, Novo Nordisk, Bagsvaerd, Denmark) and fourfold basal insulin (1.2 mU·kg–1·min–1; Eli Lilly, Indianapolis, IN) infusions were started through the splenic and jejunal catheters. Glucose (20% dextrose) was delivered intraportally at 22.2 µmol·kg–1·min–1, and PAH was mixed with it at a concentration allowing a delivery rate of 0.4 mg·kg–1·min–1. In addition, a primed continuous infusion of 50% dextrose was begun via the right cephalic vein at time 0 so that the blood glucose could quickly be clamped at the desired hyperglycemic level (~9.7 mmol/l). In P2, saline (SAL, n = 8) was infused intraportally, or SIN-1 (4 µg·kg–1·min–1; Cayman Chemical, Ann Arbor, MI) was delivered intraportally (PoSin-1, n = 12) or peripherally via the right saphenous vein (PeSin-1, n = 10). The peripheral glucose infusion rate was adjusted in P2 to maintain a similar hepatic glucose load to that seen in P1.

Femoral artery, portal vein, and hepatic vein blood samples were taken as described previously (26). Arterial blood pressure and heart rate were monitored by a DigiMed Blood Pressure Analyzer (Micro-Med, Louisville, KY).

Processing and analysis of samples. The collection and immediate processing of blood samples have been described previously (10). Four to eight 10-µl aliquots of plasma from each sample were immediately analyzed for glucose using the glucose analyzer (Beckman Instruments, Fullerton, CA; Analox Instruments, London, UK). Plasma insulin and glucagon concentrations were determined as previously described (29). PAH was measured in perchloric acid-deproteinized blood as described elsewhere (29). Cortisol, lactate, glycerol, and nonesterified fatty acid (NEFA) concentrations were measured as previously described (29).

Calculations and data analysis. An intraportal infusion of glucose has the possibility of mixing poorly with the blood in the laminar flow of the portal circulation. Thus it was critical to assess the mixing of the portal infusate in P1 and P2. This was accomplished by comparing the recovery of PAH (which was mixed with the portal glucose infusate) in the portal and hepatic veins using an approach described elsewhere (29). In the SAL group, 11 dogs were studied and 8 were included since they exhibited adequate mixing, as defined previously (25); 12 out of 16 dogs were included in the PoSin-1 group, and 10 out of 11 dogs were included in the PeSin-1 group. In the SAL, PoSin-1, and PeSin-1 animals that were retained, the ratio of PAH recovery in the portal vein to the PAH infusion rate was 0.9 ± 0.1, 0.9 ± 0.1, and 0.9 ± 0.1, respectively. Likewise, the ratio of PAH recovery in the hepatic vein to the PAH infusion rate was 0.9 ± 0.1, 0.9 ± 0.1, and 0.9 ± 0.1 in three groups, respectively (with a ratio of 1.0 representing perfect mixing).

Hepatic blood flow (HBF) was measured using ultrasonic flow probes and by the use of indocyanine green dye according to the method of Leevy et al. (18). The results obtained with the flow probes and dye were not significantly different, but the data reported here were calculated using the flow probes because this did not require an assumption regarding the distribution of the arterial and portal vein contribution to hepatic blood flow.

An indirect (I) method was used to assess NHGU to minimize the potential errors that arise due to any imperfect mixing of the infused glucose in the blood. Thus the Loadin was calculated as

Formula
where GA is the arterial blood glucose concentration, GIRPO is the portal glucose infusion rate, and GUG is the uptake of glucose by the gastrointestinal tract, calculated as previously described (25).

The load of a substrate exiting the liver was calculated as

Formula
where GH represents the hepatic vein glucose concentration.

Net hepatic glucose balance (NHGB) was thus calculated as

Formula
NHGB was also calculated using a direct calculation that has been described previously (33). The results obtained did not differ significantly from those obtained using the indirect calculation, but only the data calculated with the indirect calculation are reported. The average nonhepatic glucose uptake between two time points (T1 and T2) was calculated by subtracting the rate of NHGU and the change in the glucose mass from the total glucose infusion rate using the equation: nonhepatic glucose uptake rate = average total glucose infusion rate between T1 and T2 – (NHGUT1 + NHGUT2)/2 – glucose mass change in the pool between T1 and T2. In previous studies, under hyperglycemic hyperinsulinemic conditions, we measured hindlimb glucose uptake directly while at the same time determining nonhepatic glucose uptake using the above equation. We were able to show that virtually all nonhepatic glucose uptake could be accounted for by muscle glucose uptake (11).

Net hepatic carbon retention was calculated as the sum of the balances of glucose and lactate, once the latter was converted to glucose equivalents. The calculation of net hepatic carbon retention is an established approach to estimate hepatic glycogen accretion and has been described and validated previously (33). It has the advantage of not being dependent upon an estimate of the prestudy glycogen content, which must be obtained from a separate set of 42-h-fasted dogs. The calculation omits the contribution of gluconeogenic substrates other than lactate and fails to account for the glucose oxidized by the liver. However, these two rates are relatively small, quantitatively similar, and offsetting (33). The net hepatic balance of lactate and glycerol as well as the hepatic sinusoidal insulin and glucagon concentrations were calculated using the direct arteriovenous difference method. Net fractional glucose extraction by the liver was calculated as the ratio of NHGB to Loadin.

For all glucose balance calculations, glucose concentrations were converted from plasma to blood values by using previously determined (15, 23) conversion factors (the mean of the ratio of the blood value to the plasma concentration). The use of whole blood glucose values ensures accurate hepatic balance measurements regardless of the characteristics of glucose entry in the erythrocyte.

Statistical analysis. All data are presented as means ± SE. Time course data were analyzed with two-way repeated-measures ANOVA, and one-way ANOVA was used for any comparisons of other mean data. Post hoc analysis used the Student-Newman-Keul's method. Statistical significance was accepted at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Hemodynamic response. Systolic blood pressure did not change significantly over the course of the experiment in response to saline infusion. The systolic blood pressures in the PeSin-1 and PoSin-1 groups were similar to those seen in SAL in the basal period and P1 but dropped significantly during P2 (Table 1). Diastolic blood pressure also fell significantly during P2 in the PeSin-1 and PoSin-1 groups (Table 1). The average heart rate did not change over time in SAL but it increased (P < 0.05) during P2 in both the PeSin-1 and PoSin-1 groups (Table 1).


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  		Table 1. Heart rate and systolic and diastolic blood pressure during the basal and experimental periods in conscious 42-h-fasted dogs given saline or SIN-1 infused in the portal vein or in a peripheral vein

 
Hormone concentrations. The arterial and hepatic sinusoidal insulin levels increased three- to fourfold and remained stable during P1 and P2 in all groups (Table 2). Arterial and hepatic sinusoidal plasma glucagon concentrations remained basal in all groups (Table 2).


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  		Table 2. Hormone concentrations during the basal and experimental periods in conscious 42-h-fasted dogs given saline or SIN-1 infused in the portal vein or in a peripheral vein

 
Hepatic blood flow, blood glucose concentrations, and hepatic glucose load. Portal vein blood flow decreased ~20% during P1 in all groups in response to somatostatin infusion (Table 3). There was a concomitant small increase in hepatic arterial flow, and, as a consequence, total hepatic blood flow did not change significantly during the first experimental period. There were small increases in portal vein and hepatic arterial blood flow in all groups during P2 such that total flow was completely restored to baseline values in each group, and there were no significant differences among groups.


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  		Table 3. Average hepatic arterial, portal, and total hepatic blood flow during the basal and experimental periods in conscious 42-h-fasted dogs given saline or SIN-1 infused in the portal vein or in a peripheral vein

 
Arterial blood glucose levels increased in all groups from a basal value of 4.6 ± 0.1 to 9.7 ± 0.2 mmol/l during P1 and P2 (Fig. 1). The hepatic glucose loads increased proportionally and did not differ significantly among groups at any time (Fig. 1).


Figure 1
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  		Fig. 1. Arterial blood glucose and hepatic glucose loads in 42-h-fasted conscious dogs during the basal (–30–0 min) and experimental [period (P) 1, 0–90 min; P2, 90–240 min] periods. Somatostatin was infused peripherally and insulin (4-fold basal) and glucagon (basal) were given intraportally, whereas glucose was delivered intraportally (22.2 µmol·kg–1·min–1) and peripherally at a variable rate to increase the hepatic glucose load 2-fold basal during P1 and P2. The saline (SAL) group (n = 8) received intraportal saline during P2; PoSin-1 group (n = 12), received intraportal 3-morpholinosydnonimine (SIN-1, 4 µg·kg–1·min–1) during P2. PeSin-1 group (n = 10) received peripheral SIN-1 (4 µg·kg–1·min–1) during P2. Data are means ± SE.

 
NHGB and change of net hepatic fractional extraction of glucose from baseline. All groups exhibited a similar rate of net hepatic glucose output during the basal period. Coincident with the start of the experimental period (4x basal insulin, basal glucagon, and hyperglycemia), all groups switched from net production to net uptake of glucose, with the rates not being significantly different among groups during the last 30 min of P1 (Fig. 2). Subsequently, the rate of NHGU remained relatively stable in SAL and PeSin-1 (31.0 ± 2.4 and 29.9 ± 2.0 µmol·kg–1·min–1), whereas in PoSin-1 it was reduced to 23.7 ± 1.7 µmol·kg–1·min–1 during P2 (P < 0.05 vs. SAL and PeSin-1). The change of hepatic fractional extraction of glucose from baseline followed a similar pattern, falling significantly in response to intraportal SIN-1 infusion (Fig. 2).


Figure 2
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  		Fig. 2. Net hepatic glucose uptake and change of net hepatic fractional extraction of glucose from baseline in 42-h-fasted conscious dogs during the basal and experimental periods. See Fig. 1 for description of study conditions. Data are means ± SE. P < 0.05 compared with SAL group (*) and compared with the PeSin-1 group ({dagger}).

 
Glucose infusion rates, nonhepatic glucose uptake. The glucose infusion rate in all three groups increased over time. There was a tendency for the total glucose infusion rate to be lower in PoSin-1, in line with the reduction in NHGU seen in response to portal SIN-1 infusion (Fig. 3). Nonhepatic glucose uptake increased modestly over time in all groups, but did not differ among groups (Fig. 3).


Figure 3
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  		Fig. 3. Glucose infusion rate and nonhepatic glucose uptake in 42-h-fasted conscious dogs during the basal and experimental periods. See Fig. 1 for description of study conditions. Data are means ± SE.

 
Lactate metabolism and net hepatic carbon retention. The arterial blood lactate concentrations rose in all groups during P1 and P2 relative to the basal period. After the experimental period began, net hepatic lactate balance changed from uptake to output, and output remained evident in all groups during P2 (Table 4). Net hepatic carbon retention did not differ between PeSin-1 and SAL at any time but was reduced in PoSin-1 during P2 (21.4 ± 1.2 in PoSin-1 vs. 27.1 ± 1.5 in SAL and 26.1 ± 1.0 µmol·kg–1·min–1 in PeSin-1, respectively, P < 0.05).


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  		Table 4. Average lactate, glycerol, and NEFA concentration and net hepatic balance during the basal and experimental periods in conscious 42-h-fasted dogs given saline or SIN-1 infused in the portal vein or in a peripheral vein

 
Glycerol and NEFA metabolism. Arterial blood glycerol concentrations and net hepatic glycerol uptake were reduced 65–75% by hyperglycemia and hyperinsulinemia and remained suppressed in all groups throughout P1 and P2. The suppression of glycerol was, however, partially reversed during P2 in the PeSin-1 and PoSin-1 but not the SAL group (Table 4). Arterial NEFA concentrations and net hepatic NEFA uptake changed in a pattern similar to glycerol, decreasing 80–90% during P1 in all groups. There was, however, no rebound during P2 in the PeSin-1 or PoSin-1 groups (Table 4).


   DISCUSSION
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The rate of hepatic glucose uptake in vivo depends on a complex set of variables, including neural, hormonal, and substrate signals (28). In the present study, we took great care to control these inputs so as to allow precise assessment of the effect of SIN-1 delivery on NHGU. To our knowledge, this study is the first in vivo investigation to explore the effects of the NO donor SIN-1 on NHGU in the presence of portal glucose delivery, as well as elevated plasma insulin and glucose levels. Under these conditions, portal infusion of the NO donor SIN-1 caused an ~24% decrease in NHGU and hepatic glycogen accumulation. We were unable to detect any effect of portal SIN-1 on glucose uptake by nonhepatic tissues or whole body glucose utilization. Peripheral SIN-1 infusion was without effect on either hepatic or nonhepatic glucose utilization.

Although there are several NO donors available [e.g., S-nitroso-N-acetylpenicillamine (SNAP), V-PYRRO/NO, DEA/NO], we specifically chose to use SIN-1 in light of its metabolic fate. In vivo, SIN-1 decomposes nonenzymatically in a two-step reaction, with the second step yielding NO and superoxide, which can readily form peroxynitrite (27). NO or peroxynitrite can then nitrosylate glutathione to generate S-nitrosoglutathione (GSNO) (16). According to Lautt's (17) HISS hypothesis, GSNO can act as an endogenous NO reservoir and serve as an intermediate between NO synthesis and guanylate cyclase activation in the HISS pathway (13). In addition, we found SIN-1, in contrast to the other NO donors, has little effect on total hepatic blood flow, so that total hepatic blood flow did not differ among groups in the current study.

In contrast to the liver, where blood flow was minimally altered, there was a marked overall hemodynamic response to SIN-1 infusion whether it was given in a peripheral vein or the hepatic portal vein. This was undoubtedly due to the well-known vasodilatory effects of NO. Systolic and diastolic blood pressure both decreased significantly when the NO donor was infused, regardless of the route of infusion, whereas the heart rate rose secondary to hypotension. The drop in blood pressure and the rise in heart rate tended to be modestly greater when SIN-1 was given peripherally. Although markers of NO levels were not measured in the present study, this drop may have been associated with higher levels of SIN-1 in peripheral blood when the compound was infused through a peripheral vein as opposed to the hepatic portal vein.

There are several possible ways that SIN-1 could affect the liver. First, it may act directly on hepatocytes to reduce NHGU. In vivo, hepatocytes are exposed to autogenously derived NO as well as NO derived from nonparenchymal cells (37). Both iNOS and eNOS are expressed in hepatocytes; additionally, iNOS has been found in hepatic Kupffer and Ito cells in various species, whereas nNOS has been localized in some nerve fibers in the rat liver (3, 9, 38). Horton et al. (14) demonstrated that the NO donors SNAP and SIN-1 inhibit gluconeogenesis from isolated rat hepatocytes in a time- and dose-dependent manner. They suggested that the mechanism by which this occurs involves a decrease in the amount of phosphoenolpyruvate carboxykinase protein. On the other hand, Sprangers et al. (36) showed that glycogen synthesis from glucose in rat hepatocytes was inhibited by the NO donor SNAP due to decreased glycogen synthase activity (less conversion of glycogen synthase b into a by synthase phosphatase). They also found that glycogen synthesis is more sensitive to inhibition by NO than is gluconeogenesis (36). Borgs et al. (6) observed that NO infusion (34 µmol/l) increased the rate of glucose output in the perfused rat liver approximately threefold due to a stimulation of glycogenolysis that occurred as a result of activation of glycogen phosphorylase. Similarly, an in vivo study carried out by Ming et al. (21) showed that portal SIN-1 infusion potentiates norepinephrine-induced glucose output from the liver in cats, and this potentiation is blocked by inhibition of guanylate cyclase, a key signaling molecule downstream of NO. Although the underlying mechanisms still remain to be elucidated, it appears that the regulation of hepatic metabolism by NO may involve downstream signals such as protein nitrosylation and/or multiple cGMP-dependent pathways.

It is also possible, however, that SIN-1 reduces NHGU through an indirect effect on the liver. Such an indirect effect could have come about as a result of an increase in sympathetic drive to the liver secondary to the hypotension resulting from intraportal SIN-1 infusion. The fact that an increase in sympathetic drive occurred during SIN-1 infusion is supported by our finding that the heart rate increased in response to the drug and by our observation that lipolysis increased in response to SIN-1, as indicated by the increase in the arterial blood glycerol level. Increased sympathetic input to the liver would have been expected to reduce NHGU (7). In our earlier study, eliminating sympathetic input to the liver by selective sympathetic denervation augmented the NHGU seen in response to elevated glucose and insulin levels (7). The PeSin-1 group was therefore included to assess the possibility that SIN-1 could have an indirect effect on the liver. In the PeSin-1 group, SIN-1 was given via a leg vein to bring about a similar or greater hemodynamic response to that seen with portal vein SIN-1 infusion. In this way, sympathetic input to the liver was increased by an amount equal to or greater than when SIN-1 was given intraportally. In the PeSin-1 group, NHGU averaged ~30 µmol·kg–1·min–1 during P1 and P2 and did not differ from the rate evident during saline infusion, indicating that increased sympathetic input to the liver, if it occurred, did not reduce NHGU. This then indicates that the decrease in NHGU seen when SIN-1 was infused portally was not secondary to hypotension. The increases in heart rate and lipolysis seen in P2 in response to SIN-1 infusion actually tended to be slightly greater when SIN-1 was given in a peripheral vein than when it was given in the portal vein. Despite this, there was no decrease in NHGU or hepatic glycogen synthesis, suggesting that NO regulates hepatic glucose metabolism through a direct action on the liver. Further studies need to be performed to determine the mechanism by which this regulation comes about.

Major extrahepatic glucose consumers include skeletal muscle, adipose tissue, brain, etc. In our previous studies, in which insulin and glucose were elevated in conscious dogs, we found that muscle glucose uptake (measured using a hindlimb balance technique) accounted for a significant portion of the increase in nonhepatic glucose uptake (11). In the present experiments, nonhepatic glucose uptake in response to intraportal SIN-1 infusion was not greater than when saline was given or SIN-1 was infused via a leg vein.

Lautt (17) proposed that the liver produces HISS that the latter enhances muscle glucose uptake and insulin sensitivity. HISS generation is reported to involve the hepatic parasympathetic nervous system, the production of NO, and the interaction of NO with glutathione in the liver (12, 13). In a study carried out by Sadri et al. (32), intraportal, but not intravenous, administration of L-NAME, a nonselective NOS antagonist, significantly reduced whole body insulin sensitivity as measured under euglycemic conditions using the rapid insulin sensitivity test in rats. This suggests that NOS inhibition in the liver resulted in a drop in hepatic NO, which resulted in peripheral insulin resistance. This insulin resistance was reversed by SIN-1 infusion in the portal vein but not in a peripheral vein (31). Thus the insulin resistance that was induced by the inhibition of NOS in the liver was reversed by providing NO directly to the liver. However, our present data do not support the concept that hepatic NO action promotes glucose uptake in peripheral tissues under hyperglycemic conditions. There are several possible explanations as to why this was the case. First, HISS was thought to be active only in the fed state in rodents (34). Thus failure to observe an effect of portal SIN-1 infusion on nonhepatic glucose uptake in 42-h-fasted dogs could be due to the fast duration issue or a species difference. In addition, most HISS experiments have been conducted under euglycemic conditions (12, 13, 17, 24, 31, 32), whereas in the present study, a hyperinsulinemic hyperglycemic clamp was used, and as a result, muscle glucose uptake may have reached a rate that would make a response to a hepatic NO signal hard to detect. The current study sheds no light on the mechanism by which HISS works or the nature of the putative signaling substance "HISS."

In line with our current finding that intraportal infusion of the NO donor SIN-1 reduced NHGU in the presence of hyperinsulinemia and hyperglycemia, normal dogs that received an intraportal infusion of the NOS inhibitor L-NAME exhibited enhanced NHGU under hyperinsulinemic and hyperglycemic conditions (22).

In conclusion, we demonstrate for the first time that intraportal infusion of an NO donor, SIN-1, reduced NHGU and net hepatic carbon retention under hyperglycemic and hyperinsulinemic conditions in conscious 42-h-fasted dogs. The lack of changes in NHGU and glycogen storage when the same hemodynamic response was seen in response to peripheral SIN-1 administration suggests that the effects of portal SIN-1 probably came about as a result of a direct effect of NO on hepatocytes. The molecular mechanism by which NO brings about its hepatic effect needs to be elucidated. The effects on nonhepatic glucose uptake in response to intraportal infusion of SIN-1 were not detected so that total body glucose disposal tends to be reduced by hepatic NO elevation. These findings indicate that hepatic NO may play a role in directing the disposition of glucose reaching the liver via the portal vein, and, by extension, an oral glucose load, into liver.


   GRANTS
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This research was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant RO1 DK-43706 and Vanderbilt University Diabetes Research and Training Center Grant P60 DK-020593.


   ACKNOWLEDGMENTS
 
We appreciate the assistance and support of Jon Hastings, Margaret Lautz, Tiffany Rodewald, Ben Farmer, Jason Winnick, Patsy Raymer, and the Diabetes Research and Training Center Hormone Core in these studies. We also thank Phil Williams for technical assistance.

Part of this work was presented at the 66th Annual Meeting of the American Diabetes Association, Washington DC, June, 2006.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Z. An, 702 Light Hall, Vanderbilt Univ. School of Medicine, Nashville, TN 37232-6015 (e-mail: zhibo.an{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.

* Z. An and C. A. DiCostanzo contributed equally to this work. Back


   REFERENCES
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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