Interaction of free fatty acids and epinephrine in regulating hepatic glucose production in conscious dogs

Chang An Chu, Pietro Galassetti, Kayano Igawa, Dana K. Sindelar, Doss W. Neal, Mark Burish, and Alan D. Cherrington

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES

To determine the effects of an increase in lipolysis on the glycogenolytic effect of epinephrine (EPI), the catecholamine was infused portally into 18-h-fasted conscious dogs maintained on a pancreatic clamp in the presence [portal (Po)-EPI+FFA, n = 6] and absence (Po-EPI+SAL, n = 6) of peripheral Intralipid infusion. Control groups with high glucose (70% increase) and free fatty acid (FFA; 200% increase; HG+FFA, n = 6) and high glucose alone (HG+SAL, n = 6) were also included. Hepatic sinusoidal EPI levels were elevated (Delta 568 ± 77 and Delta 527 ± 37 pg/ml, respectively) in Po-EPI+SAL and EPI+FFA but remained basal in HG+FFA and HG+SAL. Arterial plasma FFA increased from 613 ± 73 to 1,633 ± 101 and 746 ± 112 to 1,898 ± 237 µmol/l in Po-EPI+FFA and HG+FFA but did not change in EPI+SAL or HG+SAL. Net hepatic glycogenolysis increased from 1.5 ± 0.3 to 3.1 ± 0.4 mg · kg-1 · min-1 (P < 0.05) by 30 min in response to portal EPI but did not rise (1.8 ± 0.2 to 2.1 ± 0.3 mg · kg-1 · min-1) in response to Po-EPI+FFA. Net hepatic glycogenolysis decreased from 1.7 ± 0.2 to 0.9 ± 0.2 and 1.6 ± 0.2 to 0.7 ± 0.2 mg · kg-1 · min-1 by 30 min in HG+FFA and HG+SAL. Hepatic gluconeogenic flux to glucose 6-phosphate increased from 0.6 ± 0.1 to 1.2 ± 0.1 mg · kg-1 · min-1 (P < 0.05; by 3 h) and 0.7 ± 0.1 to 1.6 ± 0.1 mg · kg-1 · min-1 (P < 0.05; at 90 min) in HG+FFA and Po-EPI+FFA. The gluconeogenic parameters remained unchanged in the Po-EPI+SAL and HG+SAL groups. In conclusion, increased FFA markedly changed the mechanism by which EPI stimulated hepatic glucose production, suggesting that its overall lipolytic effect may be important in determining its effect on the liver.

gluconeogenesis; glycogenolysis


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES

A RISE IN PLASMA EPINEPHRINE stimulates adipose tissue lipolysis, muscle glycogenolysis, and hepatic glucose production (9, 11, 20, 25, 29). The stimulatory effect of epinephrine on hepatic glucose production is thought to arise both from its direct action on the liver and from its indirect actions on adipose tissue and muscle. The latter results in increased availability of gluconeogenic precursors and free fatty acids (FFA) to the liver (13, 28, 29).

Our recent studies (4, 5) have shown that the direct effect of epinephrine on hepatic glucose production arises from a selective stimulation of glycogenolysis. When epinephrine was infused intraportally so that portal vein epinephrine increased in the absence of a rise in arterial epinephrine, hepatic glycogenolysis rose markedly, whereas gluconeogenesis failed to change. In contrast, when epinephrine was infused peripherally, both arterial and portal vein epinephrine increased, and as a result the catecholamine simultaneously stimulated the liver, muscle, and adipose tissue. The effect of epinephrine on adipose tissue and muscle in turn increased the delivery of FFA and gluconeogenic precursors to the liver. As a result, even though the hepatic sinusoidal epinephrine concentration was the same as when the catecholamine was given portally, it was then able to increase gluconeogenic flux, albeit at the cost of a reduced glycogenolytic effect.

Earlier in vitro studies (32, 33) showed that FFA can increase glucose production by stimulating hepatic gluconeogenesis. Studies of the relationship between FFA elevation and glucose production have also been carried out in humans (2, 3, 17, 24, 26, 30). Saloranta et al. (26) showed that an increase in FFA availability resulting from an infusion of Intralipid increased gluconeogenesis and glucose production in type 2 diabetic patients. In agreement with this, Boden and colleagues (2, 3) showed that, in both normal and type 2 diabetic subjects, increasing and decreasing plasma FFA stimulated and inhibited gluconeogenesis, respectively. Recently Stingl et al. (30) showed that an increase in plasma FFA decreased glycogenolysis in normal men, although plasma insulin rose, making interpretation of the data difficult. The aim of the present study, therefore, was to determine the effect of elevated FFA and glycerol on the glycogenolytic and gluconeogenic actions of portally infused epinephrine.


    METHODS AND MATERIALS
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ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES

Experiments. Experiments were carried out on 24 18-h-fasted conscious mongrel dogs (20-30 kg) of either sex that had been fed a standard diet of meat and chow, as described elsewhere (5). The animals were housed in a facility that met American Association for the Accreditation of Laboratory Animal Care guidelines, and the protocols were approved by the Vanderbilt University Medical Center Animal Care Committee.

A laparotomy was performed 16-18 days before each experiment to implant catheters and Doppler flow probes into or around appropriate blood vessels, as described elsewhere (5). Each dog was used for only one experiment. All dogs studied had 1) a leukocyte count <18,000/mm3, 2) a hematocrit >35%, 3) a good appetite, and 4) normal stools.

Each experiment consisted of a 100-min tracer equilibration and hormone adjustment period (-140 to -40 min), a 40-min basal period (-40 to 0 min), and a 180-min test period (0 to 180 min). In all studies, a priming dose of purified [3-3H]glucose (42 µCi) was given at -140 min, followed by a constant infusion of [3-3H]glucose (0.35 µCi/min), [U-14C]alanine (0.35 µCi/min), and indocyanine green (ICG; 0.1 mg · m-2 · min-1). An infusion of somatostatin (0.8 µg · kg-1 · min-1) was started at -130 min to inhibit endogenous insulin and glucagon secretion. Concurrently, intraportal replacement infusions of insulin (300 µU · kg-1 · min-1) and glucagon (0.5 ng · kg-1 · min-1) were started. The plasma glucose level was monitored every 5 min, and euglycemia was maintained by adjusting the rate of insulin infusion. The final alteration in the insulin infusion rate was made >= 30 min before the start of the basal period, and the rate of insulin infusion (mean of 245 µU · kg-1 · min-1) remained unchanged thereafter. The study included four groups: those maintained on a pancreatic clamp in the presence of portal epinephrine infusion (8 ng · kg-1 · min-1) and the presence [portal (Po)-EPI+FFA, n = 6] or absence (Po-EPI+SAL, n = 6) of peripheral Intralipid infusion and control groups with high glucose (70% increase) and FFA (HG+FFA; 200% increase, n = 6) and high glucose alone (HG+SAL, n = 6) (Fig. 1). Epinephrine (8 ng · kg-1 · min-1), in a solution of 0.07% ascorbic acid, was infused during the test period via the splenic and jejunal vein catheters in the Po-EPI+SAL and Po-EPI+FFA groups. Data from some of the dogs (5 of 6) in the Po-EPI+SAL and HG+SAL groups were published previously (4). In the Po-EPI+FFA and HG+FFA groups, Intralipid (20%, 0.02 ml · kg-1 · min-1) and heparin (0.5 unit · kg-1 · min-1) were infused via a saphenous vein during the epinephrine and glucose infusions, respectively. Arterial glucose levels in the Po-EPI+FFA, HG+FFA, and HG+SAL groups were clamped to the level in the Po-EPI+SAL group by an infusion of exogenous glucose (20% dextrose) given through the right cephalic vein. Blood pressure and heart rate were measured using methods described elsewhere (5).


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Fig. 1.   Protocol. Po, portal; EPI, epinephrine; SAL, saline; FFA, free fatty acid; Po-EPI+SAL and Po-EPI+FFA, 18-h-fasted conscious dogs maintained on a pancreatic clamp in the presence and absence, respectively, of peripheral Intralipid infusion; HG+FFA and HG+SAL, control groups with high glucose (HG) plus FFA and high glucose alone, respectively.

Plasma [3H]- and [14C]-, and [12C]glucose, blood lactate, glycerol, beta -hydroxybutyrate (BOHB), alanine, glutamine, glutamate, glycine, serine, threonine, and plasma FFA were determined using previously described methods (5). Plasma glucose was converted to blood glucose, as previously described, before calculation of net hepatic glucose balance (NHGB) (5). The levels of insulin, glucagon, cortisol, epinephrine, and norepinephrine were also determined as described elsewhere (5).

Doppler flow probes and ICG were used to estimate total hepatic blood flow (5). Because the infusion of Intralipid impaired the ICG absorbance, the data in the figures and tables were those calculated with Doppler-measured flows. The NHGB and fractional extraction of blood glucose, lactate, glycerol, BOHB, alanine, other gluconeogenic amino acids, and plasma FFA in the present study were calculated using arteriovenous difference (a-v) methods described elsewhere (5). The average inflowing hepatic sinusoidal plasma epinephrine levels were calculated using Doppler-determined plasma flow, as previously described (5).

Total glucose production (Ra) and utilization (Rd) were determined using both one- and two-compartment models, as previously described (4, 6). The results were similar regardless of which approach was employed because the deviations from steady state were minimal. The Ra and Rd data shown in the figures and tables are those calculated using the two-compartment method. Endogenous glucose production was calculated by subtracting the glucose infusion rate from Ra. It should also be noted, because the kidneys produce a small amount of glucose, that the rate of endogenous glucose production determined by the tracer method slightly (0.2 mg · kg-1 · min-1) overestimates total hepatic glucose release (18). Because epinephrine was administrated intraportally in the Po-EPI+SAL and Po-EPI+FFA groups and the rise in arterial plasma epinephrine was not significant, epinephrine infusion can be concluded to have had no effect on renal glucose production. Therefore, renal glucose production should have been minimal and equal in the four groups.

Gluconeogenesis, as classically defined, is the synthesis and subsequent release of glucose from noncarbohydrate precursors. Carbon produced from flux through the gluconeogenic pathway does not necessarily have to be released as glucose; it can also be stored in glycogen. Therefore, we make a distinction between gluoneogenic flux to glucose 6-phosphate (G-6-P; conversion of precursors to G-6-P, G-6-P-neogenesis) and gluconeogenesis (release of glucose derived from gluconeogenic flux). In the present studies, we estimated hepatic gluconeogenic flux to G-6-P (in glucose equivalents), net hepatic gluconeogenic flux (in glucose equivalents), and net hepatic glycogenolytic flux.

Gluconeogenic flux to G-6-P (4, 6) was determined by summing the net hepatic uptake rates of the gluconeogenic precursors (alanine, glycine, serine, threonine, glutamine, glutamate, glycerol, lactate, and pyruvate) and dividing by two to account for the incorporation of three carbon precursors into the six-carbon glucose molecule. The validation of this approach has been described elsewhere (14).

Net hepatic gluconeogenic flux was determined by subtracting the summed net hepatic output rates (when such occurred) of the substrates noted above (in glucose equivalents) and hepatic glucose oxidation (HGO) from gluconeogenic flux to G-6-P. HGO was assumed to be 0.2 mg · kg-1 · min-1 in each experiment on the basis of data from our earlier studies in the overnight-fasted dog (15). This parameter was not directly measured, because it is difficult to differentiate between the small signal and the high inherent noise in the measurement and because it changes minimally in response to a variety of signals.

Net hepatic glycogenolytic flux (6, 7) was estimated by subtracting net hepatic gluconeogenic flux from NHGB. A positive number therefore represents net glycogen breakdown, whereas a negative number indicates net glycogen synthesis.

Ideally, the gluconeogenic flux rate would be calculated using unidirectional hepatic uptake and output rates for each substrate, but this would be difficult, as it would require the simultaneous use of multiple stable isotopes, which could themselves induce a mild perturbation of the metabolic state. Therefore, NHGB was used instead, necessitating consideration of the limits of this approach. There is little or no production of gluconeogenic amino acids or glycerol by the liver, so in this case the compromise is of little consequence. Such is, however, not the case for lactate. Our estimate of the rate of gluconeogenic flux to G-6-P will be quantitatively accurate only if we assume that lactate flux is unidirectional at a given moment (i.e., either in or out of the liver). In a given cell, this does not seem like an unreasonable assumption in light of the reciprocal control of gluconeogenesis or glycogenolysis. It has been suggested, however, that there is spatial separation of metabolic pathways so that gluconeogenic periportal hepatocytes synthesize glucose and glycogen primarily from lactate and other noncarbohydrate precursors, whereas glycolytic perivenous hepatocytes generally consume plasma glucose, which is predominantly oxidized or released as lactate. Therefore, it is possible that, under normal nutritional conditions, in a net sense, hepatic gluconeogenic flux and glycolytic flux occur simultaneously, with lactate output or uptake occurring in different cells. To the extent that flux occurs in both directions simultaneously, the arteriovenous (a-v) difference method will underestimate the absolute gluconeogenic flux to G-6-P. It should be noted, however, that net hepatic gluconeogenic flux and net hepatic glycogenolytic flux can be calculated accurately regardless of whether the assumption related to whether or not simultaneous gluconeogenic and glycolytic substrate fluxes occur is valid or not. To the extent that intrahepatic proteolysis or lipolysis contributes to gluconeogenic flux to G-6-P, our approach will underestimate the process and thus overestimate glycogenolysis. Our previous work (15) has shown that the contribution of these two processes in the overnight-fasted dog is minimal.

Gluconeogenic efficiency was assessed using a double-isotope technique described elsewhere (5). Because the conversion of [14C]alanine to [14C]glucose by the kidney is minimal (19), [14C]glucose production was almost exclusively attributable to the liver.

Statistical analysis. All statistical comparisons were made using repeated-measures ANOVA with post hoc analysis by univariate F-tests or the paired Student's t-test where appropriate. Statistical significance was accepted at P < 0.05. Data are expressed as means ± SE.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES

Hormone levels. The arterial plasma levels of insulin, glucagon, norepinephrine, and cortisol remained at basal values in all groups throughout the study (Table 1). The arterial plasma levels of epinephrine did not change significantly during the portal infusion of epinephrine or saline (Fig. 2). The increment in portal vein plasma levels of epinephrine was indistinguishable in the Po-EPI+SAL (32 ± 10 to 687 ± 89 pg/ml; P < 0.05) and the Po-EPI+FFA (50 ± 14 to 659 ± 50 pg/ml; P < 0.05) groups. Similarly, the increments in the hepatic sinusoidal levels of epinephrine were identical in the Po-EPI+SAL group (42 ± 14 to 568 ± 77 pg/ml; P < 0.05) and the Po-EPI+FFA group (57 ± 14 to 527 ± 37 pg/ml; P < 0.05).

                              
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Table 1.   Arterial plasma insulin and glucagon during basal and test periods for Po-EPI+SAL, Po-EPI+FFA, HG+SAL, and HG+FFA groups in the presence of a pancreatic clamp in conscious 18-h-fasted dogs



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Fig. 2.   Arterial (A), portal (B), and hepatic sinusoidal plasma (C) levels of epinephrine during the basal and test periods in experiments conducted using a pancreatic clamp in conscious 18-h-fasted dogs. Values are means ± SE. *P < 0.05 vs. its own basal.

Hepatic blood flow, arterial blood pressure, and heart rate. Hepatic blood flow remained basal and stable in all groups (Table 2). Likewise, the mean arterial blood pressure and heart rate did not change significantly in any group (Table 2).

                              
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Table 2.   Hepatic blood flow, mean arterial blood pressure, and heart rate during basal and test periods for Po-EPI+SAL, Po-EPI+FFA, HG+SAL, and HG+FFA groups in the presence of a pancreatic clamp in conscious 18-h-fasted dogs

Arterial plasma level, net hepatic uptake, and hepatic fractional extraction of FFA. The arterial plasma FFA level remained unchanged in the Po-EPI+SAL and HG+SAL groups (Fig. 3). Peripheral infusion of Intralipid and heparin increased the arterial plasma FFA level from 613 ± 73 to 1,633 ± 101 µmol/l and from 746 ± 112 to 1,898 ± 237 µmol/l (both P < 0.05) by the end of the study in the Po-EPI+FFA and the HG+FFA groups, respectively (Fig. 3).


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Fig. 3.   Arterial plasma level (A) as well as net hepatic uptake (B) and fractional extraction (C) of FFA during the basal and test periods in experiments conducted using a pancreatic clamp in conscious 18-h-fasted dogs. Values are means ± SE. *P < 0.05 vs. its own basal.

Net hepatic uptake of FFA remained unchanged in the Po-EPI+SAL and HG+SAL groups but increased from 2.5 ± 0.4 to 5.3 ± 0.7 µmol · kg-1 · min-1 and from 2.5 ± 0.4 to 4.4 ± 0.8 µmol · kg-1 · min-1 (both P < 0.05) in the Po-EPI+FFA and HG+FFA groups, respectively (Fig. 3). Net hepatic fractional extraction of FFA did not change significantly in any group (Fig. 3).

Arterial blood level and net hepatic balance of glycerol and BOHB. The arterial blood glycerol level remained unchanged in the Po-EPI+SAL and HG+SAL groups (Fig. 4). Peripheral infusion of Intralipid and heparin increased arterial blood level of glycerol from 67 ± 5 to 202 ± 14 µmol/l and 76 ± 8 to 182 ± 10 µmol/l (both P < 0.05) in the Po-EPI+FFA and HG+FFA groups, respectively. Net hepatic glycerol uptake remained unchanged in the Po-EPI+SAL and HG+SAL groups but increased from 1.5 ± 0.2 to 3.7 ± 0.6 µmol · kg-1 · min-1 and from 1.5 ± 0.3 to 3.2 ± 0.3 µmol · kg-1 · min-1 (both P < 0.05) in the Po-EPI+FFA and HG+FFA groups, respectively (Fig. 4).


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Fig. 4.   Arterial blood levels and net hepatic balances of glycerol and beta -hydroxybutyrate (BOHB) during the basal and test periods in experiments conducted using a pancreatic clamp in conscious 18-h-fasted dogs. Values are means ± SE. *P < 0.05 vs. its own basal.

The arterial blood level of BOHB remained unchanged in the Po-EPI+SAL and HG+SAL groups throughout (Fig. 4). Peripheral infusion of Intralipid increased arterial blood BOHB from 15 ± 3 to 33 ± 6 (120 min) µmol/l and from 28 ± 6 to 46 ± 13 (90 min) µmol/l (both P < 0.05) in the Po-EPI+FFA and HG+FFA groups, respectively, after which it remained stable. Net hepatic output of BOHB remained unchanged in the Po-EPI+SAL and HG+SAL groups (Fig. 4). On the other hand, it increased from 0.5 ± 0.1 to 1.2 ± 0.4 µmol · kg-1 · min-1 (P < 0.05) in the Po-EPI+FFA group but did not change significantly (0.9 ± 0.3 to 1.2 ± 0.4 µmol · kg-1 · min-1) in the HG+FFA group (Fig. 4).

Arterial blood levels and net hepatic balances of lactate and gluconeogenic amino acids. The arterial blood lactate level did not change significantly in any protocol. In response to glucose infusion alone, the liver gradually switched to net lactate production. In response to portal epinephrine infusion alone, the liver immediately switched to net lactate output and remained producing lactate throughout the experiment. In the presence of glucose infusion, Intralipid infusion increased net hepatic lactate uptake to 6.1 ± 1.6 µmol · kg-1 · min-1 (Fig. 5; P < 0.05). In the presence of portal epinephrine infusion, Intralipid infusion caused an even greater increase in net hepatic lactate uptake (8.6 ± 1.5 µmol · kg-1 · min-1, P < 0.05; Fig. 5).


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Fig. 5.   Arterial blood levels (A) and net hepatic balances (B) of lactate during the basal and test periods in experiments conducted using a pancreatic clamp in conscious 18-h-fasted dogs. Values are means ± SE. *P < 0.05 vs. its own basal.

The fractional extraction of alanine increased 41 and 61% (both P < 0.05) in the Po-EPI+SAL and Po-EPI+FFA groups, respectively, during the 3-h test period (Table 3). Neither the arterial blood alanine level nor the net hepatic alanine balance changed significantly in any group (Table 3). Similarly, the arterial blood levels, the net hepatic balances, and the fractional extractions of glutamate, glutamine, glycine, serine, and threonine were not significantly changed at any point in any group (Table 3).

                              
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Table 3.   Arterial blood levels and net hepatic balances of alanine, glutamate, glutamine, glycine, serine, and threonine during basal and test periods for Po-EPI+SAL, Po-EPI+FFA, HG+SAL, and HG+FFA groups in the presence of a pancreatic clamp in conscious 18-h-fasted dogs

Glucose kinetics. The increment in the arterial blood glucose level was similar in all four groups (Fig. 6). Hyperglycemia per se decreased net hepatic glucose output from 2.1 ± 0.2 to 0.7 ± 0.4 mg · kg-1 · min-1 within 60 min (P < 0.05), where it remained. Net hepatic glucose output decreased to a similar extent (2.1 ± 0.2 to 0.8 ± 0.3 mg · kg-1 · min-1; P < 0.05) in the HG+FFA group. Portal infusion of epinephrine increased net hepatic glucose output from 2.1 ± 0.3 to 3.1 ± 0.6 mg · kg-1 · min-1 within 30 min (P < 0.05), after which it fell back to the basal rate by 60 min. In the presence of Intralipid infusion, the increment in portal epinephrine increased net hepatic glucose output only from 1.9 ± 0.2 to 2.5 ± 0.3 mg · kg-1 · min-1 within 30 min, after which it did not change. The changes in tracer-determined endogenous glucose production mirrored those observed in net hepatic glucose output (Fig. 6).


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Fig. 6.   Arterial blood level (A) and net hepatic output of glucose (B) as well as tracer-determined glucose production (C) during the basal and test periods in experiments conducted using a pancreatic clamp in conscious 18-h-fasted dogs. Values are means ± SE. *P < 0.05 vs. its own basal.

Tracer-determined glucose utilization increased from 2.6 ± 0.3 to 3.8 ± 0.4 (P < 0.05), from 2.5 ± 0.3 to 3.1 ± 0.2 [not significant (NS)], from 2.4 ± 0.2 to 3.5 ± 0.3 (P < 0.05), and from 2.8 ± 0.3 to 3.8 ± 0.4 mg · kg-1 · min-1 (P < 0.05) in the Po-EPI+SAL, Po-EPI+FFA, HG+SAL, and HG+FFA groups, respectively (Table 4). The whole body glucose clearance tended to decrease in the presence of an elevated FFA level (Delta 0.5 ml · kg-1 · min-1 in the Po-EPI+FFA and HG+FFA groups) but remained unchanged in the absence of FFA infusion (Table 4).

                              
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Table 4.   Tracer-determined Rd and clearance during basal and test periods for Po-EPI+SAL, Po-EPI+FFA, HG+SAL, and HG+FFA groups in the presence of a pancreatic clamp in conscious 18-h-fasted dogs

Gluconeogenesis and glycogenolysis. Hepatic gluconeogenic flux to G-6-P did not change significantly at any time in either the Po-EPI+SAL or the HG+SAL group (Fig. 7). Peripheral infusion of Intralipid and heparin increased hepatic gluconeogenic flux to G-6-P from 0.7 ± 0.2 to 1.6 ± 0.3 mg · kg-1 · min-1 by 90 min and from 0.6 ± 0.2 to 1.2 ± 0.2 mg · kg-1 · min-1 by 150 min (Fig. 7, both P < 0.05) in the Po-EPI+FFA and HG+FFA groups, respectively. By the end of the study, net hepatic gluconeogenic flux was 0.2 ± 0.1 and 0.2 ± 0.2 mg · kg-1 · min-1 in the HG+SAL and Po-EPI+Sal groups, respectively, indicating that flux up the gluconeogenic pathway and down the glycolytic pathway were essentially in balance. FFA increased net hepatic gluconeogenic flux to 1.0 ± 0.2 and 1.2 ± 0.3 mg · kg-1 · min-1 in the HG+FFA and EPI+FFA groups, respectively, indicating a significant shift in favor of net gluconeogenic flux. Hepatic gluconeogenic efficiency did not change significantly in either the Po-EPI+SAL or the HG+SAL group (Fig. 7). On the other hand, peripheral infusion of Intralipid increased hepatic gluconeogenic efficiency by 160 ± 10 and 75 ± 6% (both P < 0.05; Fig. 7) in the Po-EPI+FFA and HG+FFA groups, respectively. Overall hepatic gluconeogenic flux to G-6-P contributed 116 ± 14 mg/kg during the 3-h test period in the HG+FFA group. Portal infusion of epinephrine failed to have an impact on the rate (127 ± 21 mg/kg for 3 h). Peripheral infusion of Intralipid, on the other hand, increased overall hepatic gluconeogenic flux to G-6-P by 119 ± 18 mg/kg (103%; P < 0.05) and 67 ± 15 mg/kg (58%; P < 0.05) in the Po-EPI+FFA and HG+FFA groups, respectively (Fig. 8).


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Fig. 7.   Hepatic gluconeogenic flux rate (A), change in gluconeogenic efficiency (B), and net hepatic glycogenolysis (C) during the basal and test periods in experiments conducted using a pancreatic clamp in conscious 18-h-fasted dogs. Values are means ± SE. *P < 0.05 vs. its own basal.



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Fig. 8.   Increase in overall gluconeogenic and glycogenolytic rates as well as the change in net hepatic glycogenolytic rate (at 30 min) during the test period in experiments conducted using a pancreatic clamp in conscious 18-h-fasted dogs. Values are means ± SE. *P < 0.05 vs. HG group. #P < 0.05 vs. other groups in the same panel. G-6-P, glucose 6-phosphate.

Hyperglycemia per se decreased the net hepatic glycogenolytic rate from 1.6 ± 0.2 to 0.7 ± 0.2 mg · kg-1 · min-1 within 30 min (Fig. 7, P < 0.05), after which it slowly drifted back to 0.9 ± 0.2 mg · kg-1 · min-1 by the end of the study. Peripheral infusion of Intralipid in the presence of hyperglycemia abolished net hepatic glycogenolysis (from 1.7 ± 0.2 to -0.1 ± 0.1 mg · kg-1 · min-1, P < 0.05) by the end of the study. Portal infusion of epinephrine increased the net hepatic glycogenolytic rate from 1.5 ± 0.3 to 3.1 ± 0.4 mg · kg-1 · min-1 within 30 min (P < 0.05), after which it slowly drifted back to 1.9 ± 0.3 mg · kg-1 · min-1 (Fig. 7). When Intralipid infusion was added to portal epinephrine infusion, the net glycogenolytic rate rose from 1.8 ± 0.2 to 2.1 ± 0.3 mg · kg-1 · min-1 and then fell to 1.2 ± 0.2 mg · kg-1 · min-1 by 180 min (Fig. 7). Overall net hepatic glycogenolysis in the HG+SAL group contributed 121 ± 15 mg/kg over the 3-h test period. Relative to this, it increased by 253 ± 31 mg/kg (209%; P < 0.05) and by 97 ± 11 mg/kg (80%; P < 0.05), respectively, in the Po-EPI+SAL and Po-EPI+FFA groups (Fig. 8). It decreased by 38 ± 6 mg/kg (31%; P < 0.05) in the HG+FFA group (Fig. 8).


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES

Previous studies (9, 25, 29) have shown that epinephrine simultaneously increases lipolysis and hepatic glucose production. Normally, a rise in the hepatic sinusoidal epinephrine level is accompanied by a concomitant rise in arterial epinephrine and, as a result, by an increase in the plasma FFA level. In recent studies (4, 5), we showed that a selective increase in the hepatic sinusoidal epinephrine level (occurring without an increase in arterial epinephrine) increased net hepatic glycogenolysis but had no effect on hepatic gluconeogenesis. The aim of the present study was to assess the effects of a simulated rise in lipolysis on epinephrine's ability to increase net hepatic glycogenolysis and/or gluconeogenesis in vivo. The arterial levels of insulin, glucagon, epinephrine, norepinephrine, and cortisol remained at basal values in all four groups. Additionally, the arterial blood glucose concentrations were kept at a similar hyperglycemic level in each group, thereby simplifying data interpretation. Finally, the increments in the hepatic portal and sinusoidal epinephrine levels were indistinguishable in the Po-EPI+SAL and Po-EPI+FFA groups. As a result, we were able to clearly examine the effects of a rise in FFA and glycerol on epinephrine's hepatic glycogenolytic and gluconeogenic actions.

Intralipid and heparin infusion increased the arterial level and net hepatic uptake of FFA ~2.5- and 1.8-fold, respectively, in the presence of hyperglycemia (HG+FFA). Hepatic gluconeogenic flux to G-6-P and gluconeogenic efficiency were increased by ~100% (0.6 ± 0.2 to 1.2 ± 0.2 mg · kg-1 · min-1, P < 0.05) and 75% (33 ± 5% to 58 ± 10%, P < 0.05), respectively. Neither hepatic gluconeogenic flux to G-6-P nor gluconeogenic efficiency was significantly changed in the presence of hyperglycemia alone. It is clear, therefore, that Intralipid infusion had a stimulatory effect on hepatic gluconeogenesis in the presence of hyperglycemia in vivo. This is in line with previous in vitro findings (32, 33), which indicated that a rise in the availability of FFA in liver increased gluconeogenesis. It should be noted that, in the present study, the arterial blood level and net hepatic uptake of glycerol increased from 76 ± 8 to 182 ± 10 µmol/l (P < 0.05) and from 1.5 ± 0.3 to 3.2 ± 0.3 µmol · kg-1 · min-1 (P < 0.05), respectively, during the infusion of Intralipid. However, even if all of the extra glycerol taken up by the liver were entirely converted to glucose (Delta 1.7 µmol · kg-1 · min-1 glycerol approx  Delta 0.15 mg · kg-1 · min-1 glucose), it could account for only 25% of the observed increase in net gluconeogenic flux (0.6 ± 0.2 to 1.2 ± 0.2 mg · kg-1 · min-1). Therefore, the data clearly indicate that the increase in net hepatic uptake of FFA results in a stimulation of hepatic gluconeogenic flux to G-6-P even in the presence of hyperglycemia. This enhancement of gluconeogenic flux to G-6-P is also evident from the observation that the increase in net hepatic uptake of FFA was associated with a significant increase in net hepatic lactate uptake.

Hyperglycemia per se increased liver glycolysis and thus increased net hepatic lactate output. Addition of Intralipid switched net hepatic lactate balance from output to uptake. Because both the arterial levels and net hepatic output of BOHB increased during Intralipid infusion, it is apparent that beta -oxidation of FFA in the liver increased. Increased beta -oxidation could stimulate gluconeogenic flux by producing more ATP for the support of gluconeogenesis, by increasing the availability of the NADH for the glyceraldehyde-3-phosphate dehydrogenase reaction, and by activating pyruvate carboxylase via an increase in acetyl-CoA and other thioesters (1, 22, 23). In addition, the increase in citrate that occurs in response to increased FFA could inhibit phosphofructokinase (PFK), thus at the same time limiting glycolysis (22). It should be noted that an inhibition of glycolysis would increase the net flux of carbon to G-6-P even if hepatic lactate production and uptake do occur simultaneously, as suggested by Radziuk and Pye (21). It has also been suggested that increased intracellular fatty acid metabolites (fatty acyl-CoAs, diacylglycerol, ceramides) can cause defects in insulin signaling in the liver (27).

Because neither hepatic gluconeogenic flux to G-6-P, net hepatic gluconeogenic flux, nor overall hepatic gluconeogenic efficiency was significantly changed in the Po-EPI+SAL group, the present data are consistent with our earlier finding (4) that a selective increase in hepatic sinusoidal epinephrine alone has no effect on hepatic gluconeogenesis. The addition of Intralipid infusion to portal epinephrine infusion, however, resulted in an increase in hepatic gluconeogenic flux to G-6-P from 0.7 ± 0.2 to 1.6 ± 0.3 mg · kg-1 · min-1 (230%, P < 0.05). Because the increment in overall (3 h) gluconeogenic flux to G-6-P was greater (119 ± 18 mg/kg) in the Po-EPI+FFA group than in the HG+FFA group (67 ± 15 mg/kg), even though both groups had similar increases in net hepatic FFA uptake, it is clear that the increase in FFA caused a gluconeogenic effect of epinephrine to be manifested. It is possible that epinephrine stimulated beta -oxidation (26, 30) and thereby further increased gluconeogenic flux. Indeed, the change in ketogenesis was greater in the Po-EPI+FFA group than in the HG+FFA group. In addition, both net hepatic lactate uptake and the hepatic fractional extraction of alanine increased to the greatest extent in response to Intralipid infusion in the presence of portal epinephrine infusion. Once again, an increase in net hepatic glycerol uptake can explain only part of the increase in gluconeogenic flux. Even if all of the extra glycerol taken up by the liver were converted to glucose (Delta 2.2 µmol · kg-1 · min-1 glycerol approx  Delta 0.2 mg · kg-1 · min-1 glucose), it could account for only 22% of the increase in net gluconeogenic flux (0.7 ± 0.2 to 1.6 ± 0.3 mg · kg-1 · min-1). Therefore, the present data strongly suggest that the ability of increased lipolysis to allow expression of epinephrine's gluconeogenic action at the liver results from the stimulatory effect of FFA.

The increment in hepatic sinusoidal epinephrine increased net hepatic glycogenolysis by 1.5 ± 0.2 mg · kg-1 · min-1 by 30 min. Because hyperglycemia per se decreased net hepatic glycogenolysis by 0.9 ± 0.2 mg · kg-1 · min-1 at that time, the effect of the selective increase in hepatic sinusoidal epinephrine on net hepatic glycogenolysis was 2.4 ± 0.2 mg · kg-1 · min-1 (30 min; Figs. 7 and 8). In the presence of Intralipid infusion, the same increment in hepatic sinusoidal epinephrine raised net hepatic glycogenolysis above baseline by only 0.3 ± 0.1 mg · kg-1 · min-1 at 30 min. Therefore, Intralipid infusion diminished the effect of the increment in hepatic sinusoidal epinephrine on net hepatic glycogenolysis to 1.2 ± 0.2 mg · kg-1 · min-1 (50% inhibition). The overall (3-h) net glycogenolytic effect attributable to the rise in portal epinephrine was 253 ± 31 mg/kg. However, this was diminished to 97 ± 11 mg/kg when Intralipid infusion was added to the rise in portal epinephrine. Therefore, a 62% inhibition was observed when the overall (3-h) impact was examined. One can thus conclude that a rise in the circulating FFA level can inhibit the glycogenolytic effect of epinephrine on hepatic glucose production by more than one-half. This suggests that, when plasma epinephrine increases due to adrenal secretion, its effect on the adipose tissue significantly reduces the glycogenolytic effect that it would otherwise have on the liver. Because glycogen and fat are the two major fuel stores used by the body for energy, the physiological significance of this inhibition might be to preserve the liver glycogen content to allow a timely response to an unexpected need by the central nervous system when glucose is scarce, such as during fasting, hypoglycemia, or prolonged exercise.

The next question is how a high level of FFA can bring about an inhibitory effect on glycogen breakdown. Previous in vivo studies in the dog (10, 12) and human (16, 34) showed that infusing gluconeogenic precursors, i.e., alanine, lactate, and glycerol, increased hepatic gluconeogenesis but did not change hepatic glucose production. They therefore suggested that a rise in gluconeogenesis itself may trigger an inhibition of hepatic glycogenolysis (8). Because the initial change (i.e., 30 min) in hepatic gluconeogenic flux was minimal, regardless of the protocol (the time at which the glycogenolytic effect was fully manifested), it seems likely that the inhibitory effect of high FFA levels on epinephrine's glycogenolytic action resulted from a direct inhibition associated with the rapid increase in fat oxidation by the liver. Indeed, an increase in FFA oxidation would increase citrate levels, raising the possibility that it might inhibit PFK and blunt glycogen breakdown.

In conclusion, the data from the present studies indicate that epinephrine's lipolytic action limits its glycogenolytic effect and augments its gluconeogenic/glycolytic effects on the liver in vivo. Clearly, the impact of epinephrine on the liver is a function of both its direct actions on the liver and the metabolite changes that accompany the catecholamine to the liver.


    ACKNOWLEDGEMENTS

We are grateful to Drs. Mary Courtney Moore, David Wasserman, Dale Edgerton, and Owen P. McGuinness for valuable comments and careful review of this manuscript. We specially appreciate assistance from Jon Hastings, Melanie Scott, Maya Emshwiller, Pam Venson, Wanda Snead, Paul Flakoll, and Yang Ying.


    FOOTNOTES

This research was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grants 2RO1 DK-18243 and 5P60 DK-20593 (Diabetes Research and Training Center), and National Institutes of Health Grant 2P30 DK-26657 (Nutrition Center).

Address for reprint requests and other correspondence: A. D. Cherrington, Dept. of Molecular Physiology and Biophysics, 702 Light Hall, Vanderbilt University School of Medicine, Nashville, TN 37232-0615 (E-mail: alan.cherrington{at}mcmail.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.

10.1152/ajpendo.00565.2001

Received 28 December 2001; accepted in final form 19 August 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES

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