Effects of fasting and glucocorticoids on hepatic gluconeogenesis assessed using two independent methods in vivo

Transcription

1 Am J Physiol Endocrinol Metab 283: E946 E957, 2002; /ajpendo Effects of fasting and glucocorticoids on hepatic gluconeogenesis assessed using two independent methods in vivo RICHARD E. GOLDSTEIN, 1 LUCIANO ROSSETTI, 3 BRETT A. J. PALMER, 1 RONG LIU, 3 DUNA MASSILLON, 3 MELANIE SCOTT, 2 DOSS NEAL, 2 PHILLIP WILLIAMS, 2 BENJAMIN PEELER, 1 AND ALAN D. CHERRINGTON 2 1 Department of Surgery, Vanderbilt University, and the Nashville VA Medical Center; 2 Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37232; and 3 Division of Endocrinology, Department of Medicine, Albert Einstein College of Medicine, New York, New York Received 16 July 2002; accepted in final form 17 July 2002 Goldstein, Richard E., Luciano Rossetti, Brett A. J. Palmer, Rong Liu, Duna Massillon, Melanie Scott, Doss Neal, Phillip Williams, Benjamin Peeler, and Alan D. Cherrington. Effects of fasting and glucocorticoids on hepatic gluconeogenesis assessed using two independent methods in vivo. Am J Physiol Endocrinol Metab 283: E946 E957, 2002; /ajpendo The purpose of this study was to compare the assessment of gluconeogenesis (GNG) in the overnight- and prolonged-fasted states and during chronic hypercortisolemia using the arteriovenous difference and [ 14 C]phosphoenolpyruvate-liver biopsy techniques as well as a combination of the two. Two weeks before a study, catheters and flow probes were implanted in the hepatic and portal veins and femoral artery of dogs. Animals were studied after an 18-h fast (n 8), a 42- or 66-h fast (n 7), and an 18-h fast plus a continuous infusion of cortisol (3.0 g kg 1 min 1 ) for 72 h (n 7). Each experiment consisted of an 80-min tracer ([3-3 H]glucose and [U- 14 C]alanine) and dye equilibration period ( 80 to 0 min) and a 45-min sampling period. In the cortisoltreated group, plasma cortisol increased fivefold. In the overnight-fasted group, total GNG flux rate (GNG flux), conversion of glucose 6-phosphate to glucose (GNG G-6-P3Glc ), glucose cycling, and maximal GNG flux rate (GNG max ) were , , , and mg kg 1 min 1, respectively. In the prolonged-fasted group, they were , , , and mg kg 1 min 1, whereas in the cortisol-treated group they were , , , and mg kg 1 min 1. These results demonstrate that GNG G-6-P3Glc and GNG max were almost identical. However, these rates were 15 38% lower than GNG flux generated by a combination of the two methods. This difference was most apparent in the steroid-treated group, where the combination of the two methods (GNG flux ) detected a significant increase in gluconeogenic flux. fasting; cortisol THE QUEST TO DEVELOP accurate methods for determining the rate of gluconeogenesis in vivo is ongoing. Many early studies used tracer methods or arteriovenous Address for reprint requests and other correspondence: A. D. Cherrington, 710 RRB, Vanderbilt Univ. Medical Center, Nashville, TN ( Alan.Cherrington@mcmail.vanderbilt.edu). E946 (a-v) difference techniques to assess the gluconeogenic rate in vivo (12, 23, 27, 28). In 1977, Chiasson et al. (5) combined these two approaches for the assessment of gluconeogenesis in the dog. The latter approach yielded two estimates of gluconeogenesis, the gluconeogenic efficiency (percent extracted gluconeogenic carbon converted to glucose) and the gluconeogenic conversion rate (the rate of glucose production from the gluconeogenic precursor given). Both represented minimal estimates of gluconeogenesis due largely to dilution of the tracer in the oxaloacetate (OAA) pool of the liver (25). This approach was further extended by calculating the minimal and maximal rates of gluconeogenesis from circulating gluconeogenic precursors (3, 16, 51). The maximal rate was derived by measuring the net hepatic uptakes of all gluconeogenic precursors using the a-v difference technique and assuming that they were quantitatively converted to glucose. The minimal rate was derived by multiplying the maximal rate of gluconeogenesis by gluconeogenic efficiency (which is itself a minimum). With both of these rates known, the true gluconeogenic rate from circulating precursors could be bracketed. It should be noted that this approach underestimates the actual maximal rate of hepatic gluconeogenesis by an amount equal to the contribution of precursors derived from within the liver, namely from intrahepatic proteolysis and/or lipolysis. Recently, Giaccari and Rossetti (13) and Rossetti et al. (47) described a method which permits one to quantify hepatic gluconeogenesis in a more direct manner. Like that of Cherrington et al. (3), the method utilizes the infusion of a labeled gluconeogenic precursor, such as [ 14 C]alanine, but also requires a biopsy of hepatic tissue. HPLC analysis is used to determine the intrahepatic specific activities of uridine diphosphoglucose (UDPglucose) and phosphoenolpyruvate (PEP) (14). 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.

2 ASSESSMENT OF GLUCONEOGENESIS IN VIVO E947 The direct assessment of PEP specific activity overcomes the major limitation of tracer methods previously used to estimate gluconeogenesis, the dilution of tracer in the OAA pool. This information is also used to calculate the rates of hepatic glucose cycling and the net glycogenolytic contribution to hepatic glucose release. The requirement for a liver biopsy limits the applicability of the method; nevertheless, it represents a viable and independent alternative method for determining the rate of hepatic gluconeogenesis in experimental animals. It should be noted, however, that it measures hepatic gluconeogenesis from lactate, pyruvate, and amino acids (both circulating and intrahepatic) but does not account for gluconeogenesis from glycerol. Thus it also underestimates the total hepatic gluconeogenic rate to a certain extent. The first aim of the present study, therefore, was to compare the results obtained using the a-v difference technique with those obtained simultaneously using the methodology of Rossetti and Giaccari ([ 14 C]PEP technique) under conditions of overnight fasting, which favors glycogenolysis, and more prolonged fasting, which favors gluconeogenesis. The second aim was to combine information gained from these two methodologies to develop a new, more powerful approach to quantify hepatic gluconeogenesis in vivo. Goldstein et al. (15) previously reported that chronic hypercortisolemia caused only a marginal increase in gluconeogenesis as assessed using the combined tracer a-v difference technique but that it increased glucose cycling drastically. This finding was surprising in view of in vitro observations (8, 11) and in vivo data (11, 24) that characterize cortisol as a gluconeogenic hormone. Therefore, an additional aim of the present study was to assess the gluconeogenic effects of hypercortisolemia in vivo using the combined a-v difference-[ 14 C]PEP approach and to determine whether the combination of the methodologies would yield further insight into the intrahepatic gluconeogenic fluxes associated with hypercortisolemia. RESEARCH DESIGN AND METHODS Animals and surgical procedures. Experiments were performed on a total of 22 mongrel dogs (16 27 kg) of either gender that had been fed a weight-maintaining diet of meat and chow (Purina Mills, St. Louis, MO and KalKan, Vernon, CA; 31% protein, 52% carbohydrate, 11% fat, and 6% fiber based on dry weight once daily). Animal preparation has been extensively described in previous articles (1, 15). In brief, under general endotrachial anesthesia, a sampling catheter was placed directly in the portal vein, and its tip was positioned 1 in. caudal to the portal vein s entry into the liver. Sampling catheters were also placed in the left common hepatic vein and the left femoral artery. Ultrasonic perivascular flow probes (Transonic Systems, Ithaca, NY) were placed around the portal vein (7.0 mm in diameter) and the common hepatic artery (3.0 mm in diameter). An infusion catheter was placed in the left jugular vein of all dogs that were to receive chronic cortisol infusion. Three days before an experiment in those animals designated to receive cortisol, the jugular infusion catheter was exteriorized from its subcutaneous pocket through a small incision made under local anesthesia (2% xylocaine; Astra Pharmaceutical Products, Worcester, MA). It was then connected to an infusion pump (Walkmed 2000; Infumed Medex, Broomfield, CO), and the pump was placed in a jacket worn by the dog while it moved freely about its cage. On the day of the experiment, the ends of the catheters and flow probes were exteriorized from the subcutaneous pocket under local anesthesia (2% xylocaine, Astra Pharmaceutical Products). Angiocaths were inserted into the cephalic veins for peripheral infusion of tracers. The conscious dogs were then allowed to stand calmly in a Pavlov harness for 30 min before the start of the experiment. Animals in this study were maintained, and experiments performed, in accordance with the guidelines of the Animal Care Committee of Vanderbilt University in a facility accredited by the American Association for Accreditation of Laboratory Animal Care. Experimental procedures. Each experiment consisted of an 80-min tracer and dye equilibration period ( 80 to 0 min) followed by a 45-min sampling period. At time 80 min, a primed (63 Ci), constant infusion of [3-3 H]glucose (0.88 Ci/min) and a constant infusion of [U- 14 C]alanine (6.5 Ci/ min) were started in the cephalic vein and continued throughout the experiment. In addition, at 80 min, a constant infusion of indocyanine green (0.1 mg m 2 min 1 ) was started via the cephalic vein. Animals were studied after an overnight (18 h) fast (Overnight Fast; n 8), after a prolonged (42 or 66 h) fast (Prolonged Fast; n 7), or after an 18-h fast plus a chronic cortisol infusion (18-h Fast Cortisol; n 7). In the last protocol, a continuous intravenous infusion of hydrocortisone (3.0 g kg 1 min 1 ) was begun 3 days before the experiment and continued throughout the experiment. It should be noted that four dogs were fasted for 42 h and three dogs were fasted for 66 h with no discernible differences; thus the data were combined. Blood from the femoral artery, hepatic vein, and portal vein was sampled six times during the 45-min sampling period. Immediately after the final sampling time, the animals were anesthetized with pentobarbital sodium but not killed. They were removed from the Pavlov harness while the tracers continued to infuse. A midline laparotomy incision was made, the liver was exposed, and two clamps precooled in liquid nitrogen were used to simultaneously obtain biopsies in situ from the two largest hepatic lobes (within 2 min of anesthesia). The hepatic tissue was immediately removed, placed in liquid nitrogen, and stored at 70 C. The animals were then euthanized using pentobarbitol sodium (390 mg/ml) and necropsied to confirm proper positioning of the sampling catheters. Collection and processing of blood samples. Plasma glucose concentrations were determined using the glucose oxidase method in a Beckman glucose analyzer (Beckman Instrument, Fullerton, CA). Plasma glucose radioactivity ( 3 H and 14 C) was determined by liquid scintillation counting after deproteinization with barium hydroxide and zinc sulfate (49), glucose isolation with cation and anion exchange resins, and reconstitution in 1 ml of distilled water and 10 ml of Ecolite (ICN, Costa Mesa, CA). Concentrations of indocyanine green were determined spectrophotometrically (805 nm) in arterial and hepatic vein plasma samples. Whole blood glucose values were assumed to equal 73% of plasma values on the basis of extensive comparisons between whole blood and plasma glucose in the dog (1). This yielded an accurate estimate of net hepatic glucose output (NHGO) regardless of glucose exchange between red blood cell and plasma water. Whole blood lactate, alanine, glycerol, and -hydroxybutyrate concentrations were determined in samples deproteinized with 4%

3 E948 ASSESSMENT OF GLUCONEOGENESIS IN VIVO perchloric acid according to the method developed by Lloyd et al. (26) for the Technicon AutoAnalyzer. Whole blood glutamine and glutamate concentrations were determined according to methods developed for the AutoAnalyzer (54). Nonesterified fatty acid (NEFA) concentrations were determined using the Wako NEFA C reagent kit (Wako Chemicals, Richmond, VA). Immunoreactive insulin was measured using a double-antibody RIA (39). Immunoreactive glucagon was measured using a modification of the double-antibody insulin method (39). Insulin and glucagon antibodies and 125 I tracers were obtained from Linco Research (St. Louis, MO). Cortisol was determined by RIA (Clinical Assays Gamma Coat Radioimmunoassay Kit). Plasma epinephrine and norepinephrine levels were determined by HPLC (37). Labeled and unlabeled plasma alanine and lactate concentrations were determined with a short-column ion exchange chromatographic system that has been described previously (4). Amino acid concentrations were measured by reverse-phase HPLC using a modified version of the methods of Bidlingmeyer et al. (2) and Heinrikson and Meredith (18). UDPglucose and PEP concentrations and specific activities in the liver were obtained in the Rossetti laboratory through two sequential chromatographic separations by a modification of a method previously described (13, 47). The major modifications from the original method (14) were in the HPLC columns, mobile phases, and run conditions. Two C 18T columns (Supelco) were used in series for all analyses. The mobile phase (1 ml/min) was a 200 mm potassium phosphate buffer at ph 4.5 for PEP and a 100 mm potassium phosphate buffer at ph 6.5 for UDPglucose. The ion-pairing agent was tetrabutyl hydrogen sulfate at 5 mm. Materials. [3-3 H]glucose (New England Nuclear, Boston, MA) was used as the glucose tracer (500 Ci/0.005 mg), and [U- 14 C]alanine (Amersham, Chicago, IL) was used as the labeled gluconeogenic precursor (171 mci/mmol). Indocyanine green was purchased from Hynson, Westcott, and Dunning (Baltimore, MD) to measure hepatic blood flow (HBF). The [3-3 H]glucose infusate contained cold glucose so that its final concentration was 1 mg/ml. The indocyanine green infusate was prepared with sterile water, and the [U- 14 C]alanine and [3-3 H]glucose infusates were prepared with 0.82% NaCl. Calculations. In these studies, ultrasonic flow probes were used to directly determine hepatic artery and portal vein blood flow. When flow probes were not functioning properly (i.e., n 3), indocyanine green dye was used to assess total hepatic plasma flow (HPF). When indocyanine green dye was used, the proportion of HBF provided by the hepatic artery was assumed to be 20% on the basis of data compiled in our laboratory over many years with the use of Doppler flow probes in the dog. In the case of indocyanine green, HBF was derived from the HPF: (HBF HPF/1 hematocrit). Net hepatic balances of glucose, alanine, lactate, glycerol, glutamine, glutamate, -hydroxybutyrate, and gluconeogenic amino acids were determined by the formula [(F a C a) (F p C p) (F h C h)], where F a,f p, and F h represent blood flow (ml kg 1 min 1 ) in the hepatic artery, portal vein, and hepatic vein, respectively, and C a,c p, and C h represent the blood metabolite concentrations in the three respective vessels. Net hepatic glucose balance was determined as above after the plasma glucose levels were converted to blood glucose levels (1). Net hepatic fractional extraction of a metabolite was determined by the formula [(Fr a C a) (Fr p C p) C h]/[(fr a C a) (Fr p C p)], where Fr a and Fr p represent the fraction of total HBF contributed by the hepatic artery and portal vein, respectively. All net hepatic uptakes are depicted as positive values. Glucose production (R a) and glucose utilization (R d) were determined by the equations for isotope dilution during a constant infusion of radioactive glucose [3-3 H]glucose, as modified by DeBodo et al. (6). The [ 14 C]glucose production rate was determined using the tracer technique described by Chiasson et al. (5). This production rate is assumed to represent hepatic [ 14 C]glucose production, although it technically reflects whole body conversion of [ 14 C]alanine to [ 14 C]glucose. Because [ 14 C]glucose is virtually all derived from the liver, one can neglect the small amount of conversion of [ 14 C]alanine to [ 14 C]glucose in the kidney (29) [R a actually reflects both hepatic glucose production (HGP) and renal glucose production (RGP)]. Therefore, hepatic glucose release (HGR) was defined as NHGO hepatic glucose uptake (HGU) HGR NHGO HGU (1) HGU was determined as described by Goldstein et al. (15), where tracer-derived HGU was calculated by dividing the net hepatic 3 H balance by the inflowing [ 3 H]glucose specific activity. This represents the [ 3 H]glucose that is taken up by the liver and metabolized (as opposed to being recycled to plasma glucose). Implicit in this approach is the assumption that uptake of glucose occurs before the addition of glucose to the blood. However, even if this assumption is not true, the drop in [ 3 H]glucose specific activity across the liver is so small as to have little or no quantitative impact. A maximal estimate of hepatic gluconeogenic flux (GNG max) was determined by calculating the net hepatic uptake of the circulating gluconeogenic precursors (lactate, glycerol, and the gluconeogenic amino acids), assuming that there was 100% conversion to glucose, and expressing the data as glucose equivalents (mg kg 1 min 1 ). Pyruvate was not measured and was not entered into the calculation, as previous data demonstrated that it represented only onetenth the contribution of lactate (46). The product of efficiency and GNG max yields a minimal rate of hepatic gluconeogenic flux (GNG min). The minimal and maximal rates of net hepatic glycogenolysis were calculated as net Gly min mg kg 1 min 1 HGR NHLP (2) GO HGU GNG max net Gly max mg kg 1 min 1 HGR NHLP (3) GO HGU GNG min where HGR is hepatic glucose release, NHLP is the net hepatic lactate production, and GO is hepatic glucose oxidation. It should be noted that NHLP 0 when net hepatic lactate uptake occurs. Under that circumstance, lactate uptake by the liver is considered a part of gluconeogenesis. Hepatic GO was assumed to be equal to 0.3 mg kg 1 min 1 for all studies. This rate is based on data from our previous studies and our observation that it changes little under various physiological conditions (17). NHLP, GO, and GNG max were expressed in glucose equivalents. RGP was directly determined using renal vein sampling catheters and flow probes in the last three animals in each protocol. To estimate it in the other studies, RGP was calculated as the difference between tracer-determined R a and HGR. The gluconeogenic rate was also determined by a modification of the method of Rossetti et al. (47) and Giaccari and Rossetti (13). The method measures the relative contributions (%) of plasma-derived glucosyl units [glucose cycling (GC)] and PEP-derived glucosyl units (PEP-gluconeogenesis) to the hepatic glucose 6-phosphate (G-6-P) pool. Thus, as

4 ASSESSMENT OF GLUCONEOGENESIS IN VIVO E949 originally described, total glucose production (TGO) HGP GC. When RGP is zero, HGP equals the tracerderived R a rate determined using [3-3 H]glucose. Thus TGO R a GC. However, because RGP was expected to be nonzero under our experimental conditions, HGR was substituted for R a. The formula then becomes TGO HGR GC. GC was calculated as TGO multiplied by the percentage of the G-6-P pool derived from arterial plasma glucose. Thus GC TGO [3 H]UDPglucose SA (4) [ 3 H]plasma glucose SA TGO HGR / 1 [3 H]UDPglucose SA (5) [ 3 H]plasma glucose SA The contribution of PEP-gluconeogenesis to hepatic glucose- 6-phosphatase flux (i.e., from G-6-P to glucose) was determined by multiplying TGO by the fractional contribution of PEP to the G-6-P pool. This is the gluconeogenic conversion of G-6-P to glucose (GNG G-6-P3Glc), and it was originally designated PEP-gluconeogenesis by Giaccari and Rossetti (13) and Rosetti et al. (47). Under the experimental conditions used, G-6-P and UDPglucose are in equilibrium; thus GNG G-6-P3Glc TGO [14 C]UDPglucose SA (6) 2 [ 14 C]PEP SA This approach assumes that the contribution of plasma [ 14 C]glucose to [ 14 C]UDPglucose is negligible. Indeed, when hepatic [ 14 C]glucose uptake was calculated (using [ 3 H]glucose uptake by the liver), such was shown to be the case. One can also calculate the glycogenolytic contribution to TGO as Gly TGO TGO GNG G-6-P3Glc GC (7) The method of Giaccari and Rossetti (13) can also be used to provide an estimate of overall hepatic gluconeogenic flux from PEP to G-6-P by accounting for all carbons leaving the G-6-P pool that were derived from PEP. This involves combining their method and the a-v difference technique to yield the overall hepatic gluconeogenic flux rate (GNG flux) reflecting gluconeogenic flux from PEP to G-6-P hepgng flux TGO NHLP H Glc oxid 14 C UDPglucose SA 2 14 C PEPSA NHGlyU netglydep where NHLP is net hepatic lactate production, NHGlyU is net hepatic glycerol uptake, and netglydep is the net deposition of glucose carbons from G-6-P into glycogen. In the present experiment, all animals were studied in the postabsorptive period under euglycemic conditions, and netglydep was assumed to be negligible. This latter assumption is supported by recent data from this laboratory (7), which showed that net hepatic [ 14 C]glucose uptake was negligible under euglycemic hyperinsulinemic conditions. Another assumption in this calculation is that all of the glycerol taken up by the liver is converted to G-6-P. Whereas glycerol accounted for at most 0.21 mg kg 1 min 1 glucose (see RE- SULTS), the amount of glycerol that was oxidized can be considered to be negligible. In addition, by use of these methods, the net rate of hepatic glycogenolysis can be estimated as net hepatic glycogenolysis HGR NHLP (9) GO HGU GNG flux (8) Data are reported as means SE. Statistical analyses were performed using Student s t-test and one-way ANOVA to compare values between groups. Data from the prolongedfasted group and the cortisol group were compared with data obtained from the overnight-fasted group. Statistical calculations were performed using SPSS 6.1 for Macintosh. A P value 0.05 was used to define statistical significance among the prolonged-fasted group, the cortisol group, and the overnight-fasted group. RESULTS Arterial plasma hormone concentrations. The plasma cortisol level was g/dl after an overnight fast (Fig. 1). There was no significant change in this level after prolonged fasting. However, the 3-day cortisol infusion increased the arterial plasma cortisol level sixfold (P 0.01). The plasma insulin level was 10 1 U/ml in the overnight-fasted animals and decreased slightly in response to prolonged fasting to 7 1 U/ml (P 0.05). There was a significant increase in the plasma insulin level to 16 3 U/ml in the cortisoltreated animals (P 0.05). The plasma glucagon levels were not significantly different in any of the groups (Fig. 1, A-C). The arterial plasma epinephrine and norepinephrine levels were similar in the overnightand prolonged-fasted groups (60 23 vs and vs pg/ml, respectively; Fig. 1, D and E). On the other hand, the arterial plasma epinephrine level was significantly decreased (26 5 pg/ml, P 0.05) by cortisol infusion, whereas norepinephrine was not changed ( pg/ml). Gluconeogenic amino acid precursors. Although prolonged fasting tended to cause a slight decrease in the net hepatic uptake of gluconeogenic amino acids, this decrease was not statistically significant (P 0.15). Cortisol treatment, however increased net hepatic uptake of the gluconeogenic amino acids (P 0.001), primarily as a result of a doubling of net hepatic alanine uptake (Table 1 and Fig. 2). Lactate and glycerol metabolism. The arterial blood lactate level was M after overnight fasting, M with prolonged fasting (P 0.05), and 3, M in the 18-h Fast Cortisol group (P 0.001). The liver showed a tendency to produce lactate after an overnight fast ( mol kg 1 min 1 ), but it switched to net lactate consumption after a more prolonged fast ( mol kg 1 min 1 ; Fig. 2). With chronic hypercortisolemia, the liver was a larger net producer of lactate ( mol kg 1 min 1, P 0.05). Thus only during prolonged fasting was lactate a net contributor to hepatic gluconeogenesis. The arterial blood glycerol level was M after overnight fasting and did not increase with prolonged fasting (97 10 M) or with steroid treatment ( M). Net hepatic glycerol uptake was mol kg 1 min 1 after overnight fasting and was not different ( mol kg 1 min 1 ) after prolonged fasting or after cortisol treatment ( mol kg 1 min 1 ).

5 E950 ASSESSMENT OF GLUCONEOGENESIS IN VIVO Fig. 1. Arterial plasma cortisol (A), insulin (B), and glucagon levels (C) in 3 groups of dogs. Also shown are arterial plasma epinephrine (D) and norepinephrine levels (E). In the Overnight Fast group (n 8), animals were studied after an 18-h overnight fast; in the Prolonged Fast group (n 7), animals were studied after h of fasting; in the 18-h Fast Cortisol group (n 7), animals were studied after an 18-h overnight fast plus chronic infusion of cortisol (3.0 g kg 1 min 1 ) for 3 days before the study. Values are means SE. Statistical analysis was performed comparing data from Prolonged Fast and 18-h Fast Cortisol with those of Overnight Fast. Glucose metabolism. Arterial plasma glucose levels in the Overnight Fast, Prolonged Fast, and 18-h Fast Cortisol groups were 106 2, 103 3, and mg/dl, respectively. Glucose R a decreased slightly from to mg kg 1 min 1 as the fast progressed, although this difference was not statistically significant (Table 2). Chronic hypercortisolemia markedly increased R a to mg kg 1 min 1 (P 0.001). Similarly, NHGO decreased from to mg kg 1 min 1 as the fast progressed (P 0.03). However, chronic hypercortisolemia caused an even more marked decrease in NHGO to mg kg 1 min 1 (P 0.001). HGU was only mg kg 1 min 1 after an overnight fast but increased to mg kg 1 min 1 with prolonged fasting (P 0.001). In the 18-h Fast Cortisol group, HGU was markedly elevated to mg kg 1 min 1 (P 0.001). Interestingly, HGR was well preserved as the fast progressed ( to mg kg 1 min 1 ), and even in the steroid-treated animals ( ). Estimated RGP (Table 2) was mg kg 1 min 1 after an overnight fast, and prolonged fasting tended to decrease it, whereas chronic hypercortisolemia tended to increase it, but these changes did not reach statistical significance given the variances in the measurement. Gluconeogenic indexes using the a-v difference technique. In the overnight-fasted group, GNG max and GNG min were and mg kg 1 min 1, respectively (Table 3). In the prolonged-fasted group, these values increased significantly to and mg kg 1 min 1, respectively (P 0.001). This was primarily due to increased net hepatic uptake of lactate. In the cortisol-treated group, the very slight increase ( mg kg 1 min 1 ) noted in GNG max compared with the overnight-fasted animals was not statistically significant, nor was the small fall in GNG min. Calculations of minimal and maximal rates of net hepatic glycogenolysis demonstrated significant de-

6 ASSESSMENT OF GLUCONEOGENESIS IN VIVO E951 Table 1. Whole blood levels, net hepatic uptakes, and net hepatic fractional extractions of gluconeogenic amino acids in Overnight Fast, Prolonged Fast, and 18-h Fast Cortisol groups of conscious dogs Overnight Fast (n 8) Prolonged Fast (n 7) 18-h Fast Cortisol (n 7) Arterial blood levels Alanine Serine Threonine Glycine Glutamine Glutamate * Net hepatic uptakes Alanine * Serine Threonine * Glycine Glutamine Glutamate Net fractional extractions Alanine Serine * Threonine * Glycine * Glutamate Values are means SE; blood levels are expressed in M, uptakes in mol kg 1 min 1. Cortisol (3.0 g kg 1 min 1 ) was infused for 3 days before the study. Statistical analysis was performed comparing data from the Prolonged Fast and 18-h Fast Cortisol groups with those of the Overnight Fast group. The specific activity (SA) of plasma alanine was , , and dpm/ mol in the Overnight Fast, Prolonged Fast, and 18-h Fast Cortisol groups, respectively. *P 0.05, P 0.01, P vs. Overnight Fast. mg kg 1 min 1 after an overnight fast and decreased significantly to mg kg 1 min 1 during prolonged fasting. Net glycogenolysis was not significantly altered by steroid treatment compared with overnight-fasted animals without steroid treatment ( mg kg 1 min 1 ). DISCUSSION The primary purpose of this study was to compare determinations of hepatic gluconeogenic rates obtained using the a-v difference technique with those obtained using the [ 14 C]PEP-liver biopsy technique under conditions of modest or increased hepatic gluconeogenesis in vivo. Hepatic gluconeogenic rates were determined after an overnight 18-h fast and compared with those rates obtained after a prolonged fast (42- to 66-h fast) and an overnight 18-h fast following 3 days of chronic hypercortisolemia. With the recognition that the [ 14 C]PEP technique specifically reflects the conversion of gluconeogenic G-6-P to glucose (GNG G-6-P3Glc ), which is a major component of the overall gluconeogenic pathway, a second aim of this study was to develop and evaluate a third methodology, in which components of the a-v difference technique and the creases as fasting progressed but no significant increase in response to steroid treatment. Gluconeogenesis and GC determined using the [ 14 C]PEP-liver biopsy technique. After an overnight fast, the hepatic GNG G-6-P3Glc rate determined using the method of Giaccari and Rossetti (13) was mg kg 1 min 1, and the GC rate was mg kg 1 min 1 (Table 3). Prolonged fasting caused a doubling in the hepatic GNG G-6-P3Glc rate (P 0.02), whereas GC decreased significantly (P 0.05). Chronic hypercortisolemia did not result in a significant increase in the hepatic GNG G-6-P3Glc but did cause a doubling in hepatic GC (P 0.005). Hepatic gluconeogenic flux to G-6-P and net hepatic glycogenolysis determined using the [ 14 C]PEP-liver biopsy technique combined with the a-v difference technique. The gluconeogenic flux rate derived by combining the [ 14 C]PEP technique and the a-v difference technique is a more accurate reflection of new glucose carbons added to the G-6-P pool. These rates are depicted in Table 3. After an overnight fast, the gluconeogenic flux rate was mg kg 1 min 1. This rate increased significantly to mg kg 1 min 1 (P 0.05) with prolonged fasting and was also increased to mg kg 1 min 1 (P 0.05) by steroid treatment. Net glycogenolysis was Fig. 2. Net hepatic uptake of gluconeogenic amino acids (A), net hepatic glycerol uptake (B), and net hepatic lactate balance (C) in 3 groups of dogs. Values are means SE.

7 E952 ASSESSMENT OF GLUCONEOGENESIS IN VIVO Table 2. Glucose production parameters Overnight Fast (n 8) Prolonged Fast (n 7) 18-h Fast Cortisol (n 7) R a NHGO * HGU HGR Estimated RGP [ 3 H]UDPglucose SA [ 14 C]UDPglucose SA [ 14 C]PEP SA Plasma [ 3 H]glucose SA Values are means SE; SA are expressed in dpm/nmol, the other parameters in mg kg 1 min 1. Cortisol (3.0 g kg 1 min 1 ) was infused for 3 days before the study. R a, glucose production; NHGO, net hepatic glucose output; HGU, hepatic glucose uptake; HGR, hepatic glucose release; RGP, renal glucose production; PEP, phosphoenolpyruvate. Statistical analysis was performed to compare data from the Prolonged Fast and 18-h Fast Cortisol groups with those of the Overnight Fast group. *P 0.05, P vs. Overnight Fast. [ 14 C]PEP-liver biopsy technique were combined to yield insights into the gluconeogenic flux from PEP to G-6-P under the three experimental conditions cited. Although there exists no gold standard to which one can compare the results of various methodologies, several major conclusions can be drawn from the present study. 1) In the overnight-fasted animal and in the prolonged-fasted animal, there was a close correlation between the maximal hepatic gluconeogenic flux (GNG max, determined using the a-v difference technique) and the directly determined GNG G-6-P3Glc (determined using the [ 14 C]PEP-liver biopsy technique); 2) although the techniques individually appeared to yield similar results, they both underestimated (by 15 38%) the total hepatic gluconeogenic flux rate (GNG flux ) determined as a result of combining the methodologies; and 3) chronic hypercortisolemia resulted in a significant increase in GNG flux, an effect that was clearly detected only by combining the two methods and determining GNG flux. In discussion of the methodology used in the present study, it is important to consider the major assumptions and potential inaccuracies of each of the methods. A limit to the a-v difference technique lies in its inability to account for intrahepatic proteolysis (40, 44) and intrahepatic lipolysis (9). In addition, GNG max is, by definition, an estimate made with the assumption that all gluconeogenic substrates taken up by the liver are converted to glucose. The GNG min requires the determination of gluconeogenic efficiency, which is itself a minimal estimate due to tracer dilution in the OAA pool of the liver (44). The true gluconeogenic flux rate can be bracketed between the GNG min and GNG max only with the assumption that intrahepatic lipolysis and proteolysis contribute little to hepatic gluconeogenesis. In their initial study describing the [ 14 C]PEP-liver biopsy technique, Giaccari and Rossetti (13) used tracer-determined glucose production as the value for HGP. Given that the experiments were performed in the rat and therefore a-v sampling across liver could not be performed, R a determined with [3-3 H]glucose was the only practical approximation of HGP. However, RGP contributes to R a, and thus substituting R a for HGP would lead to some overestimation of hepatic gluconeogenesis. In the present study, HGR, which is a more precise measurement of HGP, was calculated. Another limitation to the method of Giaccari and Rossetti is that the technique cannot take into account the contribution of glycerol to gluconeogenesis. As glycerol enters the gluconeogenic pathway above PEP, its contribution to gluconeogenesis is not reflected in the determination of PEP specific activity, which will result in an underestimation of hepatic gluconeogenesis. The validity of the calculation of gluconeogenesis using the method of Rossetti et al. (47) has been examined under a number of potential conditions related to cycling of 14 C gluconeogenic precursors. If there is gluconeogenic flux from PEP to G-6-P but no flux of label back down to PEP, then GNG G-6-P3Glc would closely approximate the true gluconeogenic rate (given the caveats discussed). However, if there is cycling of label from G-6-P to PEP and back to G-6-P, the gluconeogenic rate would be overestimated by the calculation. A more robust estimation of GNG flux was defined in Eq. 8. Although the [ 14 C]PEP-liver biopsy technique, by itself, will account for the contribution of intrahepatic proteolysis to gluconeogenesis, the error resulting Table 3. Gluconeogenic parameters of the [ 14 C]PEPliver biopsy technique, the a-v difference technique, and the combined technique Overnight Fast (n 8) Prolonged Fast (n 7) 18-h Fast Cortisol (n 7) [ 14 C]PEP-liver biopsy technique TGO * GC * GNG G-6-P3Glc * Gly TGO * a-v Difference technique GNG max GNG min Gly max * Gly min * Combined [ 14 C]PEP plus a-v difference technique GNG flux * * Net glycogenolysis Values are means SE expressed in mg kg 1 min 1. Cortisol (3.0 g kg 1 min 1 ) was infused for 3 days before the study. TGO, total glucose production; GC, glucose cycling; GNG G-6-P Glc, conversion of glucose 6-phosphate to glucose; Gly TGO, glycogenolytic contribution to total glucose production; GNG max and GNG min, maximal and minimal gluconeogenic flux rates, respectively; Gly max and Gly min, maximal and minimal net hepatic glycogenolysis rates, respectively; GNG flux, total gluconeogenic flux rate. Statistical analysis was performed comparing data from the Prolonged Fast and 18-h Fast Cortisol groups with those of the Overnight Fast group. *P 0.05, P 0.01, P vs. Overnight Fast.

8 ASSESSMENT OF GLUCONEOGENESIS IN VIVO E953 from not accounting for the contribution of glycerol can be minimized by adding net hepatic glycerol uptake to the gluconeogenic estimate. Likewise, one can also account for the G-6-P derived from PEP that goes down the glycolytic pathway either to exit the liver as lactate or to be oxidized in the tricarboxylic acid cycle. Equation 8 also accounts for the G-6-P derived from PEP that contributes to new glycogen formation. One of the other modifications made to the original method of Giaccari and Rossetti (13) and Rosetti et al. (47) was to continuously infuse the [ 14 C]alanine during the entire course of the study rather than to bolus the tracer over the last 10 min of the study. This change raised the possibility that plasma [ 14 C]glucose could reenter the hepatic UDPglucose pool and lead to an overestimation of the calculated gluconeogenic rate. Calculations were made to determine the maximal overestimation in the gluconeogenic rate that might result. This determination was based on estimating the [ 14 C]glucose counts that entered the liver during the study and comparing those counts to the [ 14 C]UDPglucose content of the liver. In the overnight-fasted animals, gluconeogenesis could have been overestimated (data not shown) by a maximum of 4%, whereas in the prolonged-fasted animals this overestimation could have been as much as 14%. The largest possible error from this source could have occurred in the steroidtreated animals, and even then the overestimate would have been no more than 20%. Nevertheless, on the basis of these calculations, it is recommended that future applications of this technique employ an infusion of [ 14 C]alanine only during the last 20 min before liver biopsy. Previous data derived under similar conditions but analyzed using a different approach failed to reveal significant enhancement in gluconeogenesis by steroid treatment (15). This conclusion remained largely at odds with in vitro data demonstrating stimulatory effects of cortisol on phosphoenolpyruvate carboxykinase (PEPCK) activity (44) and PEPCK mrna transcription (10). In the former study (15) and in the present study, neither gluconeogenic efficiency nor the GNG max was significantly increased by steroid treatment. Furthermore, gluconeogenic conversion of alanine to glucose was only modestly increased by the steroid (15). However, by use of the combination of methodologies, a significant stimulatory effect of glucocorticoids on gluconeogenic flux was detected in vivo, even in the complex setting of enhanced GC. To the extent that [ 14 C]glucose derived from gluconeogenic flux accumulated in hepatic glycogen (which was not measured), GNG flux would have been underestimated. Each of the two methods also yielded independent information on different aspects of hepatic GC. Goldstein et al. (15) previously described a significant difference between R a and NHGO associated with chronic hypercortisolemia. This difference roughly equaled net HGU, which resulted from steroid treatment. The [ 14 C]PEP-liver biopsy technique detected enhanced steroid-induced GC through the G-6-P pool. This accounted for the additional glucose production that was undetected due to the use of [3-3 H]glucose rather than [2-3 H]glucose (51, 57). As previously shown, if one used [2-3 H]glucose as the tracer, R a determined using this tracer should equal TGO (47). Thus the combination of the two methodologies detected a significant inward movement of glucose carbons, even in the 18-h-fasted state. In the present study, RGP was determined as the difference between tracer-determined glucose production and HGR. This indirect assessment of RGP is imprecise but nevertheless suggested that the kidneys contributed at most 0.3 mg kg 1 min 1 to TGO in overnight- and prolonged-fasted dogs. In the 18-hfasted steroid-treated group, the indirect measurement of RGP suggested that the contribution of RGP to R a might be increased two- to threefold. In the last few animals studied in each group and in additional animals studied under similar experimental conditions for other purposes, RGP was directly assessed as described by McGuinness et al. (36). In those animals, the directly determined RGP was mg kg 1 min 1 after an overnight fast (n 6). This was not different from the indirectly determined value and is not different from that reported by McGuinness et al. ( mg kg 1 min 1 ). During chronic hypercor- Fig. 3. Flux rates into and out of the hepatic glucose 6-phosphate (G-6-P) pool after an 18-h overnight fast (Overnight Fast, n 8) are summarized in this figure. Total glucose output (TGO) and glucose cycling (GC) were derived using the [ 14 C]phosphoenolpyruvate (PEP)-liver biopsy technique. The gluconeogenic rate (GNG) is the gluconeogenic flux (GNG flux) determined using the combined [ 14 C]PEP-liver biopsy technique plus the arteriovenous (a-v) difference technique. All other rates into the liver from the plasma compartment were determined solely by use of the combined-tracer a-v difference technique. These include hepatic glucose uptake (HGU), net hepatic glucose output (NHGO), hepatic glucose release (HGR), net hepatic glycerol uptake (Glycerol), net hepatic lactate balance (Lactate), and the net hepatic uptake of amino acid gluconeogenic (amino acid precursors). Net hepatic glycogenolysis was calculated as the difference between the known glucose carbons entering and leaving the G-6-P pool. All rates are in mg kg 1 min 1.

9 E954 ASSESSMENT OF GLUCONEOGENESIS IN VIVO Fig. 4. Flux rates into and out of the hepatic G-6-P pool after a 42- or 66-h fast (Prolonged Fast, n 7) are summarized in this figure. See legend of Fig. 3 for explanatory details. tisolemia (n 5), directly assessed RGP was mg kg 1 min 1, whereas after prolonged fasting (n 6) it was mg kg 1 min 1. Thus, in the animals in which RGP was directly assessed, it was 10% or less of TGO. In the present study, hepatic GNG flux was equal to 37% of TGO (R a ) after an 18-h fast, and this percentage increased to 57% after more prolonged fasting. Although the steroid treatment increased hepatic gluconeogenesis, the treatment also caused a significant increase in R a, thus accounting for the failure of hepatic GNG flux (39%) to increase as a percentage of total R a. Hellerstein and colleagues (19, 20, 21) and Neese and colleagues (41, 42) have recently assessed gluconeogenesis by mass isotopomer distribution analysis by use of [2-13 C]glycerol, [3-13 C]lactate, or [1-13 C]lactate. In rats, progressive fasting increased the ratio of gluconeogenesis as a percentage of glucose production from 50% at 5 6 h to 80% at h (41). Hellerstein et al. (21) further developed this methodology and have recently incorporated the glucuronate probe technique to measure the rate of glucose entry into hepatic UDPglucose by the direct pathway. In a group of overnight-fasted human subjects, gluconeogenesis accounted for 36% of glucose production (19). After a 60-h fast, this percentage increased to 78%. A similar rate was recently reported by Tayek and Katz (52) for overnight-fasted human subjects. In their study, gluconeogenesis accounted for 40% of glucose production. In a separate group of overnight-fasted human subjects, these same authors reported that gluconeogenesis accounted for 47% of glucose production (54). Recently Landau et al. (34) estimated the contribution of gluconeogenesis to TGO in human subjects by use of 2 H 2 O. The amount of 2 H bound to carbon-6 of glucose relative to that bound to carbon-2 provides a measure of the fraction of glucose formed via gluconeogenesis (30, 45). Using this methodology, Landau et al. (32) reported that gluconeogenesis represented 23 42% of total glucose production in overnight-fasted human subjects and that this increased to 59 85% after 42 h of fasting. Further refinement of the technique by Landau and colleagues (31, 33) utilized the ratio of 2 H bound to carbon-5 of glucose relative to that bound to carbon-2. By use of this technique, data indicated that gluconeogenesis accounted for 47% of glucose production at 14 h and increased to 67% at 22 h and to 93% after 42 h of fasting in human subjects. Fig. 5. Flux rates into and out of the hepatic G-6-P pool after an 18-h fast during chronic hypercortisolemia (18-h Fast Cortisol, n 7) are summarized in this figure. See legend of Fig. 3 for explanatory details.

10 ASSESSMENT OF GLUCONEOGENESIS IN VIVO E955 These data of Landau et al. (32), Tayek and Katz (52), and Hellerstein et al. (19) obtained from human subjects are consistent with the overnight-fast data obtained in the present study despite the species difference. Because the assessment of gluconeogenesis in human subjects necessitates a less invasive methodology than can be employed in animal models, Rothman et al. (48) have pioneered 13 C nuclear magnetic resonance (NMR) spectroscopy to assess hepatic gluconeogenesis in humans. In their studies, gluconeogenesis accounted for 64% of TGO at 22 h of fasting and for 96% of glucose production by 68 h of fasting. When the methodology was applied to type 2 diabetic subjects, gluconeogenesis accounted for 88% of TGO after 23 h of fasting (35). The gluconeogenic rates obtained by Rothman et al. are higher than those obtained in the present study and higher than those obtained in the human studies discussed above. It is possible that the protocol design may have accounted for the higher gluconeogenic rates that those authors observed. For instance, the meal that subjects were fed before the official start of the fast was relatively small. Thus the subjects may have entered the fast still relatively glycogen depleted and thus had already progressed to a more gluconeogenic state. However, additional data from the same group obtained using 13 C NMR spectroscopy to assess hepatic gluconeogenesis in humans indicated a gluconeogenic rate of 60% in human subjects after an overnight fast (49). It remains unclear why the gluconeogenic rates obtained using NMR spectroscopy were slightly higher than those obtained using other techniques. It should be remembered, however, that the approach of Rothman et al. involves the measurement of the net glycogenolytic rate, which is subtrated from tracer-determined glucose production. Any RGP will thus manifest as hepatic gluconeogenesis. In addition, it should be noted that, if glycogen synthesis were to be augmented, the NMR technique would detect a decrease in gluconeogenesis that was not the result of a diminution in the flux of pyruvate to G-6-P but was instead due to an augmentation of glycogen synthesis. One must be careful when comparing the results from the different gluconeogenic methods, because they measure slightly different things. Figures 3, 4, and 5 summarize the glucose carbon fluxes within the liver. GNG represents the gluconeogenic flux from PEP to G-6-P. After an overnight fast (Fig. 3), net glycogenolysis was 1.46 mg kg 1 min 1. After a prolonged fast (Fig. 4), net glycogenolysis decreased to 0.26 mg kg 1 min 1. With steroid treatment (Fig. 5), net glycogenolysis was 1.25 mg kg 1 min 1. The derived glycogenolytic rates after an overnight fast and during prolonged fasting are consistent with rough approximations of the glycogenolytic rates derived from published hepatic glycogen measurements compiled by Moore et al. (38) and Hendrick et al. (22) at various fasted states. If one plots their data and assumes that the decline in glycogen mass is linear over time, the net glycogenolytic rate is 2.0 mg kg 1 min 1 at 20 h and 0.7 mg kg 1 min 1 at 40 h of fasting. Results from the two methods used in this study can also be combined to yield estimates of the contribution of hepatic proteolysis to the PEP pool under the three experimental conditions studied hepatic proteolysis (mg kg 1 min 1 ) GNG flux net uptake of gluconeogenic amino acids and glycerol net hepatic lactate uptake (9) where GNG flux is the gluconeogenic flux determined using the combined methodology. With this equation, the rate of hepatic proteolysis after an overnight fast was calculated to be mg kg 1 min 1. Prolonged fasting had little effect on this rate ( mg kg 1 min 1 ). However, chronic steroid treatment significantly increased this rate to mg kg 1 min 1. In summary, the results of the present study demonstrated that combining the a-v difference technique with the [ 14 C]PEP-liver biopsy technique yielded a more complete picture of hepatic glucose carbon flux than what one obtained using either method alone. The combined technique did not merely decrease the limitations of the a-v difference and [ 14 C]PEP-liver biopsy techniques but, in some cases, significantly changed conclusions such that the in vivo data reconciled with the in vitro data. We thank Kareem Jabbour for technical help in the laboratory. Some of the data in these studies were presented at the American Diabetes Association, Atlanta, GA, June These studies were supported in part by a Veterans Affairs Merit grant, National Institute of Diabetes and Digestive and Kidney Diseases Grant RO1 DK-18243, and the Diabetes Research and Training Center (P60 DK-20593). REFERENCES 1. Adkins BA, Myers SR, Hendrick GK, Stevenson RW, Williams PE, and Cherrington AD. Importance of the route of intravenous glucose delivery to hepatic glucose balance in the conscious dog. J Clin Invest 79: , Bidlingmeyer BA, Cohen SA, and Tarvin TL. Analysis of amino acids using pre-column derivatization. J Chromatogr B Biomed Appl 336: , Cherrington AD, Stevenson RW, Steiner KE, Connolly CC, Wada M, and Goldstein RE. Acute hormonal regulation of gluconeogenesis in the conscious dog. In: New Concepts in the Pathogenesis of NIDDM. New York: Plenum, 1993, p Chiasson JL, Liljenquist JE, Lacy WW, Jennings AS, and Cherrington AD. Gluconeogenesis: methodological approaches in vivo. Fed Proc 36: , Chiasson JL, Liljenquist JE, Sinclair-Smith BC, and Lacy WW. Gluconeogenesis from alanine in normal postabsorptive man intrahepatic stimulatory effect of glucagon. Diabetes 24: , DeBodo RC, Steele R, Altszuler N, Dunn A, and Bishop JS. On the hormonal regulation of carbohydrate metabolism: studies with C14 Glucose. Recent Prog Horm Res 19: , Edgerton DS, Cardin S, Emshwiller M, Neal D, Chandramouli V, Schumann WC, Landau BR, Rossetti L, and Cherrington AD. Small increases in insulin inhibit hepatic