INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

RECENT STUDIES HAVE DEMONSTRATED that high-sucrose (SU) and high-fat (HF) diets increase the basal rate of gluconeogenesis in vivo (23, 26) and the capacity for gluconeogenesis in vitro (6, 21, 24). The fact that the gluconeogenic adaptation is retained in vitro suggests that a portion of this adaptation resides within the hepatocyte and functions independently of acute hormone action.

Two enzymes that contribute to the control of gluconeogenic flux are phosphoenolpyruvate carboxykinase (PEPCK) and pyruvate kinase (PK). PEPCK represents a critical and, perhaps, rate-determining step in gluconeogenesis (13), whereas PK contributes to the amount of phosphoenolpyruvate (PEP) that is available for conversion to glucose. In addition, the mitochondrion plays an important role in the regulation of gluconeogenesis. Mitochondrial fuel oxidation provides the energy required for gluconeogenesis from precursors that pass through pyruvate. The mitochondrial enzyme pyruvate carboxylase (PC) catalyzes the first step in gluconeogenesis from pyruvate (i.e., conversion of pyruvate into oxaloacetate; Ref. 3). Thus gluconeogenesis from precursors that pass through pyruvate involves the transport of pyruvate into and oxaloacetate out of the mitochondrion. The mitochondrial enzyme pyruvate dehydrogenase (PDH) can influence gluconeogenesis via its ability to compete for pyruvate (14).

Diet composition can modify the activity of the enzymes involved in pyruvate metabolism and gluconeogenesis in the liver. Previous studies have observed increased PC and PEPCK activity (2, 32) but decreased PDH (29) and PK (8) activity after exposure to HF diets. In contrast, SU diets appear to increase PEPCK and PK activity (15, 19, 23) and high fructose diets (fructose is a major component of an SU diet) increase the activity of PDH (25). Thus although both HF and SU diets increase gluconeogenesis from precursors that pass through pyruvate, the adaptations in pyruvate metabolism that lead to increased gluconeogenesis may be different.

Fatty acids increase gluconeogenesis by providing fuel to support gluconeogenesis and via stimulation of PC (12). HF and SU diets increase endogenous lipid stores within the hepatocyte (22, 28). The extent to which changes in intracellular lipid supply contribute to the diet-induced increase in gluconeogenesis is presently unknown.

The goals of the present study were to 1) identify the site(s) contributing to the HF- and SU-induced increase in gluconeogenesis by determining flux rates through enzymes that contribute to pyruvate metabolism, namely PDH, PC, PEPCK, and PK; 2) to determine the extent to which these diets induce mitochondrial adaptations; and 3) to characterize the effect of fatty acid supply on these fluxes.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Animals and feeding. Male Sprague-Dawley rats were obtained from an institutional breeding stock weighing ~180 g. All animals were housed individually in a temperature-controlled room with a 12:12-h light-dark cycle and free access to food and water. All procedures for animal use were approved by the Institutional Animal Care and Use Committee at Arizona State University. On initiation of the study, all animals were provided free access to a semipurified high-starch (ST) diet (%total calories: 68 cornstarch, 20 protein, 12 fat) for a 2-wk baseline period. Food intake was measured daily, and body weight was recorded weekly. After the 2-wk baseline period, rats were switched to either an SU diet (%total calories: 68 sucrose, 20 protein, 12 fat), HF diet (%total calories: 45 fat, 20 protein, 35 carbohydrate), or remained on the ST diet for 1 wk. During this week, rats were fed 95% of the average food intake recorded during the second week of baseline feeding. Feeding 95% of baseline calories during the experimental feeding period results in rats with similar rates of weight gain and body composition (23). Complete diet composition is presented in Table 1.

Hepatocyte isolation. Hepatocytes were obtained from 24-h fasted rats by collagenase perfusion of the liver as described by Berry and Friend (5) and modified by Blackmore and Exton (7). Briefly, rats were anesthetized with an intramuscular injection of ketamine (50 mg/kg), xylazine (10 mg/kg), and acepromazine (5 mg/kg). The abdominal cavity was opened, and the portal vein was cannulated. The liver was perfused with calcium-free Krebs-Ringer bicarbonate buffer containing 11 mM glucose and 5 mM pyruvate that had been equilibrated with 95% O₂-5%CO₂ at 37°C and pH 7.4. Once the liver was cleared of blood, ~50 ml of the initial perfusate was allowed to drain to waste. Collagenase (Type I, Worthington Biochemicals, 0.3 mg/ml) was then added to the perfusion system and recirculated until the liver was appropriately digested (~10-12 min). The liver was carefully removed, and the capsule was gently peeled off. The liver was then shaken in 50 ml of Krebs-Ringer bicarbonate buffer containing 2.5 mM calcium, 1% BSA (fatty acid free) to release hepatocytes. Cells were washed three times in the Krebs-Ringer buffer with BSA and suspended at 30 mg/ml (wet weight). The initial quality of the cell preparation was assessed by trypan blue exclusion (0.2% final concentration). Only preparations with >90% dye exclusion were used in cell incubations.

Hepatocyte incubations. Hepatocytes (30 mg/ml final concentration) were incubated for 30 min in Krebs-Ringer buffer containing 2.5 mM CaCl₂, 1% BSA (fatty acid free). For measurement of PEPCK and PK flux, cells were incubated in 5 mM lactate, 0.5 mM pyruvate, Na H¹⁴CO₃ (2 µCi/ml), and varying concentrations (in µM: 0, 250, 500, 750, 1,000) of palmitate (bound to BSA) in a final volume of 3.0 ml. After 30 min a 1.0-ml aliquot of each, incubation was stopped by addition of 1.0 ml ice-cold 8% HClO₄. This sample was neutralized to pH 2.8 using 3.5 N KOH and used for ion-exchange chromatography. Another 1.0-ml sample of the incubation was centrifuged to pellet the cells. The supernatant was removed, frozen, and used for analysis of metabolites (glucose, lactate, and free fatty acids), and the cell pellet was frozen in liquid nitrogen and used for determination of glycogen. For measurement of PDH and PC fluxes, cells were incubated with either 1- or 3-[¹⁴C]pyruvate in the presence of lactate, pyruvate, and palmitate as described above (1, 10). Scintillation vials were fitted with rubber stoppers containing a plastic center well. The center well contained chromatography paper (Wattman, 3MM) saturated with Hyamine hydroxide and methanol (1:1 vol/vol). Immediately after the 30-min incubation, 2.7 M perchloric acid was injected and ¹⁴CO₂ produced was collected over the next 60 min. Inclusion of Na H¹⁴CO₃ standards demonstrated a ¹⁴CO₂ recovery of >90%. A portion of the perchloric acid-treated cell suspensions was neutralized for ion-exchange chromatography as described below.

Flux measurements. For determination of radioactivity incorporated into metabolites (glucose, lactate, and pyruvate) the pH 2.8 supernatant was separated by ion-exchange chromatography as described by Rognstad (27). Briefly, 1.0 ml of sample was consecutively passed through AG 50W-X8 (H⁺ form) and AG 1-X8 (acetate form) chromatography columns obtained from Bio-Rad (Hercules, CA). Glucose was eluted from the columns with H₂O while pyruvate and lactate were eluted with 2 N acetic and 2 N formic acid, respectively. Recovery of labeled glucose, lactate and pyruvate was quantified using [³H]glucose, [¹⁴C]lactate, and [¹⁴C]pyruvate standards. Portions (0.5 ml) of the eluates were added to scintillation vials and assayed for radioactivity in a Beckman LS-6500 liquid scintillation counter (Fullerton, CA). Gluconeogenic flux (J_GNEO) was calculated as the ratio of [¹⁴C]glucose to NaH¹⁴CO3 specific activity, whereas PK flux (J_PK) was calculated as the ratio of [¹⁴C]lactate + [¹⁴C]pyruvate relative to NaH¹⁴CO₃ specific activity as previously described (17). J_GNEO is expressed as nanomoles of PEP converted into glucose per hour per milligram of cell wet weight. J_PK is expressed as nanomoles PEP converted into lactate and pyruvate per hour per milligram of cell wet weight. PEPCK flux (J_PEPCK) was calculated as the sum of J_GNEO and J_PK (17). PC (J_PC) and PDH (J_PDH) fluxes were calculated as described previously (17). Briefly, J_PDH was calculated as the difference between ¹⁴CO₂ produced from 1-[¹⁴C]pyruvate and the sum of J_PK, J_PEPCK, and ¹⁴CO₂ produced from 3-[¹⁴C]pyruvate. J_PC was calculated as the difference between J_PDH and the incorporation of 1-[¹⁴C]pyruvate into ¹⁴CO₂ and [¹⁴C]glucose (i.e., total flux through PDH and PC; Ref. 1).

Pyruvate carboxylation in isolated mitochondria. Liver mitochondria were prepared essentially as described by Johnson and Lardy (16). The final mitochondrial pellet was suspended in 220 mM mannitol + 70 mM sucrose at a concentration of 40-60 mg mitochondrial protein/ml. The carboxylation of pyruvate by intact mitochondria was estimated according to the method of Walter and Stucki (30), with some modification. A 100-µl aliquot of mitochondrial suspension, containing 4-6 mg protein, was added to vials equilibrated at 37°C in a shaking water bath. Each vial contained 1.9 ml of a medium comprised of (in mM) 220 sucrose, 10 MgSO₄, 6.6 K^.PO₄, 6.6 triethanolamine, 4.0 ATP, 6.0 pyruvate, and 10 KHCO₃, pH 7.40. Metabolism was stopped by adding 0.5 ml of 25.0% HClO₄ either immediately after the addition of mitochondria or after a 20-min incubation. The acidified samples were centrifuged for 1.0 min at 14,000 g, and the supernatants were neutralized with 2 N KOH + 0.3 M MOPS. Citrate, malate, and fumarate were assayed spectrophotometrically (4) using reactions coupled to the oxidation or reduction of NADH/NAD. Under these assay conditions, the sum of accumulated citrate, malate, and fumarate accounts for >90% of the pyruvate carboxylated (30). Pyruvate carboxylation rate was found to be linear with time and mitochondrial addition over the ranges used in this study.

Mitochondrial respiration. Mitochondrial oxygen consumption was measured at 37°C in a respiration chamber as described previously (31). Mitochondria, 1.0-1.5 mg protein, were added to a total volume of 2.0 ml of respiration medium, and maximal (state 3) respiration rate was elicited with a bolus addition (1.0 umol) of ADP. Resting (state 4) respiration rate and the ADP/O ratio were also evaluated.

Analytic procedures. Cell glycogen content and radioactivity was measured using the procedures of Chan and Exton (9). Glucose (18) was measured by standard enzymatic methods. Lactate was measured using a kit according to the manufacturer's instructions (Sigma, St. Louis, MO). Free fatty acid concentration in the incubation medium was assayed using the Wako NEFA-C kit (Wako Chemicals, Dallas, TX).

Statistical analysis. Data were analyzed using a one-way ANOVA or repeated-measures ANOVA where appropriate. If the overall F was significant, comparisons between mean values were made using a Student-Newman-Keuls test. Significance was set at P < 0.05 for all comparisons. All data are presented as the mean ± SE.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

General results. After 1 wk on the respective diets there were no differences in body weight (in g: SU = 343.4 ± 3.5, ST = 345.1 ± 4.3, HF = 340.2 ± 4.3) or energy intake (in kcal/day: SU = 107.5 ± 0.5, ST = 107.6 ± 1.0, HF = 106.2 ± 0.6) among the diet groups. On hepatocyte isolation, initial liver glycogen content was similar in all groups (<2 µg/mg cell wet wt in all groups). Additionally, there was no significant incorporation of label into glycogen during the course of any of the incubations in any of the groups.

J_GNEO. The tracer estimated rate of J_GNEO was elevated in hepatocytes from SU and HF at all concentrations of palmitate, compared with the ST animals (Fig. 1A). Palmitate stimulation of J_GNEO was not significantly different among groups. In addition to the tracer estimated rate of J_GNEO, glucose release into the incubation medium was also measured. Similar to the tracer estimates of J_GNEO, glucose release was elevated in the SU and HF compared with the ST group (Fig. 1B).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   A: dose-dependence curves for gluconeogenic flux (JGNEO) on palmitate concentration in the incubation medium. Isolated hepatocytes were incubated with 5 mM lactate + 0.5 mM pyruvate and 2 µCi/ml Na14HCO3, and JGNEO was calculated as described in METHODS. B: dose-dependence curves for glucose release on palmitate concentration in isolated hepatocytes measured enzymatically. Number of hepatocyte preparations for each diet group is given in parentheses. Data are presented as the means ± SE. *High-starch (ST) diet-fed group significantly different from high-sucrose (SU) and high-fat (HF) diet-fed groups (P < 0.05).

J_PC and J_PEPCK. SU and HF diets increased J_PC in isolated hepatocytes (Fig. 2A). Palmitate stimulated J_PC, with the pattern of stimulation being similar for all diet groups. To examine if the increased flux through PC was the result of a diet-induced mitochondrial adaptation, pyruvate carboxylation by isolated intact mitochondria was assessed. Pyruvate carboxylation by intact mitochondria was not different among groups (Table 2).

View larger version (18K): [in this window] [in a new window]   Fig. 2.   A: dose-dependence curves for pyruvate carboxylase flux (JPC) on palmitate concentration in the incubation medium. Isolated hepatocytes were incubated with 5 mM lactate + 0.5 mM pyruvate and either 1-or 3-[14C]pyruvate, and JPC was calculated as described in METHODS. B: dose-dependence curves for phosphoenolpyruvate carboxykinase flux (JPEPCK) on palmitate concentration in the incubation medium. Isolated hepatocytes were incubated with 5 mM lactate + 0.5 mM pyruvate and 2 µCi/ml Na14HCO3, and JPEPCK was calculated as described in METHODS. Number of hepatocyte preparations is given in parentheses. Data are presented as the means ± SE. *ST significantly different from SU and HF groups (P < 0.05).

J_PK and J_PDH. J_PK was not significantly different among groups or across palmitate concentrations (Table 3). J_PDH was low, accounting for only 1% of the total pyruvate flux, and was not different among any of the groups across all palmitate concentrations (Table 3).

Effect of bromopalmitate. Bromopalmitate binds to CPT I and inhibits long-chain fatty acid transport and thus oxidation in the mitochondria. When hepatocytes were incubated with 5 mM lactate + 0.5 mM pyruvate as gluconeogenic precursors, the addition of 0.1 mM bromopalmitate abolished differences in J_GNEO, J_PC, and J_PEPCK (Fig. 3).

View larger version (16K): [in this window] [in a new window]   Fig. 3.   JGNEO, JPC, and JPEPCK in the absence and presence of bromopalmitate (BP; 0.1 mM final concentration). For JGNEO and JPEPCK, hepatocytes were incubated with 5 M lactate + 0.5 mM pyruvate, 2 µCi/ml Na14HCO3 in the absence (no fatty acids present) and presence of 0.1 mM BP. For JPC, hepatocytes were incubated 5 mM lactate + 0.5mM pyruvate and either 1- or 3-[14C]pyruvate in the absence (no exogenous fatty acids) or presence of 0.1 mM BP, and flux was measured and calculated as described in METHODS. Number of individual hepatocyte preparations is 4 or 5 per diet group. Data are presented as the means ± SE. *ST significantly different from SU and HF groups (P < 0.05).

Mitochondrial respiration. Isolated mitochondrial preparations demonstrated adequate integrity based on average respiratory control ratios (RCR) of 5.6-5.8 with palmitoyl-carnitine as substrate and 4.4-5.9 with pyruvate as substrate. In addition, ADP/O ratios were 1.4-1.8. Neither RCR nor ADP/O ratios were significantly different among groups. In mitochondria isolated from HF-, SU-, and ST-fed animals, there were no differences in the capacity to oxidize fat or pyruvate as indicated by the state III rates in the presence of palmitoyl-carnitine (200 µM) + malate (1 mM) or pyruvate (1 mM) + malate (Table 4). Additionally, there were no differences in state IV respiratory rates or mitochondrial content among any of the diet groups (Table 4).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

SU and HF diets increase the basal rate of gluconeogenesis in vivo (23, 26) and the capacity for gluconeogenesis in vitro (6, 21, 24). In the present study, feeding rats an HF or SU compared with an ST diet increased the capacity for J_GNEO, J_PC, and J_PEPCK in isolated hepatocytes (Fig. 4). Thus, in vitro, there appears to be a strong proportionality between the diet-induced increase in J_GNEO and flux through PC and PEPCK. This suggests that the steps in the gluconeogenic pathway from pyruvatePEP are potentially critical to the SU and HF diet-induced adaptation in gluconeogenesis from pyruvate-linked precursors.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Summary of flux measurements obtained in the present study. Rates are given in nmol · min1 · mg cell wet wt1. Bold arrows represent significantly greater flux rate then the ST-fed group. OAA, oxaloacetate; PEP, phosphoenolpyruvate; JPK, pyruvate kinase flux; JPDH, pyruvate dehydrogenase flux.

It has previously been demonstrated that SU and HF diets increase the maximal activity of the enzyme PEPCK (23, 32). Thus the observation that J_PEPCK was increased in hepatocytes from SU and HF was not surprising. However, J_PEPCK is dependent not only on enzyme concentration but also the supply of oxaloacetate, a substrate provided by the mitochondrial enzyme PC. In the present study, PC flux in hepatocytes and mitochondrial pyruvate carboxylation were assessed to gain insight into the nature of the diet-induced adaptation in gluconeogenesis. Results demonstrated that PC flux was increased in hepatocytes isolated from SU and HF compared with ST. In contrast, the rate of pyruvate carboxylation by isolated mitochondria under saturating substrate concentrations was not affected by diet. Thus SU- and HF-induced increases in J_PC in hepatocytes do not appear to result from mitochondrial adaptations that increase the amount of PC protein. In addition, the capacity for fat oxidation and mitochondrial protein content were not different among groups, providing further support for the absence of diet-induced mitochondrial adaptations. It is also unlikely that increased J_PC resulted from an increased capacity for pyruvate transport into the mitochondria, because pyruvate-stimulated mitochondrial respiration was not different among groups.

Increased delivery/use of fatty acids can increase PC activity (11, 20). In the present study, palmitate stimulation of J_GNEO, J_PC, J_PEPCK were not significantly different among groups. In addition, palmitoyl-carnitine-stimulated mitochondrial respiration was not significantly different among groups. Thus the capacity to use fatty acids or transport fatty acids across the mitochondrial membrane does not appear to be increased in SU or HF. Hepatic lipid concentration is increased after 1 wk on SU and HF diets (22, 28). Exposure of hepatocytes to bromopalmitate, in the absence of exogenous fatty acids, reduced flux through PC and subsequent measured steps (J_PEPCK, J_GNEO) in the gluconeogenic pathway, abolishing all effects of diet on gluconeogenesis. Taken together, these results suggest that use of endogenous lipids contributes to the diet-induced increase in gluconeogenesis. We hypothesize that diet-induced changes in the maximal activity of PEPCK are coupled to endogenous lipid-mediated stimulation of PC flux. Important to this hypothesis is the observation that bromopalmitate had little effect in hepatocytes from starch-fed rats.

SU diets can potentially increase and HF diets decrease the content of PK in the liver (8, 15). Because PK will influence the amount of PEP available for glucose production, we were interested in determining J_PK in the present experiments. J_PK was low in all groups relative to gluconeogenic flux and was not significantly different among groups. We also reasoned that flux through PDH might also influence J_GNEO by decreasing pyruvate availability. J_PDH was also very low and not significantly different among groups (Fig. 4). Thus, at least under the conditions in the present study, any diet-induced adaptations in PK and PDH do not appear to influence flux through these two enzymes.

The increases in J_PC and J_PEPCK seen in the HF- and SU-fed animals lead to an increase in PEP production without an increase in J_PK (Fig. 4). These data suggest that additional adaptations upstream from PEP serve to pull PEP toward glucose and contribute to the increased gluconeogenesis in HF- and SU-fed animals. In support of this, a recent study by Andrikopoulos and Proietto (2) demonstrated that feeding mice a high-fat diet increased both PC and fructose 1,6, bisphosphatase (F16BP) activity in the liver. Thus high-fat diets have the potential to produce multiple adaptations in the gluconeogenic pathway that include an increased ability to produce PEP (increased PC, PEPCK) and to divert the PEP toward glucose production (increased F16BP). The SU diet used in the present study also increased the capacity for glucose production from dihydroxyacetone in perfused livers, a precursor that enters the gluconeogenic pathway at the triose phosphate level (i.e., bypasses PC and PEPCK) (21). This SU-induced adaptation appears to involve an increase in glucose-6-phosphatase activity (unpublished observations). Thus dietary nutrients appear to influence the gluconeogenic pathway at multiple sites.

In the present study, pyruvate carboxylation in isolated mitochondria was estimated based on the accumulation of citrate, malate, and fumarate. These intermediates constitute >90% of all Krebs cycle intermediates (30), and their measured accumulation is considered a valid estimate of J_PC (11). This is in part due to the negligible production of PEP by rat liver mitochondria (11).

In summary, the results of the present study confirm that increased flux through PC and PEPCK are important contributors to the increased gluconeogenesis in sucrose and high-fat fed animals. Additionally, these studies suggest that hepatic lipid stores may mediate the diet-induced increase in J_PC.