Metabolism and Hormonal Regulation

MARTHA KALOYIAHNI* ANDR. A. FREEDLAND Department of Physiological Science, Veterinary Medicine School, University of California, Davis, CA 95616 and *Lab of Animal Physiology, Science School, Aristotelian University of Thessaloniki, Thessaloniki 54006, Greece sidic stores, primarily amino acids, lactate and glycerol. Therefore, gluconeogenesis from lactate/pyruvate and amino acids is a major and essential function of the liver in the maintenance of the supply of glucose for the nervous system at times when dietary carbohydrate is lacking and glycogen reserves are low. Amino acids are major sources for replacing glucose, which is metabo lized to COi- The majority of the amino acid-degrading enzymes are inducible by nutritional factors (2-4). The capacities of aminotransferases and amino acid catabolizing enzymes in various tissues are dependent on nutritional and hormonal factors. Fivefold differences in liver cytosolic enzyme activity and rates of enzyme synthesis occur as adaptive changes to dietary and hor monal stimuli (1-3). There are many reports on the control of gluconeo genesis by the diet (5-8). The control of gluconeogenesis may occur in peripheral tissues to alter the supply of glucose precursors to the liver or may operate in the liver to control the uptake of the precursors and their conver sion to glucose (9-11). The complexities of the multienzyme system are such that the sites of dietary regulation of this important metabolic process have not been identified. Several studies have been conducted with one or more glucose precursors at high levels (e.g., 10 HIM)(12). However, relatively few studies have exam ined the rates of gluconeogenesis with physiological substrate levels as well as the contribution of each of the precursors (13). To our knowledge, none of these studies was related to nutritional status of the animals. In the present study, we investigated the effect of starvation and of feeding a nonpurified, a high protein or a high fat diet on gluconeogenesis from various amino acids and lactate in the isolated hepatocytes. The con tribution of each of the amino acids to gluconeogenesis via the incorporation of 14C-labeled amino acids into

ABSTRACT The contribution under various nutri tional regimens of several amino acids and láclate to gluconeogenesis was estimated by measuring the glu cose formation from 'C-labeled substrates. Isolated rat hepatocytes were incubated for 60 min in a KrebsRinger bicarbonate buffer pH 7.4 containing lactate, pyruvate and all the amino acids at concentrations similar to their physiological levels found in rat plasma, with one precursor labeled in each flask. In all condi tions, lactate was the major glucose precursor, provid ing over 60% of the glucose formed. Glutamine and alanine were the major amino acid precursors of glu cose, contributing 9.8% and 10.6% of the glucose formed, respectively, in hepatocytes isolated from starved rats. Serine, glycine and threonine also con tributed to gluconeogenesis in the starved liver cells at 2.6,2.1 and 3.8%, respectively, of the glucose formed. The rate of glucose formation from the isolated hepa tocytes of the starved rats and those fed either high protein or high fat was higher than that from rats fed a nonpurified diet. J. Nutr. 120:116-122, 1990. INDEXING KEY WORDS: •gluconeogenesis •hepatocytes •amino acid metabolism •high protein diet •high fat diet •starvation

The capacity of mammals to synthesize glucose is crucial to the maintenance of a supply of glucose for the brain and for anaerobic energy production, as well as for providing glucose to the cells for biosynthesis. This is particularly critical when food intake is restricted and liver glycogen stores are depleted. Gluconeogenesis also plays an important role in the utilization of lactate produced by glycolyzing tissues, such as erythrocytes and muscles (1). When the glycogen stores of the liver are exhausted after several hours of fasting, the supply of glucose is dependent on biosynthesis from nonglyco-

0022-3166/90 $3.00 ©1990 American Institute of Nutrition. Received 20 June 1989. Accepted 14 September 1989.

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Contribution of Several Amino Acids and Lactate to Gluconeogenesis in Hepatocytes Isolated from Rats Fed Various Diets

117

AMINO ACIDS, DIETS AND GLUCONEOGENESIS

glucose was estimated by incubating the isolated liver cells in a medium with an amino acid concentration similar to that of the rat plasma.

MATERIALS AND METHODS

and light for 12 h each day; the cells were prepared at the start of the light period. Isolated cells were prepared according to the method of Berry and Friend (15)as modified by Cornell et al. (16). Four milliliters of the isolated hepatocyte suspension containing approximately 30 mg of cells were incubated in 25-ml Erlenmeyer flasks in a 37°Cshaking water bath for 60 min. The incubation media contained glucose precursors at concentrations similar to those of the plasma of rats fed a nonpurified diet: 1.38 mM lactate, 132 uM pyruvate, 180 UMtaurine, 30.5 UMaspartic acid, 28.5 UM hydroxyproline, 270 UM threonine, 267 UM serine, 10 UMasparagine, 100 UMglutamic acid, 665 UM glutamine, 161 UM proline, 284 UM glycine, 388 UM alanine, 55 UMcitrulline, 163 UMvaline, 56 UMmethionine, 80 UMisoleucine, 164.5 UMleucine, 76.5 UMtyrosine, 71.5 UM phenylalanine, 71.5 UM tryptophan, 104 UMorni thine, 511 UMlysine, 575 UMhistidine, 155 UM arginine, 95 UM cysteine and 60 UM cystine (17). Each flask contained a mixture of the physiological levels of amino acids, lactate, pyruvate and one u14C-labeled amino acid or lactate suspended in 1% albumin in Krebs-Ringer bicarbonate buffer pH 7.4. For the determination of the equilibrium of the radio active substrates intra- and extracellularly, 1 mL of the incubation mixture from the flasks containing the ra dioactive substrates was withdrawn at the end of the 60-min incubation period and was gently layered over 0.35 mL of silicon oil (density 1.035 g/cm3) that had been layered on top of 0.15 mL of 4% HC1O4 in a 1.5-mL microcentrifuge tube. Tubes were immediately centrifuged for 1 min at 12,000 x g in an Eppendorf 5412 microcentrifuge (Brinkmann Instruments, Westbury, NY) at room temperature. In order to obtain estimates of total contents, synchronous samples of the incuba tion mixture were deproteinized with HC1O4. These

in the case of methionine, proline, glutamine, leucine, ornithine and glutamic acid, 40% of 14COis lost; and in the case of histidine and threonine, 50 and 38% of 14C is lost, respectively. These are based on metabolic path ways assuming the intermediates leave the tricarboxylic acid cycle before being converted to citrate. Differences in absolute contribution due to nutri tional treatment were determined using a one-way anal ysis of variance and Duncan's multiple-range test.

RESULTS At the 60-min period all amino acids, with the excep tion of glutamate and aspartate, had an equal or higher number of dpm/uL of water in the cells than in the surrounding medium (data not presented). Thus, in these studies permeability generally was not the limit ing factor. Lactate had a somewhat greater number of dpm per uL of medium water than per uL of cell water, but due to the rapid usage of lactate this did not appear to be a limitation. It should be noted that our measure ments of the distribution were at equilibrium or nearequilibrium conditions (after 60 min of incubation) and were not related to diffusion or transport rate. The contribution of several amino acids and lactate to gluconeogenesis was estimated by measuring glucose production from lactate, pyruvate and a mixture of amino acids at concentrations similar to that found in plasma of rats fed a nonpurified diet. Isolated hepatocytes from rats fed a nonpurified diet, a high protein diet, and a high fat diet as well as from rats starved for 16 h were used for these estimations. The rates of glucose production from the glucose precursors were main tained during a 60-min incubation. With all substrates used, except lactate, less than half of the substrate was

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Female Sprague-Dawley rats (Charles River Breeding Laboratories, Wilmington, MA) weighing 200-300 g were used in all experiments. The rats were subjected to one of the following experimental treatments: 1}fed a nonpurified diet (Purina Mills, St. Louis, MO); 2) fed a nonpurified diet and starved 16 h; 3) fed a high protein diet (80% casein, 10% glucose, 5% corn oil, 4% salts, 1% vitamin mix); and 4} fed a high fat diet (65% marga rine, 25% casein, 5% corn oil, 4% salts, 1% vitamin mix). The vitamin and salt formulation used for these diets has been described previously (14). The experimen tal diets were fed for 9 d prior to cell isolation. The animals were housed in individual hanging-bottom cages at 20-21°C. The animal room was dark for 12 h

tubes were also centrifuged. The top layer of the first set of tubes was withdrawn and deproteinized and was considered to be the extracellular fluid and contents. The acid layer represented the intracellular contents of the cell with some extracellular contamination (18). In the flasks containing isotope each cell extract was neutralized and then applied to a Dowex 50 (FT form AG 50 x8) cation exchange resin (Bio-Rad, Richmond, CA) in a pasteur pipette, placed above a Dowex 1 (acetate form AGI x8) anión exchange resin (Bio-Rad, Rich mond, CA) in a pasteur pipette. Glucose was eluted with double-distilled water until 12 mL was collected. Aliquots of each fraction were counted using a liquid scin tillation spectrophotometer (Packard Instrument, Downers Grove, IL). Radioactivity is lost due to randomization of carbon atoms during the conversion of pyruvate to phosphoenolpyruvate (10). The amount of glucose from lactate, alanine, glycine and serine based on radioactivity was estimated by taking a loss of 17% 14Cinto account. In the case of aspartate and asparagine, 25% of 14Cis lost;

118

KALOYIANNI AND FREEDLAND TABLE1 Rate of gluconeogenesis in isolated hepatocytes incubated with various precursors1 Nutritional condition2 Fed nonpurified diet (control]

Starved

Fed high protein diet

0.20.36± ±0.120.04 0.030.08 ± 0.05"0.27 ± 0.02a0.53 ± 0.13'1.4 ± 0.35a29.2 ± ±4.1"6.6 Total

45

±3.6a

0.50.41± 0.040.04 ± 0.040.09 ± 0.09a0.52 ± 7ab1.6±0.1 0.15"2.2 ± O.lb41.3± ±4.2"8.3 61

±4.2h

0.500.71 ± 0.190.05 ± 0.050.06 ± 0.06a1.3 ± 0.2C2.8 ± O.lc2.3± 0.5"61.2± ±6.7b6.6 91

±6.0C

0.3h0.20 ± 0.070.16 ± 0.050.52 ± 0.03h4.3 ± 1.6a1.1 ± 0.10.16± 0.080.20 ± ±0.120.47 0.25"0.85 ± 0.24b1.3 ± 0.1"2.9± 0.7b55.9± ±5.0" 75

±4.8h

'Results are in umol glucose formed per g of hepatocytes per h. See text for methods and concentrations. Values are means ±SEMfor 4 rats. 2Studies with similar animals in our laboratory have had the following liver weight per 100 g of body weight: fed, -4.57; starved, -3.07; fed high protein, -5.31; fed high fat, -4.29. For 9 d, rats were fed either a nonpurified diet (controll, the nonpurified diet and starved for at least 16 h before cells were isolated, a high protein diet or a high fat diet. 'Numbers with different superscripts in the same row (i.e., with the same substrate) are significantly different at P < 0.05, using one way ANOVA.

utilized for gluconeogenesis over the time period. The rates of gluconeogenesis were higher overall after a 16-h starvation than during ad libitum intake of a nonpurified diet (Table 1). The starvation period in cluded the overnight dark period, and because food intake was somewhat lower during the light period, the effective starvation period could have been slightly longer than 16 h. The only three substrates that showed significant increases in gluconeogenic rates after the starvation period were serine, threonine and alanine. These three alone would not have formed the basis for the significant overall increase. Although it was not statistically significant, the greatest absolute rate of increase was due to gluconeogenesis from lactate. When all substrates were taken as a total there was a signifi cant increase. These observations are similar to those of Exton and Park using perfused liver and 10 mM sub strates (12). The observation of increased rates with serine and threonine is consistent with reported in creases in rates from 10 mM serine after starvation and observed increases in serine dehydratase activity after starvation (20). The rats fed the high protein diet demonstrated the greatest synthesis of glucose from precursors; the rate of gluconeogenesis from serine in these rats was signifi cantly higher than in those fed, starved or fed the other diets. This observation is consistent with the observed higher enzymatic activity of serine dehydratase after

intake of a high protein diet vs. starvation (20).Threo nine, lactate and alanine also formed glucose more rapidly in hepatocytes from rats fed the high protein diet relative to animals fed the nonpurified diet. The rate of gluconeogenesis from glutamine in the rats fed high protein was more rapid than that observed with either the starved rats or those fed high fat but was not signif icantly different from this rate in rats fed the nonpurified diet. The results from animals fed a high fat diet, as might be expected, showed strong similarities to those of starved animals, both of which may have utilized lipids as a major source of energy. The rate of glucose forma tion from serine, threonine, lactate, proline and alanine, as well as the overall rate of glucose formation, was significantly higher in these animals than in those fed the nonpurified diet but was not significantly different from the rate observed in starved animals. Ornithine was converted to glucose more rapidly in the rats fed high fat diets than in any of the other groups, although the absolute rate of glucose formation was still low from this amino acid. Glutamate was converted slowly to glucose relative to some of the other amino acids; how ever, gluconeogenesis from glutamate occurred signifi cantly faster in rats fed the high fat diet than in those fed the nonpurified diet. In all groups the very high rates of gluconeogenesis from glutamine, compared with those from glutamate, indicate that the hepatocytes

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0.8a30.17 ± 1.5"0.34 ± 1.5"0.53 ± AlanineAspartateAsparagineGlutamateGlutamineGlycineHistidineMethionineOrnithineProlineSerineThreonineLactate3.7 0.080.12 ± 0.070.25 ± ±0.150.41 0.040.22 ± 0.040.36 ± 0.100.42 ± 0.05a8.4 ± 6ab6.0±0.1 2ab10.1±0.1 0.8ab0.6 ± 1.0a1.3 ± 2.4h1.2 ±

Fed high fat diet

119

AMINO ACIDS, DIETS AND GLUCONEOGENESIS TABLE 2

Relative percentage contribution of substrates toward gluconeogenesis with hepatocytes from rats under various nutritional conditions1 Nutritional condition Fed nonpurified diet (control)

Starved

Fed high protein diet

0.70.3 ± 0.20.2 ± 10.7± 0.25.7 ± 0.5C1.5 ±

0.60.8 ± 0.1a0.1 ±

0.60.7 ± 0.2"0.1 ±

1.20.8 ± 0.1a0.1 ±

0.20.2 ± 0.2b0.3 ±

0.10.2 ± 0.20.6 ± 0.31.2 ± 0.3a3.1 ±

0.10.1 ± 0.10.9 ± 0.12.6 ± O.lb3.8 ±

0.10.1 ± 0.11.4 ± 0.43.1 ± l.lb2.5 ±

0.20.6 ± 0.30.9 ± 0.21.7 ± 0.6ah3.9 ±

0.465 ± ±2.910.8

0.867.7 ± ±4.27.3

0.467.3 ± ±4.28.8

'Results are in the percentage of glucose formed from the indicated substrate. Values are means ±SEMfor 4 rats. 2Numbers with different superscripts in the same row (i.e., with the same substrate) are significantly different at P

0.774.5 ± ±0.9

0.05, using one way

ANOVA.

were intact and viable, as indicated by perfused liver studies (21) in which glutamine (10 mM) was rapidly converted to glucose and 10 mM glutamate was found to be a relatively ineffective glucose precursor. The relative contributions of various substrates to glucose production are given in Table 2. Serine, as might be expected by the increase in serine dehydratase with starvation and feeding of the high protein diet, did make a larger overall contribution to the percentage of gluco neogenesis in the starved rats and in those fed high protein than in rats fed the nonpurified diet. Both the percentage contribution and overall total rate of gluco neogenesis must be considered when relating percent age contribution to the absolute rates. The percentage of gluconeogenesis from histidine was significantly lower in the rats fed high fat than the other three groups. The rates of gluconeogenesis from histidine (Table 1)by animals receiving various feeding regimens was not statistically significant because of high variation, but this variation was reduced when glucose production was calculated as a percentage contribution. One of the more striking observations was the lower percentage contri bution due to glutamine in all three experimental groups compared with the animals fed the nonpurified diet. The percentage contribution, particularly in the rats fed high protein, differs markedly from absolute rates due to the much higher (i.e., doubled) rate of overall gluconeogenesis in the rats fed high protein vs. those fed the nonpurified diet. The starved rats and those fed high fat contributed significantly less glutamine to overall gluconeogenesis than did rats fed the nonpurified diet. Starved rats and those fed the high fat diet also demon

strated the lowest absolute rate of glutamine contribu tion to gluconeogenesis. Lysine and leucine failed to incorporate any measurable radioactivity into the glu cose fraction in any of the groups studied.

DISCUSSION Several factors control the rate of conversion of amino acids to glucose: the rate of cellular transport, the activity and Km for the degradative enzymes for the amino acid, and any end-product inhibition of the de grading enzyme. Catabolism of most indispensable amino acids is regulated at the site of the initial reac tions in the degradative pathway. The activity of many enzymes is associated with nutritional and hormonal status. The changes in amino acid metabolism and the enzyme for the regulated step of their degradation in response to different dietary conditions are complex and unique (5). For animals at approximately zero nitrogen balance, high protein diets will result in higher rates of amino acid degradation than will low protein diets. Starved animals and animals fed high fat diets must maintain reasonable rates of gluconeogenesis to main tain blood glucose levels. However, with time, lipid metabolism aids the glucose economy of the animal by decreasing peripheral utilization, by increasing rates of gluconeogenesis in the liver (22, 23) and by producing ketone bodies that can be used by the brain to substitute, at least in part, for the glucose that would normally be utilized for energy (24-26). This change in substrate availability may account for the lower total gluconeo-

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1.30.6 ± AlanineAspartateAsparagineGlutamateGlutamineGlycineHistidineMethionineOrnithineProlineSerineThreonineLattate8.2 1.70.4 ± 2.00.6 ± 0.10.3 + 0.30.4 ± 0.20.5 ± 0.10.5 ± 0.40.5 ± 0.60.6 ± 0.39.8 ± 0.411.1 ± 0.218.7 ± 0.6b2.1 ± 0.9b1.3 ± 0.9a21.3 ±

Fed high fat diet

120

KALOYIANNI AND FREEDLAND

ute 37% of the carbon for glucose formation in a physi ological mixture of substrates in the perfused liver (12). Van Holt et al. (30) estimated that 50% of the glucose formed in the fasted rat is from resynthesis of degrada tion products of glucose, principally lactate and pyruvate. Alanine, which has been shown to be an important carrier of nitrogen and carbon from the muscle during protein degradation, particularly of branched-chain amino acids (31), might be expected to have a greater role when protein catabolism is in excess than when protein is adequate or limiting. In the case of the starved animal, for a short period there is a marked increase in protein catabolism and increased gluconeogenesis. The magnitude of this response decreases with the length of starvation, as is illustrated by the decrease in total nitrogen excreted in the urine with increasing starva tion time (32). The increased rate of gluconeogenesis from alanine was commensurate with an increase in the overall rate of gluconeogenesis. The percentage contri bution of alanine to glucose was not affected by dietary treatment. The increase in alanine catabolism in rats fed high fat was unexpected; it was thought that the animals would have adapted to a high fat metabolism and de creased their glucose needs after 9 d of feeding. Perhaps alanine mixes with the lactate pool due to transamination, because an increase in the absolute rate of gluco neogenesis from alanine was associated with a marked increase in glucose synthesis from lactate. The decreased utilization of glutamine for gluconeo genesis seen in starved rats and in those fed high fat diets may be related to a decrease in the liver's use of gluta mine and to the shunting of synthesized glutamine to the kidney. In the kidney the carbon of the glutamine could be converted to glucose and the nitrogen to am monia for excretion into the urine. This would be par ticularly important during starvation and periods of high fat intake, both conditions under which an acidotic condition might develop. It has been shown that during acidosis, and particularly during starvation, the activity of glutaminase in the kidney is elevated (33-36) and the percentage of nitrogen in the urine as ammonia com pared to urea increases markedly (32). Decreased utili zation of glutamine and production of glutamine by liver would allow a decrease in urea synthesis and further protect the animal from acidosis (32, 33). How ever, glutamine synthesized or not utilized by the liver would not be lost to the gluconeogenic process because it could be converted to glucose by the kidney cortex (36,37). This would allow for ammonium ion excretion, which would help alleviate acidosis. Consistent with this idea, it is tempting to suggest that the increased ornithine catabolism seen in animals fed the high fat diet may be a mechanism to deplete urea cycle interme diates and decrease urea synthesis. If the nitrogen that was not used for urea synthesis was used for glutamine synthesis by the liver, this would help alleviate the

I

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genesis in the rats fed high fat compared with those fed high protein. Rates of gluconeogenesis under various nutritional conditions have been examined (25); however, usually only one or two substrates were examined, and research ers have often used very high, unphysiological concen trations of 5-10 mM. Under these conditions, the interaction of other potential glucose precursors and energy sources would be minimized. Adding amino acids and lactate at physiological levels as in the present study, does not force hepatocytes to utilize a single substrate, and gluconeogenic rates can be estimated with normal concentrations and relationships of poten tial substrates. In studies using the perfused liver and other isolated cell preparations, rates ofgluconeogenesis (umol glucose formed/time) from single substrates in high concentration were more rapid than rates we ob served when using physiological levels of the substrate, especially when other potential glucogenic substrates were also present (27, 28). Lactate may be the only exception to this observation. Concentration is impor tant both to cell uptake and to metabolism, as has been shown for alanine (13).The interaction and stimulation of gluconeogenesis by one amino acid on other sub strates seen at higher concentrations may also be absent in these studies (29). Many of the enzymes associated with amino acid catabolism have been shown to have relatively high Km and markedly rise in activity, especially when a high protein diet is fed (3). In most cases, the rise in enzy matic activity is greater than the observed increase in gluconeogenic rate, indicating that the first enzyme of the pathway, though it may be critical in determining flux, is not the only factor involved. These other factors that determine flux include permeability into the cell and other fates of the amino acid after it has passed through the first, supposedly unidirectional, step of its catabolism. Regardless of the diet fed, lactate, glutamine and alanine were the major precursors of glucose, with ser ine and threonine in a secondary position. All other amino acids, although contributors, were not quantita tively important for net glucose synthesis. Lactate, which can be produced in the intestine, peripheral tis sues, red blood cell and other cells was rapidly converted to glucose. This was not unexpected, because its role as an intermediate for glucose synthesis and the Cori cycle (i.e., glucose to lactate followed by lactate to glucose) has been known for a number of years. Furthermore, many of the amino acids, such as serine and alanine, actually traverse into the lactate-pyruvate pool of the hepatocyte before being converted to glucose. Thus, it would be expected that neither of these, nor the combi nation of the two, could proceed any faster or as fast as lactate to glucose formation. The contribution of lactate in these studies was found to be higher than in earlier studies in which lactate-pyruvate was found to contrib

AMINO ACIDS, DIETS AND GLUCONEOGENESIS

FRIEDMANN, N. & PARK,C. R. (1970) Role of adenosine 3',5'-

12.

13.

14.

15. 16.

17. 18.

ACKNOWLEDGMENTS The authors wish to express their appreciation to Ernest Avery for his excellent technical assistance and to Barbara Washburn for her suggestions in the writing of this manuscript.

19.

20. 21.

22.

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acidotic condition that is seen in rats fed high fat diets. This marked change in glutamine use was not observed to the same degree in the starved animal in this experi ment. If the animals were starved for 48 or 72 h, how ever, an increase in gluconeogenesis from ornithine might be observed. Finally, it is important to remember that these experiments were conducted with isotopie tracers; therefore, it is possible for some compounds that show very little conversion to glucose to enter the citric acid cycle as acetyl-CoA and proceed to label glucose, without net gluconeogenesis. This was not an extensive process, as indicated by our observations, since we saw no label incorporated into glucose from lysine, which is catabolized by the liver. Furthermore, leucine, which is poorly catabolized by the liver, produced no label into glucose, indicating that artifacts due to separation of compounds were not playing a major role.

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Its possible role in regulation of renal ammonia production. /. Clin. Invest. 45: 612-619. 37. KREBS,H. A., BENNETT,D. A. H., DEGASQUET,P., GASCOYNE, T. & YOSKIDA, T. (1963) Renal gluconeogenesis. The effect of diet on the gluconeogenic capacity of rat-kidney cortex slices. Biochem. J. 86: 22-27.

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Contribution of several amino acids and lactate to gluconeogenesis in hepatocytes isolated from rats fed various diets.

The contribution under various nutritional regimens of several amino acids and lactate to gluconeogenesis was estimated by measuring the glucose forma...
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