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Article

Low Glucokinase Activity and High Rates of Gluconeogenesis Contribute to Hyperglycemia in Barn Owls (Tyto alba) after a Glucose Challenge¹

Department of Animal Sciences, University of California, Davis, CA 95616

²To whom correspondence should be addressed.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Barn owls (Tyto alba) and leghorn chickens were fed a low protein high glucose (33.44% protein, 23.67% glucose) or a high protein low glucose (55.35% protein, 1.5% glucose) diet. After an intravenous glucose infusion, the peak in plasma glucose was not affected by diet in either species and was 22.6 and 39.4 mmol/L in chickens and barn owls, respectively. Glucose levels returned to normal within 30 min in chickens, but remained elevated for 3.5 h in barn owls. An oral glucose challenge also resulted in greater and longer hyperglycemia in barn owls than in chickens. The activities of hepatic glucokinase, malic enzyme and phosphoenolpyruvate carboxykinase of barn owls were 16, 35, and 333% of the levels in chickens. Malic enzyme (P = 0.024) was less affected by dietary glucose level in barn owls than in chickens. Cultured hepatocytes from chickens produced 43% more glucose from lactate than hepatocytes from barn owls and, conversely, barn owl hepatocytes produced 87% more glucose from threonine than chickens (P = 0.001). Gluconeogenesis from lactate was greatly suppressed by high media glucose in chicken hepatocytes but not in those of barn owls (P = 0.0001 for species by glucose level interaction). When threonine was the substrate, gluconeogenesis was suppressed by increased glucose in both species but to a greater relative extent in chickens (P = 0.007 for species by glucose level interaction). Owls were glucose intolerant at least in part because of low hepatic glucokinase activity and an inadequate suppression of gluconeogenesis in the presence of exogenous glucose, apparently because they evolved with large excesses of amino acids and limited glucose in their normal diet.

KEY WORDS: • carnivores • gluconeogenesis • amino acid catabolism • owls • chickens

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Many species of avian carnivores consume a diet consisting almost exclusively of animal prey. Anatomical and physiologic adaptations that permit the consumption of vertebrate prey include a distinctively hooked beak, enlarged esophagus for swallowing whole prey, egestion of indigestible components (e.g., hair, feathers, scales, large bones or claws) by regurgitation and efficient autoenzymatic digestion that is facilitated by the reflux of digesta between the duodenum, proventriculus and ventriculus (Duke et al. 1976 , Duke 1997 ). Vertebrate prey are very high in protein and lipid but their carbohydrate content is very low and consists mainly of glycogen and free glucose. A slow rate of passage of digesta results in very complete digestion of the protein and lipid components of the food (Barton and Houston 1993 , Kirkwood 1979 , Tabaka et al. 1996 ). When compared across species, the rate of intestinal glucose absorption generally matches the glucose content of the natural diet, and glucose is absorbed more slowly in carnivorous and insectivorous species than in omnivorous ones (Diamond 1991 , Karasov and Levey 1990 ).

Although an understanding of digestive adaptations and trade-offs of avian carnivores is emerging, relatively little is known about their intermediary metabolism. Presumably, carnivorous species have enhanced capacity to use amino acids for energy and for gluconeogenesis relative to omnivorous species. Habitually low levels of dietary carbohydrate consumption and the consequent relaxed selection pressure on the metabolic pathways for dietary glucose disposal might be predicted to result in poor glucose homeostasis in response to a glucose challenge. In fact, red-tailed hawks (Dobbs 1985 ) and penguins (Chieri et al. 1972 ) take three to four times longer to reestablish baseline glucose levels after an intravenous glucose challenge than do chickens, but the enzymatic basis is not known. Nonavian carnivores such as house cats (Kettelhut and Migliorini 1980 ), rainbow trout (Palmer and Ryman 1972 ), white sturgeon (Hung 1991 ) and American alligators (Coulson and Hernandez 1983 ) are also glucose intolerant as indicated by prolonged glucose tolerance curves relative to omnivorous species such as chickens, rats and humans. In alligators, glucose intolerance is especially pronounced, and several days are required for normal blood glucose levels to be obtained after an intravenous glucose challenge. Rainbow trout are glucose intolerant due in part to the lack of hepatic glucokinase (GK;³ hexokinase type IV, EC 2.7.1.1), which is responsible for phosphorylating glucose and facilitating its uptake into hepatocytes (Palmer and Ryman 1972 ). Cats also lack hepatic glucokinase and are poor at down-regulating amino acid catabolism and gluconeogenesis when fed low protein diets, indicating obligatory gluconeogenesis. For example, feeding cats a 17.5% protein diet resulted in little change in the activity of hepatic enzymes phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.31), alanine aminotransferase (ALT; EC 2.6.1.2), aspartate aminotransferase (AST: EC 2.6.1.1), and fructose-bisphosphatase (FBP: EC 3.1.3.11) compared with cats fed a 70% protein diet (Rogers et al. 1977 ). Conversely, in the omnivorous rat (Eisenstein and Strack 1971 , Kettelhut and Migliorini 1980 , Peret et al. 1981 ) and Japanese quail (Featherston and Freedland 1973 ), PEPCK and amino acid transaminases are very adaptable to changes in dietary protein or glucose content.

Threonine is one of most gluconeogenic of the essential amino acids, but it is relatively deficient in many plant proteins, causing omnivores to alternate between periods of threonine insufficiency and excess, depending upon the composition of their diets. When dietary threonine is at or below the requirement of chickens, they use this amino acid with high efficiency for protein accretion (Edwards et al. 1997 ), but when it is in excess, it is available for use as a gluconeogenic precursor. For carnivorous species, dietary threonine would always be expected to be in excess of needs for protein accretion; this excess is oxidized or used as a substrate for gluconeogenesis. Lactate is also a good substrate for gluconeogenesis in chickens (Tinker et al. 1986 ) and the carnivorous common murre (Herzberg et al. 1988 ). The endogenous production of lactate as a result of anaerobic glucose oxidation during physical exertion makes this metabolite a common substrate in all species regardless of their customary diet. Thus, threonine represents a substrate whose surplus for oxidation or gluconeogenesis is highly dependent upon customary diet, whereas the availability of lactate is independent of diet.

The purpose of these studies was to compare glucose tolerance and dietary plasticity of hepatic glucose–metabolizing enzymes in barn owls (Tyto alba) and chickens fed either a low glucose, high protein diet or a high glucose, low protein diet. We also compared the use of threonine and lactate for oxidation and glucose formation in hepatocytes isolated from these two species.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Husbandry conditions and all experimental protocols were approved by the Animal Care and Use Committee of the University of California, Davis. Chickens and owls were gradually introduced to their meat-based experimental diets and were then subjected to either an oral or an intravenous glucose challenge; liver biopsies were taken for enzymatic analysis. A separate set of chickens and owls was used as a source of liver hepatocytes for examining the metabolism of lactate and threonine.

Diet formulation.

A high protein, low glucose diet (HPLG) and a low protein, high glucose diet (LPHG) were made from ground chicken carcasses. Male broiler-type chickens (Ross x Hubbard; n = 8), obtained from Peninsula Hatchery (Santa Cruz, CA) were grown to 8 wk of age and then killed by cervical dislocation. Chickens were skinned and their intestines, gizzards, head, necks, abdominal fat depots and feet were removed. Half of the chicken carcasses were used as the basis of HPLG. The protein content of the remaining four carcasses was decreased by removal of the leg and breast muscle groups, forming the basis of LPHG. Both groups of carcasses were ground in a meat grinder (Hobart, Troy, OH) until the meat was homogeneously mixed and most of the bones were in small (<3 mm) pieces. Any larger bones were removed and discarded.

Samples from each mixture were analyzed for the following: moisture content by overnight drying in a drying oven at 65 ^oC; lipid content by ether extraction (Tecator Soxtec System HT 1043 Extractor, Pittsburgh, PA) of the dried tissue; and protein content of the defatted material by the Kjeldahl method (Tecator Auto 1030 Analyzer, Pittsburgh, PA). Proximate analysis information was used to formulate two isocaloric diets. This was accomplished by adding back abdominal fat pads to the LPHG diet in an amount equal to the fat removed with leg and breast muscles as determined by composition analysis. In addition, 300 g/kg D-glucose (Staley, Decatur, IL) was added on a dry weight basis to the LPHG diet. Proximate analysis indicated that the LPHG and HPLG diets had similar fat contents (283.2 and 304.1 g/kg dry matter, respectively), and a protein content of 334.4 and 553.5 g/kg dry matter, respectively. Calculated metabolizable energy values were 20.5 and 20.9 MJ/kg dry matter, respectively. To facilitate consumption, diets were placed into sausage casings with the use of a manually operated sausage-making machine. Diet-filled sausage casings were tied off with string to give pellets approximating the size of a small chick (25–35 g).

Experimental animals.

Nonreleasable barn owls (n = 10) were obtained from various rehabilitation centers in central California, an area in which owls typically consume a diet made up exclusively of rodents and small birds. Barn owls (450 g average body weight) were allowed to acclimate to environmental rooms (50% humidity, 26°C, and 10 h dim light:14 h dark). The low light levels were necessary to prevent retinal injury. Owls were attached to ground perches by leather bracelets and a 3-ft leather leash and kept in groups of 2–3 per room. Owls were fed two 1-d old leghorn chicks/d. After the birds appeared comfortable with their new environment (3 d) they received two HPLG pellets each morning. Half of the birds readily ate the food pellets after several days. To accommodate those owls that did not readily accept the food pellets, pieces of skin and feathers from day-old leghorn chicks were attached to the surface of moistened pellets. One owl was unwilling to accept the modified food pellets and was not used in the study. The remaining nine owls were randomly assigned to either the HPLG diet or the LPHG diet. Owls switched to the LPHG diet were done so gradually over a 5-d period; on d 6, the owls received their experimental diets exclusively. After a further 6 d (d 11 after beginning the introduction of LPHG), three owls per dietary treatment were given an intravenous glucose tolerance test, and after 10 d (d 15 after beginning the introduction of LPHG), liver biopsies were taken for enzymatic analysis. The remaining three owls were used to determine oral glucose tolerance after receiving the HPLG diet as their exclusive food for 10 d.

Male Single Comb White Leghorn chicks (n = 30) obtained from Peninsula Hatchery were grown in battery brooders with raised wire floors. At 21 d, the chickens were moved to environmental chambers (50% humidity, 26 ^oC, and 10 h dim light:14 h dark). Chicks were fed a corn-soybean meal–based chicken starter (Purina, St. Louis, MO) ad libitum until 28 d of age and an average weight of 410 g. Chicks were randomly divided into two groups and assigned to either the HPLG diet or the LPHG diet. Because both experimental diets consisted primarily of meat, chicks were gradually introduced to each of the diets over a 5-d period. Thereafter, chicks were fed the experimental diets as the sole food source ad libitum. After a further 6 d of consuming experimental diets, three chicks per diet were used for intravenous glucose tolerance tests. After 10 d of consuming the experimental diets (d 15 since the beginning of their introduction), 10 chicks per diet were used for liver enzyme analysis and at 10 d, four chicks consuming the HPLG diet were used for an oral glucose tolerance test.

After 5 d of exclusive consumption of experimental diets, digesta samples, uncontaminated by urine, were sampled from the anterior-rectum using a syringe fitted with flexible tubing (5 mm diameter). The rectum of barn owls and chickens is relatively short (8 cm), facilitating easy access to the area of the ileocecal junction. Rectal digesta samples were taken from three owls and three chicks per diet 8 h after the morning feeding. This time period coincided with completion of consumption of the day's meal. The samples were extracted with 5 vol of water, centrifuged (10,000 x g for 10 min) and analyzed for glucose concentrations (kit 16–20; Sigma Chemical, St. Louis MO).

Glucose challenge tests.

Chickens and barn owls were subjected to an intravenous glucose tolerance test 12 h after food removal. Blood was taken from the brachial vein of the bird to determine baseline glucose levels. Birds were then weighed and given an intravenous injection of 5 mL/kg body weight of 1.1 mol/L D-(+)-glucose (Sigma Chemical) in the opposite brachial vein over a period of 15 s. Approximately 100 µL of blood was collected at 10, 25, 60, 120, 210 and 360 min after glucose injection, placed in heparinized hematocrit tubes and stored on ice. Blood plasma was stored in a -20°C freezer before analysis of glucose concentrations.

Twelve hours before an oral glucose tolerance test, the HPLG diet was removed. Birds received a slurry of 22.2 mmol glucose/kg body weight in 10 mL of water by gavage. This amount of glucose was equivalent to the amount consumed in 40 g of LPHG, a typical morning meal for an owl. Blood samples were taken at 0, 30, 60, 90, 120, 180 and 240 min after gavage.

Liver sampling.

On the morning before surgery for liver biopsy, one food pellet was offered to each barn owl and the uneaten portion was removed after 8 h. Removal of food permitted emptying of the upper gastrointestinal tract so that the birds could be ventilated adequately during surgery. A liver biopsy was taken 16 h after food removal. Barn owls were anesthetized with isoflurane via an endotracheal tube. After laparotomy, a 1- to 2-g piece of the liver was removed and immediately frozen between two aluminum plates in liquid N₂ and then stored at -20°C.

For chickens, feed was also removed 16 h before liver collection. Chicks were anesthetized with isoflurane gas; livers were removed from the body cavity, freeze-clamped in liquid N₂ and stored at -20 °C. Immediately after liver removal, chickens were killed by cervical dislocation.

Liver enzyme activity.

Frozen liver samples were prepared for enzyme analysis by placing them on dry ice and using a hammer to break them into pieces no larger than 6–7 mm across in a -20°C cold room. Liver samples were weighed into test tubes and 9 parts of ice-cold 0.14 mol/L KCl was added for ALT, AST, pyruvate kinase (PK; EC 2.7.1.40), malic enzyme (ME; EC 1.1.1.40) and FBP assays. For PEPCK assays, 9 parts of ice-cold deionized water was added. Samples were homogenized on ice with a Polytron (Brinkmann Instruments, Westbury, NY) twice at half-maximum power for 15 s. Homogenates were centrifuged for 30 min at 14,000 x g in a 5 ^oC cold room in an Eppendorf centrifuge (Brinkmann). For GK assay, 1 part liver was added to 5 parts 0.15 mol/L KCl, 0.005 mol/L sodium EDTA and 5 mmol/L MgCl₂, pH 7.0, then homogenized with a Teflon pestle twice for 15 s. The homogenate was centrifuged (Sorvall RC 100, DuPont Wilmington, DE) for 1 h at 105,000 x g and 4°C and the supernatant was assayed for GK activity as described by Dipietro and Weinhouse (1960) . The assays for ALT and AST were according to procedures described by Segal and Matsuzawa (1970) . The assays for PEPCK and FBP were according to procedures described by Opie and Newsholme (1967) . The assays for ME and PK were by the techniques described by Ochoa (1955) and Llorente et al. (1970) , respectively. Enzyme activity was measured in a multicell thermostatically controlled spectrophotometer (Shimadzu, Kyoto, Japan) and was expressed as substrate consumed per minute per milligram of protein. Protein was determined using a protein assay kit (#5656; Sigma Chemical).

Hepatocyte culture.

Two orphaned and nonreleasable barn owl chicks and single comb white leghorn chicks were fed day-old leghorn chicks and chicken starter, respectively, to an age of ~6 wk. After exsanguination, the liver was excised and rinsed in a petri-dish containing cold Hanks' balanced salt solution (without Ca²⁺ or Mg²⁺) with penicillin-streptomycin (10⁵ U penicillin and 100 mg streptomycin/L); hepatocytes were isolated by the procedure described by Laurin and Cartwright (1993) using type IV collagenase (Sigma Chemical). Washed cells were resuspended in cold Eagle's minimum essential media (#7399, Sigma Chemical) with 11.1 mmol/L glucose or in Eagle's with 19.4 mmol/L of glucose. Viability was determined with 4 g/L trypan blue and was >95%. Final cell concentrations were adjusted to 0.35 x 10⁹ cells/L for barn owls and 1.1 x 10⁹ cells/L for chickens. Differing cell concentrations were necessary to give similar cell protein concentrations because of the cell size differential between species. Aliquots of the final cell suspensions were taken for protein determination (Protein Assay Kit 5656, Sigma Chemical).

Cell suspension (2 mL) was added to 25-mL Erlenmeyer flasks containing 2 mL of one of the four treatment media designated as L/L, L/H, H/L and H/H [where the first letter signifies glucose concentration of 11.1 (L) or 19.4 (H) mmol/L and the second letter signifies threonine or lactate level of 2 mmol/L and 14.8 kBq (L) or 4 mmol/L and 29.6 kBq (H)]. Threonine and lactate were U-¹⁴C with a specific activity 4.85 and 3.1 TBq/mmol, respectively (ICN Biomedical, Costa Mesa, CA). The final specific activity of the substrate in the media was the same at both the high and low levels of substrate.

Treatments were replicated in four flasks per bird, giving a total of eight observations per treatment for each species. Flasks with cells and media were oxygenated with 95:5 O₂/CO₂ for 30 s and fitted with a septum stopper with a hanging center well (Kontes Vineland, NJ), which contained a piece of fluted filter paper for trapping CO₂. Measurement of substrate oxidation to CO₂ and conversion to glucose was as described by Beliveau and Freedland (1982) . The amount of substrate converted to CO₂ and glucose was calculated from the specific activity of label in the precursor and the products and were expressed as µmol product formed/(g protein · h).

Statistical analysis.

Data were analyzed by ANOVA using the General Linear Models procedure of SAS (SAS Institute, Cary, NC) for main effects and their interactions. Main effects for the intravenous glucose tolerance experiment were species, time and diet, and for the oral glucose tolerance experiment, they were species and time. Single degree of freedom contrasts were used to compare glucose values of chickens and barn owls at individual time points. Main effects for hepatic enzyme activities were species and diet. Main effects for the hepatocyte metabolism experiment were animal, species, glucose level and substrate level. Because the main effect for animal was not significant (P = 0.44), it was removed from the final model. Data are reported as means ± SEM and P < 0.05 indicated significant differences.

	RESULTS

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Once adapted, barn owls and chickens accepted the HPLG and LPHG diets well and did not develop any apparent physical or behavioral changes during the course of the experiment. Food intake of chickens and barn owls fed the two diets was not different. There was no difference in glucose concentrations in digesta samples taken from the anterior rectum (0.24 ± 0.32 and 0.35 ± 0.31 mmol/L in chickens and owls, respectively), indicating that substantial quantities of glucose did not escape the ileum.

Glucose tolerance.

Initial (0 min) plasma glucose concentrations, taken 12 h after food deprivation, were greater (P = 0.004) in chickens fed HPLG than in those fed LPHG (Fig. 1 ). In owls, initial plasma glucose concentrations were not significantly different due to diet (P = 0.14). Initial glucose concentrations for HPLG-fed birds were 14.87 and 14.14 mmol/L for chickens and owls, respectively, and final concentrations for HPLG-fed birds were 11.97 and 13.98 mmol/L. Initial glucose concentrations for LPHG-fed birds were 11.58 and 12.54 mmol/L for chickens and owls, respectively, and final concentrations were 10.32 and 13.04 mmol/L. The lack of significant time by diet interaction (P > 0.5) indicates that, within a species, the shapes of the glucose tolerance curves were not markedly affected by dietary treatments.

View larger version (12K): [in this window] [in a new window]   Figure 1. Comparison of plasma glucose levels in barn owls and chickens given an intravenous glucose challenge. Birds were fed a low protein high glucose (LPHG; 33.44% protein, 23.67% glucose; panel A) or a high protein low glucose (HPLG; 55.35% protein, 1.5% glucose; panel B) diet for 8 d and then injected intravenously with 5 mL/kg body weight of 1.1 mol/L glucose. Values are means ± SEM, n = 3. Significant main effects due to species (P < 0.001), diet (P < 0.001), time (P < 0.001) and species by time interaction (P = 0.019) were determined by ANOVA.

Plasma glucose concentrations after an oral glucose challenge were also very different between the two species (Fig. 2 ). Maximal glucose concentration was greater (26.5 and 17.6 mmol/L, respectively) and occurred later (90 and 30 min, respectively) in barn owls than in chickens. At 240 min, the plasma glucose concentration of barn owls were still significantly higher than initial levels (P = 0.002), whereas levels in chickens had returned to initial levels between 120 and 180 min.

View larger version (18K): [in this window] [in a new window]   Figure 2. Comparison of plasma glucose levels in barn owls and chickens given an oral glucose challenge. Birds were fed a high protein low glucose (HPLG; 55.35% protein, 1.5% glucose) diet for 10 d and then gavaged with 22.2 mmol glucose/kg body weight. Values are means ± SEM, n = 4. Significant main effects due to species (P < 0.001), time (P < 0.001) and species by time interaction (P < 0.001) were determined by ANOVA.

Hepatic ALT activity averaged 27.6% higher in HPLG-fed chickens and barn owls compared with LPHG-fed birds (Table 1 ). Owls and chickens fed the LPHG diet had significantly higher activities of ME compared with HPLG-fed birds, but the extent of this difference was species dependent (significant species by diet interaction). Owls exhibited only 25% greater ME activity when fed LPHG compared with HPLG, whereas chicken ME activity was 106% greater when LPHG was fed. There was a trend for a diet by species interaction for PEPCK (P = 0.08), with HPLG, resulting in a 14.9% greater activity than with LPHG in chickens, but little difference in barn owls.

Hepatocyte metabolism.

Owls oxidized more than three times as much lactate to carbon dioxide than did chickens (Table 2 ). Glucose level also significantly affected the rate of lactate oxidation, with 39% lower rates at the high glucose concentrations than at the low concentration. High levels of lactate (4 mmol/L) caused a 50% greater rate of lactate oxidation compared with low levels of lactate (2 mmol/L). However, owls and chickens did not adjust to the level of lactate in a similar manner, as evidenced by a significant species by substrate interaction. In barn owls, lactate was oxidized 53% faster at the high lactate level compared with the low level, whereas chickens oxidized lactate only 37% faster, when averaged across glucose levels.

Chickens oxidized 61% more threonine to carbon dioxide than did barn owls. Across both species, the rate of threonine oxidation at high glucose levels was 77% lower than at low glucose levels. However, chickens and barn owls responded differently to changes in glucose levels (P = 0.003 for species by glucose interaction), with chickens decreasing threonine oxidation due to high media glucose to a greater extent (89%) than barn owls (54%). A significant interaction also indicates that threonine oxidation in chickens and barn owls responded differently to threonine level. Owl hepatocytes experienced a 136% increase in oxidation with increased substrate, whereas chicken hepatocytes had a 65% decrease. There was also a glucose by substrate interaction (P = 0.013); at the low glucose level, the high level of threonine resulted in 24% higher oxidation than the lower level of threonine, but at the high glucose level, the difference was 98%.

Owl hepatocytes converted 87% more threonine to glucose than did chicken hepatocytes. Glucose level also had a significant effect on the rate of gluconeogenesis. Averaged across species, hepatocytes incubated in the low level of glucose had 335% greater rates of gluconeogenesis from threonine than hepatocytes incubated in the high level of glucose. Hepatocytes from barn owls and chickens responded differently to media glucose concentration. In barn owl hepatocytes incubated in a low concentration of glucose, the rate of gluconeogenesis from threonine was 310% higher than those incubated in a high glucose concentration. For the same comparison in chicken hepatocytes, gluconeogenesis increased 505%. Averaged across species and glucose levels, increasing threonine concentration from 2 to 4 mmol/L increased the rate of gluconeogenesis from threonine by 48%. The level of substrate influenced the two species differently (P = 0.0002 for the species by substrate interaction). In barn owl hepatocytes, high media threonine resulted in a 109% greater rate of gluconeogenesis from threonine compared with low media threonine, but in chicken hepatocytes, gluconeogenesis was 17% lower at the high media concentration.

Partitioning ratios (substrate oxidized/substrate converted to glucose) in hepatocytes from barn owls and chickens are shown in Figure 3 . When averaged across glucose levels and lactate concentrations, hepatocytes from both species oxidized more lactate than they utilized for gluconeogenesis. Chicken hepatocytes partitioned considerably more lactate toward gluconeogenesis (ratio of 2.26) compared with barn owl hepatocytes (ratio of 10.15). High levels of glucose shifted the partitioning of lactate toward oxidation in chicken hepatocytes but toward gluconeogenesis in barn owl hepatocytes.

View larger version (25K): [in this window] [in a new window]   Figure 3. Effect of media glucose or substrate concentration on the partitioning of lactate (panel A) or threonine (panel B) between oxidation to CO2 vs. use for gluconeogenesis in hepatocytes isolated from young chickens or owls. Cells were incubated in media containing the indicated levels of glucose and 14C-lactate (panel A) or 14C-threonine (panel B). Values (mean ± SEM) are expressed as the ratio of the micromoles of 14CO2 formed to micromoles of 14C-glucose formed. When lactate was the substrate, there were significant main effects due to species (P < 0.001) and glucose (P = 0.028), and due to the interaction between species and glucose (P = 0.024). When threonine was the substrate, there was a significant main effect due to species (P < 0.001), and the interactions between species and glucose (P = 0.004), and glucose and threonine (P < 0.001). Means with different letters are significantly different (P < 0.05).

The sum of lactate and threonine (lactate + threonine) that was oxidized or used for gluconeogenesis was lower at high glucose concentrations than at low concentrations (Table 2) . However, a significant glucose by species interaction indicated that barn owls were less sensitive to media glucose concentration; there was 32% less oxidation of lactate + threonine in low glucose relative to high glucose for barn owls, but 85% less in chickens. A similar pattern was observed for the rate of gluconeogenesis from lactate + threonine, with the decrease due to higher glucose levels of 62% in barn owls and 83% in chickens. Thus, compared with chickens, barn owls were less capable of decreasing substrate (lactate + threonine) oxidation and use for gluconeogenesis with increasing glucose.

A significant substrate by species interaction indicates that the effect of substrate concentration on the rate of lactate + threonine oxidation was also species dependent. In the barn owl, increasing the concentration of substrate increased the rate by 78%, but in the chicken, there was a decrease of 50%. Similarly, the use of lactate + threonine for gluconeogenesis was 98% greater in the barn owl at high relative to low substrate concentrations, but was 13% lower in the chicken. Thus, the use of lactate + threonine for either oxidation or gluconeogenesis by barn owl hepatocytes was more sensitive to additional media threonine and lactate than were chicken hepatocytes.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The domestic chicken is commonly used as a reference species for comparative studies in avian nutrition and metabolism. This is because homogeneous genetic lines of domestic chickens (Gallus domesticus) that have well-defined nutritional requirements are available worldwide and because it has been the primary species used for reagent and assay development. Interspecies studies on nutrition always suffer from the inability to compare more than a few species simultaneously, and the use of a reference species is necessary to facilitate research conducted in various parts of the world at different times. Pairwise comparisons to chickens have been made for species as diverse as hummingbirds, penguins and raptors. Together, these studies permit a comparative view of avian nutrition and metabolism across species with diets as diverse as nectar and flesh (Klasing 1998 ). Chickens and the jungle fowl from which they were domesticated are omnivorous. Our data and previous studies (Klandorf 1988 ) demonstrate that this species is glucose tolerant as indicated by their relatively low peak plasma glucose levels and fast adjustment back to normal levels after an oral or intravenous glucose challenge. Free living barn owls eat animal prey exclusively and virtually no plant foodstuffs (Martin et al. 1951 ). Because their customary diet is almost devoid of glucose, it is somewhat surprising that they were capable of absorbing sufficient dietary glucose to cause a sustained elevation in plasma glucose levels. In our experiment, glucose was added at 25% of the metabolizable energy, yet we observed no apparent signs of glucose malabsorption such as weight loss, diarrhea, dehydration or glucose in digesta leaving the ileum. The capacity of aves to move solutes arriving from the ileum into the ceca where they are subjected to microbial degradation makes it difficult to be certain that all of the glucose was absorbed intact in barn owls consuming LPHG. Dietary glucose absorption of avian carnivores is not well characterized, although Loggerhead Shrikes (Lanius ludoviacianus) slowly absorb dietary glucose (Karasov and Diamond 1982 ), as apparently did the barn owls in this experiment.

Chickens and barn owls had large differences in the shapes of their glucose tolerance curves after either an oral or intravenous glucose challenge as demonstrated by a difference in the height of the glucose peak or the duration required to return to baseline glucose levels. These findings are similar to those seen by Dobbs (1985) comparing chickens and red-tailed hawks. The 6.3-fold difference that we observed in hepatic glucokinase activity may be one possible explanation for these large differences in rates of glucose clearance. Rainbow trout are similarly glucose intolerant and have low activities of glucokinase activity; yet their ability to clear a glucose load is greatly improved by the administration of insulin (Palmer and Ryman 1972 ). The relatively low levels of glucokinase and pyruvate kinase were not induced by feeding barn owls a high glucose diet for 1 wk, as would be expected if hormonal adaptation were to occur. Insulin concentrations in the avian carnivores, penguins, eagles, kestrels and red-tailed hawks, are very low relative to chickens (Basabe et al. 1975 , Minick and Duke 1991 , Minick et al. 1996 ). However, in kestrels, insulin is more responsive to glucose than in chickens (Minick 1993 ). The fact that we saw considerable glycosuria when glucose was fed in a diet containing 33% protein, which is a known secretagogue for insulin in red-tailed hawks (Minick et al. 1996 ) and chickens, further suggests that insulin responses are not the only critical factor influencing our results.

The high rate of gluconeogenesis and poor capacity to down-regulate this process may partially explain the sustained high levels of glucose in the barn owl. Relative to chicken hepatocytes, those of the barn owl were less capable of down-regulating gluconeogenesis from lactate or threonine when media glucose concentrations were high. Thus, sustained production of glucose by the liver even during a glucose challenge may contribute to the slow fall of glucose levels.

Given that barn owls normally consume a diet that is mainly devoid of glucose, it is not surprising that they possess over threefold higher levels of PEPCK relative to chickens. Migliorini et al. (1973) found a similar difference (fourfold) in hepatic PEPCK activity between fed chickens and black vultures. Furthermore, PEPCK in black vultures consuming animal carcasses is near maximally induced and does not further increase in activity during fasting (Veiga et al. 1978 ). The normal high rate of gluconeogenesis in black vultures is associated with resistance to a fall in plasma glucose concentrations during starvation (Migliorini et al. 1973 ). The barn owls in our experiment were also resistant to hypoglycemia after a 12-h period of feed withdrawal. In chickens, fasting plasma glucose was higher in those fed HPLG than in those fed LPHG. Apparently, chickens previously fed HPLG and barn owls on either diet had less of a decline in glucose levels due to the 12-h food deprivation because they were already in a highly gluconeogenic state. Rats and chickens fed a high glucose diet and then deprived of food exhibit a lag time of a few days to reestablish previous glucose baseline levels as they shift from a mostly glycolytic state to a gluconeogenic one (Eisenstein and Strack 1971 , Hazelwood and Lorenz 1959 ). As with other raptors, barn owls commonly face periods of starvation and need to catabolize large amounts of endogenous protein to sustain plasma glucose levels. The barn owl is particularly sensitive to starvation in that it is unable to decrease its existence energy requirements and it loses lean body mass rapidly (Handrich et al. 1993 ). The high rate of gluconeogenesis in barn owls in the fed state continues to protect plasma glucose levels during starvation.

It is possible that the large difference in hepatic PEPCK activity between chickens and owls represents a difference in the tissue distribution of the gluconeogenic pathway between these two species. In chickens, the kidney accounts for as much as 30% of the gluconeogenic capacity (Watford 1989 ). The kidneys of owls are considerably smaller than those of chickens (Johnson 1968 ), and the liver might be relied on more for this pathway. Additionally, the cytosolic form of PEPCK found in the kidney of chickens is considerably more responsive to dietary manipulations than the mitochondrial form found in the liver (Watford 1989 ). We found a lack of regulation of hepatic PEPCK by diet in owls as well as chickens but our tissue preparation technique precluded the independent measurement of cytosolic and mitochondrial forms. Clearly, an understanding of the contribution of PEPCK to the glucose intolerance in barn owls will require measurement of the cytosolic form in the liver and kidney.

In vitro experiments with cultured hepatocytes revealed that barn owls oxidized 214% more lactate to carbon dioxide than did chickens. However, chickens oxidized 61% more threonine than barn owls. Owl hepatocytes might be predisposed to oxidizing less threonine than those of chickens because their customary diet requires them to rely more heavily on amino acids such as threonine for gluconeogenesis. Increasing glucose in the media decreased the catabolism of both substrates, and this sparing was considerably more pronounced in chickens than in barn owls. In this study, the low glucose level chosen was 11.1 mmol/L, which was slightly below the fasting level of chickens and barn owls. The high level of 19.4 mmol/L is similar to the maximum seen in chickens after an oral glucose challenge.

Chickens made 43% more glucose from lactate than did barn owls and, conversely, barn owls made 87% more glucose from threonine than did chickens. Owls preferred to use threonine as a gluconeogenic precursor relative to lactate, presumably because they evolved with large excesses of amino acids arriving from their normal diet, whereas the omnivorous chicken does not normally have large excesses of dietary threonine. Gluconeogenesis from lactate was greatly suppressed by high levels of glucose in chickens but not in barn owls. When threonine was the substrate, gluconeogenesis was suppressed by high media glucose in both species but to a greater relative extent in chickens. When threonine concentrations were increased in the media, barn owls increased its use for gluconeogenesis, whereas chickens increased its oxidation. This provides further evidence of the efficiency and preference of barn owls for using amino acids for gluconeogenesis. When both substrates are considered together (threonine + lactate), barn owls and chickens metabolize similar amounts to CO₂ and glucose, but barn owls were less capable of decreasing oxidation and gluconeogenesis in the presence of high levels of glucose.

Hepatocyte glucose production from lactate or threonine was markedly decreased by high media glucose concentrations, but in our feeding trial, PEPCK activity was not affected by high levels of glucose. This would suggest that either PEPCK is not the primary regulatory enzyme for this pathway or that regulation occurs at a step by-passed in our assay system.

In both species, ME activity was significantly lower in birds fed the HPLG diet. Malic enzyme and lipogenesis have been shown to increase with increasing glucose or carbohydrate in the diet and decrease with increasing protein or fat (Featherston and Freedland 1973 , Rosebrough et al. 1990 ; Yeh and Leveille 1969 ). Averaged across diets, ME activity in the liver of barn owls was only 35% of that in chickens. It is conceivable that the low ME activity of barn owls results from low selection pressure on this enzyme due to the high fat content of animal prey in their customary diet. This same reasoning may help to explain why ME activity was less affected by diet in barn owls relative to chickens. In barn owls fed the HPLG diet, ME was low, and it was only 26% higher in those that consumed the LPHG diet. Hepatic ME activity in chickens was 103% higher in those fed the LPHG diet compared with those fed the HPLG diet. Thus, barn owls have a low capacity to synthesize fatty acids and are less capable of augmenting this pathway when presented with additional dietary glucose.

By feeding diets differing widely in glucose and protein content and by manipulating glucose and gluconeogenic precursors in the media of cultured hepatocytes, we determined that the flexibility of glucose metabolism of barn owls is less than that of chickens. Barn owls, like other carnivores, have greatly diminished liver glucokinase and malic enzyme activity compared with omnivores, presumably due to a lack of selection pressure for retaining these habitually idle pathways. Conversely, the high activity PEPCK and preferential use of threonine for gluconeogenesis facilitate the consumption of a high protein, low glucose diet.

	ACKNOWLEDGMENTS

 
The authors would like to thank Earnest Avery and Richard Freedland for their help with the radioisotope procedures that were used for measuring gluconeogenesis and oxidation, Alida Morzenti for help with animal husbandry and Lyndsay Phillips for performing the liver biopsies.

	FOOTNOTES

 
¹ Presented in part at the Raptor Research Meetings, Flagstaff, Arizona [Meyers, M. R. & Klasing, K. C. (1995) Metabolic basis for glucose intolerance in raptors. J. Raptor Res. 29: 63 (abs.)].

³ Abbreviations used: ALT, alanine aminotransferase; AST, aspartate aminotransferase; FBP, fructose-bisphosphatase; GK, glucokinase; HPLG, high protein, low glucose diet; LPHG, low protein, high glucose diet; ME, malic enzyme; PEPCK, phosphoenolpyruvate carboxykinase; PK, pyruvate kinase.

Manuscript received March 26, 1999. Initial review completed May 6, 1999. Revision accepted July 27, 1999.

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