In monogastric and ruminant animals, the dietary requirement for carbohydrates, primarily glucose, is solely based on the metabolism of carbohydrates to generate energy. Thus, there is not a specified dietary requirement for carbohydrates per se, other than as a source of energy. Despite this, there is an obvious metabolic requirement for glucose which necessitates either the inclusion of glucose in the diet or its synthesis de novo. To determine the dietary needs for glucose requires answers to several related questions: (1) what is the contribution of dietary glucose to whole body glucose needs; (2) what are the dietary precursors for gluconeogenesis, and (3) other than oxidation (energy), what are the fates of dietary glucose carbon?
Figure 1. 13C-Mass isotopomer distribution in plasma glucose and of hepatic Krebs cycle intermediates
in a sheep given a constant infusion (32 h) of [13C
6] glucose into the duodenum (Bequette and El-Kadi,
unpubl.). PC, pyruvate carboxylase; PDH, pyruvate dehydrogenase; PEPCK, phosphoenolpyruvate carboxykinase (c, cytosolic; m, mitochondrial).
M1 M2 M3 M6 0 1 2 3 4 M1 M2 M3 0 1 [U-13C]Glucose [M6] OAA Acetyl-CoA α-Ketoglutarate Malate Fumarate Succinyl-CoA M3: C1-C3 C2-C4 Plasma Glucose ½ 13CO2 4% from Glucose 96% from Other
Glucose entry from: Gluconeogenesis: 49% Absorption - 25% Cori cycle flux - 26%
Citrate M1 M2 M3 0 1 M1 M2 M3 0 1 M1 M2 M3 0 1 M1 M2 M3 0 1 M1 M2 M3 0 1 M1 M2 M3 0 1 ½ 13CO2 7% from Glucose 93% from Other PEPCK-c PEPCK-m PC PDH Isomerized PEP Pyruvate Succinyl-CoA [M1]-[M2] diluted by Propionate & Amino Acids 26% from Glucose Cori and Krebs
Pathways of glucose metabolism have been extensively investigated in a variety of species utilizing 13C and 2H labelled tracers by various MS (Sunny et al., 2010) and NMR (Jones et al., 2001; Jin
et al., 2004) isotopomer analytical techniques. The atoms from tracer glucose traverse through
multiple pathways before returning as newly synthesized glucose, resulting in a unique metabolic signature (isotopomer pattern) that contains rich information about glucose output, new glucose synthesis and metabolism of glucose carbon through pathways of energy metabolism. The plasma glucose 13C-mass isotopomer data in Table 1 highlights several advantages of using stable isotopes as metabolic probes, and some caveats when interpreting 13C-mass isotopomer data. The data are from a continuous duodenal infusion (32 h) of [13C
6] glucose in a sheep, and measurement of plasma glucose 13C-MID by GC-MS. Under the MS conditions that we measured glucose 13C-MID, the MS measures differences in molecular mass only and so it is not possible in this case to determine the position of the 13C label in the molecule. Under different derivatization and MS conditions it is possible to determine the 13C enrichment of the triose molecules that contribute to glucose synthesis (Neese et al., 1995). In the example, there are 3 possible positional 13C-isotopomers for the [M+1] and [M+2] glucose species, whereas for the [M+0], [M+3] and [M+6] glucose species only one isotopomer exists where all carbons contain either 12C or 13C. However, the appearance of plasma [M+6] glucose can only derive from the original [13C
6] glucose tracer and the [M+1] to [M+3] glucose species only arise from the recycling of glucose molecules, i.e. Cori and Krebs cycle (Figure 1). During the recycling process, there is loss and exchange of 13C with 12C and dilution of glucose molecules by unlabelled carbon sources (e.g. glycerol, AA) corresponding to ‘new’ glucose synthesis (i.e. gluconeogenesis), all of which has been estimated on the basis of several
Table 1. Molar tracer:tracee ratios and 13C-enrichments of intestinal mucosa intracellular AA and
plasma glucose obtained from 5-day old chicks after 4 days of ad libitum consumption of a typical
starter diet containing [U-13C] algal protein (Sunny and Bequette, unpublished).1,2
Mass isotopomer tracer:tracee ratio
[moles isotopomer per 100 moles tracee] Source of AA[%] M+1 M+2 M+3 M+4 M+5 M+6 Diet
M+n3 From diet4 Synthesis from AA5 Synthesis from other6
Leucine 0.00 0.00 0.00 0.00 0.05 4.30 5.70 75.0 - - Isoleucine 0.00 0.00 0.00 0.00 0.01 5.29 6.77 78.0 - - Glutamine 1.84 1.09 0.41 0.01 0.70 - 3.91 23.9 35.7 46.4 Glutamate 1.47 0.89 0.53 0.00 0.52 - 3.46 20.1 37.2 42.7 Aspartate 0.53 0.49 0.15 1.79 - - 10.25 23.2 6.3 70.5 Serine 1.50 1.40 1.42 - - - 6.83 27.8 27.9 44.3 Plasma glucose 1.96 0.54 0.16 0.00 0.00 0.00 0.00 - 2.6 -
1 Values are the means of chicks hatched from small (55 g eggs, n=6) and large (70 g eggs, n=6) eggs. Differences were not significant (P>0.05) between small and large eggs. On day 5 post-hatch, small egg chicks weighed 55.7 g (±5.5) and large egg chicks weighed 71.4 g (±3.7).
2 The starter diet contained (as-fed): 78% ground corn, 13% casein, 2.1% [U-13C] algal protein, 3.5% soy oil, 0.25% L-methionine, 0.1% L-lysine, 0.07% choline (60%) and a complete mineral and vitamin premix.
3 Isotope enrichment of the AA in the mixed diet, where n is the number of carbon atoms in the AA.
4 Values for Leucine and Isoleucine were calculated as: (100 × mucosal [M+n])/Diet [M+n]. The dilution factor for Leucine was used to correct other AA for dilution as: (1/0.75)(100 × mucosal [M+n])/Diet [M+n].
5 AA Synthesis from AA calculated as: % from Diet × (([M+1] + [M+2] × 2 + … + [M+n-1] × (n-1)))/[M+n] × n. Glucose synthesis from AA calculated as: ([M+1] + [M+2] + [M+3])/([M+0] + [M+1] + [M+2] + [M+3]), where [M+0] (unlabelled glucose) is 100.
theoretical models (e.g. Lee et al., 1991; Landau et al., 1998; Katz and Tayek, 1999; Haymond and Sunehag, 2000). Thus, it is possible to calculate Cori cycle flux (glucose carbon recycling) and gluconeogenesis from non-glucose sources separately, with the remainder derived from glucose absorption and glycogen breakdown.
In the study in sheep, we adopted the model of Haymond and Sunehag (2000) to quantify glucose fluxes. Using this model, Cori cycling accounted for 26% (25 g/d), gluconeogenesis 49% (47 g/d) and glucose from absorption and glycogen breakdown (if any) accounted for 25% (24 g/d) of plasma glucose entry. Another advantage of using [13C
6] glucose is that it can be used as a metabolic probe to acquire simultaneous information on individual carbon fluxes through glycolysis and the Krebs cycle (e.g. Pascual et al., 1998; Wykes et al., 1998) when combined with 13C-MID analysis of downstream metabolites. The 13C-MID in Krebs cycle intermediates in the sheep liver (Figure 1) yielded 13C-MID patterns that were predictive of known biochemistry, and which confirmed important features of ruminant liver metabolism. For example, glucose metabolism by the ruminant liver made only minor contributions to the carbon fluxes through pyruvate carboxylase (4%) and pyruvate dehydrogenase (7%), as would be expected (Armentano, 1992). Furthermore, it was notable that there was considerable dilution of 13C between α-ketoglutarate and succinyl-CoA, representing the contribution of unlabelled propionate and AA to the flux through succinyl-CoA, and thence their contribution to gluconeogenesis.
We also applied these general principles to determine gluconeogenesis, glucose carbon recycling and the proportion of AA derived from glucose in developing chicken embryos (Sunny et al., 2004; 2010). At the time of lay, the egg contains very little preformed glucose (~250 mg), so it was not surprising that we observed an abrupt, sustained increase in gluconeogenesis as embryonic development advanced (embryonic day 12, 0.12 g/d; day 14, 0.58 g/d; day 16, 0.47 g/d; day 18, 0.43 g/d). This higher glucose synthesis during development was accompanied by a higher flux through the Cori cycle, which conserves 3-carbon precursors for gluconeogenesis and limits total oxygen demands. It was notable that Cori cycling and gluconeogenesis were higher in embryos from small (51 g) compared to typical size (65 g) eggs from day 12 to 16 of embryonic development, and when taken together with the observation that blood concentrations of gluconeogenic AA (threonine, glutamine, arginine, proline, isoleucine, and valine) were lower (25 to 48%) in the small egg embryos, suggests that the small embryos diverted AA away from protein synthesis and towards gluconeogenesis. Perhaps as a consequence, the reduced embryonic growth rate of the embryos from small eggs may have been a consequence of partitioning greater supplies of AA toward gluconeogenesis.