Dietary proteins are believed to participate significantly in maintaining blood glucose levels, but their contribution to endogenous glucose production (EGP) remains unclear. We investigated this question using multiple stable isotopes. After overnight fasting, eight healthy volunteers received an intravenous infusion of [6,6-2H2]-glucose. Two hours later, they ingested four eggs containing 23 g of intrinsically, uniformly, and doubly [15N]-[13C]–labeled proteins. Gas exchanges, expired CO2, blood, and urine were collected over the 8 h following egg ingestion. The cumulative amount of dietary amino acids (AAs) deaminated over this 8-h period was 18.1 ± 3.5%, 17.5% of them being oxidized. The EGP remained stable for 6 h but fell thereafter, concomitantly with blood glucose levels. During the 8 h after egg ingestion, 50.4 ± 7.7 g of glucose was produced, but only 3.9 ± 0.7 g originated from dietary AA. Our results show that the total postprandial contribution of dietary AA to EGP was small in humans habituated to a diet medium-rich in proteins, even after an overnight fast and in the absence of carbohydrates from the meal. These findings question the respective roles of dietary proteins and endogenous sources in generating significant amounts of glucose in order to maintain blood glucose levels in healthy subjects.

Dietary proteins are believed to participate significantly in maintaining blood glucose levels, but their contribution to endogenous glucose production (EGP) remains unclear. We investigated this question using multiple stable isotopes. After overnight fasting, eight healthy volunteers received an intravenous infusion of [6,6-H2]-glucose. Two hours later, they ingested four eggs containing 23 g of intrinsically, uniformly, and doubly [N]-[C]– labeled proteins. Gas exchanges, expired CO2, blood, and urine were collected over the 8 h following egg ingestion. The cumulative amount of dietary amino acids (AAs) deaminated over this 8-h period was 18.1 6 3.5%, 17.5% of them being oxidized. The EGP remained stable for 6 h but fell thereafter, concomitantly with blood glucose levels. During the 8 h after egg ingestion, 50.4 6 7.7 g of glucose was produced, but only 3.9 6 0.7 g originated from dietary AA. Our results show that the total postprandial contribution of dietary AA to EGP was small in humans habituated to a diet medium-rich in proteins, even after an overnight fast and in the absence of carbohydrates from the meal. These findings question the respective roles of dietary proteins and endogenous sources in generating significant amounts of glucose in order to maintain blood glucose levels in healthy subjects. Diabetes 62:1435–1442, 2013

The aim of this study was to determine the contribution of dietary amino acids (AA) to energy metabolism under high protein (HP) diets, using a double tracer method to follow simultaneously the metabolic fate of α-amino groups and carbon skeletons. Sixty-seven male Wistar rats were fed a normal (NP) or HP diet for 14 days. Fifteen of them were equipped with a permanent catheter. On day 15, after fasting overnight, they received a 4-g meal extrinsically labeled with a mixture of 20 U-[15N]-[13C] AA. Energy metabolism, dietary AA deamination and oxidation and their transfer to plasma glucose were measured kinetically for 4 h in the catheterized rats. The transfer of dietary AA to liver glycogen was determined at 4 h. The digestive kinetics of dietary AA, their transfer into liver AA and proteins and the liver glycogen content were measured in the 52 other rats that were killed sequentially hourly over a 4-h period. [15N] and [13C] kinetics in the splanchnic protein pools were perfectly similar. Deamination increased fivefold in HP rats compared to NP rats. In the latter, all deaminated AA were oxidized. In HP rats, the oxidation rate was slower than deamination, so that half of the deaminated AA was non-oxidized within 4 h. Non-oxidized carbon skeletons were poorly sequestrated in glycogen, although there was a significant postprandial production of hepatic glycogen. Our results strongly suggest that excess dietary AA-derived carbon skeletons above the ATP production capacity, are temporarily retained in intermediate metabolic pools until the oxidative capacities of the liver are no longer overwhelmed by an excess of substrates.

A high protein meal is considered to activate gluconeogenesis with a potential link with the satiating power of proteins. However, the contribution of dietary proteins to gluconeogenesis has never been directly determined. We used a multiple tracer methods, including the intrinsic and uniform labeling of egg proteins with 15N and 13C. After fasting overnight, eight healthy volunteers were intravenously infused with [6,6‐2H]‐glucose. Two hours later, they ingested 4 uniformly labeled eggs, cooked as an omelette (23g protein). The metabolic fate of 15N and 13C was followed through metabolic pools, and the endogenous production of glucose was determined for 8 h. After 4 h, oxidation of dietary AA was only the half of deamination while they were similar after 8 h, reaching 18 % of ingested proteins (i.e. 4.1 g). The endogenous production of glucose did not increase after meal ingestion, and significantly decreased after 6 h. The contribution of dietary amino acid to glucose was maximal between 3 and 5 h (representing 5% of produced glucose). On the 53 g of glucose that were produced trough 8 h, 1.8 g originated from dietary proteins (i.e. 3.4% of produced glucose and 44% of deaminated AA). We conclude that after fasting overnight and in the absence of glucose in the meal, a significant part of dietary AA‐derived carbon skeletons from deamination are converted to glucose but total dietary proteins poorly contributes to glucose production.

The aim of this study was to produce intrinsically and uniformly doubly (15)N-(13)C-labeled proteins. These proteins can be used as intrinsic tracers of dietary amino acids, both α-amino groups and carbon skeletons, during postprandial metabolic utilization. Two (Rhodes) laying hens were fed for 16 days with a standard poultry diet supplemented with 0, 0.2% or 0.4% of a mixture of 20 doubly (15)N-(13)C-labeled AAs. A third hen was given a non-enriched diet, as the control. The eggs laid were collected over 24 days, from 3 days before to 4 days after supplementation. The (15)N and (13)C enrichments in proteins from white and yolk were measured by EA-IRMS and GC-C-IRMS for enrichment in individual amino acids. After 10 days of supplementation, the (15)N enrichment reached an isotopic plateau at 1500 to 3000 ‰, depending on the supplementation level, in both white and yolk while the (13)C enrichment was 220 to 650 ‰ in white and was 100 to 250 ‰ in yolk. The (15)N enrichment was similar among the amino acids, except for the aromatic ones in which the enrichment was lower. The δ(13)C values were variable among amino acids in both white and yolk, ranging from 77 ‰ for tyrosine to 555 ‰ for proline with the 0.2 % supplementation level. In conclusion, the incorporation of 0.2 % labeled amino acids in the hen diet allowed us to achieve sufficient enrichment for metabolic studies. However, due to the non-homogeneity of the (13)C labeling, adequate (13)C enrichment of individual amino acids must be considered depending on the investigated metabolic pathway.

We developed a complete method to measure low [(13)C] enrichments in glycogen. Fourteen rats were fed a control diet. Six of them also ingested either [U-(13)C] glucose (n=2) or a mixture of 20 [U-(13)C] amino acids (n=4). Hepatic glycogen was extracted, digested to glucose and purified on anion-cation exchange resins. After the optimization of methylboronic acid derivatization using GC-MS, [(13)C] enrichment of extracted glucose was measured by GC-C-IRMS. The accuracy was addressed by measuring the enrichment excess of a calibration curve, which observed values were in good agreement with the expected values (R=0.9979). Corrected delta values were -15.6+/-1.6 delta(13)C (per thousand) for control rats (n=8) and increased to -5 to 8 delta(13)C (per thousand) per thousand and 12-14 delta(13)C (per thousand) per thousand after the ingestion of [U-(13)C] amino acids or [U-(13)C] glucose as oral tracers, respectively. The method enabled the determination of dietary substrate transfer into glycogen. The sequestration of dietary glucose in liver glycogen 4 h after the meal was 35% of the ingested dose whereas the transfer of carbon skeletons from amino acids was only 0.25 to 1%.

We previously proposed that under high protein feeding, dietary amino acids (AA) were partly involved in liver glycogen synthesis. We hypothesized that the entry of dietary AA into glycogen occurred early during the postprandial phase. Two groups of 24 rats were fed a normal (NP) or a high (HP) protein diet for 2 weeks and then sacrificed 1h, 2h, 3h or 4h after the ingestion of a 4 g meal containing U‐ 13C AA. Blood was sampled in the portal vein, vena cava and aorta, and the liver was collected. Hepatic glycogen content increased within the first h after the meal (18.6±8.1 mg/g) and then remained stable in NP rats, whereas in HP rats, the maximum was obtained at 3 h (13±6.7 mg/g). The apparent overall synthesis of glycogen was 170mg in NP vs. 92 mg in HP rats. Both hepatic glucose uptake and plasma insulin to glucagon ratio were significantly lower in HP than in NP rats, whereas the net liver uptake of alanine and proline was significantly increased in HP rats. The 13C enrichment measured in liver glycogen indicated a low uptake of dietary AA carbon skeletons. Considering that under HP feeding, dietary carbohydrate is entirely oxidized within the 4 postprandial hours (as previously obtained from a calorimetry study), we postulate that most of the liver glycogen in HP rats is synthesized from whole body alanine and proline.

BACKGROUND Compared with slow proteins, fast proteins are more completely extracted in the splanchnic bed but contribute less to peripheral protein accretion; however, the independent influence of absorption kinetics and the amino acid (AA) pattern of dietary protein on AA anabolism in individual tissues remains unknown. OBJECTIVE We aimed to compare the postprandial regional utilization of proteins with similar AA profiles but different absorption kinetics by coupling clinical experiments with compartmental modeling. DESIGN Experimental data pertaining to the intestine, blood, and urine for dietary nitrogen kinetics after a 15N-labeled intact (IC) or hydrolyzed (HC) casein meal were obtained in parallel groups of healthy adults (n = 21) and were analyzed by using a 13-compartment model to predict the cascade of dietary nitrogen absorption and regional metabolism. RESULTS IC and HC elicited a similar whole-body postprandial retention of dietary nitrogen, but HC was associated with a faster rate of absorption than was IC, resulting in earlier and stronger hyperaminoacidemia and hyperinsulinemia. An enhancement of both catabolic (26%) and anabolic (37%) utilization of dietary nitrogen occurred in the splanchnic bed at the expense of its further peripheral availability, which reached 18% and 11% of ingested nitrogen 8 h after the IC and HC meals, respectively. CONCLUSIONS The form of delivery of dietary AAs constituted an independent factor of modulation of their postprandial regional metabolism, with a fast supply favoring the splanchnic dietary nitrogen uptake over its peripheral anabolic use. These results question a possible effect of ingestion of protein hydrolysates on tissue nitrogen metabolism and accretion. This trial was registered at clinicaltrials.gov as NCT00873951.

Though we recently showed that, compared to casein, soluble milk protein were more extracted in the splanchnic bed but contributed less to peripheral protein accretion, the specific influence of the absorption kinetics of dietary protein, independently of their amino acid (AA) profile, on their further anabolism in individual tissues remains unknown. Clinical experiment was coupled with compartmental modeling to investigate the postprandial utilization of proteins of similar composition: intact (CA) and hydrolyzed (CH) casein. Experimental data were obtained in intestine, blood and urine for meal nitrogen (Nm) kinetics after a 15N‐labeled CA or CH meal and analyzed using a 13‐compartment model to predict the cascade of Nm absorption, transfer and regional metabolism. Compared to CA, CH had a similar whole‐body postprandial retention of Nm, but a faster rate of absorption, resulting in earlier and stronger hyperaminoacidemia and hyperinsulinemia. The faster kinetics of Nm absorption after CH favored both its catabolic (+25%) and anabolic (+40%) utilization in the splanchnic bed and lowered its further peripheral availability, which felt from 22% to 13% of the dietary N absorbed. The kinetics of dietary AA delivery thus constituted an independent factor of modulation of their regional metabolism in the postprandial phase, a fast supply favoring the splanchnic Nm uptake over its peripheral anabolic use.