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Original Studies |
Section of Endocrinology, Metabolism, and Nutrition (M.C.G., F.Q.N.), Metabolic Research Laboratory, Veterans Affairs Medical Center, Minneapolis, and Departments of Medicine (M.C.G., F.Q.N.) and Food Science and Nutrition (M.C.G.), University of Minnesota, Minneapolis, Minnesota 55417
Address correspondence and requests for reprints to: Mary C. Gannon, Ph.D., Director, Metabolic Research Laboratory (111G), Veterans Affairs Medical Center, One Veterans Drive, Minneapolis, Minnesota 55417. E-mail: ganno004{at}tc.umn.edu
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Abstract |
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Protein ingestion resulted in an increase in circulating insulin,
C-peptide, glucagon, 
amino and urea nitrogen, and triglycerides; a
decrease in nonesterified fatty acids; and a modest increase in
respiratory quotient.
The total amount of protein deaminated and the amino groups
incorporated into urea was calculated to be 
20–23 g. The net amount
of glucose estimated to be produced, based on the quantity of amino
acids deaminated, was 11–13 g. However, the amount of glucose
appearing in the circulation was only 2 g. The peripheral plasma
glucose concentration decreased by 1 mM after ingestion
of either protein or water, confirming that ingested protein does not
result in a net increase in glucose concentration, and results in only
a modest increase in the rate of glucose disappearance.
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Introduction |
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It has been known for many years that 50–80 g glucose can be derived from 100 g ingested protein (2). The amount of potential glucose produced depends on the amino acid composition. For beef skeletal muscle protein, this has been calculated to be 56 g glucose/100 g protein (2). However, it also has been demonstrated that ingestion of proteins results in little or no increase in circulating glucose concentration in nondiabetic people or in people with type 2 diabetes mellitus (3, 4, 5, 6, 7, 8, 9, 10). The reason for this remains unclear. In normal young men, we previously have reported that the lack of a rise in glucose is due to the production of less glucose than predicted (9). Subsequently, we wanted to determine whether this also was the case in people with type 2 diabetes. The present data indicate that even less glucose is produced in these subjects. Part of these data have been presented previously in abstract form (11).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The volunteers gave written informed
consent, and the study was approved by the Veterans Affairs
Medical Center, Minneapolis, and the University of Minnesota Committees
on Human Subjects. All volunteers had ingested a diet containing at
least 200 g carbohydrate/day, as well as their usual food energy
intake for the 3 days before the study.
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0.11 µCi glucose/min and 0.52 µCi
14C HCO3/min, respectively.
This was continued until 1600 h. At 0800 h, either 50 g beef protein (tenderloin, <5% fat, 236 g raw weight), or water alone was given in random order. This was consumed within 15 min. During the subsequent 8-h period, the volunteers were allowed to drink water ad libitum. Arterialized blood samples were drawn from the hand hourly from 0300–0700 h and then every 15 min until 0800 h. Subsequently, blood samples were drawn at 15-min intervals for 90 min, then at 30-min intervals for 150 min, and every hour for the final 4 h of the study. Subjects were studied over an 8-h period to assure complete absorption of the 50 g protein. The time interval between studies was at least 2 weeks to allow adequate time for the isotopes to be cleared from the body, but less than 3 months.
Plasma glucose was determined by a glucose oxidase method using a
Beckman glucose analyzer with an O2 electrode
(Beckman Coulter, Inc., Fullerton, CA). Serum
immunoreactive insulin was measured by a standard double-antibody RIA
method using kits produced by Endotech (Louisville, KY). Glucagon was
determined by RIA using 30-K antiserum purchased from Health Sciences
Center (Dallas, TX). Serum nonesterified fatty acids (NEFAs) were
determined by the colorimetric assay of Duncombe (12).
Triglycerides were determined using an EktaChem analyzer (Eastman Kodak Co., Rochester, NY). Plasma lactate was determined by the
method of Hohorst using lactate dehydrogenase (13). Plasma
and urine urea nitrogen were determined using the Ortho Vitros 950
instrument. The total -amino nitrogen was determined by the method
of Goodwin (14). Amino nitrogen concentration is a
measure of the total concentration of amino acids in serum.
The amount of protein oxidized was determined by quantifying the urine urea nitrogen excreted over the 8 h of the study in association with the change in amount of urea nitrogen retained endogenously. The latter was calculated by determining the change in plasma urea nitrogen concentration at 8 h and correcting for plasma water by dividing by 0.94. In this calculation it is assumed that there is a relatively rapid and complete equilibration of urea in total body water. Total body water as a percentage of body weight was calculated using the equation of Watson et al. (15). The overall assumption is that a change in plasma urea concentration is indicative of a corresponding change in total body water urea concentration. As indicated later, this may or may not be entirely correct. The sum of total urea nitrogen in urine and body water was divided by 0.86 to account for 14% lost to metabolism in the gut (16).
A respiratory quotient (RQ) was determined for periods of 10 min or more at 0730 and 0830 h, then at half-hour intervals until 1200 h and hourly intervals until 1600 h, by measuring the O2 consumed and the CO2 produced using a Deltatrac instrument. A protein RQ of 0.83 was used based on the RQ of individual amino acids (17) and the amino acid composition of beef muscle (18). The O2 consumed and CO2 produced as a result of carbohydrate and fat oxidation were calculated using the nomogram produced by Lusk (19).
Rates of peripheral glucose appearance (Ra) were calculated using the nonsteady-state equations of Steele et al. (20) as modified by deBodo et al. (21). To correct for noninstantaneous mixing of glucose, a correction factor of Vp = 0.65 was used (22). The volume of distribution for glucose was considered to be 26% of body weight. In humans the volume has been variously reported to be from 24–37% (23). Volume distributions over this range have little effect on the final Ra (see Ref. 24). The fasting baseline data used in presentation of the results represents the mean of the data obtained from the four blood samples obtained from 0700 to 0800 h for each individual. The steady-state equation was used for calculation of Ra over the 0700- to 0800-h baseline period. Quantitation of the subsequent 8-h integrated glucose Ra was determined as the area above or below the mean of the fasting Ra over the 0700- to 0800-h time frame. The area was calculated by the trapezoid rule (25) using a program developed for this purpose in our laboratory (26). The glucose disappearance rate (Rd) was calculated using the equation Rd = (Ra) - (rate of change of the glucose pool) (27).
The rate of gluconeogenesis was estimated by determining the incorporation of 14C from infused 14C-NaHCO3 into glucose as described by McMahon et al. (28). There is debate regarding how accurately this method, as well as other tracer methods, reflects the actual gluconeogenic rate. The method used generally is considered to underestimate the gluconeogenic rate. Potential problems in interpretation of the data using this and other methods have been reviewed previously by others (28, 29, 30, 31, 32). However, because each subject served as his or her own control, the data can be used for comparative purposes regardless of the potential limitations of the method.
Statistics were done by multivariate ANOVA using the Minitab computer program, followed by post hoc t tests when data were significantly different. Because of the large number of comparisons at individual time points, and the concern over type I errors, the criterion of significance was set at P of 0.01 or less.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
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3-fold). In contrast to the prompt
return of the glucose concentration to the fasting control value at
2.5 h, the insulin concentration was still at a maximum at that
time. The insulin concentration did not return to a fasting value until
7 h after the meal (Fig. 2). The
C-peptide curve (Table 2) was similar to
that of the insulin curve (Fig. 2).
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).
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). Beginning after the first half hour,
the glucose appearance rate tended to be slightly higher after protein
ingestion.
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). There was a gradual increase over the
8-h period of the study, both when the subjects were fasting (control)
and when they received protein. The rate was modestly higher after
protein ingestion, but it was not statistically significant.
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-amino nitrogen concentration decreased slightly after only
water was ingested (Fig. 6). After
protein ingestion, it increased rapidly indicating a rapid digestion of
the protein and absorption of the resulting amino acids. It returned to
near the control value by the 7th h, indicating that digestion,
absorption, and metabolism were largely completed.
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). Interestingly, the uric
acid concentration also increased after beef ingestion (Table 2
).
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The NEFA concentration decreased after protein ingestion (Table 2), and
this was largely the mirror image of the insulin concentration (Fig. 2).
The RQ decreased modestly when only water was ingested. It increased
modestly following the ingestion of protein. The curve was similar to
the insulin curve and was the mirror image of the NEFA concentration,
indicating a substitution of carbohydrate and/or protein for fatty
acids in the fuel mixture being oxidized (Fig. 8).
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(19), the integrated mean nonprotein
fuel mixture oxidized over the 8 h of the study when only
water was ingested was 27% carbohydrate and 73% fat. When
protein was ingested it was 31% carbohydrate and 69% fat.
There also was a late rise in triacylglycerol concentration when
protein was ingested (Table 2). In the absence of protein ingestion the
mean concentration was stable.
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Discussion |
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In 1971, it was suggested that protein ingestion did not raise the circulating glucose concentration because an increased production and release of glucose from the liver was balanced by an increased uptake and utilization of glucose by peripheral tissues (34). The mechanism proposed was that an increased circulating glucagon concentration, resulting from the ingestion of protein, would stimulate glucose production from amino acids in the liver. The increased insulin concentration resulting from the ingestion of protein then would stimulate peripheral tissues, primarily skeletal muscle, to remove the glucose produced and to store it as glycogen (34). The latter is a well known effect of high concentrations of insulin. However, using direct hepatic vein catheterization techniques, a significant increase in glucose production in the splanchnic bed after protein ingestion could not be demonstrated either in dogs (35) or in humans (36).
In a previous study in which normal young males ingested 50 g
protein in the form of casein, we reported that the ingested protein
did increase the glucose appearance rate, but this was considerably
less than expected from the amount of amino acids deaminated and the
resulting amino groups synthesized into urea (9). In the
present study, the increase in glucose appearance rate in subjects with
untreated type 2 diabetes was even less when 50 g beef protein was
ingested (Fig. 4). Based on the glucose appearance rate data integrated
over 8 h, the calculated amounts of glucose entering the
circulation were a mean of 70.8 g and 68.2 g, when protein or
water were ingested, respectively. This difference was statistically
significant (P < 0.001). Thus, the amount of glucose
appearing in the circulation due to protein ingestion was 2.6 g.
In normal young males ingesting cottage cheese protein, 10 g
glucose were calculated to have appeared over the 8 h of the study
(9). Thus, isotope dilution data as well as direct
catheterization data indicate that protein ingestion does not result in
a major glucagon-stimulated increase in glucose production and release
into the circulation with a consequent increased glucose removal rate
due to an increased insulin concentration. However, a small increase in
glucose appearance does occur. The RQ data (Fig. 8) suggest that the
additional small amount of glucose appearing in the circulation was
largely used as fuel.
To calculate the mass of urea retained at the end of the study, the concentration of urea nitrogen in serum water was multiplied by total body water. This was added to the urea nitrogen excreted to calculate the amount of protein catabolized. In these older, obese subjects the mean total body water was estimated to be 47 ± 2 L or 50 ± 0.6% of body weight using the formula of Watson et al. (15).
The calculated mean total amount of protein metabolized was 20 g after water ingestion and 42 g after protein ingestion, or a net amount of 22 g metabolized (deaminated) as a result of the ingestion of the protein. The change in urea concentration in body water based on the change in plasma concentration made up a significant proportion of the total calculated change in urea flux.
Use of the final plasma urea nitrogen concentration to estimate the
amount of urea retained in the body water assumes that the rate at
which the additional urea produced equilibrates in body water is rapid
and does not significantly affect the calculations (i.e. it
represents the maximal amount of urea that could be retained). In the
present study, the urea equilibration rate was not determined. It has
been estimated to be 40 min. However, two thirds of the final
equilibration occurred within 5 min (37). In dogs
(38) and in cats with the ureters ligated, it also was
30–45 min (39). The equilibration rate is similar to that
of D2O (40). Thus, use of changes in
serum urea concentration to calculate changes in protein oxidation
rate, as used here and by others (41), may represent a
modest overestimation of the rate at which proteins are being
deaminated. If we assume a urea pool size of 40% of body weight to
allow for incomplete mixing of urea in total body water, the mean
amount of protein-derived amino acids catabolized would be 20 g,
instead of 23 g. Thus, these are likely to represent minimum and
maximum values for this process (i.e. 20 g and 23
g).
Based on the above calculations, and assuming that for each gram of
protein deaminated, 0.56 g of glucose may be produced
(2), the expected glucose production would be 11–13 g.
This is considerably more than the amount of glucose appearing in the
blood as a consequence of the ingested protein. The gluconeogenic rate
as estimated by the incorporation of 14C
HCO3 into glucose was nearly the same whether
water or protein was ingested (Fig. 5). Again, the net amount of
glucose produced because of the protein meal, as calculated using the
incorporation of 14C HCO3
into glucose, was only 2 g. Even assuming entrance into the
gluconeogenic pathway of some amino acids not detected by
14C HCO3 incorporation,
etc. (1, 29, 30, 32, 42, 43), a maximal
increase in protein-stimulated gluconeogenesis is likely to have
yielded less than 4 g over the 8-h period. Because there was only
a very modest increase in glucose appearance in the circulation, the
ingested protein-derived amino acids most likely replaced other
gluconeogenic substrates.
The switching from endogenous gluconeogenic substrates to absorbed gluconeogenic substrates has been observed after iv administration of gluconeogenic substrates (44, 45) and after ingestion of fructose (24, 46) and galactose (47). In addition to protein, the latter are the other major gluconeogenic substrates derived from dietary sources. Their ingestion also results in only a modest increase in appearance of glucose in the circulation. Diversion of glucose into glycogen cannot be ruled out but would be unlikely in the presence of a high glucagon concentration. In rats, intragastric administration of a large amount of protein actually resulted in glycogenolysis (48).
The modest increase in glucose concentration and the intracellular switching from one gluconeogenic substrate to another occurs even though the stimulated rise in insulin and in glucagon was considerably different following ingestion of these fuels (24, 46, 47). The mechanism remains to be determined.
In overnight fasted normal subjects, it has been reported that
35–50% of the glucose being produced comes from gluconeogenesis
(30, 32, 43). It also has been reported that the gradual
decrease in glucose production with fasting is due to a progressive
reduction in the rate of glycogenolysis. There was little change in the
rate of gluconeogenesis (43). In the present study, the
initial proportion of the glucose appearance rate due to
gluconeogenesis as estimated by the incorporation of
14C-HCO3 into glucose, also
was 35% in these overnight fasted subjects with mild type 2
diabetes, but gradually increased over the 8 h of the study. As
indicated, the method used underestimates the true gluconeogenic
rate.
With continued starvation there was a progressive decrease in glucose
appearance rate and an increase in relative gluconeogenesis rate. Thus,
the results are different than those reported in nondiabetic subjects.
Also, in contrast to nondiabetic subjects where starvation for an
additional 8 h beyond the overnight fast had only a minor effect
on glucose concentration (9) in the present study, the
glucose concentration decreased continuously, indicating the importance
of glycogenolysis in maintaining the elevated, overnight fasting
glucose concentration and glucose appearance rate in people with type 2
diabetes (Fig. 1). An indirect estimate of the importance of
glycogenolysis in the maintenance of an elevated glucose concentration
in people with type 2 diabetes with short-term fasting also has been
reported from our laboratory previously (33).
The decrease in glucose concentration in association with a decrease in
glucose production rate when only water was ingested occurred in
association with an increase in NEFA concentration but in the absence
of a significant change in insulin concentration. These changes suggest
development of a modest degree of hepatic and adipose insulin
resistance due to fasting over the 8-h time period. This downward trend
was only temporarily interrupted by protein ingestion, even though the
insulin concentration remained elevated (Fig. 2). An increase in
insulin concentration would have been expected to decrease the glucose
production rate (49). Presumably, an inhibiting effect on
glucose production by insulin was just balanced by the elevated
glucagon concentration. However, the precision of this balance is
surprising and suggests that other factors are playing a role.
The glucose disappearance rate also decreased progressively with fasting, and this was modestly greater than the decline in glucose appearance rate. It was calculated that the difference in glucose Ra and Rd resulted in 5 g glucose being used in excess of the glucose appearance rate over the 8 h of the study, both when the subjects were given protein or only water. The decrease in Rd indicated a progressive conversion from a carbohydrate-based fuel mixture to a more fatty acid-based mixture. This was confirmed by the RQ data.
The increase in uric acid concentration after beef ingestion is
intriguing. It may have been due to the rapid oxidation to uric acid of
purines or their derivatives present in the beef muscle, although an
accelerated rate of endogenous purine metabolism cannot be ruled out.
In beef muscle, purine nitrogen has been estimated to be 0.06% by
weight (50). An increased glucagon concentration has been
reported to increase the rate of uric acid excretion, whereas a high
insulin concentration has been reported to decrease it. However,
neither resulted in a change in plasma uric acid concentration
(51, 52). Ingestion of protein sources free of nucleic
acids also did not result in a change in plasma uric acid
concentration, but uric acid excretion was stimulated
(53). Thus, the results were similar to those observed
with a raised glucagon concentration (52). Unfortunately,
urine uric acid excretion was not quantified in the present study.
The increase in triacylglycerol concentration induced by beef ingestion also is of interest. It may have been due to the small amount of fat present in the beef. However, based on literature data, this is unlikely. An amount of fat more than twice that present in the beef ingested did not raise the triacylglycerol concentration (54). In addition, an increase in triacylglycerol concentration was noted after ingestion of egg white (fat free) and very low-fat cottage cheese (8). Thus, ingested protein per se may result in a rise in triacylglycerol concentration. Whether a change in the synthesis or removal rate is primarily responsible for the change remains to be determined.
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Acknowledgments |
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Footnotes |
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Received September 28, 2000.
Revised November 6, 2000.
Accepted November 9, 2000.
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References |
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0.01); 
,
statistically different from the 0800-h time point
(P 
