Metabolic

Response

to Cottage Cheese or Egg White Protein, With or Without Glucose, in Type II Diabetic Subjects

Mary C. Gannon,

Frank Q. Nuttall, James T. Lane, and Lynn A. Burmeister

Test meals with 25 g protein in the form of cottage cheese or egg white were given with or without 50 g glucose to male subjects with mild to moderately severe, untreated, type II diabetes. Water was given as a control meal. The glucose, insulin, C-peptide, alpha amino nitrogen (AAN), glucagon, plasma urea nitrogen (PUN), nonesterified fatty acid (NEFA), and triglyceride area responses were determined using the water meal as a baseline. The glucose area responses following ingestion of cottage cheese or egg white were very small compared with those of the glucose meal, and were not significantly different from one another. The serum insulin area response was 3.6-fold greater following ingestion of cottage cheese compared with egg white (309 v 86 pmol/L.h). The simultaneous ingestion of glucose with cottage cheese or egg white protein decreased the glucose area response to glucose by 11% and 20%. respectively. When either protein was ingested with glucose, the insulin area response was greater than the sum of the individual responses, indicating a synergistic effect (glucose alone, 732 pmol/L.h; glucose with cottage cheese, 1,637 pmol/L.h; glucose with egg white, 1,213 pmol/L.h). The C-peptide area response was similar to the insulin area response. The AAN area response was approximately twofold greater following ingestion of cottage cheese compared with egg white. Following ingestion of glucose, it was negative. When protein was ingested with glucose, the AAN area responses were additive. The glucagon area response was similar following ingestion of cottage cheese or egg white protein. Following glucose ingestion, the glucagon area response was negative. Glucose coingestion had a modest effect on the glucagon area response to cottage cheese (-20% decrease), but markedly attenuated the response to egg white. The amount of ingested protein metabolized was calculated using the change in PUN concentration and the amount of urea nitrogen excreted in the urine. Following ingestion of cottage cheese it was 81%. and for egg white it was 52%. Ingestion of glucose with cottage cheese reduced the amount metabolized and attributable to urea formation to 56%. Coingestion of glucose reduced the amount of egg white metabolized to just 12%. In conclusion, the metabolic response to two common dietary proteins was very different. Coingestion of glucose with these proteins resulted in decreased urea formation. A close correlation was observed between the amount of protein metabolized and the glucagon area response (I = .94). Thus, the amount of ingested protein metabolized mav be mediated, at least in part, bv the circulating glucagon concentration. Copyright 0 1992 by W.B. Saunders Co&any .

PREVIOUSLY REPORTED that 50 g protein given in the form of very lean beef was just as potent as 50 g glucose in increasing the circulating insulin concentration in subjects with type II diabetes.’ In addition, when beef protein was ingested with glucose, there was a strong synergistic effect on insulin secretion between the two. Subsequently, we determined that 25 g of six other commonly ingested proteins, and beef protein, also acted synergistically with glucose in stimulating an increase in circulating insulin concentrations in type II diabetic subjects.: Generally, the S-hour insulin area response to the ingested protein from these various sources was similar. However, it was greatest with cottage cheese protein (360%) and least with egg white protein (190%) when compared with glucose ingested alone (loo%).? The greater increase in insulin resulting from cottage cheese ingestion was associated with a greater increase in total amino acid concentration when compared with that observed after egg white ingestion. This suggested that either the egg white was digested more slowly or less well, or that the absorbed amino acids were metabolized more rapidly. It also was possible that the ingested glucose modified the digestion of the protein and the absorption and/or metabolism of the absorbed amino acids in a differential fashion. The purpose of the present study was to determine whether differences existed in the metabolism of cottage cheese and egg white in type II, middle-aged or older, modestly obese, diabetic subjects when ingested without glucose. More importantly, we were interested in determining the possible modification in metabolism of these proteins when glucose was ingested simultaneously. We were

w

Metabolism,

Vol 41. No 10 (October), 1992: pp 1137.1145

particularly interested in whether the increase in plasma total amino acid concentration and/or the amount of absorbed amino acids accounted for by deamination and formation of urea would be affected by the ingested glucose. MATERIALS

AND METHODS

Seven, male, untreated,

mild to moderately severe, type II diabetic subjects were studied in a metabolic unit. All subjects met the National Diabetes Data Group criteria for the diagnosis of type II diabetes mellitus? The patient characteristics are given in Table 1. Written informed consent was obtained from all subjects, and the study was approved by the Department of Veterans Affairs Medical Center and the University of Minnesota Committee on Human Subjects. All participants had ingested a diet containing at least 200 g carbohydrate per day with adequate food energy for 3 days before testing. None of the subjects were treated with either oral hypoglycemic agents or insulin before the study.

From the Metabolic Research Laborator?, and the Section of Endocrinology, Metabolism, and Nutrition, Veterans Affairs Medicul Center, Minneapolis; and the Departments of Medicine and Food Science and Nutrition, University of Minnesota, Minneapolis, MN. Supported by Merit Review Research Funds ,fiom the Veteruns Administration, and byfinds from the National Dairy Board, administered in cooperation with the National Dairy Council. J. T.L. and L.A.B. were Fellows in Endocrinology. Address reprint requests to Mar?/ C. Gannon. PhD. Nutritional Biochemist, Director. Metabolic Research Laboratory. 11IG. VA Medical Center, Minneapolis, MN 55417. Copyright 0 1992 by W.B. Saunders Compaq 0026-0495192141 IO-OOlS$O3.OOJO

1137

1138

GANNON

Table 1. Patient Characteristics Fasting Time Since Subject Age Glucose BMI HbA, Diagnosis NO. (yr) (mmol/L) (kg/m*) (“4 of Diabetes 1

70

8.0

26

8.3

6vr

Concomitant Diseases Hx of peptic ulcer disripheral vascular disease with a hx of claudication

71

8.6

33

14.2

New

Chronic lymphocytic leukemia,

hx of ele-

vated hemoglobin with normal red cell mass, chronic obstructive

pulmonary

disease 3

67

6.9

33

7.6

2 vr

Hypertension,

hiatal

esophagitis,

athero-

sclerotic coronary vascular disease with stable exertional angina 4

60

5.8

27

7.1

15yr

Congestive

heart fail-

ure, atherosclerotic cardiovascular

dis-

ease 5

73

8.2

28

13.0

4mo

Hx of gallstone

pan-

creatitis, hypertension, hx of chronic bronchitis 6

56

7.9

35

7.1

3mo

Peripheral vascular disease, hx of coronary artery disease, cerebrovascular disease, Bell’s palsy

7

76

8.2

30

8.7

2 vr

Hypertension,

coro-

nary artery disease, B-12 deficiency Mean SEM

68 2.7

7.7 0.4

30 1.3

9.4

-

1.1

-

Following each study period, the patient consumed a regular hospital diet ad libitum for the rest of the day. Plasma glucose level was determined by a glucose oxidase method using a Beckman glucose analyzer with an 02 electrode (Beckman Instruments, Fullerton, CA). Serum immunoreactive insulin was measured by a standard double-antibody radioimmunoassay (RIA) method using kits produced by Endotech (Louisville, KY). C-peptide was measured using a double-antibody radioimmunoassay method with kits produced by Incstar (Stillwater, MN); the antibody to C-peptide has only a 4% reactivity with proinsulin. Hemoglobin Al (HbA1) level was determined using boronate agarose affinity columns (Isolab, Akron, OH). Glucagon level was determined by RIA using 30K antiserum purchased from Health Science Center (Dallas, TX). The level of serum nonesterified fatty acids (NEFA) was determined using a NEFA C kit (Wako Chemicals, Dallas, TX). Alpha amino acid nitrogen (AAN) was determined by the method of Goodwin.4 Triglycerides and urea nitrogen were determined using an EktaChem analyzer (Eastman Kodak, Rochester, NY). The areas under the curves were calculated using the trapezoid rule.” The concentration of the respective hormone or metabolite, measured following ingestion of water, was used as a baseline. Urea appearance, which represents the amount of urea excreted in the urine plus the amount retained in the body, was determined. The urine urea nitrogen excreted during each 5-hour study period was measured. The amount of urea nitrogen excreted during the 5-hour starvation period (water control) was subtracted from the amount excreted during the 5 hours after the meal. Next, the difference in plasma urea nitrogen (PUN) concentration between that measured at 0 hours and that at 5 hours after the test meal and after water ingestion was determined. The decremental urea concentration value for the water control was then subtracted from the increment of that of the test meals, ie, the difference between the concentration of the test meals and the water control at 5 hours was corrected for the concentration at zero time in each subject. This concentration was divided by 0.93 to account for the fact that 93% of plasma is water. The resultant concentration was multiplied by 60% of the body weight of the study subjects, since the urea dilution volume is reported to be 60% of body weight.6 This represents the amount of retained urea. The amount of ingested protein metabolized was determined using the urea appearance data and multiplying by 6.25 for egg white and 6.38 for cottage cheese, to convert nitrogen to protein. Egg white protein is 16.0% nitrogen, whereas cottage cheese protein is 15.7% nitrogen.’ Statistics were determined using either Student’s f test for paired variates or ANOVA, as appropriate. with the Statview 512+ program (Brain Power, Calabasas, CA) for the Macintosh computer (Apple Computer, Cupertino, CA). A P value of < .05 was the criterion for significance. Data are presented as the mean + SEM. collections.

ease, cataracts, pe-

2

ET AL

After an overnight fast of 8 to 10 hours, an indwelling catheter was inserted into an antecubital vein. During the sampling period, the catheter was kept patent with intravenous saline. Test meals were randomized and given as a single breakfast meal at 8:00 AM. All subjects consumed all test meals. The meals consisted of 50 g glucose alone, 25 g protein alone, or 50 g glucose + 25 g protein. The subjects response to starvation over this time period also was studied as a control. The overnight fast was continued for an additional 5 hours following ingestion of 480 mL water at 8:00 AM. This was the same amount of liquid ingested with the protein meals. Blood was obtained before and %, 1, 2, 3, 4, and 5 hours after the beginning of the meal. Twenty-five grams protein was given in the form of either cottage cheese or egg white. The cottage cheese was 147 g, grade A, dry, and not more than 0.5% milk fat (Old Home, Minneapolis. MN). It was served with salt, pepper, and an herb packet. The egg white meal was prepared to resemble scrambled eggs. Six drops of yellow food coloring were added to 230 g (raw weight) egg white. This was beaten slightly and cooked in a microwave oven. The meals were served with 2 cups (-480 mL) decaffeinated coffee, and were consumed in approximately 15 minutes. During the remainder of the 5-hour period, the subjects were encouraged to consume water to ensure accurate urine

RESULTS

When subjects were starved during the 5 hours of the test (water controls), serum glucose concentrations declined steadily, as reported previously.8 The decrement was approximately 1.1 mmol/L (20 mg/dL) (Fig 1A). Following ingestion of cottage cheese alone or egg white alone, the glucose concentration also decreased. However, the decrease was slightly less than that following the water alone. Following ingestion of 50 g glucose, the serum glucose concentration increased rapidly and reached a peak at 60 minutes. It was essentially unchanged at 120 minutes and subsequently returned to the control value at 5 hours. When either of the proteins was ingested with glucose, the serum glucose

EFFECTS OF DIETARY GLUCOSE AND PROTEIN

7

Qlucoss

Glucose

Fleeponas - 120 ---x

6

Am

Response

B

20

T

5

300

4 33 52

200 I. E” 1

-21 0

. 64

. (20

0

LO 160

240

CCh

300

CCh +Glu

Egg

Egg +Glu

Glu

Miwtes aftu Moel heulln

Respenw

IneullnArea Responw

2500 -

-3%

D _ 300 .

Fig 1. (A) Glucose response to meals. Incremental change in plasma glucose was determined for 5 hours after ingestion of meals. Meals consisted of 480 mL water (control), 25 g protein in the form of cottage cheese or egg white, 50 g glucose, or 25 g protein + 50 g glucose. The mean fasting plasma glucose was 138 2 3.5 mg/dL (7.7 + 0.4 mmol/L). (6) Glucose area response to meals. Areas under the curves in top panel were determined using the trapezoid rule, with the water (control) meal as a baseline. Areas are significantly different (P < .05) if they do not share a common letter. (C) Insulin response to meals. The mean fasting insulin concentration was 25 f 1.1 pU/mL (180 f 8 pmol/L). (D) Insulin area response to meal. (E) C-peptide response to meals. The mean fasting C-peptide concentration was 2.4 f 0.1 ng/mL (0.72 f 0.03 pmol/mL). (F) C-peptide area response to meal.

-‘w t-d-yl.2o 0

64

120

CCh + Glu

2cao-

CCh

160

CCh+Glu

Es

Em+GIu

Glu

-

Minutes ehw Meal

3600 FF

12

-I-

6W

0

0 60

120

160

240

300

CCh

CuI+GIu

Egg

Eq~+Glu

GlU

h4mute~an9fMe01

concentration also reached a peak at 60 minutes. It subsequently decreased more rapidly than after ingestion of glucose alone. The glucose area responses following ingestion of cottage cheese and egg white were small and not statistically different (Fig 1B). When egg white was ingested with glucose, the glucose area response was modestly less (11%) than that when glucose was ingested alone. The area response following cottage cheese + glucose ingestion was further reduced (20%). The serum insulin concentration changed little with starvation (ie, overnight fast + 5 hours of no food). Ingestion of egg white also had little effect on the serum insulin concentration (Fig 1C). Following ingestion of cottage cheese, the serum insulin concentration increased modestly, reached a peak at 60 minutes, and returned to the control value by 4 hours. After ingestion of glucose alone, the insulin concentration reached a peak at 60 minutes and returned to the control value by 5 hours. The peak response following ingestion of glucose alone was approximately twice that following ingestion of cottage cheese. When cottage cheese or egg white was ingested with glucose, the

peak insulin concentration was much greater than that following ingestion of glucose alone, and it was delayed, occurring at 120 minutes. In addition, unlike the results obtained following ingestion of protein alone or glucose alone. the concentration had not returned to the control value by 5 hours. Following ingestion of cottage cheese alone or egg white alone, the insulin area response was 309 and 86 pmol.h/L, respectively (Fig 1D). Thus, the area response was 3.6.fold greater following ingestion of cottage cheese compared with egg white and was statistically significant. Following ingestion of glucose alone, the insulin area response was 732 pmol.h/L. When protein was ingested with glucose, there was a marked, statistically significant increase in insulin area compared with ingestion of glucose alone. Following ingestion of the glucose + cottage cheese meal, the actual area response was 1,637 pmol.h/L. The sum of the individual responses to cottage cheese alone and glucose alone was 1,041 pmol.h/L. Thus, when cottage cheese protein was ingested with glucose, the insulin area response was not additive, but synergistic. as we have reported previously.’ Following ingestion of the glucose +

1140

egg white meal, the response was 1,213 versus 818 pmol.h/L for the sum of the individual glucose and egg white response; again, synergism was demonstrated. The C-peptide curves generally were similar to the insulin curves. The C-peptide concentration changed little with starvation (Fig 1E). Ingestion of egg white had only a small effect on the C-peptide concentration. Following ingestion of cottage cheese, the C-peptide concentration increased modestly, reached a peak at 60 minutes, and returned to the fasting value by 4 hours. After ingestion of glucose alone, the C-peptide concentration reached a peak at 120 minutes. It was essentially unchanged at 180 minutes. It decreased toward baseline, but was still elevated at 5 hours after the meal. When cottage cheese or egg white was ingested with glucose, the peak C-peptide concentration was much greater than that following ingestion of glucose alone. The peak responses occurred at different times after these two meals. Following ingestion of egg white + glucose, the C-peptide concentration increased rapidly until 60 minutes after the meal. It plateaued, then gradually decreased. Following ingestion of cottage cheese + glucose, the peak concentration occurred at 120 minutes, after which the concentration decreased. Unlike the results following ingestion of protein alone, when glucose was included in the meals, the C-peptide concentration had not returned to the control value by 5 hours. The C-peptide area response was threefold greater following ingestion of cottage cheese compared with egg white (630 v 210 pmol.h/L, respectively) (Fig 1F) and was statistically significant. Following glucose ingestion, the C-peptide area response was 1,400 pmol.h/L. The C-peptide area response to the glucose + cottage cheese meal was 3,020 pmol.h/L. The sum of the individual responses to cottage cheese and glucose alone was 2,000 pmolhll. Following ingestion of the glucose + egg white meal, the actual response was 2,500 versus 1,100 pmol.h/L for the sum of the individual glucose and egg white response. Thus, when glucose was given with protein, the C-peptide area response was not additive, but synergistic and quantitatively similar to the insulin area response. Since the C-peptide and insulin responses are similar, the synergistic effect of protein on insulin area responses cannot be explained by a change in the insulin removal rate. Although we have not formally determined insulin removal rates, the ratio of insulin area response to C-peptide area response was approximately equal for each of the meals (mean, 0.47 + 0.02). In the fasted state, the AAN concentration decreased slightly during the first 3 hours of the experiment and then reached a plateau (Fig 2A). Glucose ingestion resulted in a more dramatic decrement in AAN, and the concentration remained below the fasting value at 5 hours. Following ingestion of egg white, the AAN concentration increased and reached a peak at 120 minutes. By 5 hours, the concentration was still modestly elevated compared with the fasting value. Following ingestion of cottage cheese, there was a rapid, large increase in AAN concentration that reached a peak at 60 minutes. The peak concentration was approximately three times that observed following ingestion of egg white, and occurred 1 hour earlier. By 5 hours,

GANNON

ET AL

the concentration had returned to baseline. When glucose was ingested with egg white, AAN concentrations were only modestly greater than those present during starvation, indicating that glucose in the meal attenuated the AAN response to egg white. Following ingestion of the cottage cheese + glucose meal, the AAN concentration increased more slowly than after ingestion of cottage cheese alone. The peak concentration was lower and occurred later, ie, at 120 minutes. The return toward baseline also was delayed compared with that after cottage cheese alone. The AAN concentration was higher at 4 and 5 hours after the cottage cheese + glucose meal compared with the concentration after cottage cheese alone. The integrated AAN area response was negative for glucose (-1.23 mmol.h/L), but was positive for all meals containing protein (Fig 2B). In addition, the AAN area response for egg white (1.95 mmol.h/L) was statistically significantly lower than that for cottage cheese (3.59 mmol.h/ L). Following the ingestion of the glucose + cottage cheese meal, the response was 2.72 mmol.h/L. The sum of the individual responses to cottage cheese alone and glucose alone was 2.36 mmol.h/L. Following ingestion of the glucose + egg white meal, the actual response was 0.79 versus 0.72 mmol.h/L for the sum of the individual glucose and egg white response. Thus, the amino acid response to glucose and protein was additive in each case. As with the insulin concentration, the plasma glucagon concentration was changed little when the subjects were starved over the same 5 hours of the experiment (Fig 2C). Cottage cheese ingestion resulted in a rapid and large increase in plasma glucagon concentration, which reached a peak at 60 minutes. It subsequently decreased toward baseline, but was still elevated at 5 hours. Following ingestion of egg white, the rate of the increase was slower than that following ingestion of cottage cheese, and the peak was lower. However, from 120 to 300 minutes after the meal, the mean glucagon concentrations were identical whether egg white or cottage cheese was the ingested protein. Glucose ingestion resulted in a modest increase in glucagon concentration at 30 minutes, followed by a decrease of 112 rig/L by 3 hours; it was still decreased at 5 hours. Glucose ingested with protein attenuated the glucagon response. When glucose was ingested with cottage cheese, the resultant peak in glucagon concentration was approximately 50% lower than that with cottage cheese alone. When glucose was ingested with egg white. the glucagon concentration was similar to that following ingestion of egg white alone at the 30-minute time point only. At all other time points, the concentration was considerably less than that with egg white alone; in fact, it was similar to that measured during starvation. The integrated glucagon area response was modestly but not significantly lower following the ingestion of egg white alone (563 ng.h/L) compared with cottage cheese alone (711 ng.h/L) (Fig 2D). The glucagon area response following glucose alone was negative, as expected. When glucose was ingested with protein, the glucagon area response was not additive. Glucose had less of an effect on the cottage cheese meal and a far greater effect on the egg white meal

EFFECTS OF DIETARY GLUCOSE AND PROTEIN

1.5

AAN

b

Aeeponae

AAN Aree Response

---x

.

CCh+Glu

-1 5 t

a -0

I.

0

-.

60

1..

120

-.

160

240

c 3cc

I-1

CCh

CCh+Glu

Egg

Egg+Glu

Qlucagon Area

Response

Gb

-’

Minuta, dler Mod

Qlucegon

Reeponee

Cmlrd (water) 30C

---X

0 Cal

.

cch+Glu

A

Egg+Gb

1000D

,000

1,

1

.200

-104

I

‘0

:

CCh

CCh +Glu

Egg

Egg +Glu

Glu

J -600

PUN Area Response Fig 2. [A) AAN response to meals. Legend same as Fig IA. The mean fasting AAN concentration was 4.03 k 0.06 mg/dL (2.9 f 0.04 mmol/L). (B) AAN area response to meal. (C) Glucagon response to meals. The mean fasting glucagon concentration was 390 2 26 pg/mL (390 f 25 ng/L). (D) Glucagon area response to meal. (E) PUN response to meals. The mean fasting PUN concentration was 21 2 0.9 mg/dL (7.5 + 0.3 mmol/L). (F) PUN area response to meal.

than would be predicted by adding the responses to the individual meals. Glucose ingested with cottage cheese resulted in a 34% decrease in the glucagon area response. When glucose was ingested with egg white, the glucagon area response was markedly decreased, and was nearly identical to that following the control meal (starvation). The PUN concentration decreased continuously when the subjects were starved (Fig 2E), as observed previous1y.s.” An even greater decrease occurred following the ingestion of glucose. Following cottage cheese ingestion, the PUN concentration was essentially unchanged for 60 minutes. It then increased at 120 minutes and remained elevated for the duration of the study. Following egg white ingestion, the PUN concentration also was essentially unchanged for 60 minutes, after which time it increased modestly and remained elevated. Thus, following ingestion of either protein, there was a 60-minute delay before an increase in PUN occurred. When glucose was ingested with egg white, the PUN response was attenuated. However, when glucose was ingested with cottage cheese, the PUN response was merely delayed.

I

J -4 CCh

CChiGlu

Egg

Egg + Glu

Glll

The area responses are shown in Fig 2F. Quantitative interpretation is difficult because none of the concentrations had returned to the water control value. It is of some interest that, had the overnight fasting concentration (time zero) been used as baseline, all the values would be either essentially unchanged or negative, except for the results obtained following the meals containing cottage cheese. The NEFA concentration during the 5 hours of starvation varied, but, overall, was little changed from the zero time point (Fig 3A). Following ingestion of the cottage cheese meal, there was an increase in NEFA concentration at the 30- and 60-minute time points. It subsequently decreased to a nadir at 180 minutes, and then returned to the fasting value at 5 hours. The NEFA response to the egg white meal was similar, except an increase in concentration was not observed at 30 and 60 minutes. Following ingestion of the meals containing glucose, the NEFA concentration decreased to a nadir at 120 minutes, and remained depressed at 180 and 240 minutes. By 5 hours, the concentration was returning toward the fasting value. The NEFA area response was negative for all meals (Fig

1142

GANNON

NEFA Response

---x

control(w&r) o

ET AL

NEFA Area Response

ml r _

CCh

e CCh+Glu A A n

7””

Egg

Egg + Glu Glucose

-1600

0

60

120

160

240

t

300

CCh

CCh + Glu

Egg

Egg + Glu

Glu

MinutesafterMeal Triglyceride

---x

cm1ml 0

Response

Triglyceride

0.9r D

lo

Area Response

80

(water)

70 !O

CCh + Glu

60

0

5o

ii

2 1

40

Q

5 ; * E”

30 20

10

10 -0.2t

,

,

60

120

.

,

.

,

.

, 0

20 0

100

240

300

CCh

CCh+GIu

Egg

Egg + Glu

Glu

Minutes after Meal

Fig 3. (A) NEFA response to meals. Legend same as Fig 1A. The mean fasting NEFA concentration was485 f 30 mg/L (1,717 + 108 pmol/L). (B) NEFA area response to meal. (C)Triglyceride response to meals. The mean fasting triglyceride concentration was 198 + 10 mg/dL (2.2 + 0.1 mmol/L). (D) Triglyceride area response to meal.

3B). It was more negative when glucose was ingested with proteins. However, none of the differences were statistically significant. The triglyceride concentration changed little during starvation (Fig 3C). After 120 minutes, there was a very modest increase in triglyceride concentration after those meals containing protein. Glucose had little effect on the triglyceride area response to protein. The triglyceride area response to all meals containing protein was greater than the response to glucose alone (Fig 3D). Also, the area responses were similar for both proteins regardless of whether glucose was included in the meal. When the subjects were fasted, the urine glucose excretion was 73 h 18 mg/5-h collection. This amount was subtracted from the amounts measured following the test meals. Very little glucose was present in the urine following ingestion of the cottage cheese and egg white meals, ie, 35 ? 39 and 26 k 69 mg/5 h, respectively. Following the ingestion of 50 g glucose, the urine glucose was 3.0 t 1.6 g/5-h collection; when protein was ingested with glucose, the amount of glucose excreted was decreased to 1.9 2 1.2 and 1.1 k 0.6 g/5 h following cottage cheese and egg white, respectively. During starvation, the urea nitrogen excreted in the urine was 2,414 +- 220 mg/5 h. This amount was subtracted from that measured following each of the test meals. The urea

nitrogen excreted was approximately three times greater following ingestion of cottage cheese alone (852 of:208 mg) compared with that after egg white alone (252 t 190 mg) (Fig 4). Less urea nitrogen was excreted when glucose was included in the protein meals, but this did not reach statistical significance in any case. Ingested glucose, itself, had little effect on urea nitrogen excretion. The urine creatinine was 385 k 47 mg/5-h collection in the fasting state, and was similar following ingestion of all test meals.

DISCUSSION

The current study demonstrates that the metabolic response to ingestion of two common sources of protein by individuals with type II diabetes was clearly different. The differences were observed in insulin, C-peptide, AAN, and PUN area responses. The insulin area response was 3.6 times greater following ingestion of cottage cheese compared with that following egg white. The C-peptide area response was threefold greater, AAN area response was 1.8-fold greater, and PUN area response was twofold greater. Glucagon area responses were not different (1.3fold). To determine the amount of each ingested protein metabolized, the net output of urine urea nitrogen and the net increase in the urea nitrogen retained in body water at

EFFECTS OF DIETARY GLUCOSE

A 1000

ET

1143

AND PROTEIN

Urine Urea Nitrogen

ah

CCh

CCh +Glu B

600 r

CCh

Egg

Egg+

Giu

Glu

Urine Creatinine

CCh Giu

Egg

Egg G:u

Glu

Zero

Fig 4. (A) Average urine urea nitrogen. Values represent amount excreted/5-h urine collection. Averages are significantly different (P < ,051 if they do not share a common letter. (B) Average urine creatinine. Values represent amount excreted/k&h urine collection.

the conclusion of the study were measured, and the urea production over this time interval was calculated. Using our urea production data and published data on the amount of nitrogen in cottage cheese protein and egg white protein7 the calculated amount of cottage cheese protein metabolized was 81%. For egg white, it was only 52%. The hepatic urea production rate is known to increase in response to ingestion of a protein meal. Indeed, Rafoth and Onstad6 reported a linear relationship between the average hourly serum total AAN concentration and the hourly urea production in normal males following protein meals. In our study, the amino acid concentration was integrated over 5 hours following the protein meals, and over a similar period following starvation. The data obtained during starvation were subtracted from the protein meal data to obtain a net integrated amino acid response to the protein meals. The amino acid area response to cottage cheese ingestion was 185% of that to egg white ingestion. Urea production was 156% of that following egg white. Thus, our data in diabetic subjects would support the concept of a close relationship between amino acid concentration and urea produced, as

reported in normal subjects by Rafoth and Onstad.” Also, these data suggest that urea production should be a reasonable index of the amount of amino acids absorbed following a meal. As noted by these authors, the liver has a remarkable capacity to deaminate amino acids. We previously have compared the responses to 50 g protein ingested in the form of egg white or cottage cheese in normal, young, male subjects.” The urea production over 8 hours was calculated. We could account for approximately 70% of the ingested cottage cheese, but only 47% of the ingested egg white protein, in normal subjects. That is. the urea production was 149% greater following cottage cheese ingestion when compared with that following egg white protein ingestion. Thus, the ratios were very similar in subjects with diabetes and normal subjects (156% v 149%, respectively). The duration of the study in normal individuals was 8 hours, and was only 5 hours in individuals with diabetes. However, the normal subjects also ingested twice the amount of protein. In normal individuals, the X-hour integrated amino acid area response was 207% greater following cottage cheese compared with that following egg white ingestion. This is similar to the 185% found in the subjects with diabetes. Thus, the relationship between urea production and total amino acid area response also was similar in normal and diabetic subjects. This indicates that mild to moderately severe type II diabetes does not have a major effect on metabolism of amino acids in the liver after ingestion of protein. It should be noted that the AAN area response corrclated with the PUN area response. However. the increase in PUN was delayed by approximately 60 minutes. as we reported previously.” Although the AAN and PUN area responses wcrc similar in normal subjects compared with subjects with diabetes. the insulin and glucagon area responses clearly were different. In subjects with diabetes, following the ingestion of cottage cheese, the insulin area response was 3.6fold greater compared with that following ingestion of egg white. In normal subjects, it was twofold greater. Thus, in subjects with diabetes, cottage cheese was a more potent insulin secretagogue relative to egg white protein than in normal individuals. The reason for this is unknown. In persons with diabetes, the glucagon area response to the two proteins was similar (Fig 4). In contrast, in normal subjects, the glucagon area response to cottage cheese was twofold greater than the response to egg white.” This difference can be attributed to the glucagon response to cottage cheese being only 58% of that in normal subjects (700 v 1,200 pg h/mL). The reason that the glucagon area response following ingestion of cottage cheese was less in subjects with diabetes also is unclear. In addition to determining differences in metabolic response to egg white and cottage cheese, we were interested in determining how they may be modified hy the coingestion of glucose. Following the ingestion of glucose alone, the AAN and PUN area responses were negative. These changes were nssociatcd with ;I decrease in urea production. When glucose was ingextcd with protein. the AAN and PUN area rcsponsc4 WCI-c &creased compared

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GANNON ET AL

with the results with protein alone. These changes also were associated with a decreased production of urea. The urea production was decreased 19% and 74% when glucose was added to the cottage cheese and egg white meals, respectively. The AAN area responses also were decreased 25% and 59% when glucose was added to the cottage cheese and egg white meals, respectively. As indicated previously, using urea production data, we could account for metabolism of 81% of the ingested cottage cheese protein. This was reduced to 56% when glucose was coingested with the cottage cheese. Similarly, 52% of the ingested egg white protein was accounted for when the protein was ingested alone. This was reduced to 12% when glucose was included in the meal. Following protein ingestion, urea production is closely associated with the circulating PUN concentration, which in turn is closely associated with the total AAN concentration, as noted previously. When glucose was included with the protein meal, these relationships still held. Glucose coingestion resulted in a decrease in the AAN concentration and had an attenuating effect on urea production following ingestion of a protein meal. The effect was much greater when the protein was egg white. The effect of oral glucose on urine urea nitrogen appearance was reported as early as 1890,‘OJ’ and was called the “sparing action of glucose.” Around the turn of the century, investigators were determining the effect of phlorhizin diabetes on the urine glucose and urea excretion in dogs. When glucose was given to fasted animals, there was a decrease in urine nitrogen excretion compared with that observed with fasting alone. A decrease also was observed following administration of fructose or galactose.1zJ3 Thus, it appears ingestion of any of the major dietary carbohydrates will result in a decrease in urea excretion. The mechanism for this is incompletely understood. A reduced AAN concentration may explain, at least in part, the decreased urea excretion if the total circulating AAN concentration plays a major role in urea production, as suggested by Rafoth and Onstad.6 Bloomgarden et al reported that in dogs given glucose + beef orally, the amino acid concentration in portal and arterial blood was diminished compared with the response following ingestion of been alone.14 However, the response was only modestly less when beef was given orally and glucose was given intravenously. Thus, a major effect of glucose administration in these dog studies was likely to have been an insulin-mediated decrease in amino acid release from muscle.*sJ6 That insulin decreases the total

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Percent Protein Metabolized Fig 5. Relationship between percent protein metabolized and glucagon area response. The glucagon area response for each of the four test meals is plotted against the amount of protein metabolized for each meal. r = .g?.

amino acid concentration and, particularly, the branchedchain amino acids has been known for many years.” However, an increase in insulin concentration with a consequent decrease in AAN concentration cannot readily explain the decrease in urine urea excretion that was associated with the administration of fructose and galactose to phlorhizinized dogs, since fructose and, particularly, galactose are very weak insulin secretagogues.18 Glucagon also has been reported to have an effect on amino acid metabolism in man.19 Normal human subjects were made hyperglucagonemic with a glucagon infusion. The circulating insulin concentration was controlled by simultaneous somatostatin and insulin infusion. During the period of increased glucagon concentration, the amino acid concentration (sum of 21 amino acids) was decreased and urinary excretion of urea nitrogen was increased. The mechanism proposed was that glucagon was stimulating an increased rate of gluconeogenesis from the amino acids. Our data suggest that glucagon may be playing an important role in protein metabolism. Indeed, there was a close correlation between the glucagon area response and the amount of protein metabolized for all test meals (r = .97) (Fig 5). For example, the protein metabolized following ingestion of egg white + glucose was only 12% of that ingested. The glucagon area response was similar to the water control. Thus, in the absence of a glucagon response, very little protein was metabolized. Whether this relationship will be maintained when various types and amounts of proteins are ingested with various amounts of glucose or other carbohydrates remains to be determined.

REFERENCES 1. Nuttall FQ, Mooradian AD, Gannon MC, et al: Effect of protein ingestion on the glucose and insulin response to a standardized oral glucose load. Diabetes Care 7:465-470, 1984 2. Gannon MC, Nuttall FQ, Neil BJ, et al: The insulin and glucose respom,es to meals of glucose plus various proteins in type II diabetic subjects. Metabolism 37:1081-1088, 1988 3. National Diabetes Data Group: Classification and diagnosis of diabetes mellitus and other categories of glucose intolerance. Diabetes 28:1039-1057,1979 4. Goodwin JF: The calorimetric estimation of plasma amino acid nitrogen with DFNB. Clin Chem 14:1080-1090, 1968

5. Fuller G, Parker RM: Applications 13-16. Approximate integration, in Analytical Geometry and Calculus. Princeton, NJ, Van Nostrand, 1964, pp 367-368 6. Rafoth RJ, Onstad GR: Urea synthesis after oral protein ingestion in man. J Clin Invest 56:1170-1174, 1975 7. United States Department of Agriculture (USDA): Agricultural Handbook No. 8-l. Washington, DC, Agricultural Research Service, 1976, Items No. 01-016 and 01-124 8. Gannon MC, Nuttall FQ, Westphal SA, et al: Effects of dose of ingested glucose on plasma metabolite and hormone responses in type II diabetic subjects. Diabetes Care 12:544-552, 1989

EFFECTS OF DIETARY GLUCOSE AND PROTEIN

9. Nuttall FQ, Gannon MC: Metabolic response to egg white and cottage cheese protein in normal subjects. Metabolism 39:7497X,1990 10. Lusk G: Ueber den Einfluss der KJohlehydrate auf den Eiweisszerfall. Zeitschr f Biol27:459-481, 1890 11. Ringer AI: Protein metabolism in experimental diabetes. J Biol Chem 12:431-445, 1912 12. Nash TP Jr, Benedict SR: On the mechanism of phlorhizin diabetes. J Biol Chem 55:759-767, 1923 13. Deuel HJ Jr, Chambers WH: The rate of elimination of ingested sugars in phlorhizin diabetes. J Biol Chem 65:7-20, 1925 14. Bloomgarden QT. Liljunquist JE, Lacy WWU: Amino acid disposition by the liver and gastrointestinal tract following protein and glucose ingestion. Am J Physiol241:E90-E99, 1981

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15. Flakoll PJ, Kulaylat M, Frexes-Steed M. et al: Amino acids augment insulin’s suppression of whole body proteolysis. Am J Physiol257:E839-E847,1989 16. Abumrad NN, Williams P. Frexes-Steed M, et al: Interorgan metabolism of amino acids in vivo. Diabetes Metab Rev 5:213-226, 1989 17. Zinneman HH, Nuttall FQ, Goetz FC: Effect of endogenous insulin on human amino acid metabolism. Diabetes 15:5-B, 1966 18. MacDonald I, Keyser A, Pacy D: Some effects in man of varying the load of glucose, sucrose, fructose or sorbitol on various metabolites in blood. Am J Clin Nutr 31:1305-1311. 1978 19. Boden G, Rezvani I, Owen OE: Effects of glucagon on plasma amino acids. J Clin Invest 73:785-793, 1984

Metabolic response to cottage cheese or egg white protein, with or without glucose, in type II diabetic subjects.

Test meals with 25 g protein in the form of cottage cheese or egg white were given with or without 50 g glucose to male subjects with mild to moderate...
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