The protein-sparing

action of protein feeding:

absence
of relationship
to insulin secretion1
Richard

L. Landau,

Hyman

Rochman,

ABSTRACT
beef

protein

normal

Petra

The

influence

hydrolysate

per

young

men.

10 to 12 g daily

During

in each

these

levels

disclosed

protein-sparing

secretion.
KEY

the

the

protein

WORDS

J. Cliii.
Protein,

just

feeding.
during
of

Nutr.

protein
34:

protein-sparing

The American

Journal

of Clinical

Arthur

compared

with

excretion

fact

that

fasting

and

feeding

this

of a modified
insulin

sensitive
feeding
be

ascribed

nitrogen

was

of C-peptide
base-lines

and

balance

g
of

in each

dropped
profoundly
remained
at about
of insulin

provides

(1.5
in three

approached

indicator
to

fast

secretion

a negative

equilibrium

protein
cannot

that
and

indicated

nitrogen

by the urinary
excretion
13 to 22% of the control
The

H. Rubenstein

metabolism

feeding

1300-1304,

Blackburn
and his associates have called a
diet consisting of only protein or a complete
mixture
of amino
acids
a modified
fast; this
is an apt descriptive
phrase.
Qualitatively
the
metabolic
mixture
available
to the body is the
same
as that which
supports
totally
fasting
organisms. After the first hours
on both regimens
the only carbohydrate
available
to the
organism
is that derived
from gluconeogensis.
Body
fat is the principal
source
of calories,
the remainder
being
derived
from protein
in
both
the total
and
the modified
fast.
The
factors
that regulate
the rate of protein
utilization,
fat mobilization
and utilization,
etc.
are complex
and remain interesting subjects
of speculation.
The finding
of Blackburn
and
his associates
(1, 2) that the feeding
of only a
complete
protein
in quantities
of from
1.0 to
1.5 g/kg/day
could
reduce
or spare
the loss
of tissue
protein
was an important
discovery.
Aside from medical
applications,
the modified
fast offers
an obvious
opportunity
to
study the factors
responsible
for the metabolic
mix during
fasting.
How does protein
administration
alone
spare
tissue
protein?
Blackburn
et a!. (1) proposed
that the tissue
protein
sparing
effect
of protein
administration
was
the indirect
result
of diminished
insulin
secretion
and activity.
It was shown
that
plasma
insulin
levels
were
lowered
by
protein
administration
when no carbohydrate
1300

was
nitrogen

urea

protein

as indicated
averaged

difference

on

urinary

With

influence
Am.

fast

fast

wt)

and

strong
the

secretion

evidence

decline

that

in

insulin

1981.
effect,

insulin

was given.
They
proposed
that the presumed
lowering
of insulin
activity
enhanced
free
fatty
acid
mobilization
making
more
available for hepatic
metabolism,
ketogenesis,
and
tissue
utilization
for energy.
The sparing
of
body
protein
was presumed
to be the result
of the greater
availability
and ultilization
of
fat and ketones.
This
explanation
has been
questioned
by others
(3, 4). An alternate
explanation
would
relate
the sparing
of tissue
proteins
directly
to the metabolic
influence
of
amino
acids derived
from administered
protein.
The
primary
purpose
of the study
reported
here was to test the insulin
hypothesis
in a different
manner.
Rather
than just measuring
plasma
insulin
concentrations
at fixed
points,
the 24-h urinary
C-peptide
excretion
was measured
sequentially
as an integrated
quantitative
index
of insulin
secretion.
Methods
The subjects
were normal
young
men who cbrried
on
their
usual
activities
as students
but resided
in the Clinical Research
Center.
All were of normal
weight
for their
height
(Fig.
I). Each
subject
selected
a constant
diet
calorically
adequate
to maintain
sedentary
activity.
After

From
the Section
of Endocrinology,
Chicago,
Chicago,
Illinois
60637.
2 Supported
by Fisher
Endocrinology
Institutes
Research

Nutrition

of Health
(AM
Center
(USPHS

34: JULY
1981

13941)
5 MO1

University
Fund,

National

and General
RR0005).

Clinical

1981, pp. 1300-1304.

American
Society
for

Printed
Clinical

in U.S.A.
Nutrition

of

Downloaded from www.ajcn.org by guest on February 13, 2012

the

during
no significant

of a 2-day
kg body

subject.

instance.
lnsuhin
secretion
with fasting
to levels which

Blix-Gruber,

PROTEIN

SPARING

ACTION

OF

PROTEIN

1301

FEEDING

H.

GmUreaN
20
5

16.6
Diet N=2l.8
Control

Mcg

16.0
Diet
Beef

Fast

DietN2l8

N=169
Protein

Control

C-Peptide

I0

7A7

-8.25

4
2
0
Gm Creatinine
2.0
I

.0

ti

20

GmUreaN

F.

#{149}ea.:;

l3.8

-12.9
-9-3

Diet

DietN:0

N2l

Diet
Beef

Fast

Control

Diet

N 11.0
Protein

N-2l

Control

Mcg C-Peptide
8

6
4

2
0
2.0

Gm Creatinine
#{149}-S---..

I.0

20

[Gm

Urea

#{149}.-*-__.

-.

#{149}

-._S.--*

K.

I0
5

F
Diet Nl6
Control

DietN.0
Fast

Diet
Beef

Diet

N-14.5
Protein

N-16.5

Control

Mcg C- Peptide

,-NA

396

4
2
.

=!:-#{149}----

Gm

St.

Creatinine
S

#{149}.-_-S.---S---#{149}

.#{149}-S

-_#{149}.-#{149}

F
FIG.
1. The effects
of fasting
and of feeding
only
1.5 g/kg
body
wt on urinary
urea
nitrogen,
C-peptide,
and
creatinine
in the three
normal
young
men,
H, F, and K. Vertical
lines delineate
periods
of fasting
and the protein
hydrohysate
diet.
Broken
horizontal
lines depict
averages
of daily
control
values
for urinary
urea nitrogen
and Cpeptide
and the averages
of urea nitrogen
excretion
during
the fast, the protein
diet and the posttreatment
control
period.
H was 20 yr old, 73 in tall, and weighed
70.5 kg. His control
diet contained
311 g carbohydrate,
136 g protein,
119 g fat, 2786 cal. F was 27 yr old, 63 in tall, and weighed
45 kg. His control
diet contained
240 g carbohydrates,
133
g protein,
74 g fat, 2146 cal. K was 24 yr old, 67 in tall, and weighed
60 kg. His control
diet was 214 g carbohydrate,
103 g protein,
65 g fat, 1820 cal.

a few days for metabolic

equilibration,
quantitative
24h urine
collections
were begun,
and venous
blood
specimens
were
drawn
almost
every
day according
to a predetermined
schedule.
The control
period
was followed

by a 2-day
fast during
which
water
was supplied
ad
libitum
and a multiple
vitamin
capsule
was given
each
day. For the next 6 days the subjects
ate only a complete
amino
acid mixture
(beef
protein
hydrohysate)
in a dose

Downloaded from www.ajcn.org by guest on February 13, 2012

I0

1302

LANDAU

of 1.5 g/kg,
the total daily amount
being
equally
divided
into fourths
given
at the usual
meal times and at 10 PM.
The subjects
then returned
to the original
control
diet.
Since
urinary
urea
comprises
the major
portion
of
total urinary
nitrogen
and is the normal
end product
of
protein
catabolism,
the determination
of urinary
urea by
a urease
method
was accepted
for the purpose
of this
investigation
as an approximation
of total urinary
nitrogen and
a satisfactory
indicator
of protein
utilization.
Both urea and creatinine
were determined
by the Vickers
multi-channel
analysis
system
(5, 6). Fecal
nitrogen
was
not determined,
but was assumed
to be constant
and
minimal
in these
normal
young
men.
The protein
and
caloric
value
of diets
was
calculated
from
published
tables
and
the stated
content
of the purchased
amino
acid mixture.
Urinary
C-peptide
levels
were
measured
by a modification
(7) of the C-peptide
radioimmunoassay
described
by Heding
(8). The antiserum
used was raised
in
a guinea
pig by immunizing
the animal
with synthetic

Results
During the initial control periods all 3 subjects
(Fig.
1) were
in approximate
nitrogen
equilibrium
as indicated
by urinary
urea nitrogen
excretion.
The
negative
protein
balances
during
the 2-day
fasts
were
similar.
Averge daily urinary
urea outputs during the
fast were 11.4, 9.25, and 9.3 g, respectively in
H, F, and K. During the feeding of the amino
acid
mixture,
urinary
urea
shifted
to new
levels,
in each case slightly
higher
than during
the fast. The
metabolic
mixture
during
the
fasts and modified
fasts were in rather
close
approximation.
However,
all of the urea excreted
during
fasting
was derived
from
the
catabolism
of tissue
protein.
When
amino
acids
were
fed, urea
nitrogen
excretion
approached
the amount
of amino
acid nitrogen

AL.

eaten.
The subjects
were
much
closer
to nitrogen
equilibrium;
tissue protein
was spared.
The urea output during the second
control
or
recovery period was in each instance slightly
lower than the original
period,
probably
reflecting
the compensatory
recovery
of protein
lost during
the fast.
The
urinary
C-peptide
excretion
was remarkably
consistent
while
the subjects
were
consuming
their
full constant
diets.
In each
instance
the daily excretion
dropped
abruptly
to levels which averaged
1.2, 1.6, and 0.44
mcg in H, F, and K, respectively,
during
the
fast. C-peptide
excretion remained
approximately
the same during
the 6 days of protein
feeding
in H and K. However,
in F the excretion of C-peptide
actually
rose to over 4.0 jig
during
the first 3 days of amino
acid feeding
before
again
dropping
to near
1.6 jig. In all
three
of the subjects
the C-peptide
output
increased
moderately
during
the first day of
refeeding the constant diet and rose to near
the original level during the remainder
of the
recovery period.
Fasting
plasma
insulin values dropped
from
10 to 0-2.5
iiU/m!
during
the fast and
modified
fast in H. In K they were almost
unmeasurable
during
the control
period
and
remained
very low with the fast or modified
fast. In F the values dropped
from 13 to 8
jiU/ml with no difference between the values
obtained during the complete
fast and the
protein feeding interval.
The plasma
concentrations
of cortiso!
and
growth hormone
were not significantly modified
by
the
experimental
regimen.
The
plasma
glucagon
concentrations
rose during
the fast as anticipated,
returning
to base-line
promptly
in F, but remaining
elevated
during
the protein feeding
in H and K.
Discussion
It is well established that proinsulin
in the
pancreatic
$cell
is enzymatically
split to insulin and the connecting
peptide
(C-peptide),
and that the two components
are secreted
in
equimolar
amounts
(17). The kidney
appears
to be the major site of C-peptide
removal,
the
mechanism
involving both tubular uptake
and metabolism
and urinary
excretion
(18).
Since
C-peptide
and insulin
are secreted
in
equimolar
quantities,
it seemed
reasonable
to
assume
that urinary C-peptide measurements

Downloaded from www.ajcn.org by guest on February 13, 2012

benzyloxycarbonyh
human
C-peptide
coupled
to human
albumin
(9) (kindly
provided
by Dr. C. Binder,
Hvidore
Hospital,
Klampenborg,
Denmark).
Synthetic
tryosyh-Cpeptide
iodinated
with
251 by a modification
(10) of the
method
of Hunter
and Greenwood
(11) was used as the
tracer,
and natural
C-peptide
(12) was used as the standard.
Ahiquots
of the 24-h urine
samples
were
neutralized
(pH 7.0) with ammonium
hydroxide
and frozen
before
C-peptide
analysis.
Preliminary
experiments
have shown
that C-peptide
is fully recovered
from
urine
without
the
need
to add albumin
to the container.
To determine
the
urinary
C-peptide
immunoreactivity,
100 .th of antiserum,
diluted
appropriately,
was incubated
at 4#{176}C
with
100 i.th
of standard,
diluted
urine,
or buffer.
Twenty
hours
later,
100 1il of 25I-tyr-C-peptide
was
added.
Following
a
second
twenty
hour incubation
at 4#{176}C,
the bound
tracer
was precipitated
with ethanol
and counted.
Plasma
insulin (13), glucagon
(14) and growth
hormone
(15) were
determined
by radioimmunoassays;
cortisol,
by a specific
protein
binding
method
(16).

ET

PROTEIN

SPARING

ACTION

PROTEIN

FEEDING

1303

rate of insulin
secretion
coupled
with ours in
which
urinary
C-peptide
provided
a much
more
sensitive
and reliable
indication
of the
24-h secretion
of insulin
permit
the assumption that insulin
plays
little or no active
role
in regulating
the metabolic
mixture
during
starvation
or the protein
sparing
influence
of
protein
feeding
during
a fast. Since
shifts
in
plasma
levels
of cortisol,
growth
hormone,
and glucagon were inconsequential
or inconsistent
during
the modified
fast, it is reasonable
to assume
that
protein
sparing
in this
circumstance
is not mediated
by an endocrine
product
and
is the effect
of the increased
availability
of amino
acids.
El
References
1. Blackburn
EL, Fhatt JP, Chowes
GHA,
ODonnell
TE.
Peripheral
intravenous
feeding
with
isotonic
amino
acid solutions.
Am J Surg
1973;25:447-54.
2. Bistrian
BR, Blackburn
GL, Flatt JP, Sizer J, Scrimshaw
NS,
Sherman
M. Nitrogen
metabolism
and
insulin
requirements
in obese
diabetic
adults
on a
protein-sparing
modified
fast. Diabetes
1976:25:494504.
3. Freeman
JB, Stegink
LD, Wittine
MF, Danney
MM,
Thompson
RG.
Lack
of correlation
between
nitrogen balance
and serum
insulin
levels
during
protein
sparing
with and without
insulin.
Gastroenterology
1977;73:3
1-36.
4. Marhiss
EB, Murray
FT. Nakhooda
AF.The
metabolic response
to hypo caloric
protein
diets in obese
man.
J Chin Invest
1978;62:468-79.
5. Heinegard
D, Tiderstron
G. Determination
of serum
creatinine
by a direct
cohorimetric
method.
Chin
Chem
Acta
1973;43:305-10.
6. Richterich
R. In: Clinical
chemistry.
New
York:
Academic
Press,
1969:253.
7. Faber
OK, Binder
C, Markussen
J, et al. Characterization
of
seven
C-peptide
antisera.
Diabetes
27(supph.
1): 1978:170-7.
8. Heding
LG. Radioimmunohogical
determination
of
human
C-peptide
in serum.
Diabetologia
1975;11:
541-48.
9. Faber
OK,
Markussen
J, Naithani
VK,
Binder
C.
Production
of antisera
to synthetic
benzylozycarbonyh-D-peptide
of human
proinsuhin.
Hoppe-Seyhers Z. Physiol
Chem
1976;357:75l-7.
10. Freychet
P. Roth
J, Neville
DM.
Monoiodoinsuhin:
demonstration
of its biological
activity
and binding
to fat cells and liver membranes.
Biochem
Biophys
Res Commun
197 l;43:400-8.
11. Hunter
WM,
Greenwood
FC.
Preparation
of iodinet:11
labelled
growth
hormone
of high
specific
activity.
Nature
(Lond)
1952;194:495-6.
12. Oyer
PE, Cho 5, Peterson
JD, Steiner
DF. Studies
on human
proinsuhin.
Isolation
and amino
acid sequence
of human
pancreatic
C-peptide.
J Biol Chem
197 1;246: 1375-86.
13. Starr
JI, Horwitz
DL, Rubenstein
AH,
Mako
ME.
Insulin,
proinsuhin
and C-peptide.
In Jaffe
BD and

Downloaded from www.ajcn.org by guest on February 13, 2012

would
provide
a quantitatively
reliable
mdicator of the rate of insulin
secretion
in a finite
interval
(9).
Urinary
C-peptide
measurements
have
been
shown
to correlate
highly
with
frequently
measured
plasma
insulin
levels during
circumstances
such as a glucose
tolerance
test in which
the rate
of insulin
secretion
is known
to vary
(17, 19). It was
thus considered
that the 24-h C-peptide
excretion
would
provide
a reliable
and sensitive
indicator
of pancreatic
beta cell function
for
a metabolic
study
of the sort reported
here.
The immediate
drop
in urinary
C-peptide
during
the 2-day
fast emphasized
the decline
in insulin
secretion
suggested
by the mild
lowering
of plasma
insulin
concentrations.
The decline in C-peptide excretion
of 85% in
H, 78% in F, and 87% in K probably reflected
a similar
drop
in the secretion
of insulin
during
the 24-h
periods.
It is possible,
of
course,
that starvation
(both
regimens)
modified the renal
handling
of C-peptide
so that
these
changes
may not reflect
solely
the rate
of insulin
secretion.
However,
the fact that
the full effect
was manifested
within
24 h,
before
one would
ordinarily
anticipate
significant
alterations
in renal function,
strongly
suggests
that these
results
accurately
reflect
the 24-h insulin
secretion.
Moreover,
the fact
that urinary
C-peptide
remained
at the same
low level or rose slightly
during
the 6 days of
modified
fast, indicates
that in this circumstance
protein
feeding
alone
does not materially influence
the rate of insulin
secretion.
It is apparent
that although
the rate of urea
excretion
in all three
subjects
was at about
the same
level during
the fast and the subsequent
6 days
of protein
feeding,
the actua!
nitrogen
balances
were
markedly
different.
Whereas
all of the urea excreted
each day of
the fast represented a net loss of tissue protein, almost
all of the protein
catabolism
represented
by urea excretion
during
amino
acid
feeding
was in essence
provided
by the ingested
amino
acids.
Since
insulin
secretion,
as indicated
by C-peptide
excretion,
was the
same or even slightly
higher
during
the modified fast, the sparing
of body
protein
by the
protein
hydrolysate
cannot
be accounted
for
by the effects
of diminished
insulin
secretion.
As previously
noted,
others
(3, 4) arrived
at the same
conclusions
by somewhat
different routes.
Their
studies
which
used plasma
insulin
concentrations
as a correlate
of the

OF

1304

14.

15.

16.

LANDAU
Behrman
HR,
eds. Methods
of hormone
radioimmunoassay
New
York:
Academic
Press,
1979:61342.
Kuku
SF, Jaspan
iB, Emmanoueh
DS, Zeidler
A,
Katz
Al, Rubenstein
AH.
Heterogeneity
of plasma
ghucagon.
J Chin Invest
1976;58:742-50.
Odell
WD,
Rayford
PL, Ross GT: Simple,
paritahhy
automated
method
for radioimmunassay
of human
thyroid
stimulating
growth
huteinizing
and
follicle
stimulating
hormones.
J Lab and Chin Med
l967;70:
973-80.
Murphy
BEP.
Some
studies
of the protein-binding
of steroids
and their application
to the routine
micro
and ultra
micro
measurement
of various
steroids
in

ET

AL.
body
say.

17.

18.

19.

fluids

by competitive

protein-binding

radioas-

i Chin

Endocrinol
Metab
1967;27:973-90.
Horwitz
DL,
Starr
ii, Mako
ME,
Blackard
WG,
Rubenstein
AH.
Proinsuhin,
insulin
and C-peptide
concentrations
in human
portal
and
peripheral
blood.
i Chin Invest
1975;55:1278-83.
Katz Al, Rubenstein
AH. Metabolism
of proinsulin,
insulin
and
C-peptide
in the
rat.
J Chin
Invest
h973;52:l
13-21.
Kuzuya
T, Matsuda
A, Saito T, Yoshida
5: Human
C-peptide
immunoreactivity
(CPR)
in blood
and
urine-evaluation
of a radioimmunoassay
method
and its clinical
applications.
Diabetohogia
1976;12:
5 11-18.

Downloaded from www.ajcn.org by guest on February 13, 2012