Pituitary peptides and fat mobilization

Pituitary Peptides and Fat Mohilization By

HE~HY FmF.5EN

H E SEAHCH for a specific pituitary hormone which regulates fat mobilization has continued for several decades. Anselmino and Hoffmann' as early as 1931 reported that a pituitary extract when injected into rats caused ketonemia. Several years later, Best and Carnpbellv" showed in a series of experiments that administration of crude extracts of anterior pituitary to mice, rats and guinea pigs produced a rapid increase inlipid content of the liver and a decrease in carcass fat. In 1947 \Veil and Stetten! prepared an extract from urine of fasting rabbits which increased liver fat and reduced carcass fat and they introduced the term "adipokinin" for the active principle. Subsequently Rosenberg" found that oxycel-purified corticotropin had adipokinetic activit)' in mice, but at that time "it appeared unlikely that such diverse phenomena as mobilization of depot fat and stimulation of adrenal cortex should be attributable to the same substance." As highly purified, and more recently, synthetic preparations of various hormones become available, many were indeed shown to have adipokinetic activity. Growth hormone, corticotropin, thyrotropin and intermedin all enhanced fat mobilization in one or another species though the amount and concentration required were probably unphysiologic in some cases. Investigation therefore continued, seeking a substance whose primary function was the mobilization of fat; In 1958, Rudman et a1. 6 prepared a crude extract from sheep, beef and hog pituitaries which produced lipemia in rabbits, whereas the six recognized anterior lobe hormones when tested individually, had little such effect. In subsequent studies they confirmed and amplified these observations and concluded that the lipemia was produced by a substance different from the recognized pituitary hormones." My remarks shall be confined mainly to recent work which has been carried out in Dr.. Astwood's laboratory concerning the role of pituitary peptides and fat mobilization. The more general topic of pituitary hormones and fat metabolism has been summarized in a comprehensive review by Engel and KostYO.8 In 1959, Landolt and Astwood? found that the crude extract of porcine anterior pituitaries made with hot glacial acetic acid and from which corticotropin had been removed, caused mobilization of free fatty acids in rabbits (ng. 1). Following an injection of this material, the sequence of changes observed suggested that there was an initial mobilization of free fatty acids from the depots, then an accumulation of fat in the liver and finally an increase in total lipids in the serum some 16 to 18 hours later (fig. 2). After neutralizing the acid extract with ammonium hydroxide, 80 per cent of the solids precipitated with little loss of lipolytic activity.

T

From the Pratt Clinic-Ncu: England Center Hospital and the Departmcnt of Medicine, Tufts Unicersity School of Medicine, Boston, Mass.

1214 METADOLlS~I, VOL.

13, No.

lO-PART

2

(OCTODEn),

196·!

PITUITAIlY PEPTIDES

o

", 900 !lOO

100

4

8

A~D

12

HOURS fOLLOWING

FAT ;\[ODLLlZATlOX

/6

20

1215

24

INJECTION

(AI SERUIol-UNESTEPlflED fATTY ACIDS

........ -------------_..---. O--<>PIlUIlARV EXTRACT "-4SAI.INE

900

r.\

~ TOTAl. SERUM LIPIDS

!lOO 100 l....l:>-<:;)o"'CL

I00

----- ..

Fig. I.-Blood lipids, liver fat and adipose tissue activity with and WWIout the administration of pituitary extract. One hundred per cent is the preinjection value.

Nl;.:l~P"---

300 100

300 100

100

o 200 0'--

_

Our attempts to isolate and identify the lipolytic factor in the acetic exract were expedited by the method of starch gel electrophoresis developed by Ferguson and Wallace.!? Using this method, Barrett et a1.11 showed that the acetic acid extract could be resolved into a great many different components (fig. 3). Following electrophoresis of this extract, strips of the gel were preserved unstained by freezing while similar strips were developed with nigrosin. The frozen strips were divided into segments containing the desired components by placing the stained a nd the frozen pieces parallel to each other and cutting the latter according to the pattern on the stained gel. The gel was eluted by slneresis and the eluant from each segment was assayed by measuring the increase of free fatty acids in tissue and media during the incubation

1216

IIE:->HY FRIESE:-;-

% ~-------. 0----0 t· rVE R E X~ER I "E m • - B· B ODO l( IMUS COMTRO Fig. 2.-Lipid accumulation in liver and blood following pituitary extract. Average hourly lipid increase calculated from the change during each interval.

2 3 4 5 6 7 8 9 10 II

12 13

PE TIDE EP IDE

OR IG IN

Fig. 3.-Concentration of the two peptides using an anion exchange resin. Ten L. of the pH 6.5 soluble crude extract was passed through a column 3.3 x 18 cm. of Dowcx lX2. After washing with ammonium acetate buffer pH 4.0 and 0.05 N in acetic acid, elution was effected with 0.1 N acetic acid. Channels 1 and 13, the crude extract; 2 and 3, first and last samples of unabsorbed material showing complete removal of the two peptides; 4, buffer eluate; 5 to 12, successive fractions eluted with 0.1 N acetic acid. Fractions including those shown in 6 to 8 were combincd for further purification. of pieces of rabbit mesenteric fat in Krebs-Hinger phosphate buffer. In' figure 4 one can see the eluants from almost every segment had some lipolytic activity but segment IX was very potent with maximal lipolysis when as little as .02 cc. of the eluant was added to the incubation flask. -By comparing the electrophoretic mobility of pituitary hormones known to have fat mobilizing activity in' rabbits, namely a- and ,B-intermedin and corticotropin, with the components of the acetic acid extract, the bands in segment IX were not seen in any of

1217

l'ITUITARY PEl'TIDES AND FAT MOBILIZATlO:-;

Free Forty Acids Released peq Igms.lhr.

Crude Pituifary E llrocl Eluales Added

14 13 12 II 10 9

15llllllll.02c.c.

c:::::J

8 7 6 5 4 3

- -

Conlrol • 2 1

St9menl No. •

I

l!

Im I

I2:

II ~

t

-

-

f-

zr

I

-

)ZI[

I----

I,n

I +

]X

:x

I I

I

.2 e.e,

Poolide

JZII!

I

Origin

Fig. 4.-Schematic drawing of starch gel after electrophoresis of the crude pituitary extract shown in figure 3. The gel was divided into 10 segments from the cathode toward the anode and 0.02 to 0.2 cc. of the eluates from each of the segments were added to the incubation flasks. The segment containing pcptides I and II is marked. Release of free fatty acids is expressed as pEq./Gm./hr. The horizontal line represents the release of free fatty acids in the controls. DOWEX

A2nl

I.

Z

COLUMN 12.43 em

pH

350mQ ACID fRACTION

6.0

4.~

_ 302.4ml

3.0

t-------- 0 O~N

HO Ae --~.-J.I ON ""77'"'"----

r- 50 4.0

pH or ffflUfNT

2.0

30 2.0

/.0

.8 .6 .4

.2 5

/0

IS

20

25

30

35

50

55

60

TUBE NUMBER

Fig. 5.-Column chromatography of a concentrate of the peptides on an anion exchange resin. The circled fractions were found by electrophoresis to contain predominantly peptide I. The subsequent fractions eluted by 0.05 N acetic acid contained peptide II contaminated with peptide I.

1218

I1E:-:ny FIHF$EX

2 "

I TID MAJ IMP

RIG IN Fig. 6.-Separation of the two peptidcs by making use of the larger capacity of the resin for pcp tide I. Eight.tenths Cm. of an acidic concentrate applied to 4 Cm. of Dowex 1 x 2 acetate in 4 ml. of water pH 4.0. Channels 1 and 2 show the first and last fraction of unadsorbed material; channels 3, 4 and 5 are successive eluates with 0.1 N acetic acid. The spreading of the two main peptidcs was caused by the large amount of material used to show the minor components.

the other preparations. These bands were named pcp tides I and II and their purification was then undertaken. Purification was achieved by 'several methods.'? Firstly, the acidic components were adsorbed on' short columns of Dowex IX2 (200-400-mesh) in the acetate form at pH 7:0; the bulk of the less acidic components passed through the resin unretarded. After washing the column with water, the desired material was eluted with 0.05-0. 1 N acetic acid. The eluates obtained arc shown in figure 3. Secondly, using column chromatography with the same resin, more highly purified material was obtained. The details of the column and conditions are shown in figure 5. Thirdly, pcptides I and II were most easily separated by taking advantage of the greater capacity of Dowex IX2 for peptide I (fig. 6). Fourthly, separation of peptidcs I and II was also achieved using columns of Sephadex G-50 on which peptidc I was· rctarded to a greater extent than peptide II (fig. 7). Finally, using columns of DEAE cellulose and 0.17 M NH 4HC03 bufler, it was possible to obtain peptide I in virtually pure form. Chemical studies on peptide I showed it to be highly' soluble in water even at the pH of minimal SOlubility (pH 4.0-4.6). Electrodiffusion of the two pcp-

PITUITARY PEPTIDES Al'I:D FAT

1219

~IOIlILI7.ATIO:'\

2 I

:t

4

,;

PEPTIDE IDE I

3

, T

.... 1.

/'

'

..

IN

Fig. 7.-Partial sep arat ion of the two peptides using Sep hadex G-50. One-half Gm. of an acidic 'concentrate appli ed to a column 1.2 x 54 em. solvent 0.1 N acetic acid. Channel I, small amount of unr etarded mat erial; 2, peptide II contaminated with a trac e of 1 and a less anionic component; 3, mixture of I and II ; 4 to 6, nearl y homogeneous I.

\

\ 0.4

\ \ \

w

\

<.)

Z
03

'-

Fig. B.-Ultraviolet absorption spectra. (1) Peptide I, 0.2 mg./ml..in water pH 4.0; (2) peptide T, 0.2 mg./ ml. in 0.1 N sodium hydroxide.

CD

a::

a(f) CD

0 .2


0 .1

240

260

280

300

A

1220

llEXIIY FRIESEN

1

I~

12

14 13 12 II

m

t

6

·

10

F.F.A. Riliotl J,ltq/e m:/ hr•

8

9

e 7 6

8

·

e· 4 3 2 I

o Cone.of PtptidlS Jlllm./mi. p

5

m

7

~

.0 1

.02

.05

40

~ J

n o

.10

.20,

>0.1 <02 <.001 .001 .0 01

.50

1.0

2.0

10.0

.00 1 .001

Fig. 9.-Effeet of pituitary pcptides on release of free fatty acids in vitro. Peptides I and II (0 .01-10 Itg.) were added to 1.0 ml. Krebs-Ringer phosphate buffer. Free fatty acids in tissue and media were determined after 3 hours incubation and expressed as fLEq./Gm./hr. The brackets indicate S. E. of the mean; the number of experiments is indicated at the top Ofeach column.

tides through cellophane membranes of calibrated porosity by the method of Pierce and Free'" suggested that peptide I has a molecular weight of less than 10,000 and peptide II a molecular weight between 10,000 and 20,000. The ultraviolet absorption spectrum of peptide I had a maximum at 278 and a minimum at 256 (fig. 8). The biological action of peptides I and II has been studied extensively in rabbits. 14 Hudman and his colleagues!" have made a careful survey of the effects in a number of other species using "Fraction H," a preparation which contains mainly peptide II. The lipolytic activity of these pituitary polypeptides was assayed in vitro by incubating rabbit mesenteric fat in phosphate buffer as mentioned earlier. As shown in fig. 9, as little as .02 fLg'/CC. of peptide I caused a significant increase in the release of free fatty acids. Following intravenous injections of this material there was a prompt rise in plasma Free fatty acids which lasted a variable period of time depending on the dose givcn'. With a small dose, e. g., 10-25 Itg., the increase was seen within 5 minutes and lasted 45--60 minutes (fig. 10) . With larger doses, 100-500 Itg., the effect was more sustained and continued for several hours . Fasting greatly potentiated these effects. After subcutaneous injections of larger doses (2-8 mg.), there was a pronounced increase in total lipids in serum, frequently giving rise to gross

1221

PITUITARY PEPTIDES AXD FAT '-lODlLIZATlO:-':

Plasma F.F.A. P.eq/L

2400 2200 2000 1800 1600 1400 1200 1000 800 600 400 200

x---x

,I

"'

I

r

'X

. "-

,I

,I

"

'

-,""

''x

Fosted IO)Jgm . IV Fed

-----0----0

IO,L/gm. I .V.

" Canlrol- Saline

5 10 15

30

45

60

90

Time- Mins. Fig. IO.-Plasma fatty acids in fed and fasted rabbits following intravenous inject ion of pituitary pcptides.

lipemia. The lipemia usually began in 8 to 12 hours and diminished by 24 hours (table 1). Within a few hours of these large doses, the rabbits developed tachycardia, breathed rapidly, became limp and apathetic and some died, a point which will be mentioned later. When large amounts of pep tides I and 11, 10-20 mg., were injected into dogs or human subjects, there was only' a slight increase in' serum free fatty acids 1 1/2 hours later (fig. 11). No subjective effects were noted by the patients. By applying the same method of purification to. an extract from human pituitaries, a preparation containing two components with similar electrophoretic mobility to peptides I and II was obtained (fig. 12). An injection of 10-20 mg. of this preparation similarly failed to cause a significant increase in free fatty acids in man but it was fully effective in the rabbit. In an attempt to learn 'whether peptides I and II were related to any pituitary hormones, antis erum was prepared in rabbits by weekly injections Table 1 Plasma levels (mEq/liter) No. of Experiments

Do se

- - - - -. __. .. -- ----_._- -- _ 4 5 6

Time (hr) after injection

mg

. .._._--

0 .25 -1.0 1.5 -3.0' 5 .0 - 8. 0'

• Mean value in 9 rabbits. ± -SE of the me an . + Given in divided dose.

__ ._--- _. - - - - - - - - - - - -

- _._.

O·

15

24

7 +1 7±1

7±1.2 20 ±8.8 60 ±9.7

47 ±13.0 31±18.3

7±i

1222

HENRY FRIESE."

1000

Chongo

in Plasma Folly Acids eq/L

I , II -

P pti

~-

25 mgl .

alln - I e.e.

900 BOO 100 600 500

40 0 300 200 100

o - 100

- 200 \ 650 MI S.

60

120

180

0

60

120

Fig. H.-Effect of pituitary pcptldes on plasma free fatty acids in man. (All injections were given subcutaneously.)

2 Fig. 12.-Starch gel stained with nigrosin showing (1) pattern obtained with an alcohol-soluble extract from human pituitaries, (2) purified peptides I and II derived from this extract (indicated by the markers).

of preparations containing the pep tides mixed with Freund's adjuvant.l'' \Vith antiserum in the center well, precipitin lines Formed in Ouehterlony plates when either the pcptidcs or pituitary extract were in the outer wells (flg. 13), and an equal number of lines were seen after immunoelectrophoresis. Two of these appear to be specific for pep tides I and II respectively; the other two were related to contaminants in the preparation used as the antigen. No cross-reaction was seen with a number of other "lipolytic" hormones: a-~ISH, ,B-MSH, corticotropin and growth hormone. Also, there was no crossreaction on immunoelectrophoresis with other components of the acetic acid extract of pituitary which served as the source of peptides I and II. There was a cross-reaction, however, between the material isolated from human pituitary tissue and antiserum to the porcine peptides. This similarity of biological action, electrophoretic mobility, and immunologic cross-reactivity suggests that the human pituitary contains substances very similar to pcptides I and II.

PITUITARY PE1'TIDES

A~D

FAT },IOBlLIZA"flO:-;

1223

Fig. 13.-IJrecipitation lines in agar plates. The center well contains the antiserum to porcine peptidcs I ·and II. The peripheral wells cont ain 50 fig. of: (A) peptides I and II from porcine anterior pituitary glands; (B) Fraction II; (D) pure peptide I; (E) peptides I and II from human anterior pituitary glands.

1

Fig. I4.-Starch gel stained with nigrosin showing th e patterns of (2) crude porcine pituitary extract; (3) Fraction II (Rudman) and (4) "pure" peptide I. The location of peptidcs I and II In the crude extract arc indicated by the markers. Peptide I is not present in fraction H. 0 indicates origin.

As noted above, Rudman et at in' their extensive work in this area have prepared Irom .porcine pituitaries several lipolytic extracts, one of the purer and more potent of which is "Fraction H:'17 A sample of this material provided by Dr. D. Hudman was compared with peptldcs I and II by starch gel electrophoresis (fig. 14). Several components were seen, the principal one migrating in the same region as peptide II; there was no peptide lor, at most, a trace, Precipitin' lines formed with antiserum against peptides I and II, but the one against peptide I was very faint. TIle specificity of the antiserum was also demonstrated by its capacity to

1224

HE~RY

FRlESE~

;)

i

15 1 13 12

I

12

I

t.

I

6

8

I

Gl

II

.

Antiserum Absent

0. mal Ro:' b:t Serum Addcd 0 .1- 0.2 e e

4 r

4

3 2

o

~Anl i serum Added 0 .1- 0.2 c e

f

II

10 FREE 9 FATTY ACIDS 8 JI,q l om/ hr. 7 6 5

I

40

3

o

0 .1

n ~

s

~ 0. 2

3

~ 0 .5

2.0

io

5.0

MICROGRAMS- PEPTIDES I old II:

Fig. 15.-The release of free fatty acids by adipose tissue incubated with and without antiserum to peptides I and II. Incubation was carried out for 3 hours in Krebs-Ringer phosphate buffer containing 5 per cent fat-free albumin. The brackets indicate standard error of the mean. At the tops of the columns are the number of experiments performed.

2 3

4

5

6

7 8 9

10

+ PE PT IDE :II: PEPT IDE I

-

RE SID E

OR IGI

Fig. 16.-Concentration of basic peptides using the cation exchange resin Dowex 50 x 2. Channels 1--3, unadsorbed material. Channels 4 and 5, water wash. Channels 6-10, successive fractions eluted with 0.1 N ammonium hydroxide.

PlTUITAl\Y PEPTIDES AXD FAT

~lOlliLIZATlOX

2

3 4 5 6

1225

7 8 91

11 12

ORIGIN

BASIC PEP IDE

Fig. 17.-Furthcr purification of hasic peptidcs on Dowex 50 x 2 using 0.01 N and 0.03 ammonium hydroxide-channels 1-6.

neutralize in vitro the lipolytic activity of pcptides I and II, Fraction Hand human peptides I and II, but not of other pituitary polypeptides with lipolytic activity (fig.I5). Eluants from most segments of starch gel after electrophoresis of the acetic acid extract of porcine anterior pituitaries had some lipolytic activity (fig. 4). In particular, the region on' the cathode side. of the origin corresponding to segment III and IV appeared to be very active. Corticotropin-A, is probably in segment V and a-1ISII in segment I, but no other hormones with lipolytic activity were known to migrate to segments III and IV. During the past ycar, Dr. Paul Miller and I attempted to purify the active compon'ents in this region. The bulk of the components migrating towards the cathodealong with several other components moving towards the anode in the pH 7.0-s01uble acetic extract were adsorbed on Dowex 50 X 2 (200-400 mesh) in the hydrogcn form . The column was washed with water and eluted stepwise with 0.01 N, .03 Nand 0.1 N ammonium hydroxide. The eluants were lyophilized and examined by starch gel electrophoresis (fig. 16). When injected subcutaneously into rabbits, the fraction eluted with 0.03 Nand 0.1 N ammonium hydroxide produced lipemia, and when tested in vitro, cau sed a large increase in release of free fatty acids. The one component which was purified, marked "basic peptide," shown' in figure 17, had only slight lipolytic activit)', certainly not enough to account for the lipemia produced by the less pure fraction. 11lC residue of the acetic extract, after adsorption by Dowex IX2 and Dowex

1226

I1E:-;ny FRIESE;.,'

2

3

4

5

6

7

8

+ PEPT IDE II PEPTI E I

ORIGI

Fig. lB.-Electrophoretogram of (1) fresh porcine pituitary homogenatechannels I and 2; (2) peptide I-channels 5-7; (3) crude acetic acid extract of porcine pituitaries----ehannel 8.

50, had the following components when examined on starch gel (fig. 16). No peptide I and II, a-~ISH, corticotropin At, or other cathodal components were seen; but when' this crude extract, from which all these known lipolytic factors were removed, was injected into rabbits, pronounced lipemia resulted. Whether a single component or the summation of several active fractions caused the lipemia is not clear, but it is apparent that other, hitherto unrecognized pituitary peptidcs mobilize fat in the rabbit. Recent studies using aqueous or alkaline (ammonium hydroxide) extracts of fresh frozen porcine pituitary glands have shown a small number of major components on starch gel electrophoresis instead of the 50 or more easily visible bands in the acetic acid extract (fig. -18 ). One of the major components corresponded to peptide II, but virtually no peptide I was detected aJld no components migrated toward the cathode. When fresh frozen pituitaries or acetone-dried pituitary powder were extracted with either hot glacial acetic acid or 0.1 N acetic acid, the yield of both acidic and basic peptides was greater than in the alkaline extract. Incubation at room temperature in weak

1227

PITUITARY PEPTlDES AND FAT MODILIZATIOX

1. Fat Mobilization in Rabbit s II.

Melanocyte Stimulating Activity

III.

Positive Chronotropic Effect on the Iuola t ed Mammalian Heart

IV.

Aqueous Flare Response in the Rabbit Eye

V.

Hypocalcemia in Rabb its

Fig. 19.-Biological effects of peptides I and II. Serum Calcium - mg. C/o Hours Ilg·

0

1-1/2

0

11.6

II. 1

lOO

12.3

8. 7

50

12.8

10.3

100

12 .9

9.7

a-MSH

50

11. 9

9.6

I3-MSH

50

12 . l

10.2

Basic peptides

50

12. 1

10.5

Control* Peptides I and II Peptide I ACTH (oxycc1 purified)

>:<

Rabbits fasted 4 days. Injections given subcutaneously.

Fig. 20.-Semm calcium in rabbits following an injection of different pituitary p cp tides.

acid seemed particularly favorable to the formation of new components but it was not clear from which of the initial substances these were derived. Further work will be necessary to determine which peptides are present in the fresh pituitary and which are produced by the extraction procedure. Thus far, only the Iii)olytic effect of pep tides I and II in rabbits has been reviewed. Rudman et a!. have tested Fraction II (peptide II) as well as other lipolytic factors in several species. A summary of their data 18 is shown in table 2. Fraction H was effective only in' the rabbit and to a much lesser extent in the guinea pig. It had no effect in the hamster, rat, pig or dog. It was suggested from this evidence that proteolytic enzymes which inactivate "Fraction H" are contained in adipose tissue of those species whieh fail to respond." Besides their lipolytic effect, pep tides I and II have a number of other actions (fig. 19). They have melanocyte-stimulating activity, 25 per cent of the potency of corticotropin.t" they exert a positive chronotropic effect on the

... l-:l

b'5

Table 2 Hormone ~peries

Hahhit Guinea pig Hnmster Hat Pig Dog

ACTH

a-:'.lSH

0.1 Ilg/ml 3.0 Ilg/ml 10.0 Ilg/ml 0.1 Ilg/ml ::-;Rt

O. 1 Ilg/ml 10.0 IlK/ml

NR

XH Xn. XU <10.0 Ilg/mlt

{3-:\1::;H O.Olll~/ml

10.0 IJK/ml KR

Vasopressin 0.1 Ilg/ml :L 0 Il~/ml

KIt

TSH

Fraction II

XorEpinephrine

epinephrine

~H.

XH. XIt

NR 1.0 IJg/ml !\R 0.1 IlK/ml

O. a Il~/ml )00 IlK/lIll ~R

O. 1 Ilg/ml 0.1 J.lg/nl!

XR NR

<0.1 ,uK/ml t

XH.

~R

:\R

:\R

NH

<10.0 J.lg/lllit

~H

<10.0 ,ug/mlt

XR

KIt

!'IR

o.a

IJg/ml lUll IlK/llll NIt <0.1 IJg/ml t

• The minimal effective dose is the smallest conccntration of hormone that produces a stutisticnlly signifleant (p < 0.05) iru-reuse in FFA production. Experiments with rabbit, guinea pig, and hamster adipose tissue were performed with the albumin-free assay system; t hose with rat, pig, lind dog adipose tissue employed thc nlbumin-containing assay system. t Sn. indicates no response to the highest. concentration test cd. The highest concentrations tested were 100 IJg/ml for rahhit, guinea pig, and hamster; 10 J.l~/ml for rat, pig, lind dog. t A statistically significant effect was produced at thls concentrutiou, which was the srunllest (~oncentration of this hormone tested.

...t;; ~ ~ 0,:

::::

~tTl Yo

l'ITUlTAHY PEPTIPES Ar\D FAT ~rOBIL1ZATlOX

1229

isolated pcrfused dog heart-lung pr eparation." they produce an aqueous flare rcsponsc in thc rabbit eyc;:!:! and they cause a sharp drop in serum calcium in the rabbit (fig. 20) .2 :1 All these cffects are properties not only of peptldcs I and II , hilt also of ACTH, a-~ISII and .B-~rSII, all of which also cause mobilization of frec Fatty acids. The timc course of the free fatty acid rcsponse differ from the hypocalcemic response, but the two phenomena may bc related. The cffect on serum calcium is accentuated by fasting, for while 1 mg. subcutaneously caused a decrease in serum calcium in fed rabbits, only 50-100 Itg. was required in the fasting animals. Larger doses produced an evcn' greater reduction, tile largest response being from 14 to 7 mg. per cent in 2 hours, As mentioned before, some of these animal s were cold, limp and lethargic, but none developed tetany although Natcl son ct al.:!j have described both tetany and seizures from their extracts, It remains for future investigation to determine which peptides arc native to the pituitary and which are products altered by extraction methods. A hormonal function for a fat-mobilizing factor might be suggested if it could he found in the circulation and its concentration changed with fasting. Thus far, fat mobilization from these factors has not been shown to be more than a pharmacologic effect. ACKNO\"LEDG~IENT

The author wishes to thank Dr. E. D. Astwood for his helpful advice and encouragement and for his critical review of the manu script.

REFERENCES 1. Ansclrnino, K. J., and Hoffmann, F.: Klin. Wchnschr. 10:2380, 1931. 2. Best, C. H., and Campbell, J.: J. Ph ysiol. 86 :190, 1936. 3. - , and - : J. Physlol. 92:91, 1938. 4. w-n, R., and Stetten, D.: J. DioI. Ch ern. 168: 129, 1947. 5. Rosenberg, L. N.: Proc, Soc. Exp er, BioI. & Med , 82 :701, 1953. 6. Hudman, D., and Seidman. F.: Proc . Soc. Expcr, Diol. & Mcd, 99: 146, 1958. 7. - , - , and Reid, M, B.: Proc. Soc. Exp er. BioI. & Mcd . 103:315, 1960. 8. Engel, F. L., and Kostyo, J. L.: In 111c Hormones. To be published. 9. Landolt, H., and Astwood, E. B.: Am. _ J. Physlol, 200:841, 1961. 10. Ferguson, K. A., and Wallace, A. L. C.: Nature, London 190 :629, 1961. _ II. Barrett, n. J., Fri esen, II., and Astwood, E. B.: ]. BioI. Ch ern. 237:432, 1962. 12. Astwood, E. D., Barrett, H. J., and Fri esen, n.: Proc, Nat. Acad. Sc. 47: 1525, HJ61. 13. Pierce, J. G., and Fre c, C. A.: Biochirn.

et biophys. acta 48:436, 1961. 14. Fri esen, H., Barrett, H. J., and Astwood , E. B.: Endocrinology 70:579, 1962. 15. Hudman; D., Brown, S. J., and Malkin, ~1. F .: Endocrinology 72:527, 1963. 10. Friesen, rr., Irie, ~I., and Barrett, n. J.: J. Exper. Med. 115:513, 1962. 17. Hudman, D., Reid, ~1. B., Seidman, F., Girolamo, ~1. Di, Wertheim, A. R, and Bern, S.: Endocrinology 68:273, 1961. 18. - : J. Lipid Res. 4:119, 1963. 19. - , Brown, S. J., Malkin, ~1. F., and Garcia, L. A .: J. Clin. Invest, 42:974, 1963. 20. Lerner, A. B.: Personal communication. 21. Kraycr, 0., Astwood , E. D., Waud, D~ H., and Alper, M. H.: PrOf. Nat. Acad, se, 47:1227, 1961.22. Dyst er-Aas, K., and Krak au , C. E. T.: Personal communication. 23. Friesen, 11.: To he published. 2·1. Natelson, S., Pincus, J. D., and Hannazzis, G.: Crin. Chem . -9:31, 1963.