Diabetologia 3, 475-487 (!967)

ORIGINALS

The Physiology and Pharmacology of Adipose Tissue Lipolysis: Its Inhibition and Implications for the Treatment of Diabetes*

The Minkowski Award Lecture delivered on July 31, 1967 before the European Association for the Study of Diabetes at Stockholm, Sweden

E. RUDOLF :FROESCH

Metabolic Uni t , D e p a r t m e n t of Medicine, Un ive r s i t y of Zurich, Switzer land

Rece ived Sep tember 11, 1967

Summary. The regula t ion of adipose tissue lipolysis occupies a key posi t ion in the metabol ic concer t of car- bohydra te s and lipids, and insulin plays the role of the conductor . Insul in favours the s torage of fa t in adipose t issue by 3 mechan isms: -- 1. i t inhibi ts l ipolysis; - - 2. increases the glucose up take , l ipogenesis and re6steri- f icat ion of free f a t t y acids; -- 3. enhances the ac t i v i t y of l ipoprotein-l ipase, which is responsible for the up take and s torage of b lood l ipoprote ins in adipose tissue. -- On the o ther hand, basal l ipolyt ie a c t i v i t y of adipose t issue is v e r y m u c h e leva ted in ra ts which are refed af ter pro- longed s ta rva t ion . I t appears t h a t insulin induces a li- pase, which, in its presence, is nonfunct ional . This dual role of insulin -- induc t ion of a l ipase ac t iv i ty which i t inhibi ts -- would p rov ide animals wi th a v e r y efficient m e c h a n i s m of switching f rom glucose ut i l iza t ion and l ipid s torage to l ipid mobi l iza t ion and oxidat ion. - - H igh p lasma levels of free f a t t y acids decrease glucose utili- za t ion of muscle and the effects of insulin thereon. There- fore, pharmacologic inhibi t ion of lipolysis has been in- ves t iga ted in order to find new and be t t e r ways to ad- jus t the metabol ic s i tua t ion in diabetes mell i tus. We h a v e used 5-methylpyrazole-3-earboxyl ic acid as a mode l ant i - l ipolyt ie compound. This drug, nicot inic acid and pro- s tag landin E 1 no t only inhib i t lipolysis, bu t t h e y also marked ly enhance glucose up t ake of adipose t issue in vivo. 5-methylpyrazole-3-earboxyl ic acid p reven t s t he rise of the free f a t t y acids and of blood glucose acu te ly af ter the admin i s t r a t ion of ant i - insul in serum. W h e n this drug is admin is te red over a per iod of several days, adipose t issue develops an escape mechan i sm; 5-methylpyrazole- 3-carboxylie acid still effect ively inhibi ts l ipolysis and s t imula tes l ipogenesis f rom glucose, bu t only for a shor t per iod of t ime. Basa l l ipolysis is a c t i va t ed and the newly synthes ized glycerides are rapid ly spli t and released as free f a t t y acids into the blood, so t h a t soon af ter i ts ad- min i s t ra t ion the p lasma level of free f a t t y acids is in- creased above normal . These findings shed doubt on the po ten t ia l usefulness of ant i l ipolyt ie drugs in the t rea t - m e n t of d iabetes mell i tus. - - Purif ied nonsuppressible I L A of se rum is men t ioned as an an t id iabe t ie subs tance of po ten t i a l the rapeu t i c in teres t since i t increases glucose up t ake of muscle more m a r k e d l y t h a n t h a t of adipose t issue compared wi th insulin.

Physiologic et Pharmacologic de la lipolyse du tissu adipeux: son inhibition et ses implications dans le traite- ment du diab@te.

R~sum~. La rggula t ion de la l ipolyse occupe une posi- t ion centrale dans le concer t m6tabol ique des glucides et l ipides et l ' insul ine joue le r61e du chef d 'orches t re . L ' insu- line augmen te l ' a ceumula t ion de graisse dans le t issu

* The studies r epor t ed here were suppor ted by the US Publ ic H e a l t h Service (grant No. A 5'387) and by the Schweizerische Nat iona l fonds (grant No. 3'854).

ad ipeux par 3 m6chanismes : -- 1. elle inhibe la l ipolyse, -- 2. s t imule la eap ta t ion de glucose, la lipog6n6se et la r6est6rifieation des acides gras libres et - - 3. rend ac t ive la l ipoprot6ine lipase qui est responsable de la cap ta t ion des l ipoprot6ines du sang par le t issu adipeux. -- D ' a u t r e par t , la l ipolyse des rats r6al imentgs apr6s un jefine prolong@ est tr6s act ive. Ceci signifie que l ' insul ine indui t une lipase den t l ' ac t iv i t6 est inhib6e par l ' insul ine m6me. Ce double r61e de l ' insul ine -- induc t ion d 'une lipase qu 'e l le inhibe -- pe rme t k l ' an imal d 'une fagon efficace et rapide de passer de l 'u t i l i sa t ion de glucose et de la lipog6n6se la mobi l i sa t ion d 'acides gras ]ibres pour les besoins 6nerg6tiques d6s que le t a u x sanguin de l ' insul ine balsse . - - La muscu la tu re pr6f6re les acides gras libres en for te con- cen t ra t ion au glucose comme substra ts d ' o x y d a t i o n et l ' insul ine perd de son efficacit6. Les possibilit6s pha rma- cologiques d ' inh iber la l ipolyse afin de t rouve r des moyens pour mieux a jus ter la s i tua t ion m6tabol ique du diabg- t ique ont fal t l ' ob je t d ' inves t iga t ion . Dans ce bu t l ' ac ide 5-methylpyrazole-3-carbonique fur utilisg. Cet te sub- s tance ainsi que l 'ac ide n icot in ique et la p ros tag landine E 1 n ' i nh iben t pas seulement la l ipolyse mais ils augmen- t en t aussi fo r t emen t la cap ta t ion du glucose par le t issu ad ipeux in vivo. L'ac ide 5-methylpyrazole-3-carbonique emp6ehe l ' a u g m e n t a t i o n des acides gras libres ainsi que de la glyc6mie apr6s in jec t ion de s6rum ant i - insul inique. Lorsque des a n i m a u x sent t rai t6s avee ce t te subs tance pendan t plusieurs jours, la l ipolyse spontan6e basale est activ6e. L 'ac ide 5-methylpyrazole-3-carbonique est encore capable d ' inh iber la lipolyse, reals la dur6e de son effet an t i l ipo ly t ique est diminu6e. Les glyc6rides r6cemment synth6tis6s ~ pa r t i r du glucose sent r ap idemen t hydro- lys6s et ils r6apparaissent sous forme d 'ac ides gras libres dans le sang. Le t a u x de ces derniers est alors augment~ bien au dessus de celui des contrSles. Ces rdsultats ne sent pas favorables ~ l ' id6e que des substances ant i l ipoly t iques pour ra ien t se p rouver ut i les dans le t r a i t emen t des diab6tiques. -- L ' ac t iv i t6 insul inique non suppr imable ex t ra i te du s@rum et purifi@e mdri te de l'int6r@t du po in t de vue thgrapeu t ique puisque, com- parge k l ' insuline, elle agi t plus fo r t emen t sur la cap ta t ion de glucose du muscle que du t issu adipeux.

Physiologic und Pharmalcologie der .Lipolyse des _Fett- gewebes: lhre Hemmung und Bedeutung bei der Behand- lung des Diabetes mellitus.

Zusammenfassung. Die Regu la t ion der Lipolyse des Fe t tgewebes n i m m t im Stoffwechsel der K o h l e h y d r a t e und F e t t e eine zentrale Sbellung ein, in der das Insul in die l~olle des Dir igenten spielt. Insul in f6rder t die Spei- cherung yon F e t t im Fe t tgewebe durch 3 Meehanismen. -- t. Es h e m m t die Lipolyse, - - 2. fSrder t die Glucoseauf- nahme, Lipogenese und Wiederve res t e rung der freien Fe t t s~uren und -- 3. s te iger t die Ak t iv i t~ t der Lipopro- teinl ipase, welehe die Aufnahme der Lipoprote ine des

Diabetologia, Vol. 3 32

476 E. 1~. FI~OESOK: The Physiology and Pharmacology of Adipose Tissue Lipolysis Diabetologia

Blutes in das Fettgewebe bewerkstelligt. -- AnderseRs ist die spontane, basale Lipolyse des Fettgewebes yon l~atten, die nach 1/ingerem Fasten wiedergefiittert wur- den, aul3erordentlieh aktiv. Insulin scheint eine Lipase im Fettgewebe zu induzieren, deren Aktivit~t in An- wesenheit yon Insulin gehemmt ist. Diese zweifache l~olle des Insulins -- Induktion einer Lipase, die es selbst hemmt -- starter das Tier mit einer wirkungsvollen I~e- gulationsmSglichkeit des Intermedi/irstoffwechsels aus, indem es dureh Ein- und Absehalten der Insulinsekretion yon der Glucoseverbrennung und Fettspeieherung auf die Mobilisierung der Fettreserven und die Verbrennung freier Fetts/~uren iibergehen kann. -- Die 3/iuskulatur zieht freie Fetts/iuren in hoher Konzentration der Glu- cose Ms Substrat vor und Insulin verliert an Wirksam- keit. Die pharmakologisehen MSglichkeiten dcr Hemmung der Lipolyse wurden untersucht, in der Hoffnung, neue Wege ffir eine Verbesserung der Stoffwechselsituation des Diabetikers zu linden. 5-Methylpyrazol-3-carbon- s/iure wurde als sehr wirksame antilipolytisehe Substanz verwendet. Es zeigte sich, dal3 diese Substanz, Nieotin- s~iure und Prostaglandin E 1 nicht nur die Lipolyse hem- men, sondern aueh die Glucoseaufnahme des Fettge-

webes in vivo sehr stark steigern. 5-Methylpyrazol-3- carbonsgure verhindert den Anstieg der freien Fett- s/~uren und des Blutzuckers kurz nach der Injektion yon Anti-Insulinserum. Wird diese Substanz wghrcnd mehre- rer Tage verabreicht, so nimmt die spontane Lipolyse des Fettgewebes zu. Die Droge hemmt die Lipolyse und fSrdert die Glucoseaufnahme zwar immer noch, abcr die Wirkungsdaucr nimmt ab. Die neu synthetisierten Glyce- ride werden raseh wieder hydrolysiert and als freie Fett- s~uren in das Blur abgegeben, so dal3 der Spiegel der freien Fetts/turen h6her ansteigt als bei Kontrolltieren. Diese Daten lassen daran zweifeln, dal3 diese antilipoly- tisehen Substanzen in der Therapie des Diabetes mellitus einen Platz finden werden. -- Die gereinigte und nicht hemmbare Insulinaktivit/~t des Serums scheint hingegen in dieser Bezichung recht vielversprechend, weft sic, ver- glichen mit Insulin, die Glucoseaufnahme der Muskulatur mehr steigert als die des Fettgewebes.

Key-words : Diabetes, adipose tissue, lipolysis, anti-lipoly. sis, insulin, non-suppressible ILA, glucose metabolism, fructose metabolism, 5-methylpyrazole-3-carboxylic acid, nicotinic acid, prostaglandin, fasting-refeeding

Introduction

On the occasion of the first lecture held in hononr of MINKOWSKI a year ago, Dr. I~ANDLE [29] stated that an essential feature of his concept of the glucose fat ty acid cycle was the idea that deficiencies in the uptake and metabolism of glucose were not primarily responsible for the augmented release and oxidation of fat ty acids in diabetes. HAL~s and RASDL~ [20] suggested that one of the very early aberrations of metabolism in diabetes might be an elevation of the level of free fat ty acids diminishing the sensitivity of tissues to insulin and finally leading to glucose intolerance by way of an exhaustion of the fi-islet cells. There is good evidence that free fa t ty acids may be oxidized in muscle in preference to glucose, and that the level of free fa t ty acids is one of the factors that regulate overall glucose utilization [11, 27, 38, 30]. If diabetes did begin with a high level of free fat ty acids, ways should be found to bring their level back to normal. Since adipose tissue is the only source of free fat ty acids, it was reasonable to search for antilipolytic drugs which -- by way of lowering the free fat ty acid concentration in plasma -- would be expected to increase the glucose uptake and metabolism of muscle and the efficacy of insulin thereon. Acutely, this goal may be achieved. However, long term therapy with antiSpolytic drugs has not been successful owing to the very ingenious ability of Nature; in this instance, adipose tissue not allowing itself to be fooled by the pharmacologist. Before we tackle this problem, I shall quickly review some aspects of lipolysis and of its regulation.

Regulation of lipolysis by the availability of glucose, i.e. by regsterifieation of free fatty acids with c~-glycerophos-

phate Fig. 1 shows schematically the main reactions in-

volved in the regulation of the release of glycerol and free fa t ty acids by adipose tissue. Free fa t ty acid

/hsulin

I ~q/ucoee

J,

I/polu/ic "~ horrnone~

.~-----~ ins'ulin.

glL/c~ en tm'glycem'de ~ -~ glycePo/

"~ ~-glycerophce#he/e I M#y ec/d CoA ~ --

Fig. 1 : l~eactions involved in the regulation of the release of free fatty acids and glycerol by adipose tissue

release depends not only on the activity of the lipase, but also on the availability of glucose for their re~sterifi- cation with ~-glycero-phosphate (for a detailed review see VAUC~ASr and STE~BEaG [37]).

We know from studies with adipose tissue in vitro, that the free fa t ty acid release decreases as the glucose concentration in the medium is increased. This observation is physiologically not very meaningful since glucose transport in adipose tissue in vitro does not follow the same rules as glucose transport in vivo. Glucose uptake of adipose tissue in vivo ceases almost completely when anti-insulin serum is administered or when insulin secretion is stopped by the administration of mannoheptulose [18]. Thus, re~sterification of free fa t ty acids in vivo primarily depends upon the action of insulin on glucose transport and not on the glucose concentration. Without insulin glucose does not pass the barrier of the cell membrane and it remains unavailable for cellular metabolism.

I know only of one example of a fall of free fat ty acids by increased re~sterification in vivo concomittant with a decreasing plasma insulin level. Patients with hereditary fructose intolerance are unable to metabolize fructose in the liver owing to a deficiency of hepatic aldolase (for a review see FRol~soH [12]). Fructose is phosphorylated; and fructose-l-phosphate accumu-

Vol. 3, No. 6, 1967 E.R. I~ROESeg: The Physiology and Pharmacology of Adipose Tissue Lipolysis 477

lares and blocks glucose release from the liver so tha t hypoglycaemia develops. Fig. 2 shows tha t the level of free fa t ty acids falls in a patient with hereditary fructose intolerance in spite of hypoglycaemia and of a decreasing insulin concentration at a t ime when the fructose concentration in blood is high.

M. ,~ , z,c, ~ t./ s/ ,~og frucla~e /:v. 8.q glucaae Lz

1 ~.2 ~Fz2

a FFA l .O 1-1-10 m /nsuh# ,.~Er aerie pV/rnl

ae1-Fe a4 I-I-4 a2 ~ 2

I I I I I I I I I I

obloodfruc/o~e 80 rng/iOOm/ II ~

eo

" 4- .... \" 40 4 x/norg.R ~ ~

-5" 0 10 20 30 40 gO BO Z5 gO m/,'r

Fig. 2. Blood glucose and fructose, immunoreactive insu- lin and plasma free fat ty acids in a patient with heredi- tary fructose intolerance during an intravenous tolerance

test with 0.25 g fructose/kg

Only when hypoglycaemia becomes severe, does adrenergic counterregulation induce the plasma level of free fatty acids to rise again. The fall of free fatty acids, despite hypoglycaemia and low insulin levels, is due to the proper ty of adipose tissue to take up glucose and fructose by separate t ransport systems, so tha t their metabolism is additive rather than competitive as in most other tissues [15]. In the presence of glucose and fructose, total hexose uptake is considerably greater than with glucose alone, and although hypo- glyeaemia develops, the level of free fa t ty acids falls as a result of increased re6sterification. However, this is a very exceptional example of increased re~sterification regulating the free fa t ty acid concentration at a t ime when insulinaemia is low.

Some time ago, BALLu et al. [1] showed tha t glucose alone, or added together with insulin, may, under certain conditions, increase rather than decrease the rate of lipolysis in vitro. This occurs when at high rates of lipolysis free fa t ty acids accumulate in the adipose tissue cell and inhibit ]ipolysis. Addition of glucose alone or together with insulin may then relieve this inhibition by the formation of glycerides from free fa t ty acids through the supply of ~-glyeero-phosphate. This observation may be an artefact of the incubation in vitro in which the outflow of free fa t ty acids into the medium is slow.

The role of insulin in the regulation of lipolysis

Insulin inhibits hormonally-induced lipolysis in vitro. Small concentrations of ACTH and of epinephrine are effectively counteracted by insulin [23, 21]. Growth

hormone, with and without added glucocorticoids, stimulates lipolysis of adipose tissue and insulin inhibits its effect [10]. Spontaneous, basal lipolysis is also inhibited by insulin [13, 24, 36].

d C

'S

C9

r

• u • o l G l u c . / p a d / 2 h /

~ , / / 5 - - - no Insul in F . . . . . " ' " 4 . . . . . Insu l in " t _.o / 3- 1000pU/mt / ~ - -

2-1 r 0- 8- ........

}J m o t / p a d / 2 h

2

L L -

I

0

0

o

~ m o l / p a d / 2 h

/ ~ ......... I I I I ] I - ' - ~ - - - r . . . . I 0 6 12 18 2/, 30 36 42hours

norrnct[ I 72-hours! feedingl fasting I Refeeding Carbohydrate

Fig. 3. Glycerol and free fat ty acid release and glycogen content of adipose tissue of fasted -- refed rats with and without insulin stimulation as a function of the duration

of refeeding Two groups of animals with a fasting period of 72 and 120 h, respectively, were used. The tissue of 4 animals was pooled into 4 flasks, two of which contained insulin. The Krebs-Ringer bicarbonate buffer contained 4 g albumin per 100 ml and no glucose. The incubation lasted 2 h. The means of the results of two flasks and their range are

plotted. (From FROESCH et al. [13])

This is illustrated in Fig. 3. Glycerol, free fa t ty acid release as well as glycogen content of adipose tissue were studied in fed, fasted and fasted-refed rats. Adipose tissue of fasted rats releases significantly greater amounts of glycerol into the medium than adipose tissue of fed rats. Insulin exerts a slight inhibitory effect. During the refeeding period, basal glycerol release increases to a level way above tha t found during fasting, and inhibition by insulin be- comes very pronounced. Free fa t ty acid release does not parallel glycerol release during refeeding. As is shown in the top curve, glycogen accumulates in large amounts in the tissue during the refeeding period, providing ~-glyeero-phosphate for the re~sterification of the free fa t ty acids and making glucose superfluous for this purpose.

Lipolysis of adipose tissue of fasted-refed rats and its inhibition by insulin is, indeed, independent of the presence of glucose or any other substrate in the

32*

478 E. 1~. F~oEseE: The Physiology and Pharmacology of Adipose Tissue Lipolysis Diabetologia

medium. Fig. 4 demonstrates that the formation of ~-glycero-phosphate from exogenous glucose is un- important for the release of free fat ty acids in vitro since glycogen provides ample amounts of this sub- strate. Free fat ty acids may actually be taken up by the tissue from the albumin-containing medium at a time when the glycerol release is very high [13].

I - - I no Glucose F-'/] Glucose 2rag/m[

GLYCEROL-RELEASE FFA-RELEASE /Jrnoles/pad/h /u Eq/pad/h

3.00-

ZOO-

tO0-

/ / / / / / / / / / / / / 2 / / / / / /

Z / / / / / J / /

2_00

1,00

% no Insulin Insulin no Insulin Insulin

1000 ~.U/rnl 1000 ~.U/rnl

Fig. 4. Effect of insulin on release of glycerol and free fatty acids by adipose tissue of 72 h fasted -- 24 h refed

rats in the absence and presence of glucose

A pool of adipose tissue from 8 rats was used. The Krebs- Ringer bicarbonate medium contained 4 g albumin per 100 ml and the incubation lasted for 3 h. The bars repre- sent the mean of the results of two flasks, and the range

is given. (From F~OESCH et al. [13])

Thus, insulin seems to play a complex role in the regulation of lipolysis. As is demonstrated in Figs. 3 and 4, insulin inhibits lipolysis of adipose tissue in vitro most markedly when rats were refed after fasting, i.e. when extremely high rates of glucose assimilation were maintained over a prolonged period of time by an elevated insulin level in blood. On the other hand, these high rates of glucose assimilation and of lipogene- sis lead to a stimulation of basal lipase activity, which is inhibited or "non-functional" in the presence of insulin. As we shall see later, antilipolytic substances other than insulin also enhance lipogenesis in vivo and at the same time increase the basal lipase activity. Adrenergic mechanisms do not appear to be responsible for this activation of basal lipolysis by insulin for the following two reasons: 1. lipolysis of Iasted-refed rats is not subject to suppression by propranolol, a blocker of the beta-receptors of adipose tissue cells; and 2. treatment of rats with large doses of propranolol or

5-methylpyrazole-3-carboxylic acid during the refee- ding period does not alter the lipolytic response of the tissue. Table 1 shows that propranololin concentrations from 10 -s to 10 -4 M at which it blocks the response of adipose tissue to adrenaline and other ]ipo]ytic

Table i. Independence of adipose tissue lipolysis of fasted-- refed rats of the bloelcing of fl-receptors by propranolol in

vitro The figures give the mean of the results of two flasks and

their range

p r o p r a n o l o l c o n c e n t r a t i o n in vitro c o n t r o l 10 -~ 1~I 10 -~ M 10 -s ~I

glycerol release in #moles/g/h

14.0~:0.4 15.5:k0.9 15.9• 15.2-4-0.6

hormones, does not change the spontaneous glycerol release of adipose tissue in vitro of fasted-refed rats. In another series of experiments rats were treated with propranolol or 5-methylpyrazole-3-carboxylie acid during the refeeding period. The latter drug inhibits lipolysis activated by ~- and fl-receptors of adipose tissue. Basal glycerol release of adipose tissue in vitro was not inhibited by these premedications (Table 2).

Table 2. Independence of adipose tissue lipolysis of fasted- refed rats of the administration of propranolol and 5-methyl- pyrazole-3.carboxylic acid during the 24 h-refeeding period Rats were fasted for 120 h and then refed for 24 h. During the 24 h of refeeding they were treated with the fi-receptor blocking agent propranolol or with 5-methylpyrazole-3- carboxylic acid. Adipose tissue was pooled into 10 flasks and was incubated in Krebs Ringer bicarbonate buffer containing no glucose and additions as indicated. The

SEM (controls, n = 6) or the range (n ~ 2) is given

a d d i t i o n s t o i n c u b a t i o n m e d i u m :

t h e r a p y d u r i n g n o n e i n s u l i n 5 - m e t h y l - r e f e e d i n g ( s . c . ) : 1000 # U / m l p y r a z o l e - 3 -

c a r b o x y l i e a c i d , 10 -51~[

NaC1, 0.9% 1 ml/4h

propranolol in NaC1 0.5 mg/r

5-methylpyrazolc 3-carboxylic acid 0.5 mg/4h

glycerol release in #moles/g/h

7.7~1.2 2.4• i.9•

7.7~0.8 2.5~0.1 2.5•

9.5• 4.6~0.1 3.3~0.1

6 2 2

Thus, the role of insulin in the regulation of lipolysis appears to be twofold: 1. Insulin favours lipid storage in adipose tissue by 3

mechanisms : a) by inhibiting lipolysis ; b) by increasing glucose uptake, lipogenesis and

re~sterifieation;

Vol. 3, No. 6, 1967 E . g . F~OESCE: The Physiology and Pharmacology of Adipose Tissue Lipolysis 479

c) b y s t imula t ing l ipoprote in- l ipase ac t iv i ty , which is responsible for the u p t a k e of l ipoprote ins f rom blood (reviewed b y I~oBI~so~ [31]).

2. On the o ther hand, insul in induces a l ipase for the mobi l i za t ion of s to red l ipids, which becomes ac t ive as soon as insul in release stops. Teleological ly this makes sense and would provide

animals wi th feeding hab i t s such as the lion, who eats enormous amoun t s a t one t ime (equiva lent of re- feeding), and then rests un t i l hunger overcomes his s t rong inc l ina t ion towards peaceful laziness, wi th a very efficient r egu la t ion of energy metabo l i sm. As long as hype rg lyeaemia lasts a f te r feeding, insul in fills the energy stores b y t r ig lycer ide synthes is and b y inhibi- t ing l ipolysis. Lipolys is t akes off aga in as soon as the b lood glucose level falls off so t h a t insul in release stops. The h a b i t of fas t ing and refeeding p r o b a b l y was also charac te r i s t i c for men - - and sti l l is in large pa r t s of the wor ld - - before c iv i l iza t ion made a more or less cont inuous food supp ly ava i lab le to him, resul t ing in obes i ty , d iabetes , a theroselerosis and - - las t b u t no t leas t - - den t a l caries.

The d a t a in Fig . 5 show t h a t the inh ib i t ion in vitro of l ipolysis of ad ipose t issue of fas ted- re fed ra t s b y

#moles/pad GLYCEROL RELEASE 2.5-

2.0-

1.5

1.0

0.5

no insulin /

/ /

/ %

/ /

/

/ I mU insutin/ml

3'0 6'0 9'0 1�89 min. of incubation Fig. 5. Effec t of a n t i - i n s u l i n s e r u m on in su l in - induced depress ion of g lycerol re lease b y ad ipose t i ssue of 120 h

f a s t ed -- 24 h refed r a t s Apool of epididymal adipose tissue of 16 rats was used. Each flask contained 2 ml Krebs -g inger bicarbonate buffer with 3 g a lbumin per 100 ml and no glucose. Anti-insulin serum, 0.1 ml with an insulin-neutralizing capaci ty of 50 mU, was added from the side arm of the Warburg vessel 30 min after the s tar t of the incubation, as indicated by the arrow. To the control flasks 0.1 ml normal guinea pig serum was added at the same time. The brackets indicate

the results of the two flasks. (From F~OESCH et al. [13])

insulin in the absence of glucose is r ead i ly revers ible when ant i - insul in serum is a d d e d to the incuba t ion medium. Dur ing the two hours of incuba t ion v i r t ua l l y no glycerol is re leased b y the t issue t h a t is under the

influence of insulin. A t 30 min af te r the add i t i on of an t i - insul in serum the t issue s t a r t s aga in to release glycerol a t a similar, a l though no t qui te equal ra te as nons t imu la t e d tissue. A dose-response re la t ionship be tween the insul in concen t ra t ion and the inh ib i t ion of glycerol release is shown in f igure 6. Glycerol release b y adipose t issue of fas ted-refed r a t s is s ignif icant ly inh ib i ted a t an insulin concen t ra t ion of 31 FU per ml.

100 %"

80

60

40

20

100%

80

60

40

20

GLYCEROL RELEASE IN ~ OF .BASELINE

LACTIC ACID RELEASE IN % OF BASELINE

. . . . . . 3'] {~2 ]:25 250 Insulin/uU/ml. log.scale

Fig. 6. Ef fec t of insu l in on release of glycerol a n d lac t ic acid by adipose tissue of fasted -- refed rats in the ab-

sence of glucose Two pools of epididymal adipose tissue each from 12 rats were used. One group of animals was fasted for 72 h, the other for 144 h, and all animals were refed for 24 h. The Krebs-Ringer bicarbonate buffer contained 200 nag gelatin per 100 ml and no glucose. The results are ex- pressed as the percentage of the value in the absence of insulin. The means of the results of 4 flasks and the SEM

are given. (From F~OESeH et al. [13])

The insul in effect becomes nea r ly ma x ima l a t a concen t ra t ion of 250 FU per ml. The shape of the curve is i ndependen t of whe ther glucose is p resen t in the med ium or not. The o u t p u t of lact ic acid is also suppressed b y insul in in the absence of glucose in the med ium.

Pharmacologic inhibition of !ipolysis

Ever since the first observa t ions on the in te r re la t ion of the me tabo l i sm of free f a t t y acids and glucose were repor ted , and in pa r t i cu l a r since the hypothes i s was p u t fo rward t h a t d iabe tes mel l i tus migh t ac tua l ly s t a r t off wi th an e leva t ion of the free f a t t y ac id level before an a b n o r m a l i t y of glucose me tabo l i sm is

480 E.I~. F~OESCH: The Physiology and Pharmacology of Adipose Tissue Lipolysis Diabetologia

discernible [20], a great deal of work has been carried out with drugs with antilipolytie activity.

Various potent drugs which inhibit spontaneous and hormonally-aetivated lipolysis are listed in Table 3. Of

Table 3. Substances inhibiting spontaneous lipolysis of adi. pose tissue of fasted -- refed rats

The studies with nucleosides, nucleotides, nicotinic acid, 3-pyridfl-acetic acid and phospholipases have not yet been published. Glycerol release of pooled epididymal adipose tissue of 120 h fasted -- 24 h refed rats in the absence of

glucose was used as a test system

range of activity (moles/l)

1 i n s u l i n 5 X 10 - i i - - 10 -9

2 purified nonsuppressible ILA 10 - s - 10 -~ ( ? )

3 nicotinic acid 5-methylpyrazole- 3-pyridil- 3-earboxylie acid acetic acid

5 • 10 - s - 10 -~

4 prostaglandin E 1 5 X J[0 - a - 10 -7

5 nucleosides and nucleotides 10 - s - 10 -~

6 phospholipase

these, insulin is by far the most potent. I t inhibits glycerol release of adipose tissue of fasted-refed rats at concentrations as low as 10 -1~ M. Purified nonsuppres- sible ILA of serum shows exactly the same curve of dose-response relationship, but its activity is about two potencies less than tha t of insulin [16]. The phospho-lipases are of interest since they inhibit lipoly- sis and, at the same time, increase glucose uptake of adipose tissue in vitro almost as much as insulin [32, 2]. Under 3, drugs of similar structure are listed, i.e. nicotinic acid, 5-methylpyrazole-3-carboxylic acid and 3-pyridfl-acetic acid. The antilipolytic properties of nico- tinic acid have been investigated by CARLSOn, O~STMA~ and ]~AZLu among others (for review see CAI~Lso~ and BALLu [7]). 5-methylpyrazole-3-carboxylie acid is an interesting compound, which has a much longer biological half life than nicotinic acid [34]. I t s anti- lipolytie and antidiabetic effects were first observed by S~ITIt, FoRInT, D~LI~ [33] and GE~lCImSE~r and D v L ~ [19] of the Upjohn Company. These 3 drugs have a similar ring structure and have in common a carboxyl group at a relatively constant distance from the nitrogen in the ring. Small effects on lipolysis in vitro are obtained at concentrations of 5 X 10 -s M, and maximum activity is reached at a concentration of 10 -~ 1V[. Even more active are the naturally occnring prostaglandins in the concentration range of 5 x 10 -~ to 10 -~ IV/[35, 3]. Their biologic half-life is very short. Many nucleosides and nuelcotides inhibit lipolysis, but they are relatively less active than the drugs mentioned above [9, 25]. ~- and fl-receptor blocking agents are not listed in this table.

Effects of 5-methyIpyrazole-3-carboxylie acid and prosta- glandine E 1 on adipose tissue metabolism in vitro and

comparison with those of insulin

The main effects of 5-methylpyrazole-3-carboxylic acid and of prostaglandin E 1 on adipose tissue of normal fed rats are shown in Fig. 7. Both drugs increase glucose incorporation into total lipids, CO 2 and

20

18

12

$

4

s - .z-

/nsul/'n, lO00Jz Ul,,~l

5'luc~162 ] ft'/"cf~

lrlcor'florGl/~n /',7/o glycogen, lu rno/e$/,a/h

1:,wo,"por~//o/7/h,~o ~n6' 2 § mo/es/~,/h

- +

+

-- +

+ +

Fig. 7. Comparison of the effects of 5-methylpyrazole-3- carboxylic acid, prostaglandin E I and of insulin on glu- cose-U-tiC - and fructose-U-14C-metabolism of pooled epi-

didymal adipose tissue of normal, fed rats in vitro The bars give the mean of the results of two flasks

glycogen. Insulin is much more effective. Under insulin stimulation incorporation of glucose-U-14C into glycogen accounts for approximately 25% of total glucose metabolism. 5-methylpyrazole-3-earboxylic acid and prostaglandin do not affect the incorporation of fructose-U-14C into CO2 and total ]ipids, while they approximately double its incorporation into glycogen. The same phenomenon is also seen when the meta- bolism of adipose tissue of fasted-refed rats is studied (Fig. 8). Both drugs reduce the glycerol release of adipose tissue of fasted-refed rats to approximately 15% of the base line. They stimulate glucose meta- bolism and incorporation of glucose-U-14C into glycogen to a small extent. Fructose-U-14C incorporation into total lipids and CO 2 is not stimulated, whereas its incorporation into glycogen is increased manyfold.

These data favour the concept tha t fructose enters the adipose tissue cell, and tha t it is converted to glucose-6-phosphate in a compar tment which is particularly favourable for glycogen synthesis [17]. In contrast, insulin apparent ly leads to formation of glueose-6-phosphate in a compartment in which glycogen synthesis takes place only at very high rates of glueose-6-phosphate formation.

Vol. 3, No. 6, 1967 E.R. FRO~.SCH: The Physiology and Pharmacology of Adipose Tissue Lipolysis 481

On the basis of the results of BU~CH~,R et al. [5] and of J~7~AS [22] one may draw the following purely hypothetical scheme (Fig. 9) of the regulation of lipolysis and of glycogen metabolism by lipolytic

glucose - U- ~4C, fruclose - U- 14 6", ~ubs~nefe: 200mg/100ml 800rag~ lOOm/

0.8

~4

~2

/2

8

4

glgce:ol ~ole~se, ~ moles/g/h 16

12

4

5"- rnolh#/p#~azo/e - ~- car$oz#/io acid, IO-EM --

proolag/en~ E1 IO-~M

4- - -

+

Fig. 8. Effects of 5-methylpyrazole-3-carboxylic acid and of prostaglandin E 1 on glycerol release, glucose-U-l~C- and fruetose-U-ltC-metabolism of adipose tissue of fasted

- - refed rats The bars give the mean of the results of two flasks

A TP D~phospho - ~ Phosphm'#lase (b) L/polyHc u i phosphor#lase (a)

h ~ r+ ~., L J f ' ~ J Adenyl- ,~. i Phospha:'#lase-

N/~oh~ic acld+ .I. Anelogue~ (--) �9 UDP- Glucoge

Prosfeg/and/~ El J'S'-~M~ ~ (F)GIL/cogon- , ~> J| Conoenh.,~h~nan#comp, T/o#- | ~ (--) s#nlhofaae ~ menleh~alion o# 8-6-P l ~ G&oogen

Theoph#llin (--) Phoapho- I d,~,tor~,e"m~l .Aclival~slem"(?)

I Lipase inaclive D LiFa-~e acHve

�9 TG ~ Im FFA 5"'-AMP Glyce:'ol

Fig. 9. t~eactions invo lved in the regu]ation of the level of cyclic 3 ' , 5 ' - ~ P in adipose tissue and the influence of cyclic 3',5'-A~d:P on the rate of ]ipo-

lysis and on glycogen metabolism A causal relationship between the level of cyclic 3',5'-AMP and the activ- ity of these enzymes of adipose tissue has not yet been established. Levels of cyclic 3',5'-AMP have not been reported for tissue of fasted -- refed rats. This scheme is based on the experimental findings of BLTTCltEI~ et al. [5]

and JUNOAS [22]

hormones and by their inhibitors. The metabolic reactions governing the rate of lipolysis, glycogen formation and glycolysis centre around cyclic 3'5'- AMP, the so-called second messenger of Sutherland. The formation of cyclic 3'5'-AMP is regulated by the activity of adenyl-cyclase and its degradation by the activity of phospho-di-esterase (for a review see

BUTCHER and SUTttERLAIqD [6]). Lipolytic hormones activate adenyl-cye]ase, and theophylline inhibits phospho-di-esterase, thus increasing the intrace]lular concentration of cyclic 3'5'-AMP. Insulin and other antilipolytic drugs appear to inhibit the stimulatory effects of lipolytic hormones on adenyl-cyclase, thereby keeping the concentration of cyclic 3'5'-AMP low. Whereas the induction of lipolysis by lipolytic hor- mones is counteracted by proprano]ol, lipolysis of adipose tissue of fasted-refed rats does not appear to depend on the activation of the fl-receptors. Therefore, it is not yet certain whether or not cyclic 3'5'-AMP plays the same crucial role in the regulation of lipolysis and glycogen metabolism of adipose tissue of fasted- refed rats as it may play in the stimulation of lipolysis by lipolytic hormones and theophyll ine.

Effects of antilipolytic drugs on the metabolism of adipose tissue in rive

We have tested the antilipolytic effects of 3- methylpyrazole-3-carboxylie acid in rats in vivo, and were struck by the marked insulin-like effects which this drug exerts on glucose metabolism of adipose tissue in rive. 5-methylpyrazole-3-carboxylie acid stimulates the incorporation of 14C-labelled glucose into adipose tissue as markedly as a large dose of insulin, whereas it is without any effect on the in- corporation of glucose into glycogen of the diaphragm.

The experiments, the results of which are shown in Fig. 10, were carried out in the following manner:

100 g rats were anaesthetized with a barbiturate, and subsequently injec- ted intravenously with various doses of 5-methylpyrazo]e-3- carboxylic acid together with glucose-14C accord- ing to a method previously publish- ed [18, 16]. The rats were decapitat- ed 30 rain later and the blood, dia- phragm and epididymal fat pads were examined. As may be seen, 1.26 ~g of 5-methylpyrazole-3-carboxylic acid significantly lowers the free fa t ty acid level, and a maximal depression is obtained with doses of 126 to 1260 ~g per rat. The in- corporation of labelled glucose into total lipids and fa t ty acids of adi- pose tissue is significantly stimulated by 12.6 ~g of the drug per rat.

The time course of the effects of intravenously administered 5-methylpyrazole-3-carboxylic acid is demonstrated in Fig. 11. The level of free fa t ty acids is significantly depressed by 5-methylpyrazole-3- carboxy]ic acid throughout the entire period of ob- servation. Blood sugar and glucose assimilation are not affected by the drug despite its marked effects on

482 E.R. F~oEsc~: The Physiology and Pharmacology of Adipose Tissue Lipolysis Diabetologia

glucose assimilation by adipose tissue. This and the following experiments demonstrate that the importance of adipose tissue with regard to overall glucose assimilation has been grossly overestimated because of its avidity for glucose in vitro. Incorporation of glucose-6-iaC into glycogen of diaphragm is not in- creased by 5-methylpyrazole-3-carboxylic acid.

Studies on the antidiabetic properties of 5-methylpyrazole- 3-carboxylic acid

As shown in Table 5, 5-methylpyrazole-3-carbo- xylie acid blocks the hyperglycaemic response to the intravenous injection of anti-insulin serum in rats. Whereas the free fat ty acid level rises to 1.58 ~eq/ml

/ncarporehon of glucose-S- r ##a fatal h~oz~le and /og ecele feffy ecid.e of edz)~aae hL~aue in/x molea/g/JOminutea.

3 ~ 12.8~12G ;P~aOS

I I

0 12.6;PeO.O$ / / I 12.6--12G ;P<O0~

free fa#y acid~, / I

" T, / ,'T L " 1 r ' - . . . .

/ • ""~.~ 0 , �9 Z2B,'P<~OOE 0,2 / ~. Z2G'*-~'I2B ;p<LZ025"

/

I

O ~2B 12.G 126 12SO /~g of g-me/hylpyraza/e-J-cat,boxyh? acid

Fig. 10. Effects of 4 doses of intravenously administered 5-methyl-pyrazole-3-carboxylie acid on plasma free fatty acids and on the incorporation of glucose-6JaC into total

lipids and fatty acids of adipose tissue 5-Methylpyrazole-3-earboxylie acid was dissolved in physiologic saline and 1 ml was administered intravenous- ly together with a tracer dose of glucose-6-~aC. The rats were killed after 30 rain. The means of the results of 4 rats and the SEM are given. The specific activity of the blood glucose at the time of sacrifice was used to express the results of the incorporation into adipose tissue in terms of ~molcs glucose-6-~aC per g per 30 rain. (From

F~oEse~ et al. [18])

Experiments of the same type were carried out with nicotinic acid and with prostaglandin, i.e. drugs of comparable structure, or of different structure but with similar antilipolytic potency. Table 4 shows that nicotinic acid does not only depress the level of free fat ty acids, but that it shares with 5-methylpyrazole-3- carboxylio acid the stimulatory activity on glucose incorporation into total lipids and fat ty acids of adipose tissue. Nicotinic acid does not stimulate glucose metabolism of the diaphragm. The results obtained with prostaglandin E t are shown in Fig. 12. Again, glucose uptake of adipose tissue is stimulated, whereas incorporation of gluoose-6-~C into glycogen of diaphragm is not.

Thus, we may conclude that all these drugs with antflipolytio activity share a marked glucose uptake- promoting activity in adipose tissue in rive, a finding which was unexpected on the basis of the in vitro results, which had indicated only a very weak activity on glucose uptake and a marked antilipolytie activity.

f~e fa## acids /zer

2O

cpm//u mole bl0odz glucose x iO-2

x10 -$

( : - - - - : ) s

4

Otood~luooee 1oo my/ZOO ml

p~O007 p<O00i p<DOW

11%.

%% ~

K:-5 I.... ........ .........

~-_._~.--.~.~ ........

incorporat/on

d/ephregm a2 I glycogen ~i

ed/pose tissue: g/gceride- i gl#cerol

2.0

1.5

felfy ecids to

~E

of Zv. glucose-e-14C in /u molos/g into Tp>O.ls

p>~05

i / l - __~

p > O.Og p< ~Og p> a05

~ ~ - . . . . . . . . . . . . . . i P"" p<~Ol p<O00l

75 J0

minutes after i.v. injection ef~lucose-e-~4C in saline o . . . . ~ ,andwith O.Srng of g-mefhyl- pyrazolo-J-carboxylic acid.

Fig. 11. Effects of intravenously administered 5-methyl- pyrazole-3-earboxylic acid on blood glucose, plasma free fatty acids and incorporation of glucose-6-1aC into tissues

as a function of time 0.5 mg of 5-methylpyrazole-3-carboxylie acid in 1 ml saline was injected intravenously together with a tracer dose of glucose-6-1~C. Control rats were given i ml of saline. The rats were sacrificed after 15, 30 and 60 rain, respectively. The means of the results of 4 rats and the SEM are given. The specific activity of the blood glucose at the half time between injection and sacrifice was used to express the results in terms of ~moles of glucose-6-1~C

incorporated per g. (From FROESC~ et al. [18])

plasma in the rats treated with anti-insulin serum alone, it stays at the initial level of 0.98 ~eq/ml in the rats which, in addition to anti-insulin serum, received 1 mg of the pyrazole derivative.

In the experiment, the results of which are shown in Fig. 13, the antidiabetic activity of the pyrazole is further analyzed. 5-methylpyrazole-3-earboxylie acid stimulates the incorporation of glucose-6-~C into total

Vol. 3, No. 6, 1967 E.R. FI~OESCtt: The Physiology and Pharmacology of Adipose Tissue Lipolysis 483

lipids of adipose tissue in the presence of anti-insulin serum as much as in its absence. Insulin is no t a prerequisite for the effect of 5-methylpyrazole-3- carboxyHc acid in adipose tissue in vivo. The drug does not increase the glycogen format ion in muscle f rom

by muscle in spite of the tota l lack of insulin. Thus, a lack of insulin does no t necessarily lead to decreased glucose oxidat ion by muscle, provided tha t the level of free f a t ty acids is kept low. These results are in good agreement with da ta from experiments in vitro in which

TaMe 4. Effects of two doses of intravenously administered nicotinic acid on the level of free fatty acids and on the incorpora- tion of intravenously injected glueose-6-1tC into total lipide and .fatty acids of ad@ose tissue and into glycogen of the dia-

phragm Nicotinic acid was dissolved in saline and 1 ml was injected into the tail vein. The rats were killed after 30 rain. The means of the results obtained in 5 rats and the standard error of the means are given. Students t-test was applied for statistical analysis. The results, which are expressed as ~moles of glucose-6-~tC incorporated per g per h, were obtained by dividing the counts per rain in the respective metabolic indices by the specific activity of the blood glucose a~ the

time of sacrifice (From F~onse~ et al. [18]). P* = statistical analysis between control group receiving saline and the group receiving 1.2 mg of nicotinic acid.

incorpora t ion of glucose-6-~4C in pmoles/g/30" into

i .v . admin i - adipose t issue d i a p h r a g m s t ra t iou o f total lipide f a t t y acids glycogen

m e a n • SE3I p m e a n • SEM p m e a n ~= SE3s

free f a t t y acids/~eq/ml p lasma

mean • SEM p

NaC1, 9%0 , 0.234-0.06 0.074-0.04 0.064-0.01 0.73*4-0.04 1 ml <0.005 <0.01 p>0 .25

nicotinic acid, 3.074-0.68 2.134-0.58 0.05=~0.01 0.70 4-0.29 0.12 nag >0.10 >0.10 p>0 .10

nicotinic acid, 4.91 4- 1.14 4.074-1.15 0.074-0.01 0.28*4-0.02 1.2 mg

<*0.0005

Table 5. Effects of 5-methylpyrazole-3.carboxylic acid, insulin, anti-insulin serum alone and with 8-methylpyrazole-3-car- boxylic acid on blood glucose and free fatty acids

1 ml of saline with 3 g albumin/100 ml or of undiluted dialyzed anti-insulin guinea-pig sermn was injected into the tail vein with or without 0.5 mg of 5-methylpyrazole-3-carboxylic acid. The dose of insulin was 6 mU per rat. The rats were killed 30 rain after the intravenous injection. The means of the results obtained in 4 rats and the SEM are given

(From FRoEseI~ et al. [18]).

admin i s t r a t i on 30 rain pr ior to sacrifice of

a l bumin a lbumin a lbumin ant i - insul in ant i - insul in + + s e rum se rum + insul in 5-methylpyrazole-3- 5-methylpyrazole-

carboxylic acid 3-carboxylic acid

blood glucose 139.64-3.8 109.24-7.0 126.0~=5.6 203.84-1.9 145.64-3.4 rag%

P versus albumin < 0.01 0.10 0.005 0.20

free fat ty acids 1.2624-0.012 0.9144-0.033 1.0664-0.012 1.580• 0.9804-0.020

~eq/ml

P versus albumin < 0.0005 0.0005 0.0005 0.0005

glucose-6-14C. 5-methylpyrazole-3-carboxylic acid is devoid of a significant effect on total ac t iv i ty of serum glucose, i.e. it does no t significantly increase glucose turnover despite its effects on glucose metabolism of adipose tissue. However, the block on the outflow of glucose into tissues due to anti-insulin serum is b rought back to normal by 5-methylpyrazole-3-earbo- xylic acid. This can no t be explained by its act ion on glucose up take of adipose tissue since the same st imulat ion did not influence overall glucose tu rnover in the absence of anti-insulin serum. Rather , it is due to the anti-l ipolytic act ion of this drug. ]3y keeping the level of free f a t t y acids low, glucose is being oxidized

muscle metabolized and oxidized free f a t t y acids at high concentrat ions in preference to glucose [11, 27, 38, 30]. Thus, 5-methylpyrazole-3-carboxylic acid prevents hyperglycaemia in acutely-induced insulin deficiency. These promising results in acutely diabetic rats have been extended to chronically diabetic rats, where 5-methylpyrazole-3-carboxylic acid also stimulates the glucose uptake of adipose tissue and lowers the level of free f a t ty acids (FROl~SC~, unpublished data). However, when deeompensat ion of carbohydra te and lipid metabolism due to insulin deficiency lasted for sev- eral days, overall glucose utilization was no longer nor- malized by a mere depression of the free f a t t y acid level.

484 E.R. Fl~oEse~: The Physiology and Pharmacology of Adipose Tissue Lipolysis Diabetologia

Escape from the acute antilipolytic effects of 5-methyl- pyrazole-3-carboxylic acid during prolonged admini-

stration of 5-methylpyrazole-3-carboxylic acid

I t has been described tha t the inhibition of free fa t ty acid release in vivo by nicotinic acid may be followed by an overshooting llpolysis resulting in increased levels of free fa t ty acids [8, 28]. We have observed a similar phenomenon in rats t reated for 5 days with 5-methylpyrazole-3-carboxylic acid [18]. The results of such an experiment are shown in Fig. 14.

incarporalian ~f ~lur ~C inlo to/a/lipid~ ~nd ~a#~ ao/da of adipaxe tissue mnd ~l#cagea of ~phz~agrn. in/x /noles/g/IEmiz.

~9

o.F

~a

total//pid~ ~ O~50,p<.aOS

~ ]//o,~..,~,,,,,.,,,.,~ ,~- O~--~SO, p~OOS

�9 I glNcog~n ".2 "--.~ ~-*'-I~ :( ......... o ........... -0 0~-~ $0, p > ~ 10 ..... i i i

2 10 ~0 Io 8. scale

~ of pmaPa~land/n Zv:

Fig. 12. Effects of intravenously administered prostaglan- din E 1 on the incorporation of intravenously injected glueose-6-14C into total lipids and fatty acids of adipose

tissue and glycogen of the diaphragm Each dot gives the mean of the results obtained in 5 rats

The plasma level of free fa t ty acids is significantly higher in rats pretreated with 5-methylpyrazole-3- carboxylie acid than in control rats, 5 h after the last subcutaneous injection. The acute depression of the plasma free fa t ty acids by the intravenously ad- ministered drug is the same in both groups of rats, but the subsequent behaviour of the free fa t ty acid level is very different. In the group pretreated with 5-methyl- pyrazole-3-carboxylie acid it rises rapidly to the initial levels, whereas it stays low in the control group during the whole period of observation. Stimulation of the incorporation of labelled glucose into fa t ty acids and glyeeride-glycerol of adipose tissue is more marked acutely after the injection of 5-methylpyrazole-3- earboxylic acid in the premedieated rats than in the control rats. However, the glycerides synthesized under the influence of this drug seem to be hydrolyzed and released by the tissue very rapidly after their synthesis. The ~4C-aetivity in the free fa t ty acids of the plasma is significantly higher in the rats pretreated with the drug than in the control rats 60 rain after the intravenous injection of 5-methylpyrazole-3-carboxylie acid. These data indicate that under the chronic administration of pyrazole derivatives, both synthesis of glyeerides from glucose as well as hydrolysis of freshly synthesized glycerides, are act ivated to a marked degree. The net result of these two phenomena

is an actual elevation of the free fatty acid concentration above the control level. The observations on the alter- ations of adipose tissue metabolism after prolonged t reatment with 5-methylpyrazo]e-3-carboxylie acid are shown in a schematic and simplified way in Fig. 15. The acute effect of anti]ipolytic drugs consists in an inhibition of lipolysis and in an increased uptake of glucose and formation of glycerides. The newly

free fa#,,u a cida z. o /u er

14000

izooo i

ac/l'Vit# af 1oooo

blood g/ucose ,9ooo

6000

cpm/ml aerurd

blood #luceao rag~ 100 m /

.q,

incorporation ally. glucaso-8- /4Cin/~ rnelea/g/min, into

diaphragm- glgcogen a z

ad/'pose tisaue: f glgcer/'do- a I glycerole

fattb' acids a l

r~

treaCment: alburn/n "l- anti-insulin san'urn -- s 3-carb-oxglicacl'd -- insulin

+ +

+ - -

- - +

+ + - - +

Fig. 13. Comparison of the effects of 5-methylpyrazole-3- carboxylic acid with those of insulin on the metabolism of intravenously administered glucose-6-14C in normal

and acutely diabetic rats Insulin deficiency was induced by the intravenous injec- tion of 1 ml of anti-insulin serum diluted 1 : 2. The dose of insulin was 6 mU and that of 5-methylpyrazole-3- carboxylie acid 1 mg per rat. The means of the results obtained in 4 rats and the SEM are given. The specific activity of the blood glucose at the time of sacrifice was used to express the results of the incorporation in terms

of glueose-6-1~C per g per 30 rain

synthesized glycerides do not seem to mix readily with the main triglyceride depot. Somehow a lipase is induced, which splits the freshly synthesized glycerides as soon as the acute antilipolytic effect of the drug wears off. This general scheme also describes our observations on adipose tissue of fasted-refed rats. Maintenance of a high level of insulin over a prolonged period of t ime induces a rise of basal ]ipase activity, which is nonfunctional in the presence of insulin.

Vol. 3, No. 6, 1967 E.g . F~oEso~: The Physiology and Pharmacology of Adipose Tissue Lipolysis 485

Conclusions and outloo/c on the prospectives for the treat- ment of diabetes

Randle's statement, which I put at the beginning of this lecture, tha t increased release and oxidation of free fa t ty acids is not necessarily the result of a deficient uptake and metabolism of glucose [29], has been amply illustrated by experiments in living rats

plas~:oa fr~e lamb' ac/d..r /.~eq/ml plasma

~9 p<O01 Tp<O.O2E T 0.8 ~1 preireaOrnent w/In .~ o-------,~ sah~a / -k

a7 1 "- : E-methg~grazole- / 5-carboxLlhkac/d, ~,~

OG 1m$ b.Zd.

0.3

a2

I 1 1 1

100~ apm/ml plasma p> ~ 15 1 p < O . O O 2 ~

I ,

incorporation of glucose-6- IzO in cpm/g adipose ?i~su~ into

t p>aOg 3000 ~ i ~ . g l g c e r o l 2000 1 =: 1000 1

�9 I I I

~ooo t T~<oz ~. ~att~ acZd~

0 I0 60 t20 minutes attar fK i~]ectlon of l. 2G mg oP 5- methylpyrezole - 5- cerboxyh~ acid

Fig. 14. Effects of 5-methylpyrazole-S-carboxylic acid on glucose metabolism and lipolysis of adi- pose tissue after pretreatment with this Idrug for

5 days 20 rats were injected subcutaneously with 1.26 mg of 5-methylpyrazole-3-earboxylic acid b.i.d. during 6 days. A control group received 1 ml of sa, line b.i.d. 5 h after the last subcutaneous injection the rats were anesthetized and an intravenous dose of 1.26 mg of 5-meghylpyrazole-3- earboxylic acid was administered together with glucose- 6-z~C. 5 rats of each group were killed after I0, 60 and 120 min, respectively. The means of the results in 5 rats and the SEM are given. (From FI~OESO]~ et

el. [18])

[18]. Inhibition of free fa t ty acid release by 5-methyl- pyrazole-3-carboxylie acid blocks the hyperglycaemic response to acute insulin deficiency by keeping glucose oxidation by muscle normal. This concept may be broadened in such a way tha t increased glucose uptake by one tissue, i. e. adipose tissue, may eventually

become responsible for increased lipolysis, free fa t ty acid release and oxidation.

From the point of view of therapy for diabetics antilipolytic drugs do not appear to be very promising [26]. They not only inhibit lipolysis, but they also markedly stimulate glucose uptake of adipose tissue. Treatment with antiHpolytic drugs over prolonged periods of t ime would result in obesity, if adipose tissue did not develop mechanisms to get rid of the extra lipids synthesized from glucose. We have shown tha t an escape phenomenon occurs, and tha t chronic t rea tment with 5-methylpyrazole-3-earboxylic acid results in a rapid conversion of glucose to free fa t ty acids, and to an actual elevation of plasma free fa t ty acids above normal. We should expect tha t diabetics treated in this manner would become ~ and would tend ~o develop ketonuria.

Insulin also induces a lipase, but it suppresses its act ivi ty as long as it is present in blood. Thus, insulin may lead to an accumulation of fat as long as glucose is supplied and as long as the fi-eells agree to secrete insulin.

In many diabetics before and after instalment of insulin therapy, phases of hyperinsulinism and of hypoinsulinism alternate due to the untimely secretion of endogenous, or the untimely administration of exogenous insulin. I t m a y well be tha t lipolysis is act ivated by the above mentioned mechanism, resulting in difficulties in the stabilization of the metabolic situation. I t is still one of the main tasks for every physician to t ry to find the right moment in every diabetic for the administration of the right kind of insulin.

Since antilipolytic activity is always associated with glucose uptake-promoting activity into adipose

antillpolytic drags #lilPolgtic'hormones insulin I /3-recepl~

l l I ~ I glocogen glycerol I L I triglycenMe

$ 4 I" I ,epot ' '. glucose .... ~m, O-e-Pf='~ I ~ I

CA C i l v I

dual role e f insulin and i•h/b/tion of --~ L.. induch'an oP of antilipalgt/~ dFU2,~': nil'peso n (acute) Jipese" fchmonic)

Fig. 15. Alterations of adipose tissue metabolism during prolonged treatment with 5-methylpyrazole-3-carboxylic

acid and in the fasted-refed rats Triglyeeride synthesis from glucose is increased and their hydrolysis inhibited in the presence of antilipolytie sub- stances. As soon as their action wears off, hydrolysis of the newly synthesized *riglyeerides, which do not seem to mix readily with the depot triglyeerides, takes off at a rapid rate. Thus, glucose is rapidly converted to free fatty acids and a glucose to fatty acid shunt is established

in adipose tissue. (From FROESe~ et al. [18])

486 E.R. FROESCH: The Physiology and Pharmacology of Adipose Tissue Lipolysis Diabetologia

tissue, leading to increased lipolysis, we may have to go back to drugs with t ru ly insulin-l ike act ivi ty, i.e. to substances which s t imulate the glucose metabol ism of muscle as much or more so t han tha t of adipose tissue. Purified nonsuppressible ILA m a y well fulfill this requirement . I t is a peptide with a molecular weight of approximate ly 6000, which may be extracted from serum and which mimicks most of the effects of insul in [:[6, 4, 14]. As shown in Fig. 16, this substance is more

Effects of i.v. injected

,u o c Q . -

SE 8 _~

~ o_ 6

m

2 0

~176 2

-5

0

insuiin end nonsuppressibte ILA on incorporation of gtucose-6-14C into total [ipids of fat pads 10

/ r _ I [ I

3 6 10

glycogen of diaphragm

n~onsuppressib[~

///~/ t i n s u l i n

J

I I I 3 6 10 log scare

mU insutin or nonsuppressibte ILA inj. i.~/100g rat

Fig. 16. Effects of intravenously administered insulin and purified nonsuppressible ILA on the incorporation of glucose-6-1aC into the total lipids of the fat pad and the

glycogen of the diaphragm In these experiments the specific activity of the serum glu- cose at the time of sacrifice was used for calculation of the ~moles of glucose-6-~4C which were incorporated into the glycogen and total lipids. The number of rats was 20 in all bu t those groups of rats injected with 3 and 6 mU nonsuppressible ILA in which it was 4. The bars indicate the S.E. The effect of 6 mU nonsuppressible ILA on the incorporation of glucose-6-~C into glycogen was signi- ficantly greater than that of 10 mU crystalline insulin

(P < 0.05) O -- O, crystalline insulin; � 9 �9 nonsuppressible ILA.

(From FRo~seH et al. [16])

active on the diaphragm t h a n on adipose tissue compared with insulin. We are now fur ther purifying this molecule and hope to get enough mater ia l to test i t in m a n for favonrable ant i -diabet ic properties.

Acknowledgements. I should like to express my deeply felt gratitude to Professors A.E. 1%ENOLD and A. LA]3- HA~T, who have introduced me in their comprehensive, progressive and seducing way into experimental medi- eine, endocrinology, and into the art of teamwork, and for their continued interest and encouragement of our efforts in the laboratory. I have always enjoyed the some- times rather harsh but extremely helpful criticism of Dr. g . E. HUMBEL and Dr. J. Mi~LLER. The work presented today is the result of a collaborative effort of many friends who gracefully bore the hardship of all the nega- rive personal characteristics that make oneself elligible for a prize. My particular thanks go to Drs. H. Bi~RGI, A. JA~:OR, U.A. MEYEr, W.A. Mi~LT,E~, J.L. GINSBE~G and

E. RAMSEIER and to Misses M. WALDVOGEL, V. STURZEN- ECGER, S. DIEM and Ch. ~ARK. They all have kept the banner of our metabolic unit high in good and bad times. I deplore my wife's and my children's bad luck of having to bear a stubborn scientist as a husband and father. However, I am quite sure that my wife is just as happy as I am that the Minkowski prize will keep us from starving for another year. I wish to thank the European Associa- tion for the Study of Diabetes and Hoechst AG for having chosen me as this year's recipient. Finally, I should like to acknowledge the financial support of the Schweizeri- sche Nationalfonds and of the US Public Health Service, which made the realization of these studies possible.

J~eference8

[1] BALLY, P.R., H. KAPPELER, E.R. FROESCH and A. LAB~ART: Effects of glucose on spontaneous limita- tion of lipolysis in isolated adipose tissue: a potential regulatory mechanism. Ann. N.Y. Acad. Sci. 131, 143--156 (1965).

[2] BLEOttER, M.: Effects of insulin and phospholipase on glucose transportation across the plasma mere- brane of free adipose cells. Biochim. biophys. Acta 1 3 7 , 557--571 (1967).

[3] B6HLE, E., E. D6BE~T, J. AM~A~N and H. DIT- SC~UNEIT: Ober Stoffwechselwirkungen yon Pro- staglandinen. I. Der Einflul~ yon Prostaglandin E auf den Glucose- und Fettstoffwechsel des epididy- malen Fettgewebes der 1%atte. Diabetologia 2, 162-- i68 (i966).

[4] Bf)RGI, H., W.A. Mi~LLER, I~.E. HVMBEL, A. LAB- HA~aT and E.1%. FROESCH: Nonsuppressible insulin- like activity of human serum. I. Physicochemical properties, extraction and partial purification. Bio- chim. biophys. Acta 121, 349--359 (1966).

[5] BUTCHER, R.W., 1%.J. Ho, H.C. MENG and E.W. SUTHERLAND : Adenosine 3',5'-monophosphate in biological material. II . The measurement of adeno- sine 3', 5-monophosphate in tissues and the role of the cyclic nueleotide in the lipolytic response of fat to epinephrine. J. biol. Chem. 240, 4515--4523 (1965).

[6] --, and E.W. SUTHE~LAND : The effects of the cate- cholamines, adrenergic blocking agents, prostaglan- din E and insulin on cyclic 3',5'-AMP levels in the rat epididymal fat pad in vitro. Ann. IXT. Y. Acad. Sci. 139, 849--859 (1967).

[7] CARLSOX, L.A., and P.R. BALLu Inhibition of lipid mobilization. In: A.E. RENOT,D and G.F. CAHILL (Eds.), Handbook of Physiology, Section 5, Adipose tissue, p. 557--574. Washington, D.C.: American Physiological Society 1965.

[8] --, and J. OEST~AN: Inhibition of the mobilization of free fat ty acids from adipose tissue in diabetes. II . Effect of nicotinic acid and acetylsalicylate on blood glucose in human diabetics. Acta med. scand. 178, 71--79 (1965).

[9] DOLE, V.P. : Effect of nucleic acid metabolites on lipolysis in adipose tissue. J. biol. Chem. 236, 3125-- 3130 (1961).

[10] FAIN, J .W., V.P. KOVACEV and 1%.O. Scow: Effects of growth hormone and dexamethasone on lipolysis and metabolism in isolated fat cells of the rat. J. biol. Chem. 240, 3522--3529 (1965).

[ 11] FRzTz, I .B. : Effects of insulin on glucose and palmi- tare metabolism by resting and stimulated rat dia- phragm. Amer. J. Physiol. 198, 807--810 (1960).

[12] F~OE~SC~, E.t~.: Essential Fructosuria and Here- ditary Fructose Intolerance. In : STANBU~Y, FRED- RIe~:soN and WYNGAAI~DEN (Eds.), The Metabolic Basis of Inherited Disease, p. 124--140. New York: McGraw Hill Inc. 1965.

VoL 3, No. 6, 1967 E.t~. FgoEscK: The Physiology and Pharmacology of Adipose Tissue Lipolysis 487

[13] -- , H. BfTRGI, P. BALLY and A. LAB~ART: Insul in inhibition of spontaneous adipose tissue lipolysis and effects upon fructose and glucose metabolism. Mot. Pharmacol. 1, 280--296 (1965).

[14] -- -- W.A. MtTLLER, l i .E . HUlVIBEL, A. JAKOB and i . LABKA~T: Nonsuppressible insulin-like activity of human serum: Purification, physicochemical and biological properties and its relation to total serum ILA. l iecent Progr. Hormone lies. 23, 565-605 (1967).

[15] - - , and J .L. GINSBE~G: Fructose Metabolism of Adipose Tissue. I. Comparison of fructose and glu- cose metabolism in epididymal adipose tissue of normal rats. J. biol. Chem. 237, 3317--3324 (1962).

[16] -- W.A. MtJ'LLER, H. BURGI, M. WALDVOGEL and A. L~HAl~r: l~onsuppressible insulin-like activity of human serum. II. Biological properties of plasma extracts with nonsuppressible insulinlike activity. Biochim. hiophys. Acta 121, 360--374 (1966).

[17] -- M. WALDVOGEL, U.A. MEYER, A. JAKOB and A. LABHART: Effects of 5-methylpyrazole-3-carboxylic acid on adipose tissue. I. Inhibition of lipolysis, effects on glucose-, fructose- and glycogen-meta- bolism in vitro and comparison with insulin. Mol. Pharmacol. 3, 429--441 (1967).

[18] Effects of 5-methylpyrazole-3-car- boxylic acid on adipose tissue. II. Antilipolytic and hypoglycemic effects in vivo. Mol. Pharmacol. 3, 442--452 (1967).

[19] GE~RITSEN, G.C., and W.E. DULIN: The effect of 5-methylpyrazole-3-carboxylic acid on carbohydrate and free fat ty acid metabolism. J. Pharmacol exp. Ther. 150, 491--498 (1965).

[20] H~mES, C.N., and P.J . !~ANDLE: Effects of low carbohydrate diet and diabetes mellitus on plasma concentrations of glucose, non-esterified fat ty acids and insulin during oral glucose tolerance tests. Lancet 1963 I, 790--793.

[21] HEPP, D., PE. L. POFFENBERGER, J .W. ENSINCK and R.H. WIr,r.IA~S: Effects of nonsuppressible insulin- like activity and insulin on glucose oxidation and lipolysis in the isolated adipose tissue cell. Meta- bolism 16, 393--401 (1967).

[22] JVNGAS, l i .L . : liole of cyclic 3~,5'-AMP in the response of adipose tissue ~o insulin. Proe. nat. Acad. Sci. 56, 757--763 (1966).

[23] -- , and E.G. B)~LL: Studies on the metabolism of adipose tissue. XII . The effects of insulin and epinephrine on free fat ty acid and glycerol produc- tion in the presence and absence of glucose. Bioche- mistry 2, 383--388 (1963).

[24] -- -- Studies on the metabolism of adipose tissue. XVII . In vitro effects of insulin upon the metabolism of the carbohydrate and triglyceride stores of adi- pose tissue from fasted-refed rats. Biochemistry 3, 1696-- 1702 (1964).

[25] KAPPELEE, H. : Zur Pharmakologie der Lipolysehem- mung. I. Wirkungsweise adenosinhaltiger Nucleoside und Nucleotide auf die Lipolyse des Fettgewebes in vitro. Diabetologia 2, 52-- 61 (1966).

[26] MOL~AR, G.D., K.G. BeiGE, J .W. /IIOSEVAE~, W.F. MCGUOKIN and l i .W.P . ACEOR: The effect of nice-

tinic acid in diabetes mellitus. METABOLISl~I 13, 181--190 (1964).

[27] er iE, L.H., J. l i . EvAI~S and J.C. SmPr: Effect of fasting on glucose and palmitate metabolism of perfused rat heart. Amer. J. Physiol. 205, 1203-- 1208 (1963).

[28] PINTER, E.J. , and C.J. PA~EE; Biphasic nature of blood glucose and free fat ty acid changes following intravenous nicotinic acid in man. J. clan. Endocr. 27, 440--443 (1967).

[29] IIANDLE, P.J.: Carbohydrate metabolism and lipid storage and breakdown in diabetes. Diabeto]ogia 2, 237--247 (1966).

[30] -- E.A. NEWSHOL~E and P.B. GARLAND: Regula- tion of glucose uptake by muscle. 8. Effects of fatty acids, ketone bodies and pyruvate, and diabetes and starvation on uptake and metabolic fate of glucose in rat heart and diaphragm muscle. Biochem. J. 93, 652--664 (1964).

[31] IIOBINSON, D. S. : The clearing factor lipase activity of adipose tissue. In: A.E. IIENOLD and G.F. CA- HILL, Jr., (Eds.), Handbook of Physiology, Section 5, Adipose tissue, p. 295-- 299. Washington, D.C. : American Physiological Society 1965.

[32] RODBELL, M., and A.B. JONES: Metabolism of iso- lated fat cells. III. The similar inhibitory action of phospholipase C (clostridium perfringens a toxin) and of insulin on lipolysis stimulated by lipolytic hormones and theophilline. J. biol. Chem. 241, 140-- 142 (1965).

[33] SI~IT~, D.L., A.A. FORIST and W.E. DULIN: 5- methylpyrazole-3-carboxylic acid. The potent hypo- glycemie metabolite of 3,5-dimethylpyrazole in the rat. J. reed. Chem. 8, 350--353 (1965).

[34] -- J.G. WAGNER and G.C. GE~RI~SEN: Absorption, metabolism and excretion of 5-methylpyrazole-3-car- boxylic acid in the rat, dog and human. J. Phar- mocol. Sci., in press.

[35] STEINBERG, D.M., M. VAUG~A~, P .J . NESTEL, 0. STI~AUD and S. BERGS~6M: Effects of the prosta- glandins on hormone induced mobilization of free fatty acids. J. clan. Invest. 43, 1533--1540 (1964).

[36] TA~RAN% M.E., 1%. MA~LER and J. ASHMOI~E: Studies in experimental diabetes. IV. Free fat ty acid mobilization. J. biol. Chem. 239, 1714--1719 (1964).

[37] VAUGHAN, M., and D. STEINBERG: Glyceride bio- synthesis, glyceride breakdown and glycogen break- down in adipose tissue: Mechanism and regulation. In : "Handbook of Physiology" (A.E. IIENOLD and G.F. CAHILL, Jr. eds.), Section 5, Adipose tissue, p. 239--251. Washington, D.C.: American Physiolo- gical Society 1965.

[38] WILLIAMSON, J .R . : Glycolytie control mechanisms. I. Inhibi t ion of glycolysis by acetate and pyruvate in the isolated, perfused rat heart. J. biol. Chem. 240, 2308--2321 (1965).

Dr. E . l i . F~OESCK Metabolic Uni t Department of Medicine University of Ziirich CH-8006 Ziirich, Switzerland