Non-hormonal factors in the control of gluconeogenesis

NON-HORMONAL OF

FACTORS

IN THE

CONTROL

GLUCONEOGENESIS*

M. G. H ~ E R A , D. KAMM, N. RUDERMANand G. F. CAHILL,JR.t Elliott P. Joslin Research Laboratory, Department of Medicine, Harvard University Medical School, the Peter Bent Brigham Hospital and the Diabetes Foundation, Inc., Boston, Massachusetts A. T H E R O L E O F S U B S T R A T E IN T H E C O N T R O L OF HEPATIC GLUCONEOGENESIS

GLUCONEOGENESISin the total animal is a complex physiological process which entails the orderly release of glucose precursors from storage depots, their transport to the liver and kidneys, and finally, their elaboration into glucose molecules by the enzymatic machinery of these organs. It follows that the step limiting the rate of the over-all process may reside either at the site of substrate mobilization (full hepatic gluconeogenic capacity yet untapped) or at the liver itself (hepatic gluconeogenic capacity rate-limiting). The exogenous provision of protein and lactate to intact animals has been shown to do more than simply saturate the gluconeogenic pathway. These glucogenic precursors administered over a period of time also increase the activity of rate-limiting enzymes. The simplest hypothesis to explain this phenomenon would be substrate-induced, sequential expansion of the glucose forming enzymatic pathways. The effect of certain hormones on gluconeogenesis may also be related to their ability to provide the liver with substrate. The stimulatory effects of in vivo cortisol administration on hepatic purine and RNA biosynthesis were reproduced by Feigelson and Feigelson tl) by injecting exogenous amino acids into adrenalectomized animals. Weber et al. t2) found that the first detectable effect of acute triamcinolone administration to intact rats was an increase of hepatic free amino acid nitrogen which concurred with an increase in total hepatic nitrogen. This effect was evident within 2 hr, while increases in RNA synthesis and gluconeogenic enzyme activities followed later. In addition, Wells and Kendall °) have demonstrated that the provision of a casein diet to a phlorizinized-adrenalectomized rat completely restored its glycosuria. These findings suggest that the primary effect of the hormone may be to mobilize amino acids from the periphery. * Supported in part by U.S.P.H.S. Grants T1 AM 5077-09 and AM 9584-01, and by the Adler Foundation, Inc., Rye, New York. t Investigator, Howard Hughes Medical Institute. 225

226

M . G . HERRERA, D. KAMM, N. RUDERMAN AND G. F. CAHILL, JR.

Nutritional and hormonal determinants of endogenous amino acid mobilization may therefore conceivably regulate hepatic gluconeogenesis in two ways: (a) by determining the amount of substrate immediately available and (b) by increasing the activity of rate-limiting gluconeogenic enzymes by protracted loading with substrate, or diminishing their activity by a prolonged decrease in substrate provision. The experiments reported here represent a preliminary attempt to explore the effects of substrate provision and endproduct concentration on glucose production by the isolated rat liver. MATERIALS AND METHODS Male Sprague Dawley rats were fed Purina laboratory chow and water ad libitum unless otherwise specified. The animals were anesthetized with nembutal intraperitoneally (30 rng/kg). The bile duct and portal vein were cannulated and a preperfusion with oxygenated Krebs-Ringer-bicarbonate (KRB) buffer was immediately started prior to removal of the liver from the animal. The perfusion apparatus was a modification of Miller's design(4) and was equipped with a CO2 trap. The perfusion medium utilized was a KRB buffer containing 4g/100 ml albumin, 40 rag/100 ml heparin, 5 rng/100 ml penicillin, and 5 rag/100 ml streptomycin. Stable and radioactive substrates were added as noted under individual experiments. During the course of the perfusion, samples of the perfusate were withdrawn for analysis every 15 or 30 rain. Glucose was determined by the method of Somogyi and Nelson(5) or by the ferrycyanide method in the Technicon Autoanalyzer (Chauncey, New York). Glucosazones were prepared and plated on steel planchettes. Their radioactivity was measured in a gas flow counter, and the appropriate correction for self absorption was applied. Total lipids were isolated from a weighed portion of liver at the end of the perfusions by the method of Folch et al. (6) A portion of these lipids was dissolved in toluene, POP, PPOP and counted in a Nuclear-Chicago liquid scintillation counter. Another portion of the lipid extract was saponified, the fatty acids extracted with petroleum ether and their radioactivity determined in the same fashion. Glyceride glycerol was oxidized and the alpha carbons isolated as the dimedone and counted in toluene. (~) CO2 was collected in a NaOH trap, from which an aliquot was counted directly in dioxane-Cabosil. In some experiments a biopsy was obtained prior to cannulating the portal vein and again at the end of the perfusion. RESULTS AND DISCUSSION

Effect of Varying the Concentration of Perfusate Alanine on Gluconeogenesis Both isolated perfused livers and liver slices produce increased quantities of glucose when provided with increasing concentrations of glucogenic pre-

NON-HORMONALFACTORS IN THE CONTROL OF GLUCONEOGENESIS 227 cursors. Tables 1 and 2 summarize the results of 3 experiments in which fasted rat livers were perfused with 3 different L-alanine concentrations, using randomly-labeled L-alanine 14C. Net glucose production was measured by the increment in perfusate concentration; alanine incorporation into TABLE 1. Net Hepatic Glucose Production (NI-IGP)During Perfusion of Fasted Rat Livers. Effect of Varying Initial Alanine Concentrations Perfusion time in min 30 60 90 120

NHGP/oa/g dry wt 0.5 mM alanine 5.0 rnM alanine 22 30 34 35

45 68 86 88

10 rnM alanine 57 108 126 128

Results are means of 3 experiments. 100 ~ / h r linolei¢ acid infused in all experiments. TABLE2. Incorporation of L-Alanine 14C into Medium Glucose by Fasted Rat Livers. Effect of Varying Initial Alanine Concentrations Perfusion time in rain 30 60 90 120

Alanine incor ~oration into glucose/~M/gdry wt 0.5 mM alanine 5.0 mM alanine 2.7 4.0 4.2 3.9

32 55 56 62

10 mM alanine 31 80 92 87

Restflts are means of 3 experiments. 100/zM/hrlinoleic acid infused in all experiments. glucose was quantified by the appearance of 14C in perfusate glucose. Both parameters increased as perfusate alanine concentration was increased. A change in liver glycogen content accounted for less than 5 per cent of glucose production.

Effect of Perfusate Glucose Concentrations on Net Hepatic Glucose Production It is well known that high glucose concentrations in the incubation medium result in net glucose uptake by liver slices and promote glycogen deposition. Conversely, low glucose concentrations favor glycogenolysis and net glucose production. We have confirmed these findings employing the perfused rat liver. The results are summarized in Table 3. Livers of fed rats were perfused

228

M . G . HERRERA, D. KAMM, N. RUDERMAN AND G. F. CAHILL, JR. TABLE 3. Net Hepatic Glucose Production (NHGP) During Perfusion of Fed Rat Livers. Effect of Sequential Changes of Initial Glucose Concentrations Period I

II III IV

Time min 0-45 45-90 90-135 135-180

Initial perfusate NHGP/zg/min [Glucose] mg ~o per g dry wt4,S.E. 400

N

528 4- 163 723 + 20 20 + 141 6004-74

0

40O 0

No substrate other than glucose present. Difference I I - I 195 + 154 P > .2 Difference II-III 703 + 142 P < .001 Difference IV-III 580 4- 158.5 P < .01 N = number of experiments.

sequentially with a m e d i u m c o n t a i n i n g 400 mg/lO0 ml glucose, then with a m e d i u m d e v o i d o f glucose. T h e livers were then re-perfused at the high glucose c o n c e n t r a t i o n a n d t h e n once m o r e e x p o s e d to the " h y p o g l y c e m i c " m e d i u m . Very significant differences in net h e p a t i c glucose p r o d u c t i o n were observed. I n a n o t h e r series o f e x p e r i m e n t s fed r a t livers were perfused for 45 rain with each o f the following perfusate glucose c o n c e n t r a t i o n s : 400 rag/100 ml, 0 rag/100 ml, 150 mg/100 ml a n d 400 rag/100 ml. T h e results, as s u m m a r i z e d in T a b l e 4, d e a r l y show t h a t net h e p a t i c glucose p r o d u c t i o n was m o d u l a t e d b y perfusate glucose concentration.

TABLE 4. Net Hepatic Glucose Production (NHGP) During Perfusion of Fed Rat Livers. Effect of Sequential Changes of Initial Glucose Concentrations Period I

II III IV

Time min

Initial perfusate [Glucose]. mg ~o

NHGP/~g/min per g dry wt4,S.E.

0-45 45-90 90-135 135-180

40O 0 150

51.7 4- 6.52 565 4- 56.0 299 4- 55.8 567 + 64.7

0

I

Glucose was the only substrate added. Difference4.S.E. II-I 513 4- 56.4 P Difference4.S.E. II-III 266 4- 79.2 P Difference4.S.E. IV-III 268 4- 85.4 P DifferencebS.E. III-I 247 4- 56.0 P N ----number of experiments.

< < < <

.001 .01 .01 .0

N

NON-HORMONAL FACTORS IN THE CONTROL OF GLUCONEOGENESIS 229

Effect of Perfusate Glucose Concentration on NHGP from Alanine Livers o f 24-hr fasted rats were used in these experiments. Initial and final glycogen content was determined and net hepatic glucose production quantified by following the changes in perfusate glucose concentration. The organs were again sequentially perfused for 45-min periods with medium containing no glucose, 150 mg/100 ml glucose, and once again in the absence of glucose. The results are summarized in Table 5. Since glycogenolysis (as measured and calculated) could not account for the glucose appearing in TABLE5. Net Hepatic Glucose Production (NHGP) During Perfusion of Fasted Rat Livers with 20 mM Alanine. Effect of Sequential Changes of Initial Glucose Concentrations Period

Time rain

Initial perfusate [Glucose]mg %

NHGP/~g/min per g dry wt4-S.E.

N

I II III

0-45 45-90 9-135

0 150 0

319 4- 43.6 65.9 4- 42.3 240 4-22.1

9 9 9

Difference I-II 253 + 61 P < .001 Difference III-II 174 + 48 P < .01 N = number of experiments. the perfusate, the latter was in all likelihood the product o f alanine incorporation into glucose. It is there reasonable to suspect that gluconeogenesis in this system was influenced by the perfusate glucose concentration.

Effect of Provision of Fatty Acid on Hepatic Gluconeogenesisfrom Alanine Earlier investigators working with isolated perfused livers reported an enhancement of gluconeogenesis upon addition of fatty acids and phospholipids to the perfusate. More recently, Krebs and his colleagues, c8) working with kidney slices, demonstrated that the in vitro addition o f short chain fatty acids or acetoacetate to the incubation medium increased gluconeogenesis from lactate. With this in mind, the effect of a free fatty acid infusion on the conversion o f alanine to glucose by the perfused liver o f the fasted rat was studied. The initial alanine concentration in the perfusate was 20 mM. Alanine, randomly labeled with 1"C, was added to yield a specific activity o f 6.25/m/raM. Sodium linoleate, when added, was infused at the rate o f 300 #mole]hr. Net hepatic glucose production as reflected b y perfusate glucose concentrations is represented in Fig. 1. The conversion o f alanine-t*C to glucose in the presence and absence of fatty acid is compared in Table 6.

230

M . G . HERRERA, D. KAMM, N. RUDERMAN AND G. F. CAHILL, JR. SUBSTRATE L-ALANINE

20mM

14C

12¢

8C pIrRFUSAT£ 6LUCOSE CONCENTRATION 6¢

/ 2O

I

*

,e~

I

30

60

I

90

I

I

120

150

I

180

PERFUStO. Tree ~..~SUTSS FIG. 1.

(-I-S.E.) achieved in the medium by peffused livers of fasted rats.

Glucose concentration

TABLE 6. Incorporation of L-Alanine 14C into Medium Glucose by Fasted Rat Livers Perfused with 20 m~ Alanine. Effect of Linoleic Acid Infusion* Perfusion time in min

Alanine incorporated into glucose pM/g dry wt ± S.E. With FA

N

Without FA

Difference + S.E.

P

34±7.62 92-4-21.6 98±33.3 113±34.6

< .001 < .001 <.01 <.01

.

30 60 90 120

63 ,1.6.43 169,1,15.7 253 ,1,20.4 316±26.1

16 16 16 16

29,1-4.1 77-4-14.8 155,1,26.4 203±22.7

14 14

* Infusion rate 300 pM/hr. N = Number of experiments. Very significant e n h a n c e m e n t o f b o t h net glucose p r o d u c t i o n a n d a l a n i n e c a r b o n i n c o r p o r a t i o n into glucose is a p p a r e n t in the livers infused with fatty acid. A s s h o w n in Fig. 2 a n d T a b l e 7, fatty acid infusion d i m i n i s h e d the i n c o r p o r a t i o n o f alanine c a r b o n into CO2, while a greater a m o u n t o f alanine was i n c o r p o r a t e d into t o t a l lipids. T h e e n h a n c e m e n t o f t o t a l lipid specific

NON-HORMONAL

F A C T O R S IN T H E C O N T R O L O F G L U C O N E O G E N E S I $

231

£ffeet of L/no/eic Acid Inf¢#ion [ 3 0 0 y M / h r . ) : [ ] WITH ["]WITSOUT

300

to©,I ALANINI= pM per. gin. dry liver in ='~u.(2 S.E)

tO0

100

p<.OI

p • .OZ

.4L.4NINE TO SLUGOSE

. 4 L I N I N Lr

ro co~

FIG. 2. Incorporation of L-alanine into medium glucose, and CO2, by fasted rat livers perfused with 20 n ~ alanine. activity was due entirely to an increase o f radioactivity in the glycerol moiety; in fact, significantly less alanine was incorporated into the fatty acid chain. TABLE 7. Specific Activity of Total Lipids, Glyccride-glyccrol and Giyccride Fatty Acids in Fasted Rat Livers Peffused with 20 mM L-Alanine 14C. Effect of Linoleic Acid Infusion 300/~M/hr

DPM/mg total lipid =[=S.E. DPM/mg glyceride-glycerol =t=S.E. DPM/mg glyceride FA

Without FA

With FA

Diff. ~S.E.

P

306:E55

1.560-4-110 (n = 12) 1432-4-187 (n = 6) 15.8=1=5.9 (n = 4)

1.254-4-123

< .001

1274+190

<.001

38.2-4-13

<.05

( n = ]l)

158+26 (n = 5) 544-12 (n = 5)

n = number of experiments. It is not likely that the stimulatory effect o f fatty acid provision on glucose p r o d u c t i o n is due to simple sparing o f the alanine, for it occurred even at the very high a m i n o acid concentrations. It therefore appears reasonable to postulate that the fatty acid or one o f its metabolic products has influenced one or m o r e o f the gluconeogenic steps. Several studies seem to bear this statement out. Wivland et al. c9) have shown that fatty acyl-CoA blocks citrate synthetase and therefore diminishes the overall oxidation o f 2 C units

232

M . G . HERRERA, D. KAMM, N. RUDERMAN AND G. F. CAHILL, JR.

by the Krebs cycle. This plus an inhibitory effect of acetyl CoA on pyruvate decarboxylation¢1°) at least in part may explain the diminished oxidation of alanine to CO2 in our studies. In addition to this effect, fatty acid by providing an increased quantity of acetyl-CoA, may increase the activity of pymvate carboxylase, as first demonstrated by Utter and Keech.¢1:) Finally, as Wieland and his co-workers have hypothesized,¢~2) free fatty acid oxidation may enhance hepatic gluconeogenesis by simply increasing the concentrations of reduced pyridine nucleotides in the liver. Our data do not permit any conclusions as to the mechanism by which fatty acid stimulates glucose formation. However, they do suggest that nutritional and hormonal factors may affect hepatic metabolism through an action on adipose tissue as well as muscle. The above in no way detracts from the fact that hormones may have a direct hepatic effect on glucose production. In vitro glucocorticoids,~13.14) catecholamines~15) and glucagon,~15.~6) have all been shown to stimulate gluconeogenesis; however, supraphysiologic concentrations were used in many of these studies. More work is necessary to elucidate the relative importance of the peripheral and hepatic effects of these hormones in the whole animal. B.

RENAL

GLUCONEOGENESIS

It has been known for several decades that the kidneys synthesize glucose. ¢17) As described by Krebs (1s,19) the marked capacity of renal cortical slices to make glucose from amino acids is enhanced by starvation and depressed by carbohydrate feeding. Since ketoacidosis occurs in the fasting state, it seemed of interest to evaluate the effects of [H + ] and other physiological variables on renal gluconeogenesis. The following experiments were performed in our laboratory by Goodman and Fuisz. ¢2°) Adrenalectomized rats were tube fed acid (NH4CL) or alkali (NaHCO3) loads for 2 days following which cortical slices were prepared and incubated in vitro in a standard Krebs-Ringer bicarbonate buffer at pH 7.4. Glucose production from glutamine, glutamate, ~-ketoglutarate or oxaloacetate was found to be markedly increased in slices from acid-loaded animals, whereas depressed gluconeogenesis was present in slices from alkali-loaded animals. Another series of experiments was then performed in which slices from normal fed animals were utilized and the pH of the media altered by changing bicarbonate concentration. As illustrated in Fig. 3 gluconeogenesis from glutamine, glutamate, u-ketoglutarate, and oxaloacetate was depressed in slices incubated at pH 7.7 and increased when incubated at pH 7.1. To exclude the possibility that their findings were the result of increased nonionic diffusion of substrate at lower pH, slices were incubated in the same three media without substrate. As demonstrated in Fig. 4 similar results

NON-HORMONAL FACTORS IN THE CONTROL OF GLUCONEOGENESIS

24C 22C

P
~

ACID

20C

GLUCOSE

233

LINE

18C

PRODUCTION

16(

by

t4G

KIDNEY CORTEX

12c

/ m ~ l / g . D r y W t / 2 h r s toc 8O

( * I S.D.)

6o 40 20 GLUTAMINE IOmM

GLUTAMATE IOmM

KET ~ ' I ~ IOmM

OX&LAGETATE IOmM

l~o. 3. Effectof in vitro changesin pH on renal glucon~genesis. were obtained. Thus, there appears to be an intrinsic system operating in renal cortex in which increases or decreases in [H + ] are accompanied by corresponding changes in gluconeogenesis. These changes occur in vivo when animals are chronically loaded with acid or alkali, and also occur acutely following in vitro pH changes. 18 16

pc .02

14 Z

[]

ACID

B

NOm~ALKALINE

NO SUBSTRATE

Pro. 4.

Renal gluconeogenesiswithout added substrate.

Recently, Goldstein (2t) has shown that the concentration of glutamate in rat kidney rises during metabolic alkalosis and falls during acidosis in association with increased NH4 + excretion. In addition, glutamate is known to be a non-competitive inhibitor of the glutaminase I reaction with the K x of 2 × 10-3 M approaching the concentration of glutamate normally found in

234

M.G. HERRERA,D. KAMM, N. RUDERMANAND G. F. CAHILL, JR.

lddncy cortex (8 × 10 -3 M/kg fresh kidney). Glutamate therefore could be controlling renal NH 3 production by limiting the deamidation of glutamine by glutaminase I. These observations, coupled with our studies, suggest that [H +] by accelerating one or more steps in gluconeogenesis may decrease glutamate concentration and thereby increase NH 3 production. In order to characterize this physiologic process further, rats were made potassium deficient, since this procedure is associated with increased NH3 production. This process greatly enhanced gluconeogenesis from glutamine glutamate and r,-ketoglutarate, again suggesting an association between NH 3 production and glucose formation. Our recent studies have been directed towards determining the specific rate-limiting step at which [H +] affects gluconeogenesis. Preliminary work has shown that substrates below phosphoenolpyruvate arc converted more rapidly to glucose during acidosis whereas those entering the gluconeogenetic sequence above this point are less affccted. In studies designed to determine the relative influence of pH, pCO2 and HCO3, we have found gluconeogenesis to be most markedly affected by pH. ~22) However, at any given [H+], when pCO2 and [HCO3] were proportionately varied, glucose formation was higher at the lower [HCO3] and pCO2. It is well known that both hepatic and renal gluconeogenesis increase during starvation and diabetes. When diabetic or starved rats are fed alkali for 2 days, we find renal gluconeogenesis returned to normal levels. Hepatic glucose formation, on the other hand, remained elevated despite alkali loading. Thus, while renal gluconeogenesis is markedly influenced by [H +], hepatic glucose production appears to proceed independently of this physiologic variable.

SUMMARY Increasing the concentration of alanine in the medium in a perfused rat liver preparation increases net hepatic glucose production. A high perfusateglucose concentration diminishes while a low perfusate-glucose concentration enhances hepatic glucose release into the medium. This phenomenon is observed with both fed and fasted livers, suggesting a modulation of glycogenolysis and/or gluconeogenesis by the level of medium glucose. Provision of fatty acids enhances conversion of alanine to glucose and net hepatic glucose production while concurrently reducing alanine oxidation and its incorporation into fatty acids. On the other hand, gluconeogenesis in kidney appears to be also related to both acute and chronic changes in pH and acidbase balance.

NON-HORMONAL FACTORS IN THE CONTROL OF GLUCONEOGENESIS 235 REFERENCES 1. M. F~O~SON and P. F~GEtSO~, Metabolic effects of glucocorticoids as related to enzyme induction, Advances in Enzyme Re£ulation 3, 11-24 (1965). 2. G. WV.nER,R. L. SINOHALand S. K. Sn_rV~TAVA,Action of glucocorticoid as inducer and insulin as suppressor of biosynthesis of hepatic gluconeogenic enzymes, Advances in Enzyme Regulation 3, 43-75 (1965). 3. B . B . W~LLS and E. C. K ~ , ~ . L , The influence of the adrenal cortex in phlorizin diabetes, Proc. Staff Meet. Mayo Clinic 15, 565-573 (1940). 4. M. GRiN and L. L. M ~ , Protein catabolism and protein synthesis in perfused livers of normal and alloxan diabetic rats, J. Biol. Chem. 235, 3202-3208 (1960). 5. N. NEtsON, A photometric adaptation of the Somogyi method for the determination of glucose, J. Biol. Chem. 153, 375-380 (1944). 6. J. FOLCH,M. LEESand G. H. SLOA],n~STANLEY,A simple method for the isolation and purification of total lipids from animal tissues, J. BioL Chem. 226, 492-509 (1957). 7. R . E . R~vv.s, The estimation of primary carbinol groups in carbohydrates, J. Am. Chem. Soc. 63, 1476-1477 (1941). 8. H.A. K~es, R. N. S P ~ l ~ and R. HEMS, Acceleration of renal gluconeogenesis by ketone bodies and fatty acids, Biochem. J. 94, 712-720 (1965). 9. O. WIV.t~ND and L. W~ss, Inhibition of citrate synthase by palmitylcoenzyme A, Biochem. Biophys. Res. Commun. 13, 26-31 (1963). 10. P.B. GARLANDand P. ~ u ~ . Personal Communication. 11. M. F. UTr]~ and D. B. K~c~, Pyruvate carboxylase, J. BioL Chem. 238, 2603-2608 (1963). 12. O. Wn~LA~ et al. Diabetes. In press. 13. R.C. HAY~resJr., Studies of an in vitro effect of gluco¢orticoids or gluconeogenesis, Endocrinology 71, 399--406 (1962). 14. T. Azu~A and A. B. EmENS're~,Effect of adrenal steroids on carbohydrate metabolism in various species, Endocrinology 75, 521-526 (1964). 15. J.H. EXTONand C. R. P~J¢. Personal communication. 16. A. G A R ~ , J. R. WXI~JT~SONand G. F. CAmtJ~, Studies on the perfused rat liver, II. Effect of glacagon on gluconeogenesis, Diabetes. 15, 188-193 (1966). 17. R . M . ~ c ~ , The kidney as a source of glucose in the eviscerated rat, Am. J. Physiol. 140, 276-285 (1943). 18. H. A. Ks~Bs, Renal gluconeogenesis, Advances in Enzyme Regulation 1, 385--400 (1963). 19. H . A . K~Bs, D. A. H. BENNETT,P. De G~QU~T, T. G A s ¢ o ~ and T. Yostm)A, The effect of diet on the gluconeogenic capacity of rat-kidney-cortex slices, Biochem. J. 86, 22-27 (1963). 20. A.D. GOODMAN,R. E. FUISZandG. F. C~n.L, JR., Renal gluconeogenesis in acidosis, alkalosis and potassium deficiency: Its possible role in regulation of renal ammonia production, J. Clin. Invest. 45, 612-619 (1966). 21. L. GOLDSTErN, Relation of glutamate to ammonia production in the rat kidney, Am. J. Physiol. 210, 661-666 (1966). 22. D.E. KJdv~ and G. F. CAmLL, JR., Effect of changes in H + and HCO3- on renal cortical gluconeogenesis, Clin. Res. 14, 350 (1966).