Acid Tissue 4
The effect of nicotinic acid on the fatty acid metabolism of epididymal adipose tissue from alloxan diabetic rats was studied in vitro. Nicotinic acid caused an inhibition of the FFA and glycerol release from adipose tissue. Simultaneously an increased uptake of FFA and an enhanced incorporation of palmitic
iis~M.4~ acid-l-C” into the neutral tissue lipids occurred. The mechanism of the increased lipid mobilization from adipose tissue in alloxan diabetes as well as the mechanism of nicotinic acid on the lipid mobilization are discussed against the results.
HAS RECENTLY BEEN DEhlONSTRATED that nicotinic acid decreases the concentration of free fatty acids (FFA) in arterial plasma in normal man’ and inhibits the increase in the plasma FFA caused by norepinephrine in dog.] It has also been shown in vitro on rat epididymal adipose tissue that nirotinic acid inhibits the enhanced lipid mobilization induced by norepinephrine.” The increased release of FFA and glycerol produced by other hormonrs in vitro, such as ACTH and glucagon, is also inhibited bv nicotinic
It was diabetes,
therefore a state
and gl~cerolG from adipose
of interest in which
to study there
tissue in vitro.
MATERIAL .AVD METIIODS
of nicotinic release
Standard of mean (n = 4)
acid + (NG )
Individual differences: (N) (NG)
p < 0.05. not significant.
0.149 zk 0.021 ~._ at 37 C. for 2 hours. 2 per cent of human
acid + (NG)
-t 0.020 Nicotinic Glucose
at a final
zk 0.021 t -~
Individual differences: (N) (NG)
Glycerol Net Release uMol/ml./Gm. wet weiaht
of FFA and GZycerol from Adipose Tissue of Alloxan Diabetic
FFA Net Release tiEa./ml./Gm. wet weieht
of Nicotinic Acid on the Net Release
Halved enididvmal fat Dads of nonfasting alloxan diabetic rats were incubated Basal medium: 5 ml. of Krebs-Henseleit bicarbonate buffer, pH 7.4, containing concentration of lo--GM and glucose at a final concentration of 3 mg. per ml. medium
(n = 6)
Mean -+- Standard error of mean
acid was added at a final concentration
determined by the method of Dole’ and glycerol b the run in duplicate. The changes of FFA enzymatic method of Wieland,c Y both determinations and glycerol concentration in the incubation medium was calculated as the difference fronr the concentrations of medium containing no tissue hut otherwise treated in the same way. The wet weight of the tissue was estimated by weighing the flask plus content beforc FFA
and after the addition of tissue. In the esperiments where radioactivity was used the tissue was rinsed in 0.9 per cent NaCl and 1 per cent albumin solutions repeated times before homogenization and extraction by the method of Dole.7 The upper heptanc phase obtained by the addition of water and heptane to the extraction mixture was divided in four aliclrrots. Two were taken for the determination of the total radioactivity, while two others wcrc taken for titration and later separation into one acid and one neutra1 fraction. By three washings with petroleum ether after alkalinization and acidification, respectively, FFA and neutral lipids were separated. By this method aborrt 98-100 per cent of added active, tripalmitin was recovered in the neutral phase and about 1 per cent in the acid phasc~. Added palmitic acid-l-Cl* was recovered in the acid phase to about 96-97 per cent. while the neutral e1scwhere.a
phase contained 3-4 In these experiments
per cent. The methodology will bc described also aliqtxots of the heptane phase obtained
in detail front the
extraction of the medium FFA were taken for radioassay. The counting WM performcxl in a Parkard Tri Carb Liquid Scintillation Counter with a system containing 50 rng. c~f %5-diphenyloxazole and 3 mg. of 1.4-his (-4 methyl-fi-phenyloxazolyl)-benzene in ml. of toluene. Quenching was tested and corrected for by 11s~ of an internal standard.
~\~arksl’b as modified
Calculations As the percentage the net FFA uptake
decrease of medium radioactivity can not he used as an estimate of when media with varying amounts of FFA released from the fat pads
arc compared, the uptake of FFA has been calculated mrthod also the outflow of FFA was calculated.
By the same
As is shown in table 1 (exp. A) the addition of nicotinic acid to the incuhation medium significantly decreased the release of FFA and glycerol from adipose tissue. The ratio between the inhibition of FFA release to the inhibition of glycerol release, when calculated for corresponding pair of tissues was 2.89 t 0.47 (mean 2 standard error of mean). A higher uptake of FFA from the medium is observed in the presence of nicotinic acid judging from the disappearance of palmitic acid-l-C?* from the medium and the radioactivity recovered from the tissues (table 2). Even when consideration is taken to the dilution of medium FFA with unlabeled FFA released from the fat pads the uptake of FFA, calculated according to Dole, was higher in the presence of nicotinic acid. While the incorporation of palmitic acid-l-C’* into the neutral tissue lipids was increased by about 100 per cent, the final tissue FFA radioactivity was much lowered. Also the tissue FFA was lower during the incubation with nicotinic acid. The net release of FFA in this experiment was even more inhibited than in those described above (table 1). This might be explained by the preincubation used in the last series of experiments. I3 The outflow of FFA, calculated according to Dole, was, of course, markedly reduced.
Final Tissue FFA Cont. /anoles/Gm.
Final Tissue FFA Radioactivity c.p.m./Gm.
Final Radioactivity of Neutral Tissue Lipids e.p.m./Gm.
FFA Net Release pIIlOl~/G~.
FFA Out&w ~moles/Gm.
37 C., in 5 ml. of a Krebs-Henseleit buffer, pH 7.4, containing 2 per cent of human albumin medium). Palmitic acid-l-Cl4 providing a specific activity of initial medium FFA to approxi-
fat pads were incubated for 2 hours, content of 0.35 p mol FFA per ml.
178,806 367,800 88,606
FFA Uptake /.moks/Gm.
Radioactivity Disapp. from the medium c.p.m./Gm.
mately 170,000 c.p.m. per micromole FFA. Nicotinic acid was added at a concentration the contralateral tissue serving as the control.
Epididymal (with a FFA
Addition to Medium
of Nicotinic Acid on the Uptake from the Medium and on the Zncorporation in the Neutral Acid-Z-C14 during in vitro Zncubation of Adipose Tissue from Alloxan Diabetic Rats
ON FATTY ACID METABOLISM
Nicotinic acid was observed to reduce the release of FFA and glycerol from adipose tissue of alloxan diabetic rats. Simultaneously an increased uptake and esterification of FFA occurred. These two major changes, an inhibited lipid mobilization and an increased esterification, have also been observed with nicotinic acid in fasting normal ratl’ in vitro, even if the effect is less pronounced. When glucose was added to the medium a further inhibition of FFA release was noticed, which corresponds with findings in media without nicotinic acid.r5*‘” The release of glycerol was not altered which is in accord with previous reports on adipose tissue of normal rat.l~~‘x The observations suggest that nicotinic acid and glucose enhance the esterification processes in different ways in vitro. As nicotinic acid increases the esterification in alloxan diabetes, a state with presumably diminished availability of 1-alpha-glycerophosphate, this taken together with the reduced glycerol release might indicate that nicotinic acid produces an increased esterification at the mono- or diglyceride level. Whether this is the primary effect of nicotinic acid on fatty acid metabolism or a secondary phenomenon to an inhibited lipolysis, however, cannot be predicted from present knowledge about the fatty acid metabolism within the tissue. The increased mobilization of FFA from adipose tissue in diabetes mellitus has mainly been explained by a re-esterification of FFA due to a lack of lalpha-glycerophosphate” and/or a deficit of ATP. Some previous data, however, seem to indicate that also an enhanced lipolysis might exist in diabetes mellitus. The elevated release of glycerol from adipose tissue of alloxan diabetic rats” and diabetic human subjects19 as well as increased glycerol level during severe diabetic state in man2” may speak in favor of an increased lipolysis. Evidence in support of this is the observation that the pretreatment of alloxan diabetic rats with dibenzyckne, an adrenergic blocking agent, leads to a reduction of FFA release from adipose tissue in vitro.” An enhancement of the lipolysis in diabetes might in that case be produced by one or several lipolytic hormones, of which norepineprine, ACTH and glucagon are known to be counteracted by nicotinic acid in vitro. With nicotinic acid it has been possible to reduce the increased lipid mobilization in alloxan diabetic state in vitro concomitant with an enhancement of the glyceride synthesis. These findings do not answer the question whether the increased lipid mobilization in diabetes mellitus is primarily due to an enhanced lipolysis or is secondary to a decreased re-esterification. ACKNOWLEDGMENTS I am very grateful to Dr. Lars A. Carlson for his continuing interest during the study and his most valuable criticism during the preparation of the manuscript. I also wish to acknowledge the competent technical assistance of Mrs. Britt Landberger and Mrs. Inger Brrgner. This
was supported the Svenska
in part by grants from U. S. Public Health Service Diabetesforbundet and from Nordisk Insulin Fond.
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Jan iistman, M.D., Senior Resident in Internal Medicine, Department of Internal Medicine, Karolinska Hospital, Stockholm, Sweden.