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Enzymes related to lipogenesis in the adipose tissue of obese subjects
Metabolism Clinical and Eqwrimental VOL. XXV,
NO. 5
MAY
1976
Enzymes Related to Lipogenesis in the Adipose Tissue of Obese Subjects Francesco Belfiore, Vito Borzi, Elena Napoli, and Agata M. Rabuazzo In a group of ten adult obese subiects, maintained for 15 days on a normal caloric intake and balanced diet, the activity of hexokinase (K 2.7.1.1), bphosphofructokinase (8C 2.7.1.11), and ATP citratelyase (EC 4.1.3.8) in the adipose tissue was significantly increased, both on Q protein and on a fat cell number basis, compared to matched normal subiects. The activity of glucose-bphosphate dehydrogenase (8C 1.l .1.49), malote dehydrogenose (EC 1.1.1.37), and malate dehydmgenase (decorboxyloting) (NADP) (EC 1.l .1.40), on the other hand, was unchanged. Since both hexokinose and bphosphofructokinase ore rate-limiting in glycolysis, their enhanced activity would indicate the occurrence of on increased capacity to
metabolize glucose and therefore to genemte alpha-glycerophosphate. The elevation of ATP citmtolyose would suggest increased lipogenesis, owing to the regulatory role that this enzyme plays in fatty acid synthesis. The normal activity of glucose-bphosphate dehydrogenose and molate dehydrogenose (decorboxyloting) (NADP), which supply NADPH for the reduction of acetyl-CoA to fatty acids, would suggest that the change in lipogenesis is of modemte degree, thereby affecting only the most mte-limiting enzyme, ATP citrots-lyase. These data, on the whole, am consistent with the occurrenceof enhanced triglyceride formation. Whether the enzyme changes observed ore odoptive or genetic in nature remains to be clarified.
I
N THE GENETIC OBESE MOUSE increased activity of enzymes correlated with lipogenesis has been found in liver and adipose tissue, which are the main sites of fat synthesis. In the liver the reported elevations concern
From the I Clinica Medica. University of Catania Medical School. Catania. Italy. Received for publication April 19. 1975. Reprint requests should be addressed to Dr. Francesco Be&ore. I Clinica Medica Ospedale Garibaldi, 95123 Catania. Italy. Q 1976 by Grune & Stratton, Inc.
Metabolism, Vol. 25, No. 5 (May), 1976
Vniversitb.
483
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acetyl-CoA carboxylase* and fatty acid synthetase,’ ATP citrate-lyase,2*3 glucose-6-phosphate dehydrogenase,4 malate dehydrogenase (decarboxylating) (NADP),S hexokinase,5 glycerol-3-phosphate dehydrogenase (NAD),4,5 and glycerol kinase.6 Most of these changes, however, are suppressed by reducing weight-gain through dietary restrictions and exercise,5*6 or are reduced by starvation,‘s3 thus appearing to be the result of adaptive responses to hyperphagia or inactivity. In the adipose tissue increased activity has been reported for glucose-6-phosdehydrogenase (decarboxylating),5 phate dehydrogenase,4*5 phosphogluconate hexokinase,5 glycerol-3-phosphate dehydrogenase (NAD),5 and glycerol kinase,6*7 while normal values have been found for isocitrate dehydrogenase (NADP), malate dehydrogenase, and malate dehydrogenase (decarboxylating) (NADP).’ Most of these enzyme changes are not significantly affected by weight-gain control5 or starvation ,8 thus appearing to result from a genetic lesion. In addition to genetic states, dietary factors can also be responsible for the increase in lipogenesis and related enzymes.9 The effect of “meal eating” is of special interest. With this food intake pattern, pentose cycle dehydrogenases@” and malate dehydrogenase (decarboxylating) (NADP)” increase in rat adipose tissue. Pentose cycle dehydrogenases increase also in the liver,‘0,“*‘3 although the changes are smaller than in adipose tissue in some studies.” The findings previously described indicate that increased lipogenesis may occur as a result of congenital or acquired enzyme alterations. On these grounds, it seemed to us of interest to search for possible enzyme abnormalities in the adipose tissue of obese humans. The only data so far published on this subject, to our knowledge, include the reduction of glycerol-3-phosphate dehydrogenase activity14*r5 and the change in some glycolytic enzymes.16 Therefore, in the present study we investigated the behavior of several enzymes related to lipogenesis in the adipose tissue of obese subjects, maintained on a controlled regimen as far as concerns dietary intake and physical exercise.
names” suggested in *Enzymes were designated throughout the paper by the “recommended reference 26. The corresponding systematic names and EC numbers are as follows: Acetyl-CoA carboxylase, acetyl-CoA:carbon-dioxide ligase (ADP-forming), EC 6.4.1.2; ATP citrate-lyase, ATP: - acetyl-CoA; ATP dephosphorylating), EC 4. I .3.8; citrate oxalacetate-lyase (pro-3S-CH2.COOglucose-6-phosphate dehydrogenase, D-glucoseJ%phosphate:NADP+ I-oxidoreductase, EC 1.1. I .49; glycerol kinase, ATP:glycerol 3_phosphotransferase, EC 2.7. I .30; glycerol-3-phosphate dehydrogenase (NAD), sn-glycerol-3-phosphate:NAD+ 2-oxidoreductase, EC I. 1.1.8; glycerol-3phosphate dehydrogenase, sn-glycerol-3-phosphate:(acceptor) oxidoreductase, EC 1.1.99.5; hexokinase, ATPD-hexose 6-phosphotransferase, EC 2.7.1. I; isocitrate dehydrogenase (NADP), fhreoD,-isocitrate:NADP+ oxidoreductase (decarboxylating), EC I. I. 1.42; malate dehydrogenase, L-malate:NAD+ oxidoreductase, EC 1.1 .1.37; malate dehydrogenase (decarboxylating) (NADP), L-malate:NADP+ oxidoreductase (oxalacetate decarboxylating), EC I. 1.1.40; phosphoenolpyruvate carboxykinase (GTP), GTPoxalacetate carboxy-lyase (transphosphorylating), EC 4. I. 1.32; 6_phosphofructokinase, ATPD-fructose-6-phosphate I-phosphotransferase, EC 2.7.1.1 I ; phosphogluconate dehydrogenase (decarboxylating), 6-phospho-D-gluconate:NADP+ 2-oxidoreductase (decarboxylating), EC I. I. 1.44; pyruvate carboxylase, pyruvate:carbon-dioxide ligase (ADP forming), EC 6.4.1. I.
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MATERIALS
485
AND METHODS
The subjects studied included ten hospitalized obese patients, six males and four females, aged between 44 and 70, who had a body weight that exceeded the ideal value to an extent of 20%-I 10%. as well as ten matched control subjects, hospitalized for minor illnesses, In each patient obesity has appeared during the second decade, and familial history revealed high incidence of obesity. For at least 15 days before the study began, both the normal and the obese subjects were allowed a caloric intake of about 2000 Cal/d, of which 50% was derived from carbohydrates, 20% from proteins, and 30% from fats. During this period no significant changes in body weight were observed in either group. Fasting blood glucose levels and oral glucose tolerance were normal in all subjects. After consent was obtained from the subjects selected for this study, a small incision was made in the lower left quadrant of the abdomen, under local intradermal anesthesia with Recorcaina@ (diethyl-amino-ethyl-p-aminobenzoate chlorhydrate), followed by direct removal of 1.552.5 g of fat tissue. Specimens thus obtained were washed in cold (+4”C) Krebs’ solution. A portion of tissue was reserved for determination of fat cell size, which was made on cells liberated with collagenase.” The remaining portion of the specimen was homogenized by means of an Ultra-Turrax homogenizer in a ratio of I g wet weight to 2 ml of homogenization medium. Tissue from five normal and five obese subjects was homogenized in distilled water, while tissue from the remaining five normal and five obese subjects was homogenized in triethanolamine buffer 0.05 M, pH 7.4, containing Na-EDTA 0.005 M. A small amount of homogenate was taken and used for the determination of triglycerides.‘* The rest of the homogenate was centrifuged in a refrigerated centrifuge at 1500 x g for 10 min. By means of a fine Pasteur pipette, the middle aqueous layer was aspirated, while the upper fat cake and the bottom layer of cell debris were discarded. In five instances a portion of tissue specimen was homogenized in 0.25 M sucrose by a Potter-Elvehjemtype homogenizer with a Teflon pestle, and centrifuged at 12,000 x g for 15 min to obtain a homogenate without mitochondria. Once prepared, homogenates were analyzed for protein concentration by the method of Lowry et al.19 and for enzyme activities. The following enzymes were studied by the methods indicated by their respective reference number: hexokinase,*’ 6-phosphofructokinase,*’ glucose-h-phosphate dehydrogenase,** ATP 23 citrate-lyase, malate dehydrogenase, 24 and malate dehydrogenase (decarboxylating) (NADP).” For the determination of ATP citrate-lyase a blank was used that contained the complete reaction mixture, with the exception of ATP. Moreover, the determination was carried out at pH 7.6 instead of at pH 7.3 as suggested by Srere,23 because, in preliminary experiments. we observed that this is the pH optimum. According to the Recommendations (1972) of the International Union of Pure and Applied Chemistry and the International Union of Biochemistry.26 enzyme activities were expressed in Katals. One Katal is “the amount of activity that converts one mole of substrate per second.“26 Results were given as nano-Katals (nKat) and were expressed on the basis of protein content, wet weight, and fat cell number. One old enzyme unit2’ ISequal to 16.67 nKat; one nKat is equal to 0.06 U. The number of fat cells was calculated by dividing the triglyceride content of a given amount of tissue by the average triglyceride content of the fat cell. The latter parameter. in turn, was obtained from the mean cell volume and the triglyceride (triolein) specific weight (0.915).” Data were statistically analyzed with a calculating machine, according to standard statistical methods.2” Substrates, enzymes, and coenzymes used in the enzyme assays were purchased from ringer, Mannheim, West Germany, with the exception of the citrate, which was obtained Sigma, St. Louis, MO. All other chemicals used were of Analytical-Reagent grade.
Boehfrom
RESULTS
Comparison of the enzyme activities found in the tissue samples homogenized in distilled water with those obtained with tissue samples homogenized in triethanolamine-EDTA at pH 7.4 revealed no significant differences, average values for the former and the latter group of tissue homogenates being 31 & 12
486
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Table 1. Enzyme Activities
in the Adipose Tissue From Normal and Obese Subiects Obese Subjects
Normals
n=lO Enzymes
ET AL.
” = 10*
Mean f SD
Per cent Variations
P
+44
+56
<0.05
+4
>o.ao
14
+90
2981
+17
> 0.20
-12
> 0.40
+47
+63
<0.02
103
+6
> 0.70
11
+93
2440
+20
>0.20
-11
>0.70
+ia
>0.05
+27
>0.05
-15
>0.50
+56
-3
> 0.90
-28
>O.lO
Mean f SD
nKat/g protein Hexokinase 6-phosphofructokinase Glucose-bphosphate
32 f ii.5
f
9
46&
a
ia*8
194 Zlr 147
11
201 & 126
dehydrogenose ATP citrate-lyose Molate dehydrogenose Malate dehydrogenase
20*
11
5290 f 2488
3a* 6211 f llP~tB7
136 f
72
25.5 f
7
37.6 f
6.4
14.7 & 6.3
(decorboxylating) (NADP) nKat/106 fat cells Hexokinase 6-phosphofructokinose Glucose-bphosphote
9 f 155+
117
16 f
a.7
9
164 f
dehydrogenase ATP citrate-lyase Malate dehydrogenase Molate dehydrogenase
4221 ZIZ 1965 109 f
93
255 f
72
31 f 5085 f
97Zt 71
(decarboxylating) (NADP) nKot/kg tissue wet weight Hexokinase 6-phosphofructokinase Glucose-bphosphote
92 f 64 i54ah
1173
301 f
71
117&52 1316 f
a25
dehydrogenose ATP citrate-lyase
159+
Malate dehydrogenose
42214 f
Malate dehydrogenase
i 085
a7 19655
f 574
249 Z!Z 91 40682 f
19525
779 =k 569
(decarboxyloting) (NADP) *Except for 6-phosphofructokinase, which was studied in nine cases.
and 33 + 13 nKat/g protein for hexokinase, 11.9 f 9 and 11.1 f 10 for 6-phosphofructokinase, 187 f 151 and 201 f 149 for glucose-6-phosphate dehydrogenase, 18 f 12 and 22 + 13 for ATP citrate-lyase, 5370 f 2441 and 5210 f 2611 for malate dehydrogenase, and 145 f 8 1 and 127 f 66 for malate dehydrogenase (decarboxylating) (NADP) (p > 0.30 in all instances). These data refer to normal subjects, but similar differences were obtained with homogenates from obese subjects. Therefore, data were considered altogether, regardless of the homogenization medium employed. Data shown in Table 1 indicate that, both on a protein and on a fat cell number basis, the activity of hexokinase, 6-phosphofructokinase and ATP citratelyase was significantly increased in the tissue from the obese subjects as compared to that of the control subjects. Glucose-6-phosphate dehydrogenase, malate dehydrogenase and malate dehydrogenase (decarboxylating) (NADP) were not significantly different in the two groups of subjects considered. The enzyme pattern resulting from these changes in the tissue from obese subjects is depicted in Fig. 1. The fat cell diameter was significantly (p < 0.05) greater in obese patients (62 + 8 pm) than in control subjects (50 f 6 pm). Consistently, the protein
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Fig. 1. Scheme showing the functional connection between the enzymes studied as well as the changes in enzyme activities found in the obese subjects both on a protein and on a fat cell number basis.
content per gram of fresh tissue was lower in the obese (6.55 f 2.69 mg) than in normal subjects (7.98 f 2.92 mg). Consequently, when data were expressed on a wet-weight basis, differences between values in normals and in obese patients were generally changed in favor of the former group. Therefore, the increase of hexokinase and 6-phosphofructokinase activities noticed on a protein or cell number basis in the obese subjects was no longer appreciable (Table 1). Measurement of ATP citrate-lyase in mitochondria-free homogenates from normal subjects gave an average value of 22.5 f 17 nKat/g protein, which was not significantly different (p > 0.80) from that obtained with total homogenate (21 f 16 nKat). DISCUSSION
General
As shown in Table 1, our results were expressed on a protein, cell number, and wet-weight bases. However, references to wet-weight is misleading, because a substantial part of the adipose tissue weight is due to the metabolically in-
488
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active triglyceride depot, which is increased in obese subjects, as indicated also by the larger cell size and the lower protein content per gram of tissue. Therefore, the discussion that follows will be based primarily on the values expressed on a protein or cell number basis. If compared to data in the literature, our enzyme levels are, on a whole, similar to those obtained with the same assay methods and the same material (total tissue homogenate),29-32 with one exception33 in which unusually high activity is reported for hexokinase, 6-phosphofructokinase, and glucose-6phosphate dehydrogenase. Our values are, on the other hand, low if compared to some data’6,34 which, however, were obtained with isolated fat cells16J4 or different assay methods.” Glycolytic Enzymes The elevation in the activity of hexokinase in the tissue of obese subjects is in accordance with data in the obese mice,5 and could be connected with the enhanced basal glucose uptake by adipose tissue reported in human obesity.35 Since both hexokinase and 6-phosphofructokinase are rate-limiting in glycolysis activity in the tissue in the adipose tissue of man29 and rat,36 their enhanced from obese patients would suggest an increased glucose utilization through this pathway. It should be mentioned, however, that the regulatory role of 6-phosphofructokinase in glycolysis could not be detected in some conditions.37 Glycolysis is the most important source of the glycerol 3-phosphate necessary for the esterification of fatty acids, as indicated by the observation that most of the radioactivity from glucose-‘4C is recovered in glyceride-glycerol.3*s39 Other pathways for glycerol 3-phosphate synthesis have been demonstrated in the adipose tissue, which include: (1) possible phosphorylation of some of the glycerol released during lipolysis by glycerol kinase, which, contrary to some negative has been found present in the adipose tissue;6.7*43v44(2) formation findings, 29*40-22 of glycerol 3-phosphate from glycerol and pyrophosphate through a transphosphorylation reaction;4’ (3) conversi on of pyruvate to glycerol 3-phosphate (glyceroneogenesis),45*M made possible by the occurrence of pyruvate carboxylase,47-49 and phosphoenolpyruvate carboxykinase (GTP)49*50 in the adipose tissue of both rat47*48*50 and man. 49 This pathway might play a role in fasting rats45*46or in diabetic or triamcinolone treated rats,5’ but seems to be less active role of in the obese mice than in controls. 52 However, the actual physiologic these mechanisms is uncertain, and therefore, increased glucose utilization as suggested by our findings, would indicate an enhanced availability of glycerol 3-phosphate. This might result in an increased triglyceride formation, since the availability of glycerol 3-phosphate seems to be rate-limiting in lipogenesis,53 although this assumption has been questioned.54 It has been claimed that an increased availability of glycerol 3-phosphate in the adipose tissue of obese subjects could arise from a decreased oxidation of this compound by the mitochondrial glycerol-3-phosphate dehydrogenase.‘4,‘5 However, analysis of the reported datai shows that the decrease affects to an almost equal extent both the cytoplasmic glycerophosphate-forming glycerol3-phosphate dehydrogenase (NAD) (- 44%) and the mitochondrial glycero-
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409
phosphate-oxidizing glycerol-3-phosphate dehydrogenase (- 5 lx), the values being 36 & 4 mpmoles NADH/min/mg protein in lean and 20 f 2 in obese for the former enzyme, and 14.2 f 2.2 ~1 OJ30 min/mg protein in lean and 6.9 f 1.8 in obese for the latter. Is Therefore, other things being equal, these changes may have little effect on cytoplasmic glycerophosphate availability. Furthermore, it has been observed that the rate of glycerophosphate oxidation by mitochondria in the adipose tissue is too low to be metabolically significant.55.56 Enzymes Involved in Fatty Acid Synthesis It has been reported 30~49,57 that human adipose tissue, unlike that of the rat, contains little or no ATP citrate-lyase and consistently has a poor capacity to synthesize fatty acid de novo. However, other studies35*58 have shown that optimally fortified systems from human adipose tissue are, in fact, capable of readily incorporating the radioactivity of citrate-1,5-14C into fatty acids. This finding implies that ATP citrate-lyase is present and active, and is in accordance with our observation that this enzyme is contained in human adipose tissue in easily measurable amounts. In an attempt to explain the discrepancy it must be mentioned that we assayed the enzyme activity at pH 7.6, which resulted in a much higher activity than in assays at pH 7.3, as used by others.49*57 This finding is in keeping with data recently reported.59 Moreover, we calculated the activity from the reading taken between the tenth and the twentieth minute, because of the occurrence of an initial lag-period lasting up to 7 min. Pentose cycle dehydrogenases and malate dehydrogenase (decarboxylating) (NADP) are involved in the generation of NADPH required for the reduction of the extramitochondrial acetyl-CoA, formed by ATP citrate-lyase, to fatty acids.60 Consistently, the three dehydrogenases mentioned and ATP citratelyase fluctuate coordinately in response to altered physiologic states and are closely correlated to variations in the rate of fatty acid synthesis.W In this study, however, we found a discordance between the behavior of ATP citrate-lyase and that of glucose-6-phosphate dehydrogenase and malate dehydrogenase (decarboxylating) (NADP), inasmuch as the former enzyme was increased, while the latter two dehydrogenases were normal. It is worth mentioning that normal activity of malate dehydrogenase (decarboxylating) (NADP) has also been observed in obese mice,5 and that a lack of correlation between body weight and glucose-6-phosphate dehydrogenase5’v6’ and malate dehydrogenase (decarboxylating) (NADP)” has been reported in humans. In evaluating this point it must be kept in mind that ATP citrate-lyase has a low activity compared to the dehydrogenases studied. Moreover, the rate of acetyl-CoA formation (which depend primarily upon the ATP citrate-lyase activity), despite negative reports,54 seems to be rate-limiting in fatty acid synthesis62*63 together with the accumulation of cytoplasmic NADH. 56 Therefore, it appears reasonable to postulate that an increase in fatty acid synthesis of a moderate degree may be associated with an elevation of ATP citrate-lyase activity without appreciable changes in other lipogenic enzymes, thus accounting for the normal activity of glucose-6-phosphate dehydrogenase and malate dehydrogenase (decarboxylating) (NADP). Our findings disagree somewhat to data by others35 showing a
490
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normal rate of fatty acid synthesis in obese subjects, but are in accordance with the observation that fatty acid synthesis expressed per cell is enhanced in obesity.58
CONCLUSION
The data discussed above indicate that in the adipose tissue of obese subjects there is an increased activity of enzymes related to glycerol 3-phosphate and fatty acid synthesis, which is consistent with the occurrence of enhanced triglyceride formation. Concerning the mechanism of the enzyme changes observed, we have not enough evidence to decide whether they are adaptive or genetic in nature. The former possibility cannot be ruled out on the basis of the poor responsiveness of human adipose tissue enzymes to nutritional, metabolic and hormonal alterations compared to rat tissue enzymes,30*57since adaptive changes in humans might take place at a very slow rate, thus preventing their detection in relatively short-term experiments. M Hormonal factors are generally thought to be involved in human obesity,@ including hyperinsulinism and adrenal cortical hyperfunction. A role of these factors, however, seems unlikely, since studies on alloxan-diabetic47~65-68and adrenalectomized rats68 suggest that both insulin and adrenal cortical hormones affect similarly hexokinase,65-68 glucose-6-phosphate dehydrogenase,67*68 and malate dehydrogenase (decarboxylating) (NADP),47 which contrasts with the dissociated behavior found in this research for these enzymes. Thus, further work is required before any definite statement can be made on the intimate mechanism of the enzymatic alterations observed.
ACKNOWLEDGMENT We are greatly indebted to Saverio Signorelli, M.D., Professor and Chairman, Medicine, University of Catania Medical School, and Head, Postgraduate School and Metabolic Diseases, for having supported this research.
Department of of Hematologic
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sue of obese and nonobese mice. Arch Biochem Biophys 125:259-268, 1968 53. Tzur R, Tal E, Shapiro B: Alpha-glycerophosphate as regulatory factor in fatty acid esterification. Biochim Biophys Acta 84: 18-23, 1964 54. Denton RM, Halperin ML: The control of fatty acid and triglyceride synthesis in rat epididymal adipose tissue. Roles of coenzyme A derivatives, citrate and L-glycerol 3-phosphate. Biochem J 110:27-38, 1968 55. Robinson BH, Halperin ML: Transport of reduced nicotinamide-adenine dinucleotide into mitochondria of rat white adipose tissue. Biochem J 116:229-233, 1970 56. Halperin ML, Robinson BH: Mechanism of insulin action on control of fatty acid synthesis independent ofglucose transport. Metabolism 20:78-86, I97 I 57. Shrago E, Glennon JA, Gordon ES: Studies on enzyme concentration and adaptation in human liver and adipose tissue. J Clin Endocrinol Metab 27:679-685, 1967 58. Sjostrom L: Adult human adipose tissue cellularity and metabolism. Acta Med Stand 544 (Suppl): 1-52, 1972 59. Goldrick RB, Galton DJ: Fatty acid synthesis de now in human adipose tissue. Clin Sci Mol Med 46~469~479, 1974 60. Lane MD, Moss J: Regulation of fatty acid synthesis in animal tissues, in Vogel HJ (ed): Metabolic Pathways, Vol V, Metabolic Regulation. New York, Academic, 1971, p 23 61. Tjabbes T, Baars JD, Blom PS: Glucose6-phosphate dehydrogenase (G-6-PD) activity in human adipose tissue. Metabolism 16:977984, 1967 62. Del Boca J, Flatt JP: Fatty acid synthesis from glucose and acetate and the control of lipogenesis in adipose tissue. Eur J Biochem Il:l27-134, 1969 63. Saggerson ED, Greenbaum AL: The regulation of triglyceride and fatty acid synthesis in rat epididymal adipose tissue. Biochem J ll9:221-242, 1970 64. Gordon ES: Metabolic aspects of obesity. Adv Metab Disorders 4:229-296, 1970 65. Katzen HM: The effect of diabetes and insulin in vivo and in vitro on a low Km form of hexokinase from various rat tissues. Biochem Biophys Res Commun 24:531-536, 1966 66. McLean P, Brown J, Greenslade K, Brew K: ElTect of alloxan-diabetes on the glucase-ATP phosphotransferase activity of adipose tissue. Biochem Biophys Res Commun 23: 117-121, 1966
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67. Orevi M, Gorin E, Shafrir E: Adaptive changes of phosphofructokinase and aldolase in adipose tissue. Em J Biochem 30:418-426, 1972 68. Gumaa KA. Novello F. McLean P: The
pentose
phosphate
pathway
of glucose
metab-
olism. Hormonal and dietary control of the oxidative and non-oxidative reactions and related enzymes of the cycle in adipose tissue. Biochem J 115:4055417, 1969
NO. 5
MAY
1976
Enzymes Related to Lipogenesis in the Adipose Tissue of Obese Subjects Francesco Belfiore, Vito Borzi, Elena Napoli, and Agata M. Rabuazzo In a group of ten adult obese subiects, maintained for 15 days on a normal caloric intake and balanced diet, the activity of hexokinase (K 2.7.1.1), bphosphofructokinase (8C 2.7.1.11), and ATP citratelyase (EC 4.1.3.8) in the adipose tissue was significantly increased, both on Q protein and on a fat cell number basis, compared to matched normal subiects. The activity of glucose-bphosphate dehydrogenase (8C 1.l .1.49), malote dehydrogenose (EC 1.1.1.37), and malate dehydmgenase (decorboxyloting) (NADP) (EC 1.l .1.40), on the other hand, was unchanged. Since both hexokinose and bphosphofructokinase ore rate-limiting in glycolysis, their enhanced activity would indicate the occurrence of on increased capacity to
metabolize glucose and therefore to genemte alpha-glycerophosphate. The elevation of ATP citmtolyose would suggest increased lipogenesis, owing to the regulatory role that this enzyme plays in fatty acid synthesis. The normal activity of glucose-bphosphate dehydrogenose and molate dehydrogenose (decorboxyloting) (NADP), which supply NADPH for the reduction of acetyl-CoA to fatty acids, would suggest that the change in lipogenesis is of modemte degree, thereby affecting only the most mte-limiting enzyme, ATP citrots-lyase. These data, on the whole, am consistent with the occurrenceof enhanced triglyceride formation. Whether the enzyme changes observed ore odoptive or genetic in nature remains to be clarified.
I
N THE GENETIC OBESE MOUSE increased activity of enzymes correlated with lipogenesis has been found in liver and adipose tissue, which are the main sites of fat synthesis. In the liver the reported elevations concern
From the I Clinica Medica. University of Catania Medical School. Catania. Italy. Received for publication April 19. 1975. Reprint requests should be addressed to Dr. Francesco Be&ore. I Clinica Medica Ospedale Garibaldi, 95123 Catania. Italy. Q 1976 by Grune & Stratton, Inc.
Metabolism, Vol. 25, No. 5 (May), 1976
Vniversitb.
483
484
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ET AL.
acetyl-CoA carboxylase* and fatty acid synthetase,’ ATP citrate-lyase,2*3 glucose-6-phosphate dehydrogenase,4 malate dehydrogenase (decarboxylating) (NADP),S hexokinase,5 glycerol-3-phosphate dehydrogenase (NAD),4,5 and glycerol kinase.6 Most of these changes, however, are suppressed by reducing weight-gain through dietary restrictions and exercise,5*6 or are reduced by starvation,‘s3 thus appearing to be the result of adaptive responses to hyperphagia or inactivity. In the adipose tissue increased activity has been reported for glucose-6-phosdehydrogenase (decarboxylating),5 phate dehydrogenase,4*5 phosphogluconate hexokinase,5 glycerol-3-phosphate dehydrogenase (NAD),5 and glycerol kinase,6*7 while normal values have been found for isocitrate dehydrogenase (NADP), malate dehydrogenase, and malate dehydrogenase (decarboxylating) (NADP).’ Most of these enzyme changes are not significantly affected by weight-gain control5 or starvation ,8 thus appearing to result from a genetic lesion. In addition to genetic states, dietary factors can also be responsible for the increase in lipogenesis and related enzymes.9 The effect of “meal eating” is of special interest. With this food intake pattern, pentose cycle dehydrogenases@” and malate dehydrogenase (decarboxylating) (NADP)” increase in rat adipose tissue. Pentose cycle dehydrogenases increase also in the liver,‘0,“*‘3 although the changes are smaller than in adipose tissue in some studies.” The findings previously described indicate that increased lipogenesis may occur as a result of congenital or acquired enzyme alterations. On these grounds, it seemed to us of interest to search for possible enzyme abnormalities in the adipose tissue of obese humans. The only data so far published on this subject, to our knowledge, include the reduction of glycerol-3-phosphate dehydrogenase activity14*r5 and the change in some glycolytic enzymes.16 Therefore, in the present study we investigated the behavior of several enzymes related to lipogenesis in the adipose tissue of obese subjects, maintained on a controlled regimen as far as concerns dietary intake and physical exercise.
names” suggested in *Enzymes were designated throughout the paper by the “recommended reference 26. The corresponding systematic names and EC numbers are as follows: Acetyl-CoA carboxylase, acetyl-CoA:carbon-dioxide ligase (ADP-forming), EC 6.4.1.2; ATP citrate-lyase, ATP: - acetyl-CoA; ATP dephosphorylating), EC 4. I .3.8; citrate oxalacetate-lyase (pro-3S-CH2.COOglucose-6-phosphate dehydrogenase, D-glucoseJ%phosphate:NADP+ I-oxidoreductase, EC 1.1. I .49; glycerol kinase, ATP:glycerol 3_phosphotransferase, EC 2.7. I .30; glycerol-3-phosphate dehydrogenase (NAD), sn-glycerol-3-phosphate:NAD+ 2-oxidoreductase, EC I. 1.1.8; glycerol-3phosphate dehydrogenase, sn-glycerol-3-phosphate:(acceptor) oxidoreductase, EC 1.1.99.5; hexokinase, ATPD-hexose 6-phosphotransferase, EC 2.7.1. I; isocitrate dehydrogenase (NADP), fhreoD,-isocitrate:NADP+ oxidoreductase (decarboxylating), EC I. I. 1.42; malate dehydrogenase, L-malate:NAD+ oxidoreductase, EC 1.1 .1.37; malate dehydrogenase (decarboxylating) (NADP), L-malate:NADP+ oxidoreductase (oxalacetate decarboxylating), EC I. 1.1.40; phosphoenolpyruvate carboxykinase (GTP), GTPoxalacetate carboxy-lyase (transphosphorylating), EC 4. I. 1.32; 6_phosphofructokinase, ATPD-fructose-6-phosphate I-phosphotransferase, EC 2.7.1.1 I ; phosphogluconate dehydrogenase (decarboxylating), 6-phospho-D-gluconate:NADP+ 2-oxidoreductase (decarboxylating), EC I. I. 1.44; pyruvate carboxylase, pyruvate:carbon-dioxide ligase (ADP forming), EC 6.4.1. I.
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MATERIALS
485
AND METHODS
The subjects studied included ten hospitalized obese patients, six males and four females, aged between 44 and 70, who had a body weight that exceeded the ideal value to an extent of 20%-I 10%. as well as ten matched control subjects, hospitalized for minor illnesses, In each patient obesity has appeared during the second decade, and familial history revealed high incidence of obesity. For at least 15 days before the study began, both the normal and the obese subjects were allowed a caloric intake of about 2000 Cal/d, of which 50% was derived from carbohydrates, 20% from proteins, and 30% from fats. During this period no significant changes in body weight were observed in either group. Fasting blood glucose levels and oral glucose tolerance were normal in all subjects. After consent was obtained from the subjects selected for this study, a small incision was made in the lower left quadrant of the abdomen, under local intradermal anesthesia with Recorcaina@ (diethyl-amino-ethyl-p-aminobenzoate chlorhydrate), followed by direct removal of 1.552.5 g of fat tissue. Specimens thus obtained were washed in cold (+4”C) Krebs’ solution. A portion of tissue was reserved for determination of fat cell size, which was made on cells liberated with collagenase.” The remaining portion of the specimen was homogenized by means of an Ultra-Turrax homogenizer in a ratio of I g wet weight to 2 ml of homogenization medium. Tissue from five normal and five obese subjects was homogenized in distilled water, while tissue from the remaining five normal and five obese subjects was homogenized in triethanolamine buffer 0.05 M, pH 7.4, containing Na-EDTA 0.005 M. A small amount of homogenate was taken and used for the determination of triglycerides.‘* The rest of the homogenate was centrifuged in a refrigerated centrifuge at 1500 x g for 10 min. By means of a fine Pasteur pipette, the middle aqueous layer was aspirated, while the upper fat cake and the bottom layer of cell debris were discarded. In five instances a portion of tissue specimen was homogenized in 0.25 M sucrose by a Potter-Elvehjemtype homogenizer with a Teflon pestle, and centrifuged at 12,000 x g for 15 min to obtain a homogenate without mitochondria. Once prepared, homogenates were analyzed for protein concentration by the method of Lowry et al.19 and for enzyme activities. The following enzymes were studied by the methods indicated by their respective reference number: hexokinase,*’ 6-phosphofructokinase,*’ glucose-h-phosphate dehydrogenase,** ATP 23 citrate-lyase, malate dehydrogenase, 24 and malate dehydrogenase (decarboxylating) (NADP).” For the determination of ATP citrate-lyase a blank was used that contained the complete reaction mixture, with the exception of ATP. Moreover, the determination was carried out at pH 7.6 instead of at pH 7.3 as suggested by Srere,23 because, in preliminary experiments. we observed that this is the pH optimum. According to the Recommendations (1972) of the International Union of Pure and Applied Chemistry and the International Union of Biochemistry.26 enzyme activities were expressed in Katals. One Katal is “the amount of activity that converts one mole of substrate per second.“26 Results were given as nano-Katals (nKat) and were expressed on the basis of protein content, wet weight, and fat cell number. One old enzyme unit2’ ISequal to 16.67 nKat; one nKat is equal to 0.06 U. The number of fat cells was calculated by dividing the triglyceride content of a given amount of tissue by the average triglyceride content of the fat cell. The latter parameter. in turn, was obtained from the mean cell volume and the triglyceride (triolein) specific weight (0.915).” Data were statistically analyzed with a calculating machine, according to standard statistical methods.2” Substrates, enzymes, and coenzymes used in the enzyme assays were purchased from ringer, Mannheim, West Germany, with the exception of the citrate, which was obtained Sigma, St. Louis, MO. All other chemicals used were of Analytical-Reagent grade.
Boehfrom
RESULTS
Comparison of the enzyme activities found in the tissue samples homogenized in distilled water with those obtained with tissue samples homogenized in triethanolamine-EDTA at pH 7.4 revealed no significant differences, average values for the former and the latter group of tissue homogenates being 31 & 12
486
BELFIORE
Table 1. Enzyme Activities
in the Adipose Tissue From Normal and Obese Subiects Obese Subjects
Normals
n=lO Enzymes
ET AL.
” = 10*
Mean f SD
Per cent Variations
P
+44
+56
<0.05
+4
>o.ao
14
+90
2981
+17
> 0.20
-12
> 0.40
+47
+63
<0.02
103
+6
> 0.70
11
+93
2440
+20
>0.20
-11
>0.70
+ia
>0.05
+27
>0.05
-15
>0.50
+56
-3
> 0.90
-28
>O.lO
Mean f SD
nKat/g protein Hexokinase 6-phosphofructokinase Glucose-bphosphate
32 f ii.5
f
9
46&
a
ia*8
194 Zlr 147
11
201 & 126
dehydrogenose ATP citrate-lyose Molate dehydrogenose Malate dehydrogenase
20*
11
5290 f 2488
3a* 6211 f llP~tB7
136 f
72
25.5 f
7
37.6 f
6.4
14.7 & 6.3
(decorboxylating) (NADP) nKat/106 fat cells Hexokinase 6-phosphofructokinose Glucose-bphosphote
9 f 155+
117
16 f
a.7
9
164 f
dehydrogenase ATP citrate-lyase Malate dehydrogenase Molate dehydrogenase
4221 ZIZ 1965 109 f
93
255 f
72
31 f 5085 f
97Zt 71
(decarboxylating) (NADP) nKot/kg tissue wet weight Hexokinase 6-phosphofructokinase Glucose-bphosphote
92 f 64 i54ah
1173
301 f
71
117&52 1316 f
a25
dehydrogenose ATP citrate-lyase
159+
Malate dehydrogenose
42214 f
Malate dehydrogenase
i 085
a7 19655
f 574
249 Z!Z 91 40682 f
19525
779 =k 569
(decarboxyloting) (NADP) *Except for 6-phosphofructokinase, which was studied in nine cases.
and 33 + 13 nKat/g protein for hexokinase, 11.9 f 9 and 11.1 f 10 for 6-phosphofructokinase, 187 f 151 and 201 f 149 for glucose-6-phosphate dehydrogenase, 18 f 12 and 22 + 13 for ATP citrate-lyase, 5370 f 2441 and 5210 f 2611 for malate dehydrogenase, and 145 f 8 1 and 127 f 66 for malate dehydrogenase (decarboxylating) (NADP) (p > 0.30 in all instances). These data refer to normal subjects, but similar differences were obtained with homogenates from obese subjects. Therefore, data were considered altogether, regardless of the homogenization medium employed. Data shown in Table 1 indicate that, both on a protein and on a fat cell number basis, the activity of hexokinase, 6-phosphofructokinase and ATP citratelyase was significantly increased in the tissue from the obese subjects as compared to that of the control subjects. Glucose-6-phosphate dehydrogenase, malate dehydrogenase and malate dehydrogenase (decarboxylating) (NADP) were not significantly different in the two groups of subjects considered. The enzyme pattern resulting from these changes in the tissue from obese subjects is depicted in Fig. 1. The fat cell diameter was significantly (p < 0.05) greater in obese patients (62 + 8 pm) than in control subjects (50 f 6 pm). Consistently, the protein
ENZYMES
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Fig. 1. Scheme showing the functional connection between the enzymes studied as well as the changes in enzyme activities found in the obese subjects both on a protein and on a fat cell number basis.
content per gram of fresh tissue was lower in the obese (6.55 f 2.69 mg) than in normal subjects (7.98 f 2.92 mg). Consequently, when data were expressed on a wet-weight basis, differences between values in normals and in obese patients were generally changed in favor of the former group. Therefore, the increase of hexokinase and 6-phosphofructokinase activities noticed on a protein or cell number basis in the obese subjects was no longer appreciable (Table 1). Measurement of ATP citrate-lyase in mitochondria-free homogenates from normal subjects gave an average value of 22.5 f 17 nKat/g protein, which was not significantly different (p > 0.80) from that obtained with total homogenate (21 f 16 nKat). DISCUSSION
General
As shown in Table 1, our results were expressed on a protein, cell number, and wet-weight bases. However, references to wet-weight is misleading, because a substantial part of the adipose tissue weight is due to the metabolically in-
488
BELFIORE
ET Al.
active triglyceride depot, which is increased in obese subjects, as indicated also by the larger cell size and the lower protein content per gram of tissue. Therefore, the discussion that follows will be based primarily on the values expressed on a protein or cell number basis. If compared to data in the literature, our enzyme levels are, on a whole, similar to those obtained with the same assay methods and the same material (total tissue homogenate),29-32 with one exception33 in which unusually high activity is reported for hexokinase, 6-phosphofructokinase, and glucose-6phosphate dehydrogenase. Our values are, on the other hand, low if compared to some data’6,34 which, however, were obtained with isolated fat cells16J4 or different assay methods.” Glycolytic Enzymes The elevation in the activity of hexokinase in the tissue of obese subjects is in accordance with data in the obese mice,5 and could be connected with the enhanced basal glucose uptake by adipose tissue reported in human obesity.35 Since both hexokinase and 6-phosphofructokinase are rate-limiting in glycolysis activity in the tissue in the adipose tissue of man29 and rat,36 their enhanced from obese patients would suggest an increased glucose utilization through this pathway. It should be mentioned, however, that the regulatory role of 6-phosphofructokinase in glycolysis could not be detected in some conditions.37 Glycolysis is the most important source of the glycerol 3-phosphate necessary for the esterification of fatty acids, as indicated by the observation that most of the radioactivity from glucose-‘4C is recovered in glyceride-glycerol.3*s39 Other pathways for glycerol 3-phosphate synthesis have been demonstrated in the adipose tissue, which include: (1) possible phosphorylation of some of the glycerol released during lipolysis by glycerol kinase, which, contrary to some negative has been found present in the adipose tissue;6.7*43v44(2) formation findings, 29*40-22 of glycerol 3-phosphate from glycerol and pyrophosphate through a transphosphorylation reaction;4’ (3) conversi on of pyruvate to glycerol 3-phosphate (glyceroneogenesis),45*M made possible by the occurrence of pyruvate carboxylase,47-49 and phosphoenolpyruvate carboxykinase (GTP)49*50 in the adipose tissue of both rat47*48*50 and man. 49 This pathway might play a role in fasting rats45*46or in diabetic or triamcinolone treated rats,5’ but seems to be less active role of in the obese mice than in controls. 52 However, the actual physiologic these mechanisms is uncertain, and therefore, increased glucose utilization as suggested by our findings, would indicate an enhanced availability of glycerol 3-phosphate. This might result in an increased triglyceride formation, since the availability of glycerol 3-phosphate seems to be rate-limiting in lipogenesis,53 although this assumption has been questioned.54 It has been claimed that an increased availability of glycerol 3-phosphate in the adipose tissue of obese subjects could arise from a decreased oxidation of this compound by the mitochondrial glycerol-3-phosphate dehydrogenase.‘4,‘5 However, analysis of the reported datai shows that the decrease affects to an almost equal extent both the cytoplasmic glycerophosphate-forming glycerol3-phosphate dehydrogenase (NAD) (- 44%) and the mitochondrial glycero-
ENZYMES
RELATED
TO LIPOGENESIS
409
phosphate-oxidizing glycerol-3-phosphate dehydrogenase (- 5 lx), the values being 36 & 4 mpmoles NADH/min/mg protein in lean and 20 f 2 in obese for the former enzyme, and 14.2 f 2.2 ~1 OJ30 min/mg protein in lean and 6.9 f 1.8 in obese for the latter. Is Therefore, other things being equal, these changes may have little effect on cytoplasmic glycerophosphate availability. Furthermore, it has been observed that the rate of glycerophosphate oxidation by mitochondria in the adipose tissue is too low to be metabolically significant.55.56 Enzymes Involved in Fatty Acid Synthesis It has been reported 30~49,57 that human adipose tissue, unlike that of the rat, contains little or no ATP citrate-lyase and consistently has a poor capacity to synthesize fatty acid de novo. However, other studies35*58 have shown that optimally fortified systems from human adipose tissue are, in fact, capable of readily incorporating the radioactivity of citrate-1,5-14C into fatty acids. This finding implies that ATP citrate-lyase is present and active, and is in accordance with our observation that this enzyme is contained in human adipose tissue in easily measurable amounts. In an attempt to explain the discrepancy it must be mentioned that we assayed the enzyme activity at pH 7.6, which resulted in a much higher activity than in assays at pH 7.3, as used by others.49*57 This finding is in keeping with data recently reported.59 Moreover, we calculated the activity from the reading taken between the tenth and the twentieth minute, because of the occurrence of an initial lag-period lasting up to 7 min. Pentose cycle dehydrogenases and malate dehydrogenase (decarboxylating) (NADP) are involved in the generation of NADPH required for the reduction of the extramitochondrial acetyl-CoA, formed by ATP citrate-lyase, to fatty acids.60 Consistently, the three dehydrogenases mentioned and ATP citratelyase fluctuate coordinately in response to altered physiologic states and are closely correlated to variations in the rate of fatty acid synthesis.W In this study, however, we found a discordance between the behavior of ATP citrate-lyase and that of glucose-6-phosphate dehydrogenase and malate dehydrogenase (decarboxylating) (NADP), inasmuch as the former enzyme was increased, while the latter two dehydrogenases were normal. It is worth mentioning that normal activity of malate dehydrogenase (decarboxylating) (NADP) has also been observed in obese mice,5 and that a lack of correlation between body weight and glucose-6-phosphate dehydrogenase5’v6’ and malate dehydrogenase (decarboxylating) (NADP)” has been reported in humans. In evaluating this point it must be kept in mind that ATP citrate-lyase has a low activity compared to the dehydrogenases studied. Moreover, the rate of acetyl-CoA formation (which depend primarily upon the ATP citrate-lyase activity), despite negative reports,54 seems to be rate-limiting in fatty acid synthesis62*63 together with the accumulation of cytoplasmic NADH. 56 Therefore, it appears reasonable to postulate that an increase in fatty acid synthesis of a moderate degree may be associated with an elevation of ATP citrate-lyase activity without appreciable changes in other lipogenic enzymes, thus accounting for the normal activity of glucose-6-phosphate dehydrogenase and malate dehydrogenase (decarboxylating) (NADP). Our findings disagree somewhat to data by others35 showing a
490
BELFIORE
ET AL.
normal rate of fatty acid synthesis in obese subjects, but are in accordance with the observation that fatty acid synthesis expressed per cell is enhanced in obesity.58
CONCLUSION
The data discussed above indicate that in the adipose tissue of obese subjects there is an increased activity of enzymes related to glycerol 3-phosphate and fatty acid synthesis, which is consistent with the occurrence of enhanced triglyceride formation. Concerning the mechanism of the enzyme changes observed, we have not enough evidence to decide whether they are adaptive or genetic in nature. The former possibility cannot be ruled out on the basis of the poor responsiveness of human adipose tissue enzymes to nutritional, metabolic and hormonal alterations compared to rat tissue enzymes,30*57since adaptive changes in humans might take place at a very slow rate, thus preventing their detection in relatively short-term experiments. M Hormonal factors are generally thought to be involved in human obesity,@ including hyperinsulinism and adrenal cortical hyperfunction. A role of these factors, however, seems unlikely, since studies on alloxan-diabetic47~65-68and adrenalectomized rats68 suggest that both insulin and adrenal cortical hormones affect similarly hexokinase,65-68 glucose-6-phosphate dehydrogenase,67*68 and malate dehydrogenase (decarboxylating) (NADP),47 which contrasts with the dissociated behavior found in this research for these enzymes. Thus, further work is required before any definite statement can be made on the intimate mechanism of the enzymatic alterations observed.
ACKNOWLEDGMENT We are greatly indebted to Saverio Signorelli, M.D., Professor and Chairman, Medicine, University of Catania Medical School, and Head, Postgraduate School and Metabolic Diseases, for having supported this research.
Department of of Hematologic
REFERENCES I. Chang HC, Seidman I, Teebor G, Lane MD: Liver acetyl-CoA carboxylase and fatty acid synthetase: Relative activities in the normal state and in hereditary obesity. Biochem Biophys Res Commun 28:682-686, 1967 2. Spencer AF, Lowenstein JM: Citrate and the conversion of carbohydrate into fat. Citrate cleavage in obesity and lactation. Biochem J 991760-765, 1966 3. Kornacker MS, Lowenstein JM: Citrate cleavage enzyme in livers of obese and nonobese mice. Science 1441027-1028, 1964 4. Fried GH, Antopol W: Enzymatic activities in tissues of obese-hyperglycemic mice. Am J Physiol211:1321-1324, 1966
5. Martin RJ, Welton RF, Baumgardt BR: Adipose and liver tissue enzyme profiles in obese hyperglycemic mice. Proc Sot Exp Biol Med 142:241-245, 1973 6. Welton RF, Martin RJ, Baumgardt BR: Effects of feeding and exercise regimens on adipose tissue glycerokinase activity and body composition of lean and obese mice. J Nutr 103: 1212-1219, 1973 7. Treble DH, Mayer J: Glycerol kinase activity in white adipose tissue of obese hyperglycemic mice. Nature (Lond) 200:363-365, 1963 8. Anderson J, Hollifield G: Glucose-6-phosphate dehydrogenase activity in adipose tissue
ENZYMES
RELATED
491
TO LIPOGENESIS
from two types of obese mice during starvation. Metabolism 15109221097, 1966 9. Freedland RA, Szepesi B: Control of enzyme activity: Nutritional factors, in Rechcigl M Jr (ed): Enzyme Synthesis and Degradation in Mammalian Systems. Basel, Karger, 1971. p 103 10. Cohn C, Joseph D: Role of rate of ingestion of diet on regulation of intermediate metabolism (“Meal eating” vs. “Nibbling”). Metabolism 9:492-500, 1960 11. Hollifield G, Parson W: Metabolic adaptations to a “stuff and starve” feeding program. 1. Studies of adipose tissue and liver glycogen in rats limited to a short daily feeding period. J Clin Invest 41:245-249, 1962 12. Leveille GA: Glycogen metabolism in meal-fed rats and chicks and the time sequence of lipogenic and enzymatic adaptive changes. J Nutr 90449-460, 1966 13. Tepperman J, Tepperman HM: Effects of antecedent food intake pattern on hepatic lipogenesis. Am J Physiol 193:55-64, 1958 14. Galton DJ: An enzymatic defect in a group of obese patients. Br Med J 2:1498-1500, 1966 15. Galton DJ, Bray GA: Metabolism of alpha-glycerol phosphate in human adipose tissue in obesity. J Clin Endocrinol Metab 27: 1573-1580, 1967 16. Englhardt A, Gries FA, Liebermeister H, Jahnke K: Size, lipid and enzyme content of isolated human adipocytes in relation to nutritional state. Diabetologia 7:51-58, 1971 17. Di Girolamo M, Mendlinger S, Fertig JW: A simple method to determine fat cell size and number in four mammalian species. Am J Physiol 221:850-858, 1971 18. Eggstein M, Kreutz FH: Eine neue Bestimmung der Neutralfette im Blutserum und Gewebe. I Mitteilung: Prinzip, Besprechung der Method. Klin
Durchftihrung, Wschr 44:262-
267. 1966 19. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with the Folin-phenol reagent. J Biol Chem 193:265-275, 1951 20. Viiiuela E, Salas M, Sols A: Glucokinase and hexokinase in liver in relation to glycogen synthesis. J Biol Chem 238:PC 1175-l 177, 1963 21. Ling KH. Paetkau V, Marcus F, Lardy HA: Phosphofructokinase. 1. Skeletal muscle. Methods Enzymol9:425-429, 1966 22. Lohr GW, Wailer HD: Glucose-6-phosphate dehydrogenase, in Bergmeyer H-U (ed): Methods of Enzymatic Analysis. New York, Academic, 1963, p 744
23. Srere
PA:
Citrate-cleavage
enzyme.
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