International Journal of Obesity (1997) 21, 439±444 ß 1997 Stockton Press All rights reserved 0307±0565/97 $12.00
Decreased white fat cell thermogenesis in obese individuals H BoÈttcher and P FuÈrst University of Hohenheim, Institute for Biological Chemistry and Nutrition, D-70593 Stuttgart, FRG Germany
OBJECTIVE: To investigate whether white adipocyte thermogenesis and energy metabolism are reduced in obese individuals. SUBJECTS: Eight lean and 15 obese men and women; BMI 19±41. DESIGN: Isolated subcutaneous adipocytes were maintained in agarose gel for 20 h under basal conditions and subsequently for 10 h after stimulation with 1 mM isoprenaline. Direct microcalorimetry was performed continuously over 30 h while biochemical measures were obtained after 0, 20, 25 and 30 h. MEASUREMENTS: Total cellular thermogenesis, oxygen consumption, glycolysis, lipolysis, triglyceride/FFA substrate cycle, adenine nucleotides, DNA content as basis of reference. RESULTS: Under basal and stimulated conditions, thermogenesis (5.6 and 8.6 mW/mgDNA, respectively; P < 0.0001) correlated negatively (P < 0.01 and P < 0.05, respectively) with the BMI and positively with O2 and glucose consumption, lactate, glycerol, FFA release and FFA re-esteri®cation. Reduced basal lactate production with increased BMI (P < 0.05) indicates a more aerobic adipocyte metabolism in obese individuals. Negative correlation between BMI and stimulated triglyceride/FFA substrate cycle activity (P < 0.01) explains the decreased hormone induced adipocyte heat production in the obese. CONCLUSIONS: The results suggest that a reduction of total body energy expenditure, which is discussed to cause obesity, can be associated with distinct metabolic alterations at a cellular level. However, since the estimated total body fat cell thermogenesis does not exceed 7% of the resting metabolic rate, the observed decrease of adipocyte heat production in the face of augmented BMI can only in part be responsible for the development of obesity. Keywords: catecholamine-induced thermogenesis; direct microcalorimetry; energy metabolism; fat cell culture system; substrate cycle; obesity
Introduction According to the rules of thermodynamics, obesity is simply a question of energy balance. Thus, excess dietary energy intake over energy output (heat and mechanical work) will inevitably result in storage of the excess energy in the form of triglyceride in the white adipose tissue1±4 which is therefore considered as the target organ of obesity.5 Indeed, obesity is a major health problem and represents an obvious risk factor for many diseases, like diabetes, stroke, or coronary heart disease.6±8 Although overeating is considered as the major cause for the development of obesity in overweight subpopulations obesity might be induced by diminished body heat production.9±11 In contrast, a lean person is able to compensate excess energy intake by an augmented thermogenesis, thereby keeping its body weight constant.4,12,13 In the human body there are a number of `empty' biochemical mechanisms at the level of cellular metabolism which are devoted to transform chemically bound energy to heat without Correspondence: Dr H BoÈttcher. Received 23 October 1996; revised 7 February 1997; accepted 11 February 1997
work performance. These processes include uncoupling of the respiration chain and increased Na/KATPase activity as well as various substrate cycles (lipolysis and re-esteri®cation of free fatty acidstriglyceride/FFA cycle or the interorgan cycle of glycolysis and gluconeogenesis between adipose tissue and liver-glucose/lactate cycle).4,14±19 Since the metabolic characteristics of adipose tissue are mainly based on the white adipocyte,20 studies related to energy expenditure of this cell type might be helpful to understand the relation between obesity and reduced cellular thermogenesis. In previous microcalorimetric investigations with white adipocyte suspensions, reduced cellular heat production was observed in obese subjects, compared to lean donors.21 These results, however, are not supported by simultaneous biochemical investigations of cellular metabolism, making the interpretation of a reduced thermogenesis dif®cult.19 In the present study, white adipocytes obtained from lean and obese donors were cultured in agarose gel19,22 and basal and catecholamine-induced thermogenesis were measured by direct microcalorimetry. The underlying biochemical mechanisms were examined by parallel measurements of oxidative phosphorylation, glycolysis, and lipolysis, as well as the triglyceride/FFA substrate cycle.
Decreased fat cell thermogenesis in obese humans H BoÈttcher and P FuÈrst
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Methods Subjects
Subcutaneous adipose tissue was obtained from 15 obese (8 f/7 m; mean BMI 30.6; range 27.7±40.4 kg/ m2) and 8 lean (4 f/4 m; mean BMI 22.1; range 19.5± 24.7 kg/m2) patients undergoing a minor uncomplicated surgery of inguinal hernia. The patients had no identi®ed metabolic or endocrinological disorders. After an overnight fast, general anaesthesia was given with Iso¯urane, oxygen and nitrous oxide. Tissue samples were taken at commencement of the surgical procedure.
Fat cell culture
White fat cells were isolated by collagenase digestion20 and embedded in 1% agarose gel (type VII, Sigma, St. Louis, MO) containing Medium 199 (Gibco Ltd., Paisley, U.K.) and 2% human serum albumin (Behringwerke, Marburg, FRG). The gel cultures were prepared in 3 ml glass ampoules and sealed with metal caps (Macherey-Nagel, DuÈren, FRG). From each preparation, up to 20 parallel cultures were established and kept at 37 C. The procedure was described earlier in detail.19,22,23 Under these culture conditions, basal rates of fat cell thermogenesis, lipolysis, glycolysis and oxidative phosphorylation are constant for more than 72 h, while FFA release declines continuously.19,23
Microcalorimetry
Two identical microcalorimeters of the thermopile heat conduction type (2277-BioActivityMonitor, LKB, Bromma, Sweden and 2277-ThermalActivityMonitor, ThermoMetric, JaÈrfaÈlla, Sweden) were used. In this system Peltier elements are sandwiched between the measuring ampoule containing the sample and tempered heat sinks. The produced voltage is proportional to the heat production rate which is continuously monitored.22,24 In every experiment, unstimulated heat production of six parallel cultures from one patient was monitored continuously for 20 h. Subsequently, isoprenaline (®nal concentration 1076 M) was injected into the cultures without interrupting the measurement by using a special microcatheter as described previously.25 Stimulated thermogenesis was measured for a further 10 h. After isoprenaline addition the heat production rate increased, the maximum being reached after 150 50 min. 10 h after stimulation, thermogenesis was decreased by only 9 7% of the maximum value.25 In the parallel cultures not used for microcalorimetry, hormone injection was performed after 20 h with a syringe and cannula pierced through the rubber sealing of the closed vessels.
Biochemcial analyses
Aliquots of the parallel cultures were taken 0, 20, 25 and 30 h after start of the experiment for determination of oxygen content (TIA-111-LV oxygen sensor, PBI Dansensor, Tùllùse, Danmark) and subsequent analyses of glucose, lactate, pyruvate, glycerol and FFA concentrations, and cellular contents of adenine nucleotides and DNA. These methods were previously described and evaluated in detail.19,23 Re-esteri®cation rate of FFA (RRE) was calculated from the lipolytic rate (RGLL) and the rate of FFA release (RFFA) according to the formula RRE 3 6 RGLL 7 RFFA.15 Statistics
The rates of oxygen and glucose uptake as well as release of lactate, pyruvate and glycerol were estimated by regression analysis after testing the linearity. The mean FFA release and re-esteri®cation rates were calculated for the time intervals 0±20 h and 20±30 h. Comparisons between groups were made by the paired rank-test. All results are expressed as mean s.e.
Results The age of the subjects did not correlate with BMI, heat production rate or biochemical indices, and sex had no in¯uence on the results. Therefore, the combined data from men and women are evaluated. Stimulated and unstimulated thermogenesis were virtually constant throughout the experiment. Mean basal heat production was 5.64 0.44 mW/mgDNA, and correlated negatively with the BMI (r 70.56; P < 0.01). Isoprenaline induced thermogenesis (8.60 0.61 mW/m P < 0.0001) was also negatively correlated with the BMI (r 70.44; P < 0.05; Figure 1). The absolute increase of heat production after isoprenaline addition was independent from BMI; the relative increase being positively correlated with BMI (r 0.53; P < 0.01). The rates of oxygen and glucose consumption as well as lactate, pyruvate and glycerol release (Table 1) were stable not only under basal, but also under stimulated conditions (Table 2), whereas with the time elapsed, FFA release decreased and re-esteri®cation increased. The degree of increase or decrease was independent from BMI, thermogenesis or FFA concentrations. None of the metabolic events appeared to be affected by the FFA concentration. Correlations with cellular heat production rate under basal and stimulated conditions are shown in Table 3. Under basal conditions, correlations with the subjects' BMI were observed only with glucose consumption (r 70.58; P < 0.01) and lactate production (r 70.52; P < 0.05; Figure 2), while after isoprenaline stimulation a correlation with FFA reesteri®cation was observed (r 70.63; P < 0.01; Figure 3).
Decreased fat cell thermogenesis in obese humans H BoÈttcher and P FuÈrst
Cellular contents of ATP, ADP, and total adenines after 20 h of culture (3.80 0.28; 0.22 0.02 and 4.12 0.29 nmol/mgDNA, respectively) were inversely correlated with the BMI (P < 0.05), while the AMP content (0.10 0.02 nmol/mgDNA) and the ATP/ADP ratio (19.1 1.7) were independent from the BMI. Correlations with heat production rates are shown in Table 3.
Discussion Basal conditions
Figure 1 Regression of BMI and fat cell thermogenesis, d basal (r 7 0.56; P < 0.01) and s after stimulation with 1076M isoprenaline (r 7 0.44; P < 0.05).
The assessed rates of thermogenesis, oxidative phosphorylation, glycolysis, lipolysis, re-esteri®cation and the cellular adenine nucleotide contents in the present study (Table 1) were comparable with previous data
Table 1 Rates of fat cell oxygen consumption, glycolysis, lipolysis and FFA reesteri®cation; basal over 20 h and 0±10 h after stimulation with 1076 M isoprenaline [nmol/mgDNA h] O2-consumption Glucose consumption Lactate production Pyruvate production Glycerol release FFA release FFA re-esteri®cation
1076 M isoprenaline
Basal 36.8 2.5 33.1 4.2 38.6 4.9 1.23 0.30 6.74 0.82 6.26 1.13 14.0 1.9
* * * ns * * *
87.3 13.1 78.3 7.8 84.5 9.1 1.54 0.77 43.6 3.6 82.2 8.2 47.2 9.0
mean s.e.; n 23; * P < 0.0001 (paired rank-test).
Table 2 Rates of fat cell oxygen consumption, glycolysis and lipolysis, 0±5 h and 5±10 h after stimulation with 1076 M isoprenaline [nmol/mgDNA h] O2-consumption Glucose consumption Lactate production Glycerol release FFA release
0^5 h after stimulation
5^10 h after stimulation
111.5 17.8 74.5 11.4 82.5 11.9 41.1 3.9 120.7 12.8
ns ns ns ns *
63.0 13.4 82.1 12.0 86.5 12.4 46.1 4.9 43.7 7.3
mean s.e.; n 23; * P < 0.001 (paired rank-test).
Table 3 Correlations of fat cell heat production with oxygen consumption, glycolysis, lipolysis, FFA re-esteri®cation and adenine nucleotides under basal and catecholamine stimulated conditions 1076 M isoprenaline
Basal
O2 consumption Glucose consumption Lactate production Glycerol release FFA release FFA re-esteri®cation ATP ADP AMP Total adenines ATP/ADP ratio
r
P
r
P
0.59 0.83 0.71 0.66 0.28 0.68 0.88 0.69 70.14 0.89 70.12
< 0.01 < 0.0001 < 0.001 < 0.001 ns < 0.001 < 0.0001 < 0.001 ns < 0.0001 ns
0.29 0.44 0.63 0.87 0.50 0.68 0.88 0.66 70.13 0.89 0.00
ns < 0.05 < 0.01 < 0.0001 < 0.05 < 0.001 < 0.0001 < 0.001 ns < 0.0001 ns
441
Decreased fat cell thermogenesis in obese humans H BoÈttcher and P FuÈrst
442
Indeed, the reduction of glucose consumption and lactate production with increasing BMI (Figure 2) suggests a more anaerobic fat cell metabolism in lean individuals and the high adenine nucleotide contents observed at high rates of heat production (basal and stimulated; Table 3), suggest that the size of the adenine pool might be indicative for the metabolic rate. Considering that the ATP/ADP ratio was independent from the measured thermogenesis, it is reasonable to assume that the low adipocyte heat production rates in obese subjects are not caused by a decreased cell viability. b -adrenergic stimulation
Figure 2 Regression of BMI and basal lactate production (r 70.52; P < 0.05).
Figure 3 Regression of BMI and FFA re-esteri®cation after stimulation with 1076 M isoprenaline (r 70.63; P < 0.01).
derived from human adipocytes in vivo or in agarose gel cultures and suspensions.19,23,26 Considering the positive correlation of cell size and the rates of oxidative phosphorylation, glycolysis and lipolysis,5,15,27±30 it is not likely that the observed decrease of thermogenic rate with increasing BMI (Figure 1) is due to an increased fat cell size.31 Indeed, human obesity is associated with increased fat cell thermogenesis despite a larger cell size.21 Employing reference values of the standard heat of formation,32 the heat production of oxidative phosphorylation, glycolysis and the triglyceride/FFA substrate cycle might be estimated. (Yet, since there are no standard conditions in the cell, the results of these calculations might deviate from the actual heat production.19 Heat production derived from oxidative processes was 4.8 mW/mgDNA (85% of total cellular thermogenesis), while 0.6 mW/mgDNA (11% of total thermogenesis) were generated by glycolysis. The triglyceride/FFA substrate cycle14±17 produced 0.7 mW/mgDNA (12% of total thermogenesis).
In the human body, catecholamines stimulate heat production, especially by non shivering thermogenesis, and play also a role in dietary induced thermogenesis.2,3,18,33±38 In the present study, cellular thermogenesis was stimulated by the b-agonist isoprenaline and found to be, like the basal thermogenesis, decreased with increasing extent of obesity (Figure 1). The catecholamine induced stimulation of thermogenesis, oxidative phosphorylation, glycolysis, lipolysis and re-esteri®cation (Table 1), was in accordance with previous data from in vivo and in vitro studies.17±19,33,36±40 A lower heat production per mol O2 than observed under basal conditions might be due to the hormonal in¯uence as suggested previously.16,41,42 This event might be due to an increased storage of energy by formation of cAMP from ATP under catecholamine stimulated conditions.39,43 Following stimulation, glycolysis and the triglyceride/ FFA cycle show a higher contribution to the total cellular thermogenesis (15 and 27%, respectively) than under basal conditions. Consequently, the isoprenaline induced increase of cellular heat production is mainly due to the stimulation of glycolysis and the triglyceride/FFA cycle. The observed negative correlation between FFA re-esteri®cation and BMI (Figure 3) implies that the reduced fat cell thermogenesis in the face of augmented BMI might be due to a decreased activity of the triglyceride/FFA cycle. Under basal and/or stimulated conditions, numerous correlations of cellular thermogenesis with substrate metabolism (Table 3) and BMI were observed. Yet, most of the metabolic pathways revealed no correlation with the BMI. This paradoxical ®nding in our obese subjects is dif®cult to explain. In the frame of the present study fat cell size could not be assessed, but there is convincing evidence existing that obesity is associated with increased fat cell size.31 Furthermore there are several reports showing that increased fat cell size is associated with increased rates of oxygen and glucose consumption, glycerol and FFA release, as well as FFA re-esteri®cation.5,15,27±30 However, in only some of these evaluations has the factor of human obesity been contemplated. Thus, the question might be raised,
Decreased fat cell thermogenesis in obese humans H BoÈttcher and P FuÈrst
which underlying pathophysiological mechanisms in¯uence ¯ux and metabolism of substrates.
Implications for the whole body
It is notable that the estimated total body fat cell thermogenesis, as calculated from cellular heat production and known values for total body fat cell number,15 does not exceed 5% of the resting metabolic rate under basal and 7% under stimulated conditions. Indeed, these calculated values are in good agreement with data derived from in vivo studies.33,34 The results of the present study therefore suggest, that a decrease of energy turnover and consequently a reduced thermogenic capacity of white adipocytes might only in part contribute to the development of obesity. Yet, the relationship between fat cell and total body thermogenesis is made complex by the fact that massive obesity is accompanied by an increased fat cell number.15,44 According to a computer generated numerical simulation,45 an about 20% reduction of the resting metabolic rate would be necessary to make a lean person obese, provided that energy intake remains unchanged. This could be the case if the energy expenditure of all body cells would be uniformly reduced by the same extent as in the fat cells examined in the present study. This consideration is supported by reports describing a corresponding decrease of energy expenditure in about one ®fth of the obese.9,11 In a recent study it was shown, that both in lean and obese individuals overfeeding leads to an increase of resting and non-resting energy expenditure, while underfeeding causes an alteration into the opposite direction.46 This may provide another explanation for the observations made in the present study. Accordingly, decreased fat cell energy turnover in certain obese subjects might be the consequence of perpetual dieting in order to prevent a further increase of body weight. Yet, this interpretation is questioned by publications of a Swedish group, which observed that the reduced fat cell thermogenesis in obese individuals was increased after weight reduction by fasting21 or gastroplasty,47 and concluded that reduced heat production in adipocytes derived from obese persons might be a consequence rather than a cause of obesity. This deduction is however not conclusive, since even after weight loss (30 kg on an average) the patients were still massively obese (about 40% above normal weight), while their fat cell thermogenesis already showed the same values as that of the lean controls. This suggests that the observed increase of adipocyte thermogenesis during weight loss might rather be an effect of the reduced energy intake than of the reduction in body fat mass itself. Future examinations of adipocyte heat production under circumstances of over- and underfeeding, especially prior to the development of obesity, might help to solve these contradictions.
Conclusions The overall results of the present study show that reduction of total body energy expenditure can be caused by alterations of energy metabolism on cellular level. The methodology used in this work enables future studies related to cellular heat production, not only under physiological conditions, but also under the in¯uence of thermogenic drugs. Indeed, studies with other cell types like hepatocytes or muscle ®bres from lean and obese donors might be of interest and render new information related to drug therapy of obesity.
Acknowledgements
This work was supported by grants of the Deutsche Forschungsgemeinschaft (Fu-153/4-1), the International Foundation for the Promotion of Nutrition Research and Nutrition Education (ISFE) and the European Society of Parenteral and Enteral Nutrition (ESPEN). We are indebted to Dr. E. Brand and his surgical staff for the excellent cooperation, to Mrs. A. Stublinac/Liebscher for FFA analyses and to Mrs. A. Harmsen for her excellent technical assistance. References
1 Rampone AJ, Reynolds PJ. Obesity: Thermodynamic principles in perspective. Life Sci 1988; 43: 93±110. 2 Woo R, Daniels-Kush R, Horton ES. Regulation of energy balance. Ann Rev Nutr 1985; 5: 411±433. 3 Himms-Hagen J. Cellular Thermogenesis. Ann Rev Physiol 1976; 38: 315±342. 4 Hegsted DM. Energy needs and energy utilisation. Nutr Rev 1974; 32: 33±38. 5 Hallgren P, SjoÈstroÈm L, Hedlund H, Lundell L, Olbe L. In¯uence of age, fat cell weight, and obesity on O2 consumption of human adipose tissue. Am J Physiol 1989; 256: E467± E474. 6 Felber JP, Golay A, JeÂquier E, Curchod B, Temler E, DeFronzo RA, Ferrannini E. The metabolic consequences of long-term human obesity. Int J Obes 1987; 12: 377±389. 7 Landin K, Krotiewski M, Smith U. Importance of obesity for the metabolic abnormalities associated with an abdominal fat distribution. Metabolism 1989; 38: 572±576. 8 Bakris GL, Weir MR, Sowers JR. Therapeutic challenges in the obese diabetic patient with hypertension. American Journal of Medicine 1996; 101: 33S±46S. 9 Weststrate JA, Dekker J, Stoel M, Bergheijn L, Deurenberg P, Hautvast JGAJ. Resting energy expenditure in women: Impact of obesity and body-fat distribution. Metabolism 1990; 39: 11± 17. 10 Anonymous. Decreased energy expenditure as a cause of obesity in infants. Nutr Rev 1988; 46: 255±257. 11 Ravussin E, Lilliola S, Knowler WC, Christin L, Freymond D, Abbott WGH, Boyce V, Howard BV, Bogardus C. Reduced rate of energy expenditure as a risk factor for body weight gain. N Engl J Med 1988; 318: 467±472. 12 Horton ES. Introduction: An overview of the assessment and regulation of energy balance in humans. Am J Clin Nutr 1983; 38: 972±977. 13 Sims EAH, Danforth E, Horton ES, Bray GA, Glennon JA, Salans LB. Endocrine and metabolic effects of experimental obesity in man. Rec Progr Horm Res 1973; 29: 457±496.
443
Decreased fat cell thermogenesis in obese humans H BoÈttcher and P FuÈrst
444
14 Newsholme EA, Crabtree B. Substrate cycles: Their metabolic, energy, and thermic consequences in man. Biochem Soc Symp 1976; 43: 183±205. 15 Hammond VA, Johnston DG. Substrate cycling between triglyceride and fatty acid in human adipocytes. Metabolism 1987; 36: 308±313. 16 Elia M, Zed C, Neale G, Livesey G. The energy cost of triglyceride-fatty acid recycling in nonobese subjects after an overnight fast and four days of starvation. Metabolism 1987; 36: 251±255. 17 Edens NK, Leibel RL, Hirsch J. Mechanism of free fatty acid re-esteri®cation in human adipocytes in vitro. J Lipid Res 1990; 31: 1423±1431. 18 SjoÈstroÈm L. Catecholamine sensitivity with respect to metabolic rate in man. Int J Obes 1985; 9 (Suppl. 2): 123±129. 19 BoÈttcher H, FuÈrst P. Microcalorimetric and biochemical investigations of thermogenesis and metabolic pathways in human white adipocytes. Int J Obes 1996; 20: 874±881. 20 Rodbell M. Metabolism of isolated fat cells. J Biol Chem 1964; 239: 375±380. 21 SoÈrbris R, Monti M, Nilsson-Ehle P, WadsoÈ I. Heat production by adipocytes from obese subjects before and after weight reduction. Metabolism 1982; 31: 973±978. 22 BoÈttcher H, Nittinger J, Engel S, FuÈrst P. Thermogenesis of white adipocytes. A novel method allowing long-term microcalorimetric investigations. J Biochem Biophys Methods 1991; 23: 181±187. 23 BoÈttcher H, Engel S, FuÈrst P. Determination of gaseous and dissolved oxygen in a closed fat cell culture system. Anal Biochem 1995; 230: 149±153. 24 Suurkuusk J, WadsoÈ I. A multichannel microcalorimetry system. Chem Scripta 1982; 20: 155±163. 25 BoÈttcher H, FuÈrst P. A new microcatheter enables microcalorimetric assessment of thermogenesis after stimulation in cell cultures. J Biochem Biophys Methods 1996; 32: 191±194. 26 BoÈttcher H, Engel S, FuÈrst P. Thermogenesis and metabolism of human white adipocytes in gel culture. Clin Nutr 1992; 11: 53±55. 27 Hood RL, Trankina ML, Beitz DC, Best DJ. Relationship between adipocyte size and lipid synthesis in the Zucker rat. Nutr Rep Int 1983; 27: 791±797. 28 DiGirolamo M, Howe MD, Esposito J, Thurman L, Owens JL. Metabolic patterns and insulin responsiveness of enlarging fat cells. J Lipid Res 1974; 15: 332±338. 29 Marin P, Andersson B, Ottosson M, Olbe L, Chowdhury B, Kvist H, Holm G, SjoÈstroÈm L, BjoÈrntorp P. The morphology and metabolism of intraabdominal adipose tissue in men. Metabolism 1992; 41: 1242±1248. 30 Zinder O, Shapiro B. Effect of cell size on epinephrine- and ACTH-induced fatty acid release from isolated fat cells. J Lipid Res 1971; 12: 91±95. 31 Gurr MI, Jung RT, Robinson MP, James WPT. Adipose tissue cellularity in man: The relationship between fat cell size and number, the mass and distribution of body fat, and the history of weight gain and loss. Int J Obes 1982; 6: 419±436.
32 Wilhoit RC. Selected values of thermodynamic properties. In: Brown HD (ed). Biochemical Microcalorimetry. Academic Press: New York, London, 1993, pp 305±317. 33 Kurpad A, Khan K, Calder G, Coppack S, Frayn K, MacDonald I. Effect of noradrenaline on glycerol turnover and lipolysis in the whole body and subcutaneous adipose tissue in humans in vivo. Clin Sci 1994; 86: 177±184. 34 Simonsen L, Stallknecht B, BuÈlow J. Contribution of skeletal muscle and adipose tissue to adrenaline-induced thermogenesis in man. Int J Obes 1993; 17 (Suppl. 3): S47±S51. 35 Simonsen L, BuÈlow J, Madsen J, Christensen NJ. Thermic response to epinephrine in the forearm and abdominal subcutaneous adipose tissue. Am J Physiol 1992; 263: E850± E855. 36 Connacher A, Jung ART, Mitchell PE, Ford RP, Leslie P, Illingworth P. Heterogeneity of noradrenergic thermic response in obese and lean humans. Int J Obes 1988; 12: 267±276. 37 Katzeff HL, O'Connell M, Horton ES, Danforth E Jr., Young JB, Landsberg L. Metabolic studies in human obesity during overnutrition and undernutrition: Thermogenic and hormonal responses to norepinephrine. Metabolism 1986; 35: 166± 175. 38 Jung RT, Shetty PS, James WPT. Reduced thermogenesis in obesity. Nature 1979; 279: 322±323. 39 Kather H. Beta-adrenergic stimulation of adenine nucleotide catabolism and purine release in human adipocytes. J Clin Invest 1990; 85: 106±114. 40 Miyoshi H, Shulman GI, Peters EJ, Wolfe MH, Elahi D, Wolfe RR. Hormonal control of substrate cycling in humans. J Clin Invest 1988; 81: 1545±1555. 41 Nedergaard J, Cannon B, Lindberg O. Microcalorimetry of isolated mammalian cells. Nature 1977; 267: 518±520. 42 Clark DG, Brinkman M, Neville SD. Microcalorimetric measurements of heat production in brown adipocytes from control and cafeteria-fed rats. Biochem J 1986; 235: 337±342. 43 Kather H, Bieger W, Michel G, Aktories K, Jakobs H. Human fat cell lipolysis is primary regulated acting through distinct mechanisms. J Clin Invest 1985; 76: 1559±1565. 44 Hauner H, Entenmann G, Wabitsch M, Gaillard D, Ailhaud G, Negrel R, Pfeiffer EF. Promoting effect of Glucocorticoids on the differentiation of human adipocyte precursor cells cultured in a chemically de®ned medium. J Clin Invest 1989; 84: 1663± 1670. 45 Weinsier RL, Bracco D, Schutz Y. Predicted effects of small decreases in energy expenditure on weight gain in adult women. Int J Obes 1993; 17: 693±700. 46 Leibel RL, Rosenbaum M, Hirsch J. Changes in energy expenditure resulting from altered body weight. N Engl J Med 1995; 332: 621±628. 47 Olsson SA, Monti M, Sorbris R, Nilsson-Ehle P. Adipocyte heat production before and after weight reduction by gastroplasty. Int J Obes 1986; 10: 99±105.
