Decreased White Fat Cell Thermogenesis in Obese Individuals

HBoÈttcher 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 work performance. These processes include uncoup- ling of the respiration chain and increased Na/K- ATPase activity as well as various substrate cycles According to the rules of thermodynamics, obesity is (lipolysis and re-esteri®cation of free fatty acids- simply a question of energy balance. Thus, excess triglyceride/FFA cycle or the interorgan cycle of dietary energy intake over energy output (heat and glycolysis and gluconeogenesis between adipose mechanical work) will inevitably result in storage of tissue and liver-glucose/lactate cycle).4,14±19 the excess energy in the form of triglyceride in the Since the metabolic characteristics of adipose tissue white adipose tissue1±4 which is therefore considered are mainly based on the white adipocyte,20 studies as the target organ of obesity.5 Indeed, obesity is a related to energy expenditure of this cell type might major health problem and represents an obvious risk be helpful to understand the relation between obesity factor for many diseases, like diabetes, stroke, or and reduced cellular thermogenesis. In previous coronary heart disease.6±8 microcalorimetric investigations with white adipocyte Although overeating is considered as the major suspensions, reduced cellular heat production was cause for the development of obesity in overweight observed in obese subjects, compared to lean subpopulations obesity might be induced by dimin- donors.21 These results, however, are not supported ished body heat production.9±11 In contrast, a lean by simultaneous biochemical investigations of cellular person is able to compensate excess energy intake by metabolism, making the interpretation of a reduced an augmented thermogenesis, thereby keeping its thermogenesis dif®cult.19 body weight constant.4,12,13 In the human body there In the present study, white adipocytes obtained are a number of `empty' biochemical mechanisms at from lean and obese donors were cultured in agarose the level of cellular metabolism which are devoted to gel19,22 and basal and catecholamine-induced thermo- transform chemically bound energy to heat without genesis were measured by direct microcalorimetry. The underlying biochemical mechanisms were examined by parallel measurements of oxidative Correspondence: Dr H BoÈ ttcher. Received 23 October 1996; revised 7 February 1997; accepted 11 phosphorylation, glycolysis, and lipolysis, as well as February 1997 the triglyceride/FFA substrate cycle. Decreased fat cell thermogenesis in obese humans H BoÈttcher and P FuÈrst 440 Methods Biochemcial analyses Aliquots of the parallel cultures were taken 0, 20, 25 and 30 h after start of the experiment for determina- Subjects tion of oxygen content (TIA-111-LV oxygen sensor, Subcutaneous adipose tissue was obtained from 15 PBI Dansensor, Tùllùse, Danmark) and subsequent obese (8 f/7 m; mean BMI 30.6; range 27.7±40.4 kg/ analyses of glucose, lactate, pyruvate, glycerol and m2) and 8 lean (4 f/4 m; mean BMI 22.1; range 19.5± FFA concentrations, and cellular contents of adenine 24.7 kg/m2) patients undergoing a minor uncompli- nucleotides and DNA. These methods were previously cated surgery of inguinal hernia. The patients had no described and evaluated in detail.19,23 Re-esteri®cation identi®ed metabolic or endocrinological disorders. rate of FFA (RRE) was calculated from the lipolytic After an overnight fast, general anaesthesia was rate (RGLL) and the rate of FFA release (RFFA) 15 given with Iso¯urane, oxygen and nitrous oxide. according to the formula RRE 3 6 RGLL 7 RFFA. Tissue samples were taken at commencement of the surgical procedure. Statistics The rates of oxygen and glucose uptake as well as release of lactate, pyruvate and glycerol were esti- Fat cell culture mated by regression analysis after testing the linearity. White fat cells were isolated by collagenase diges- The mean FFA release and re-esteri®cation rates were tion20 and embedded in 1% agarose gel (type VII, calculated for the time intervals 0±20 h and 20±30 h. Sigma, St. Louis, MO) containing Medium 199 Comparisons between groups were made by the paired (Gibco Ltd., Paisley, U.K.) and 2% human serum rank-test. All results are expressed as mean Æ s.e. albumin (Behringwerke, Marburg, FRG). The gel cultures were prepared in 3 ml glass ampoules and sealed with metal caps (Macherey-Nagel, DuÈren, Results FRG). From each preparation, up to 20 parallel cultures were established and kept at 37C. The procedure was described earlier in detail.19,22,23 The age of the subjects did not correlate with BMI, Under these culture conditions, basal rates of fat heat production rate or biochemical indices, and sex cell thermogenesis, lipolysis, glycolysis and oxidative had no in¯uence on the results. Therefore, the com- phosphorylation are constant for more than 72 h, while bined data from men and women are evaluated. FFA release declines continuously.19,23 Stimulated and unstimulated thermogenesis were virtually constant throughout the experiment. Mean basal heat production was 5.64 Æ 0.44 mW/mgDNA, and correlated negatively with the BMI (r 70.56; Microcalorimetry P < 0.01). Isoprenaline induced thermogenesis Two identical microcalorimeters of the thermopile (8.60 Æ 0.61 mW/m P < 0.0001) was also negatively heat conduction type (2277-BioActivityMonitor, correlated with the BMI (r 70.44; P < 0.05; LKB, Bromma, Sweden and 2277-ThermalActivity- Figure 1). The absolute increase of heat production Monitor, ThermoMetric, JaÈrfaÈlla, Sweden) were used. after isoprenaline addition was independent from In this system Peltier elements are sandwiched BMI; the relative increase being positively correlated between the measuring ampoule containing the with BMI (r 0.53; P < 0.01). sample and tempered heat sinks. The produced vol- The rates of oxygen and glucose consumption as tage is proportional to the heat production rate which well as lactate, pyruvate and glycerol release (Table 1) is continuously monitored.22,24 In every experiment, were stable not only under basal, but also under unstimulated heat production of six parallel cultures stimulated conditions (Table 2), whereas with the from one patient was monitored continuously for 20 h. time elapsed, FFA release decreased and re-esteri®ca- Subsequently, isoprenaline (®nal concentration tion increased. The degree of increase or decrease was 1076 M) was injected into the cultures without inter- independent from BMI, thermogenesis or FFA con- rupting the measurement by using a special micro- centrations. None of the metabolic events appeared to catheter as described previously.25 Stimulated be affected by the FFA concentration. Correlations thermogenesis was measured for a further 10 h. with cellular heat production rate under basal and After isoprenaline addition the heat production rate stimulated conditions are shown in Table 3. Under increased, the maximum being reached after basal conditions, correlations with the subjects' BMI 150 Æ 50 min. 10 h after stimulation, thermogenesis were observed only with glucose consumption was decreased by only 9 Æ 7% of the maximum (r 70.58; P < 0.01) and lactate production value.25 In the parallel cultures not used for micro- (r 70.52; P < 0.05; Figure 2), while after iso- calorimetry, hormone injection was performed after prenaline stimulation a correlation with FFA re- 20 h with a syringe and cannula pierced through the esteri®cation was observed (r 70.63; P < 0.01; rubber sealing of the closed vessels. Figure 3). Decreased fat cell thermogenesis in obese humans H BoÈttcher and P FuÈrst 441 Cellular contents of ATP, ADP, and total adenines after 20 h of culture (3.80 Æ 0.28; 0.22 Æ 0.02 and 4.12 Æ 0.29 nmol/mgDNA, respectively) were inver- sely correlated with the BMI (P < 0.05), while the AMP content (0.10 Æ 0.02 nmol/mgDNA) and the ATP/ADP ratio (19.1 Æ 1.7) were independent from the BMI. Correlations with heat production rates are shown in Table 3.

Discussion

Basal conditions The assessed rates of thermogenesis, oxidative phosphorylation, glycolysis, lipolysis, re-esteri®cation and Figure 1 Regression of BMI and fat cell thermogenesis, d basal (r 7 0.56; P < 0.01) and s after stimulation with 1076M the cellular adenine nucleotide contents in the present isoprenaline (r 7 0.44; P< 0.05). study (Table 1) were comparable with previous data

Table 1 Rates of fat cell oxygen consumption, glycolysis, lipolysis and FFA re- esteri®cation; basal over 20 h and 0±10 h after stimulation with 1076 M isoprenaline

[nmol/mgDNA Á h] Basal 1076 M isoprenaline

O2-consumption 36.8 Æ 2.5 * 87.3 Æ 13.1 Glucose consumption 33.1 Æ 4.2 * 78.3 Æ 7.8 Lactate production 38.6 Æ 4.9 * 84.5 Æ 9.1 Pyruvate production 1.23 Æ 0.30 ns 1.54 Æ 0.77 Glycerol release 6.74 Æ 0.82 * 43.6 Æ 3.6 FFA release 6.26 Æ 1.13 * 82.2 Æ 8.2 FFA re-esteri®cation 14.0 Æ 1.9 * 47.2 Æ 9.0

mean Æ 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 Á h] 0^5 h 5^10 h after stimulation after stimulation

O2-consumption 111.5 Æ 17.8 ns 63.0 Æ 13.4 Glucose consumption 74.5 Æ 11.4 ns 82.1 Æ 12.0 Lactate production 82.5 Æ 11.9 ns 86.5 Æ 12.4 Glycerol release 41.1 Æ 3.9 ns 46.1 Æ 4.9 FFA release 120.7 Æ 12.8 * 43.7 Æ 7.3

mean Æ 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

Basal 1076 M isoprenaline

r P r P

O2 consumption 0.59 < 0.01 0.29 ns Glucose consumption 0.83 < 0.0001 0.44 < 0.05 Lactate production 0.71 < 0.001 0.63 < 0.01 Glycerol release 0.66 < 0.001 0.87 < 0.0001 FFA release 0.28 ns 0.50 < 0.05 FFA re-esteri®cation 0.68 < 0.001 0.68 < 0.001 ATP 0.88 < 0.0001 0.88 < 0.0001 ADP 0.69 < 0.001 0.66 < 0.001 AMP 70.14 ns 70.13 ns Total adenines 0.89 < 0.0001 0.89 < 0.0001 ATP/ADP ratio 70.12 ns 0.00 ns 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 In the human body, catecholamines stimulate heat Figure 2 Regression of BMI and basal lactate production production, especially by non shivering thermogen- (r 70.52; P < 0.05). esis, 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 accor- dance 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 Fol- lowing stimulation, glycolysis and the triglyceride/ FFA cycle show a higher contribution to the total Figure 3 Regression of BMI and FFA re-esteri®cation after cellular thermogenesis (15 and 27%, respectively) stimulation with 1076 M isoprenaline (r 70.63; P< 0.01). than under basal conditions. Consequently, the isoprenaline induced increase of cellular heat production is mainly due to the stimulation of glycolysis and the derived from human adipocytes in vivo or in agarose triglyceride/FFA cycle. The observed negative corre- gel cultures and suspensions.19,23,26 Considering the lation between FFA re-esteri®cation and BMI (Figure positive correlation of cell size and the rates of 3) implies that the reduced fat cell thermogenesis in oxidative phosphorylation, glycolysis and lipoly- the face of augmented BMI might be due to a sis,5,15,27±30 it is not likely that the observed decrease decreased activity of the triglyceride/FFA cycle. of thermogenic rate with increasing BMI (Figure 1) is Under basal and/or stimulated conditions, numer- due to an increased fat cell size.31 Indeed, human ous correlations of cellular thermogenesis with sub- obesity is associated with increased fat cell thermo- strate metabolism (Table 3) and BMI were observed. genesis despite a larger cell size.21 Yet, most of the metabolic pathways revealed no Employing reference values of the standard heat of correlation with the BMI. This paradoxical ®nding formation,32 the heat production of oxidative phos- in our obese subjects is dif®cult to explain. In the phorylation, glycolysis and the triglyceride/FFA sub- frame of the present study fat cell size could not be strate cycle might be estimated. (Yet, since there are assessed, but there is convincing evidence existing no standard conditions in the cell, the results of these that obesity is associated with increased fat cell size.31 calculations might deviate from the actual heat pro- Furthermore there are several reports showing that duction.19 Heat production derived from oxidative increased fat cell size is associated with increased processes was 4.8 mW/mgDNA (85% of total cellular rates of oxygen and glucose consumption, glycerol thermogenesis), while 0.6 mW/mgDNA (11% of total and FFA release, as well as FFA re-esteri®ca- thermogenesis) were generated by glycolysis. The tion.5,15,27±30 However, in only some of these evalua- triglyceride/FFA substrate cycle14±17 produced tions has the factor of human obesity been 0.7 mW/mgDNA (12% of total thermogenesis). contemplated. Thus, the question might be raised, Decreased fat cell thermogenesis in obese humans H BoÈttcher and P FuÈrst 443 which underlying pathophysiological mechanisms Conclusions in¯uence ¯ux and metabolism of substrates.

The overall results of the present study show that reduction of total body energy expenditure can be Implications for the whole body caused by alterations of energy metabolism on cellular It is notable that the estimated total body fat cell level. The methodology used in this work enables thermogenesis, as calculated from cellular heat pro- future studies related to cellular heat production, not duction and known values for total body fat cell only under physiological conditions, but also under number,15 does not exceed 5% of the resting meta- the in¯uence of thermogenic drugs. Indeed, studies bolic rate under basal and 7% under stimulated con- with other cell types like hepatocytes or muscle ®bres ditions. Indeed, these calculated values are in good from lean and obese donors might be of interest and agreement with data derived from in vivo studies.33,34 render new information related to drug therapy of The results of the present study therefore suggest, that obesity. a decrease of energy turnover and consequently a reduced thermogenic capacity of white adipocytes might only in part contribute to the development of Acknowledgements obesity. Yet, the relationship between fat cell and total This work was supported by grants of the Deutsche body thermogenesis is made complex by the fact that Forschungsgemeinschaft (Fu-153/4-1), the Interna- massive obesity is accompanied by an increased fat tional Foundation for the Promotion of Nutrition cell number.15,44 According to a computer generated Research and Nutrition Education (ISFE) and the numerical simulation,45 an about 20% reduction of the European Society of Parenteral and Enteral Nutrition resting metabolic rate would be necessary to make a (ESPEN). We are indebted to Dr. E. Brand and his lean person obese, provided that energy intake surgical staff for the excellent cooperation, to Mrs. A. remains unchanged. This could be the case if the Stublinac/Liebscher for FFA analyses and to Mrs. A. energy expenditure of all body cells would be uni- Harmsen for her excellent technical assistance. formly reduced by the same extent as in the fat cells examined in the present study. 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