Cyclic AMP and glycerol concentrations in freeze-clamped human omental and subcutaneous adipose tissues.

OBJECTIVE: To compare intracellular concentrations of cyclic AMP, GTP, ATP, AMP and glycerol in omental and abdominal subcutaneous adipose tissues. DESIGN: Samples of omental and/or abdominal subcutaneous adipose tissues of 14 women undergoing elective surgery were freeze-clamped, deproteinized with perchloric acid and neutralized. All subjects were obese since it was not possible to freeze-clamp lean individuals. MEASUREMENTS: The concentrations of ATP and GTP were determined by ion-paired high performance liquid chromatography and AMP and glycerol enzymatically. Cyclic AMP was determined by radioimmunoassay. Intracellular nucleotide concentrations were calculated from tissue weights, water contents and relative amounts of sodium and potassium. RESULTS: The intracellular concentration of cyclic AMP in omental adipose tissue (31 mumol/l) was twice as high as that in subcutaneous fat while those of ATP, GTP and AMP were similar at the two sites. Glycerol concentrations were higher in subcutaneous (472 mumol/l) than in omental (325 mumol/l) adipose tissue. CONCLUSION: The results suggest higher relative stimulation of lipolysis by cyclic AMP in omental adipose tissue than in the subcutaneous region in situ. However, probably because of differences in blood flow and possibly in the lipolytic machinery, this is not reflected as a higher glycerol concentration.

Weight loss normalizes the inhibitory effect of N6-(phenylisopropyl)adenosine on lipolysis in fat cells of massively obese human subjects.

1. Fat cells were isolated from massively obese patients at or before gastric bypass, from other patients after normalization of body weight after gastric bypass or gastroplasty (post-bypass patients) and from control subjects of a stable normal body weight. 2. The inhibition of isoprenaline-stimulated lipolysis by N6-(phenylisopropyl)adenosine in the presence of adenosine deaminase was much attenuated in cells from the massively obese patients as compared with those from normal-weight control subjects, but was normal in cells from post-bypass patients. 3. Isolated fat cells of the massively obese patients were larger (913 +/- 197 pl, mean +/- SEM) than those of the normal-weight group (437 +/- 95 pl). The volume of cells from the post-bypass patients was only 125 +/- 49 pl, although the body mass index of this group was almost exactly the same as that of the normal-weight control subjects. 4. Although epidemiological studies have suggested that genetic factors are important in the development and maintenance of obesity, these results demonstrate that the changes observed in the inhibitory regulation of lipolysis in obesity are secondary.

Pyruvate carboxylase in genetic obesity.

Immunoblotting and protein microsequencing were used to identify several adipocyte proteins expressed in an obesity-related fashion in the Zucker rat. One of these was a 116-kDa particulate protein (p116). The p116 levels in adipocytes from 5- to 7-wk-old obese Zucker rats were two- to fivefold higher on a per milligram of protein basis than levels in lean animals and decreased after the induction of streptozotocin-induced diabetes mellitus. This suggests the change may be related to the actions of insulin. Hepatic levels of p116 did not change. The p116 was purified to homogeneity from obese Zucker rat adipocytes, and polyclonal antisera were prepared against the purified protein in rabbits. Microanalysis of electroblotted p116 proteolytic fragments suggested that p116 was pyruvate carboxylase (PC). Other evidence that p116 was PC included the following: 1) p116 contained biotin, 2) p116 in particulate subcellular fractions was soluble after freeze-lysis, 3) antibodies to p116 reacted with purified hepatic PC, 4) p116 and purified hepatic PC had identical pI and relative molecular weight values, and 5) similar changes were detected in adipocyte p116 and PC enzyme activity during obesity and after the induction of streptozotocin-induced diabetes mellitus. Increased adipose tissue PC probably contributes to the increased lipogenic capacity of young obese Zucker rat adipocytes.

Attenuated adenosine-sensitivity and decreased adenosine-receptor number in adipocyte plasma membranes in human obesity.

Fat-cells were isolated from patients of body-mass indices (BMIs) ranging from 17.9 to 83.9 kg/m2. Isoprenaline-stimulated cyclic AMP accumulation in cells prepared from obese subjects as compared with normal-weight subjects, was less sensitive to inhibition by the adenosine agonist N6-(phenylisopropyl)adenosine (PIA) (P = 0.047). The inhibition of 7 beta-desacetyl-7 beta-[gamma-(N-methylpiperazino) butyryl]-forskolin-stimulated adenylate cyclase by PIA in the presence of adenosine deaminase was also much attenuated in crude plasma membranes of adipocytes prepared from massively obese patients as compared with lean controls (P = 0.0143). This difference was probably not due to different cell size, because adenylate cyclase of crude plasma membranes of large adipocytes was actually more sensitive to PIA than was adenylate cyclase of membranes of smaller fat-cells co-isolated from the same individual. The stimulatory effect of PIA on glucose uptake in the presence of adenosine deaminase was depressed in adipocytes prepared from obese subjects and correlated with BMI at r = -0.626 (P = 0.007) at 100 nM-PIA. The adenosine receptors were studied by using the adenosine antagonist 1,3-[3H]dipropyl-8-cyclopentylxanthine. The binding was rapid and proportional to protein concentration. There was no difference in the affinities of receptors in membranes of obese and normal-weight subjects; Kd values of all patients averaged 3.3 nM. Bmax values were 54 and 130 fmol/mg of protein in membranes prepared from seven obese and five control patients respectively. The Bmax values calculated per mg of protein correlated with BMI at r = -0.539 (P = 0.047). The adenosine content of adipose tissue was higher in obese than in control subjects. These results demonstrate an attenuated response of cyclic AMP accumulation, adenylate cyclase and glucose uptake to adenosine in fat-cells prepared from obese subjects, and suggest that this change is at least partly due to changes in the amount of adenosine receptors, but not their affinity. The decreased receptor number could be due to higher adenosine content. A higher adenosine concentration in adipose tissue could explain why lipolysis is inhibited in situ in obesity, and the desensitization could explain the diminished response to adenosine analogues in isolated fat-cells.