| Abstract: | OBJECTIVE—In human adipocytes, the cAMP-dependent pathway mediates signals originating from β-adrenergic activation, thus playing a key role in the regulation of important metabolic processes, i.e., lipolysis and thermogenesis. Cyclic AMP effects are mainly mediated by protein kinase A (PKA), whose R2B regulatory isoform is the most expressed in mouse adipose tissue, where it protects against diet-induced obesity and fatty liver development. The aim of the study was to investigate possible differences in R2B expression, PKA activity, and lipolysis in adipose tissues from obese and nonobese subjects.RESEARCH DESIGN AND METHODS—The expression of the different PKA regulatory subunits was evaluated by immunohistochemistry, Western blot, and real-time PCR in subcutaneous and visceral adipose tissue samples from 20 nonobese and 67 obese patients. PKA activity and glycerol release were evaluated in total protein extract and adipocytes isolated from fresh tissue samples, respectively.RESULTS—Expression techniques showed that R2B was the most abundant regulatory protein, both at mRNA and protein level. Interestingly, R2B mRNA levels were significantly lower in both subcutaneous and visceral adipose tissues from obese than nonobese patients and negatively correlated with BMI, waist circumference, insulin levels, and homeostasis model assessment of insulin resistance. Moreover, both basal and stimulated PKA activity and glycerol release were significantly lower in visceral adipose tissue from obese patients then nonobese subjects.CONCLUSIONS—Our results first indicate that, in human adipose tissue, there are important BMI-related differences in R2B expression and PKA activation, which might be included among the multiple determinants involved in the different lipolytic response to β-adrenergic activation in obesity.Cyclic AMP is implicated in the regulation of a variety of cell functions that are, at least in part, related to protein phosphorylation through the activation of protein kinase A (PKA). In addition to the control of differentiated functions, such as motility, secretion, metabolism, differentiation, synaptic transmission, and ion channel activities, cAMP inhibits or stimulates cell proliferation depending on the cell type. In human adipocytes, the cAMP-dependent pathway mediates signals originating from the activation of β-adrenergic receptors, thus playing a key role in the regulation of important metabolic processes, such as lipolysis and thermogenesis. Cyclic AMP effects are mainly mediated by PKA, a tetrameric enzyme composed of two catalytic subunits associated with two regulatory subunits. There are four different regulatory subunit genes and proteins (R1A, R1B, R2A, and R2B) expressed with a tissue-specific pattern and exerting distinct roles in cell differentiation and growth control (1). Dramatic changes in the proportion of the two PKA regulatory subunits, R1 and R2, occur during ontogenic development, differentiation processes, and neoplastic transformation, indicating distinct roles for these isoenzymes in cell homeostasis and growth control (2,3). In the past few years, many studies seem to indicate that signaling via PKA plays an important role in regulating metabolism and body weight (4). In particular, the R2B isoform has been demonstrated to be, in mice, the most expressed in three tissues known to regulate energy homeostasis, i.e., brown adipose tissue, white adipose tissue, and brain (4,5). In general, the activation of the holoenzyme PKA in fat is now thought to decrease obesity, as demonstrated in both genetically obese (ob/ob) (6,7) and diet-induced obese mice (8). As far as the R2B subunit is concerned, studies in mice lacking this specific PKA subunit have revealed an unexpected role for this protein in regulating energy balance (5). R2B knockout mice (RIIß−/−) remain remarkably lean, even when challenged with a high-fat diet (5). These animals have increased metabolic activity, manifested by increases in body temperature, uncoupling protein 1 concentration, and lipid hydrolysis. Biochemical studies have shown that loss of R2B is compensated by the increased R1A regulatory subunit, which is more sensitive to cAMP activation and results in a net increase in basal PKA activity (9).In contrast to this increasing knowledge in mice, little is known about the differential role played by the different PKA regulatory subunits in humans. Two studies from the same group have described lower R2B levels in adipose tissues from 10 normal-weight women affected with polycystic ovary syndrome (PCOS) when compared with 13 matched control women and associated this event with lipolytic catecholamine resistance and insulin resistance in these patients (10,11). We present the first study evaluating the relative expression of the different PKA isoforms in a large series of human adipose tissues. Our results indicate important BMI-related differences in R2B expression and PKA activation, which might play a role in the different lipolytic response to β-adrenergic activation in obese and nonobese subjects. |
