Catecholamine-stimulated Lipolysis Research Articles

Increased OCT3 Expression in Adipose Tissue With Aging: Implications for Catecholamine and Lipid Turnover and Insulin Resistance in Women.

Catecholamine-stimulated lipolysis is reduced with aging, which may promote adiposity and insulin resistance. Organic cation transporter 3 (OCT3), which is inhibited by estradiol (E2), mediates catecholamine transport into adipocytes for degradation, thus decreasing lipolysis. In this study, we investigated the association of OCT3 mRNA levels in subcutaneous adipose tissue (SAT) with aging and markers of insulin resistance in women. SAT biopsies were obtained from 66 women with (19) or without (47) type 2 diabetes (age 22-76 years, 20.0-40.1 kg/m2). OCT3 mRNA and protein levels were measured for group comparisons and correlation analysis. SAT was incubated with E2 and OCT3 mRNA levels were measured. Associations between OCT3 single nucleotide polymorphisms (SNPs) and diabetes-associated traits were assessed. OCT3 mRNA and protein levels in SAT increased with aging. SAT from postmenopausal women had higher levels of OCT3 than premenopausal women, and there was a dose-dependent reduction in OCT3 mRNA levels in SAT treated with E2. OCT3 mRNA levels were negatively associated with markers of insulin resistance, and ex vivo lipolysis. OCT3 SNPs were associated with BMI, waist to hip ratio, and circulating lipids (eg, triglycerides). OCT3 mRNA and protein levels in SAT increased with aging, and mRNA levels were negatively associated with markers of insulin resistance. E2 incubation downregulated OCT3 mRNA levels, which may explain lower OCT3 mRNA in premenopausal vs postmenopausal women. High OCT3 protein levels in adipose tissue may result in increased catecholamine degradation, and this can contribute to the reduction in lipolysis observed in women with aging.

CLSTN3 gene variant associates with obesity risk and contributes to dysfunction in white adipose tissue

ObjectiveWhite adipose tissue (WAT) possesses the remarkable remodeling capacity, and maladaptation of this ability contributes to the development of obesity and associated comorbidities. Calsyntenin-3 (CLSTN3) is a transmembrane protein that promotes synapse development in brain. Even though this gene has been reported to be associated with adipose tissue, its role in the regulation of WAT function is unknown yet. We aim to further assess the expression pattern of CLSTN3 gene in human adipose tissue, and investigate its regulatory impact on WAT function. MethodsIn our study, we observed the expression pattern of Clstn3/CLSTN3 gene in mouse and human WAT. Genetic association study and expression quantitative trait loci analysis were combined to identify the phenotypic effect of CLSTN3 gene variant in humans. This was followed by mouse experiments using adeno-associated virus-mediated human CLSTN3 overexpression in inguinal WAT. We investigated the effect of CLSTN3 on WAT function and overall metabolic homeostasis, as well as the possible underlying molecular mechanism. ResultsWe observed that CLSTN3 gene was routinely expressed in human WAT and predominantly enriched in adipocyte fraction. Furthermore, we identified that the variant rs7296261 in the CLSTN3 locus was associated with a high risk of obesity, and its risk allele was linked to an increase in CLSTN3 expression in human WAT. Overexpression of CLSTN3 in inguinal WAT of mice resulted in diet-induced local dysfunctional expansion, liver steatosis, and systemic metabolic deficiency. In vivo and ex vivo lipolysis assays demonstrated that CLSTN3 overexpression attenuated catecholamine-stimulated lipolysis. Mechanistically, CLSTN3 could interact with amyloid precursor protein (APP) in WAT and increase APP accumulation in mitochondria, which in turn impaired adipose mitochondrial function and promoted obesity. ConclusionTaken together, we provide the evidence for a novel role of CLSTN3 in modulating WAT function, thereby reinforcing the fact that targeting CLSTN3 may be a potential approach for the treatment of obesity and associated metabolic diseases.

Lipolysis defect in people with obesity who undergo metabolic surgery.

ObjectiveCross‐sectional studies demonstrate that catecholamine stimulation of fat cell lipolysis is blunted in obesity. We investigated whether this defect persists after substantial weight loss has been induced by metabolic surgery, and whether it is related to the outcome.Design/MethodsPatients with obesity not able to successfully reduce body weight by conventional means (n = 126) were investigated before and 5 years after Roux‐en‐Y gastric bypass surgery (RYGB). They were compared with propensity‐score matched subjects selected from a control group (n = 1017), and with the entire group after adjustment for age, sex, body mass index (BMI), fat cell volume and other clinical parameters. Catecholamine‐stimulated lipolysis (glycerol release) was investigated in isolated fat cells using noradrenaline (natural hormone) or isoprenaline (synthetic beta‐adrenoceptor agonist).ResultsFollowing RYGB, BMI was reduced from 39.9 (37.5–43.5) (median and interquartile range) to 29.5 (26.7–31.9) kg/m2 (p < 0.0001). The post‐RYGB patients had about 50% lower lipolysis rates compared with the matched and total series of controls (p < 0.0005). Nordrenaline activation of lipolysis at baseline was associated with the RYGB effect; those with high lipolysis activation (upper tertile) lost 30%–45% more in body weight, BMI or fat mass than those with low (bottom tertile) initial lipolysis activation (p < 0.0007).ConclusionPatients with obesity requiring metabolic surgery have impaired ability of catecholamines to stimulate lipolysis, which remains despite long‐term normalization of body weight by RYGB. Furthermore, preoperative variations in the ability of catecholamines to activate lipolysis may predict the long‐term reduction in body weight and fat mass.

β3-Adrenergic receptor downregulation leads to adipocyte catecholamine resistance in obesity.

The dysregulation of energy homeostasis in obesity involves multihormone resistance. Although leptin and insulin resistance have been well characterized, catecholamine resistance remains largely unexplored. Murine β3-adrenergic receptor expression in adipocytes is orders of magnitude higher compared with that of other isoforms. While resistant to classical desensitization pathways, its mRNA (Adrb3) and protein expression are dramatically downregulated after ligand exposure (homologous desensitization). β3-Adrenergic receptor downregulation also occurs after high-fat diet feeding, concurrent with catecholamine resistance and elevated inflammation. This downregulation is recapitulated in vitro by TNF-α treatment (heterologous desensitization). Both homologous and heterologous desensitization of Adrb3 were triggered by induction of the pseudokinase TRIB1 downstream of the EPAC/RAP2A/PI-PLC pathway. TRIB1 in turn degraded the primary transcriptional activator of Adrb3, CEBPα. EPAC/RAP inhibition enhanced catecholamine-stimulated lipolysis and energy expenditure in obese mice. Moreover, adipose tissue expression of genes in this pathway correlated with body weight extremes in a cohort of genetically diverse mice and with BMI in 2 independent cohorts of humans. These data implicate a signaling axis that may explain reduced hormone-stimulated lipolysis in obesity and resistance to therapeutic interventions with β3-adrenergic receptor agonists.

Glucose enhances catecholamine-stimulated lipolysis via increased glycerol-3-phosphate synthesis in 3T3-L1 adipocytes and rat adipose tissue.

During lipolysis, triglyceride (TG) are hydrolyzed into a glycerol and fatty acids in adipocyte. A significant portion of the fatty acids are re-esterificated into TG, and this is a critical step in promoting lipolysis. Although glycerol-3-phosphate (G3P) is required for triglyceride synthesis in mammalian cell, the substrate for G3P synthesis during active lipolysis is not known. A recent study showed that the inhibition of glucose uptake reduces catecholamine-stimulated lipolysis, suggesting that glucose availability is important in lipolysis in adipocytes. We hypothesized that glucose might play an essential role in generating G3P and thereby promoting catecholamine-stimulated lipolysis in adipocytes. Therefore, we determined the effect of glucose availability on catecholamine-stimulated lipolysis in 3T3-L1 adipocytes and rat adipose tissue. 3T3-L1 adipocytes and rat epididymal fat pads were cultured in a medium with/without glucose during stimulation by isoproterenol. Glycerol release was higher when adipocytes were cultured in a glucose-containing medium than that in a medium without glucose. Measurement of glucose uptake during catecholamine-stimulated lipolysis showed a slight, but significant increase in glucose uptake. We also compared glucose metabolism-related protein, such as glucose transporter 4, hexokinase, glycerol-3-phosphate dehydrogenase and lipase contents between fat tissues that play a critical role in active lipolysis. Epididymal fat exhibited higher lipolytic activity than inguinal fat because of higher lipase and glucose metabolism-related protein contents. We demonstrated that catecholamine-stimulated lipolysis is enhanced in the presence of glucose, and suggests that glucose is one of the primary substrates for G3P in adipocytes during active lipolysis.

CaV1.2 and CaV1.3 voltage-gated L-type Ca2+ channels in rat white fat adipocytes.

L-type channel antagonists are of therapeutic benefit in the treatment of hyperlipidaemia and insulin resistance. Our aim was to identify L-type voltage-gated Ca2+ channels in white fat adipocytes, and determine if they affect intracellular Ca2+, lipolysis and lipogenesis. We used a multidisciplinary approach of molecular biology, confocal microscopy, Ca2+ imaging and metabolic assays to explore this problem using adipocytes isolated from adult rat epididymal fat pads. CaV1.2, CaV1.3 and CaV1.1 alpha1, beta and alpha2delta subunits were detected at the gene expression level. The CaV1.2 and CaV1.3 alpha1 subunits were identified in the plasma membrane at the protein level. Confocal microscopy with fluorescent antibodies labelled CaV1.2 in the plasma membrane. Ca2+ imaging revealed that the intracellular Ca2+ concentration, [Ca2 +]i was reversibly decreased by removal of extracellular Ca2+, an effect mimicked by verapamil, nifedipine and Co2+, all blockers of L-type channels, whereas the Ca2+ channel agonist BAY-K8644 increased [Ca2+]i. The finding that the magnitude of these effects correlated with basal [Ca2+]i suggests that adipocyte [Ca2+]i is controlled by L-type Ca2+ channels that are constitutively active at the adipocyte depolarized membrane potential. Pharmacological manipulation of L-type channel activity modulated both basal and catecholamine-stimulated lipolysis but not insulin-induced glucose uptake or lipogenesis. We conclude that white adipocytes have constitutively active L-type Ca2+ channels which explains their sensitivity of lipolysis to Ca2+ channel modulators. Our data suggest CaV1.2 as a potential novel therapeutic target in the treatment of obesity.

Influence of Aging and Menstrual Status on Subcutaneous Fat Cell Lipolysis.

Aging is accompanied by inhibited fat cell mobilization of fatty acids through lipolysis, which may contribute to decreased energy expenditure in elderly subjects. However, the influence of menstrual status is unknown. To investigate the role of menstrual status on changes in lipolysis induced by aging. A longitudinal investigation with a mean 13-year interval. Ambulatory study at a clinical academic unit. Eighty-two continuously recruited women between 24 and 62 years of age and with body mass index 21 to 48 kg/m2 at first examination. Twenty-nine women continued to have normal menstruation, 42 developed irregular menstruation/menopause, and 11 had a perimenstrual/menopausal phenotype already at the first examination. Lipolysis measured as glycerol release from isolated subcutaneous fat cells incubated in vitro. On average, body weight/body fat mass levels did not change over time. In all 3 groups, aging was associated with a similar decrease in spontaneous (basal) and catecholamine-stimulated lipolysis. The latter was due to decreased signal transduction through stimulatory beta adrenoceptors and increased alpha-2-adrenoceptor-mediated antilipolytic effects. Gene microarray data from adipose tissue at baseline and follow-up (n = 53) showed that a limited set of lipolysis-linked genes, including phosphodiesterase-3B, were altered over time, but this was independent of menstrual status. Fat cell size also decreased during aging, but this could not explain the decrease in lipolysis. In women, the rate of fat cell lipolysis decreases during aging due to multiple alterations in spontaneous (basal) and catecholamine-induced lipolysis. This is independent of changes in menstrual status or fat cell size.

Association between Beta3-Adrenergic Receptor Trp64Arg Polymorphism and Fat Preference in Healthy Young Japanese Women.

Beta3-adrenergic receptor (ADRB3) is a mediator of catecholamine-stimulated lipolysis in humans. The Trp64Arg polymorphism with T/C transition in the ADRB3 gene has been considered to reduce lipolysis and metabolic expenditure. Here, we investigated the hitherto unknown role of the Trp64Arg substitution on food preference among healthy young adults (mean age, 24.3; n = 53, including 25 men). Preference toward four food types (bitter, sour, salty, or sweet) and greasy (high-fat) foods was examined using a self-reported questionnaire. There was no noticeable sex-difference in food preference. Incidentally, only among female subjects, the genotype frequencies of the Trp64Arg polymorphism were in accordance with the Hardy-Weinberg equilibrium. Consequently, female subjects were divided into two groups for further analyses: 18 subjects with TT genotype (Trp64Trp) (wild-type group) and 10 subjects with TC genotype (Trp64Arg) (heterozygous group). No significant difference was observed in preference for four food types between the groups. However, when sweet foods were divided into high-fat and low-fat subgroups, food preference for high-fat sweet foods in heterozygous group was significantly higher than that in wild-type group. Moreover, when subjects were divided into two classes based on preference for greasy foods (like, n = 16 or dislike, n = 12), the preference degree in heterozygous group who liked high-fat foods (n = 5) was significantly higher than that in wild-type group (n = 11), suggesting that the Trp64Arg substitution might genetically enhance high-fat food preference. Thus, understanding the relationship between ADRB3 Trp64Arg substitution and fat preference will be valuable for obesity prevention.

169 - Oxidized neutral lipid lipolysis regulates insulin signaling during acute stress

Acute hyperglycemia and systemic insulin resistance (IR) often develop after injury or surgery. This stress response, appropriately known as critical illness diabetes, leads to increased post-operative complications and mortality. Insulin administration reduces this hyperglycemia in some patients but has a risk of dangerous hypoglycemia. The underlying mechanisms driving IR are unknown, which limits the development of alternative therapies and emphasizes the need to elucidate the pathophysiology. It is, however, known that surgical animal models rapidly develop adipose IR, and impaired insulin action in adipose alone can result in whole-body IR. Therefore, we focused on how adipose contributes to stress-induced IR. We have previously demonstrated using a mouse model of surgery that the development of IR and hyperglycemia are dependent on adipose lipolysis. Furthermore, β-adrenergic stimulation of adipocytes causes inhibition and dissociation of mTORC1 and mTORC2, key steps in the insulin cascade, in a lipolysis-dependent manner. Therefore, we hypothesized that a product of adipose lipolysis was responsible for the development of IR. We found that lipid extracts isolated from forskolin-stimulated 3T3-L1 adipocytes inhibited the activity of mTORC in vitro, however “traditional” lipolytic products (DAG, MAG, fatty acids) had no effect. Interestingly, stimulating peroxidation of fatty acids further exacerbated mTORC inhibition, while antioxidants reversed this effect. This suggests that the active species is an oxidized fatty acid. In fact, incubation of mTORC with in vitro auto-oxidized fatty acids is sufficient to inhibit kinase activity. Therefore, we propose that lipolysis breaks down oxidized TAGs into oxidized fatty acids, which cause mTORC dissociation and attenuation of insulin signaling during the stress response. Our work will elucidate a novel role for oxidized TAGs and their lipolytic products in the regulation of insulin signaling. Defining the mechanism by which catecholamine-stimulated lipolysis attenuates insulin signaling will provide novel therapeutic targets for improved post-operative glucose homeostasis.

Cardiovascular risk score is linked to subcutaneous adipocyte size and lipid metabolism.

Although white adipose tissue mass and distribution correlates with cardiovascular disease, the fat cell-specific perturbations underlying this association are not known. We determined the relationship between adipocyte size and lipid metabolism with cardiovascular risk. Adipocyte size as well as spontaneous (basal) and hormone-stimulated effects on adipocyte lipid metabolism (lipolysis and lipogenesis) were investigated in abdominal subcutaneous adipose tissue of 304 men and 775 women. Subjects were classified into five categories according to Adult Treatment PanelIII (ATPIII) metabolic syndrome criteria. Adipocyte size increased with increasing ATPIII score (P<0.0001). For lipolysis, there was a gradual increase in basal and catecholamine-stimulated lipolysis and a decrease in insulin-mediated inhibition of stimulated lipolysis with ATPIII (P<0.0001). In contrast, the lipolytic action of atrial natriuretic peptide was similar between ATPIII classes. Basal and insulin-stimulated lipogenesis decreased with increasing score (P<0.0001). Circulating free fatty acid levels were 50% higher in the top risk category (4-5) compared with the lowest score (P<0.0001). Fat cell size correlated positively with increasing ATPIII score and lipolysis but negatively with lipogenesis. All these differences were independent of age, sex and body weight status (P<0.0001 to 0.02 after correction). When all functional measures were put together, maximum insulin-stimulated lipogenesis, insulin-antilipolytic sensitivity and basal lipolysis together explained about 20% in the variation of ATPIII in score. Independently of sex, age and body weight status, a high cardiovascular risk score associates with increased circulating free fatty acid levels and hormone-specific alterations of lipolysis/lipogenesis in enlarged subcutaneous fat cells.

Nuclear Perilipin 5 integrates lipid droplet lipolysis with PGC-1α/SIRT1-dependent transcriptional regulation of mitochondrial function.

Dysfunctional cellular lipid metabolism contributes to common chronic human diseases, including type 2 diabetes, obesity, fatty liver disease and diabetic cardiomyopathy. How cells balance lipid storage and mitochondrial oxidative capacity is poorly understood. Here we identify the lipid droplet protein Perilipin 5 as a catecholamine-triggered interaction partner of PGC-1α. We report that during catecholamine-stimulated lipolysis, Perilipin 5 is phosphorylated by protein kinase A and forms transcriptional complexes with PGC-1α and SIRT1 in the nucleus. Perilipin 5 promotes PGC-1α co-activator function by disinhibiting SIRT1 deacetylase activity. We show by gain-and-loss of function studies in cells that nuclear Perilipin 5 promotes transcription of genes that mediate mitochondrial biogenesis and oxidative function. We propose that Perilipin 5 is an important molecular link that couples the coordinated catecholamine activation of the PKA pathway and of lipid droplet lipolysis with transcriptional regulation to promote efficient fatty acid catabolism and prevent mitochondrial dysfunction.

Effects of Securigera securidaca Extract on Lipolysis and Adipogenesis in Diabetic Rats.

Diabetes mellitus is associated with dysregulation of adipose tissue metabolism and increased level of serum lipids. In our previous work we found that Securigera securidaca decreases cholesterol level in blood of diabetic rats. The present study was carried out to further investigate the effects of this plant on lipid metabolism, lipolysis, and adipogenesis, in diabetic rats. Female Wistar rats were rendered diabetic by intraperitoneal injection of streptozotocin. Retroperitoneal adipose tissue was removed from diabetic animals after seven days of streptozotocin injection. Effect of hydroalcoholic extract of S. securidaca seeds (100–800 μg/mL) on adipose tissue lipolysis was evaluated in ex vivo condition. Also, to evaluate adipogenesis, preadipocytes were isolated from adipose tissue and differentiated to adipocytes in the presence of the extract. The extract at concentration of 800 μg/mL decreased both basal and catecholamine-stimulated lipolysis (P < 0.05). Incubation of differentiating preadipocytes with 800 μg/mL of S. securidaca extract decreased intracellular lipid droplet accumulation as evaluated with Oil Red O staining (P < 0.001). The extract even at high concentrations had no effect on viability of preadipocytes. In conclusion, S. securidaca decreases lipolysis and adipogenesis without cytotoxicity, which makes it a good candidate for management of dyslipidemia and reduction of cardiovascular risks in diabetes.

Skeletal muscle as a target of LXR agonist after long-term treatment: focus on lipid homeostasis

The liver X receptors (LXR)α and LXRβ are transcription factors belonging to the nuclear receptor family, which play a central role in metabolic homeostasis, being master regulators of key target genes in the glucose and lipid pathways. Wild-type (WT), LXRα(-/-), and LXRβ(-/-) mice were fed a chow diet with (treated) or without (control) the synthetic dual LXR agonist GW3965 for 5 wk. GW3965 raised intrahepatic triglyceride (TG) level but, surprisingly, reduced serum TG level through the activation of serum lipase activity. The serum TG reduction was associated with a repression of both catecholamine-stimulated lipolysis and relative glucose incorporation into lipid in isolated adipocytes through activation of LXRβ. We also demonstrated that LXRα is required for basal (nonstimulated) adipocyte metabolism, whereas LXRβ acts as a repressor of lipolysis. On the contrary, in skeletal muscle (SM), the lipogenic and cholesterol transporter LXR target genes were markedly induced in WT and LXRα(-/-) mice and to a lesser extent in LXRβ(-/-) mice following treatment with GW3965. Moreover, TG content was reduced in SM of LXRβ(-/-) mice, associated with increased expression of the main TG-lipase genes Hsl and Atgl. Energy expenditure was increased, and a switch from glucose to lipid oxidation was observed. In conclusion, we provide evidence that LXR might be an essential regulator of the lipid balance between tissues to ensure appropriate control of the flux of fuel. Importantly, we show that, after chronic treatment with GW3965, SM becomes the target tissue for LXR activation, as opposed to liver, in acute treatment.

MicroRNA profiling links miR-378 to enhanced adipocyte lipolysis in human cancer cachexia

Cancer cachexia is associated with pronounced adipose tissue loss due to, at least in part, increased fat cell lipolysis. MicroRNAs (miRNAs) have recently been implicated in controlling several aspects of adipocyte function. To gain insight into the possible impact of miRNAs on adipose lipolysis in cancer cachexia, global miRNA expression was explored in abdominal subcutaneous adipose tissue from gastrointestinal cancer patients with (n = 10) or without (n = 11) cachexia. Effects of miRNA overexpression or inhibition on lipolysis were determined in human in vitro differentiated adipocytes. Out of 116 miRNAs present in adipose tissue, five displayed distinct cachexia-associated expression according to both microarray and RT-qPCR. Four (miR-483-5p/-23a/-744/-99b) were downregulated, whereas one (miR-378) was significantly upregulated in cachexia. Adipose expression of miR-378 associated strongly and positively with catecholamine-stimulated lipolysis in adipocytes. This correlation is most probably causal because overexpression of miR-378 in human adipocytes increased catecholamine-stimulated lipolysis. In addition, inhibition of miR-378 expression attenuated stimulated lipolysis and reduced the expression of LIPE, PLIN1, and PNPLA2, a set of genes encoding key lipolytic regulators. Taken together, increased miR-378 expression could play an etiological role in cancer cachexia-associated adipose tissue loss via effects on adipocyte lipolysis.

Regulation of human subcutaneous adipose tissue blood flow

Subcutaneous adipose tissue represents about 85% of all body fat. Its major metabolic role is the regulated storage and mobilization of lipid energy. It stores lipid in the form of triacylglycerol (TG), which is mobilized, as required for use by other tissues, in the form of non-esterified fatty acids (NEFA). Neither TG nor NEFA are soluble to any extent in water, and their transport to and out of the tissue requires specialized transport mechanisms and adequate blood flow. Subcutaneous adipose tissue blood flow (ATBF) is therefore tightly linked to the tissue's metabolic functioning. ATBF is relatively high (in the fasting state, similar to that of resting skeletal muscle, when expressed per 100 g tissue) and changes markedly in different physiological states. Those most studied are after ingestion of a meal, when there is normally a marked rise in ATBF, and exercise, when ATBF also increases. Pharmacological studies have helped to define the physiological regulation of ATBF. Adrenergic influences predominate in most situations, but nevertheless the regulation of ATBF is complex and depends on the interplay of many different systems. ATBF is downregulated in obesity (when expressed per 100 g tissue), and its responsiveness to meal intake is reduced. However, there is little evidence that this leads to adipose tissue hypoxia in human obesity, and we suggest that, like the downregulation of catecholamine-stimulated lipolysis seen in obesity, the reduction in ATBF represents an adaptation to the increased fat mass. Most information on ATBF has been obtained from studying the subcutaneous abdominal fat depot, but more limited information on lower-body fat depots suggests some similarities, but also some differences: in particular, marked alpha-adrenergic tone, which can reduce the femoral ATBF response to adrenergic stimuli.

Chronic AICAR-induced AMP-kinase activation regulates adipocyte lipolysis in a time-dependent and fat depot-specific manner in rats

This study investigated the effects of chronic in vivo AMP-kinase activation with 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) on lipolysis in subcutaneous inguinal, epididymal, and retroperitoneal fat pads. Male Wistar rats received daily single intraperitoneal injections of either saline or AICAR (0.7 g/kg body wt) for a period of 8 wk. The fat pads were used either to isolate adipocytes and measure basal and catecholamine-stimulated lipolysis or to assess signaling steps of lipolysis after 4 and 8 wk of AICAR treatment. Blood was sampled weekly to measure nonesterified fatty acids (NEFAs). AICAR treatment reduced basal and catecholamine-stimulated lipolysis at week 4 in adipocytes from all fat depots. However, at week 8, catecholamine-induced lipolysis significantly increased in inguinal and retroperitoneal adipocytes. Interestingly, plasma levels of NEFAs were also decreased and subsequently increased at 4 and 8 wk, respectively. The lipolytic cascade of the inguinal fat pad was the most drastically affected by the treatment, since the phosphorylation and content of most proteins involved in lipolysis were consistently undetected in this tissue after 4 and 8 wk of AICAR treatment. The enhancement of catecholamine-induced lipolysis in inguinal and retroperitoneal adipocytes after 8 wk of AICAR treatment was accompanied by increased contents of adipose triglyceride lipase (ATGL) and perilipin A in these fat depots. In summary, despite depot-specific regulation of the lipolytic cascade, catecholamine-induced lipolysis in isolated adipocytes correlated well with plasma NEFA concentrations in the course of chronic AICAR-induced AMPK activation. The mechanisms underlying these effects also involved time-dependent and depot-specific regulation of hormone-sensitive lipase, ATGL, and perilipin.

Estradiol acutely inhibits whole body lipid oxidation and attenuates lipolysis in subcutaneous adipose tissue: a randomized, placebo-controlled study in postmenopausal women

Estradiol (E(2)) promotes and maintains the female phenotype characterized by subcutaneous fat accumulation. There is evidence to suggest that this effect is due to increased anti-lipolytic α2A-adrenergic receptors, but whether this requires long-term exposure to E(2) or is an immediate effect is not clear. To study acute effects of a single dose (4 mg) of 17β-E(2) on regional and systemic lipolysis. Sixteen postmenopausal women (age, 595 years; weight, 6710 kg; and BMI, 24.82.9) were studied in a crossover design: i) placebo and ii) 4 mg E(2). Basal and adrenaline-stimulated regional lipolysis was assessed by microdialysis and substrate oxidation rates by indirect calorimetry. Tissue biopsies were obtained to assess lipoprotein lipase activity and mRNA expression of adrenergic, estrogen, cytokine, and vascular reactivity receptors. Acute E(2) stimulation significantly attenuated catecholamine-stimulated lipolysis in femoral subcutaneous adipose tissue (interstitial glycerol concentration (micromole/liter) ANOVA time vs treatment interaction, P=0.01) and lipolysis in general in abdominal adipose tissue (ANOVA treatment alone, P<0.05). E(2) also reduced basal lipid oxidation ((mg/kg per min) placebo, 0.58 ± 0.06 vs E(2), 0.45 ± 0.03; P=0.03) and induced a significantly higher expression of anti-lipolytic α2A-adrenergic receptor mRNA (P=0.02) in skeletal muscle tissue as well as an upregulation of eNOS (NOS3) mRNA (P=0.02). E(2) acutely attenuates the lipolytic response to catecholamines in subcutaneous adipose tissue, shifts muscular adrenergic receptor mRNA toward anti-lipolytic α2A-receptors, decreases whole body lipid oxidation, and enhances expression of markers of vascular reactivity.

Identification and Functional Characterization of Protein Kinase A Phosphorylation Sites in the Major Lipolytic Protein, Adipose Triglyceride Lipase

Catecholamine-stimulated lipolysis occurs by activating adenylate cyclase and raising cAMP levels, thereby increasing protein kinase A (PKA) activity. This results in phosphorylation and modulated activity of several key lipolytic proteins. Adipose triglyceride lipase (ATGL) is the primary lipase for the initial step in triacylglycerol hydrolysis, and ATGL activity is increased during stimulated lipolysis. Here, we demonstrate that murine ATGL is phosphorylated by PKA at several serine residues in vitro and identify Ser(406) as a functionally important site. ATGL null adipocytes expressing ATGL S406A (nonphosphorylatable) had reduced stimulated lipolysis. Studies in mice demonstrated increased ATGL Ser(406) phosphorylation during fasting and moderate intensity exercise, conditions associated with elevated lipolytic rates. ATGL Ser(404) (corresponding to murine Ser(406)) phosphorylation was increased by β-adrenergic stimulation but not 5'AMP-activated protein kinase activation in human subcutaneous adipose tissue explants, which correlated with lipolysis rates. Our studies suggest that β-adrenergic activation can result in PKA-mediated phosphorylation of ATGL Ser(406), to moderately increase ATGL-mediated lipolysis.

Rye bran alkylresorcinols suppress adipocyte lipolysis and hormone‐sensitive lipase activity

The effects of alkylresorcinols (ARs) isolated from rye bran on adipocyte lipolysis, hormone-sensitive lipase activity and phosphorylation and on phosphorylation of protein kinase A substrates were studied. Preincubation with ARs for 18 h suppressed catecholamine-stimulated lipolysis in 3T3-L1 adipocytes. Furthermore, phosphorylation of hormone-sensitive lipase (HSL), a key lipase responsible for stimulated lipolysis, and phosphorylation of protein kinase A substrates, were diminished after preincubation with ARs, whereas HSL protein expression was unaltered. ARs were also shown to inhibit HSL activity in an in vitro assay.

Relationship between Site-Specific HSL Phosphorylation and Adipocyte Lipolysis in Obese Women

Background/Aims: In fat cells of obese humans, basal lipolysis is increased but catecholamine-stimulated lipolysis is blunted. This is linked to decreased expression of hormone-sensitive lipase (HSL). Upon stimulation by cAMP, HSL is phosphorylated at several serine residues (P-Ser⁵⁵², P-Ser⁶⁴⁹ and P-Ser⁶⁵⁰) leading to enzymatic activation. In contrast, P-Ser⁵⁵⁴ prevents phosphorylation at Ser⁵⁵² and is thus considered an inactivating site. We hypothesized that differences in HSL phosphorylation could be linked to disturbed adipocyte lipolysis in obesity. Methods: Phosphorylation at Ser⁵⁵², Ser⁵⁵⁴, Ser⁶⁵⁰ as well as total HSL and adipose triglyceride lipase (ATGL) protein expression were assessed by Western blot in subcutaneous adipose tissue samples of 32 obese women. Basal and stimulated lipolysis in isolated fat cells were correlated to phosphorylation levels. Results: While there was no correlation between basal lipolysis and P-Ser⁶⁵⁰ or P-Ser⁵⁵⁴, there was a negative correlation with P-Ser⁵⁵² (r = 0.39; p < 0.05). In contrast, only P-Ser⁵⁵⁴ was strongly and negatively correlated with noradrenaline- (r = –0.50; p < 0.01) and dibutyryl cAMP-stimulated (r = –0.45; p < 0.05) lipolysis. There were no significant correlations between any measure of lipolysis and total levels of HSL and ATGL. Conclusion: In contrast to total HSL and ATGL levels, phosphorylation at Ser⁵⁵⁴ and Ser⁵⁵², but not at Ser⁶⁵⁰, may differentially predict adipocyte lipolysis in vitro. Posttranslational modifications of HSL may therefore constitute an important regulator of adipocyte lipolysis, at least in adipose tissue of obese women. Whether this is also relevant in lean individuals remains to be demonstrated.