HSL Phosphorylation Research Articles

A PDE1 inhibitor reduces adipogenesis in mice via regulation of lipolysis and adipogenic cell signaling

Vinpocetine, a phosphodiesterase (PDE) type-1 inhibitor, increases cAMP and cGMP levels and is currently used for the management of cerebrovascular disorders, such as stroke, cerebral hemorrhage, and cognitive dysfunctions. In this study, we first determined that vinpocetine effectively suppressed adipogenesis and lipid accumulation. However, we questioned which molecular mechanism is involved because the role of PDE in adipogenesis is still controversial. Vinpocetine decreased adipogenic cell signaling, including the phosphorylation of ERK, AKT, JAK2, and STAT3, and adipokine secretion, including IL-6, IL-10, and IFN-α. Interestingly, vinpocetine increased the phosphorylation of HSL, suggesting the induction of the lipolysis pathway. Moreover, vinpocetine increased UCP1 expression via increasing cAMP and PKA phosphorylation. The administration of vinpocetine with a normal-chow diet (NFD) or a high-fat diet (HFD) in mice attenuated body weight gain in mice fed both the NFD and HFD. These effects were larger in the HFD-fed mice, without a difference in food intake. Vinpocetine drastically decreased fat weight and adipocyte cell sizes in gonadal and inguinal white adipose tissues and in the liver in both diet groups. Serum triacylglycerol levels and fasting blood glucose levels were reduced by vinpocetine treatment. This study suggested that vinpocetine prevents adipocyte differentiation through the inhibition of adipogenesis-associated cell signaling in the early stages of adipogenesis. Moreover, upregulating cAMP levels leads to an increase in lipolysis and UCP1 expression and then inhibits lipid accumulation. Therefore, we suggest that vinpocetine could be an effective agent for treating obesity, as well as improving cognition and cardiovascular function in older individuals.

Human adipose beiging in response to cold and mirabegron.

The induction of beige adipocytes in s.c. white adipose tissue (WAT) depots of humans is postulated to improve glucose and lipid metabolism in obesity. The ability of obese, insulin-resistant humans to induce beige adipose tissue is unknown. We exposed lean and obese research participants to cold (30-minute ice pack application each day for 10 days of the upper thigh) or treated them with the β3 agonist mirabegron. We determined beige adipose marker expression by IHC and quantitative PCR, and we analyzed mitochondrial bioenergetics and UCP activity with an Oxytherm system. Cold significantly induced UCP1 and TMEM26 protein in both lean and obese subjects, and this response was not associated with age. Interestingly, these proteins increased to the same extent in s.c. WAT of the noniced contralateral leg, indicating a crossover effect. We further analyzed the bioenergetics of purified mitochondria from the abdominal s.c. WAT of cold-treated subjects and determined that repeat ice application significantly increased uncoupled respiration, consistent with the UCP1 protein induction and subsequent activation. Cold also increased State 3 and maximal respiration, and this effect on mitochondrial bioenergetics was stronger in summer than winter. Chronic treatment (10 weeks; 50 mg/day) with the β3 receptor agonist mirabegron induces UCP1, TMEM26, CIDEA, and phosphorylation of HSL on serine660 in obese subjects. Cold or β3 agonists cause the induction of beige adipose tissue in human s.c. WAT; this phenomenon may be exploited to increase beige adipose in older, insulin-resistant, obese individuals. Clinicaltrials.gov NCT02596776, NCT02919176. NIH (DK107646, DK112282, P20GM103527, and by CTSA grant UL1TR001998).

Kupffer cells activation promoted binge drinking-induced fatty liver by activating lipolysis in white adipose tissues.

Kupffer cells (KCs) have been suggested to play critical roles in chronic ethanol induced early liver injury, but the role of KCs in binge drinking-induced hepatic steatosis remains unclear. This study was designed to investigate the roles of KCs inhibitor (GdCl3) and TNF-α antagonist (etanercept) on binge drinking-induced liver steatosis and to explore the underlying mechanisms. C57BL/6 mice were exposed to three doses of ethanol (6g/kg body weight) to mimic binge drinking-induced fatty liver. The results showed that both GdCl3 and etanercept partially but significantly alleviated binge drinking-induced increase of hepatic triglyceride (TG) level, and reduced fat droplets accumulation in mice liver. GdCl3 but not etanercept significantly blocked binge drinking-induced activation of KCs. However, neither GdCl3 nor etanercept could affect binge drinking-induced decrease of PPAR-α, ACOX, FAS, ACC and SCD protein levels, or increase of the LC3 II/LC3 I ratio and p62 protein level. Interestingly, both GdCl3 and etanercept significantly suppressed binge drinking-induced phosphorylation of HSL in epididymal adipose tissues. Results of in vitro studies with cultured epididymal adipose tissues showed that TNF-α could increase the phosphorylation of HSL in adipose tissues and upgrade the secretion of free fatty acid (FFA) in the culture medium. Taken together, KCs inhibitor and TNF-α antagonist could partially attenuate binge drinking-induced liver steatosis, which might be attributed to the suppression of mobilization of white adipose tissues. These results suggest that KCs activation may promote binge drinking-induced fatty liver by TNF-α mediated activation of lipolysis in white adipose tissues.

Cinnamaldehyde induces fat cell-autonomous thermogenesis and metabolic reprogramming

ObjectiveCinnamaldehyde (CA) is a food compound that has previously been observed to be protective against obesity and hyperglycemia in mouse models. In this study, we aimed to elucidate the mechanisms behind this protective effect by assessing the cell-autonomous response of primary adipocytes to CA treatment. MethodsPrimary murine adipocytes were treated with CA and thermogenic and metabolic responses were assessed after both acute and chronic treatments. Human adipose stem cells were differentiated and treated with CA to assess whether the CA-mediated signaling is conserved in humans. ResultsCA significantly activated PKA signaling, increased expression levels of thermogenic genes and induced phosphorylation of HSL and PLIN1 in murine primary adipocytes. Inhibition of PKA or p38 MAPK enzymatic activity markedly inhibited the CA-induced thermogenic response. In addition, chronic CA treatment regulates metabolic reprogramming, which was partially diminished in FGF21KO adipocytes. Importantly, both acute and chronic effects of CA were observed in human adipose stem cells isolated from multiple donors of different ethnicities and ages and with a variety of body mass indexes (BMI). ConclusionsCA activates thermogenic and metabolic responses in mouse and human primary subcutaneous adipocytes in a cell-autonomous manner, giving a mechanistic explanation for the anti-obesity effects of CA observed previously and further supporting its potential metabolic benefits on humans. Given the wide usage of cinnamon in the food industry, the notion that this popular food additive, instead of a drug, may activate thermogenesis, could ultimately lead to therapeutic strategies against obesity that are much better adhered to by participants.

Estradiol does not directly regulate adipose lipolysis

ABSTRACTThe mechanisms by which estradiol modulates adipose lipolysis are poorly understood. We sought to measure basal and β3-stimulated indices of lipoysis (FFAs, glycerol) in vivo in E2 deficient or supplemented rats, and ex vivo with direct acute E2 exposure. For 2 weeks, ovariectomized (OVX) and OVX rats treated with a daily oral dose of E2 (OVX E2) were pairfed to SHAM controls (n = 12 per group). Adipocyte size was modestly (∼40%) increased in OVX rats, but did not reach significance (p = 0.2). After 2 weeks, half of the animals in each group received an in vivo injection of saline or 1 mg/kg of the β3 agonist CL 316, 243. Serum FFA concentrations, but not glycerol, were lower in OVX and OVX E2 rats compared with SHAM controls (p = 0.02). A significant CL response was present in all groups (p<0.001) and HSL activation was unaffected by OVX or OVX E2 in retroperitoneal (r.p.) or inguinal (iWAT) adipose depots in vivo. Ex vivo, CL increased FFA and glycerol accumulation in the media as well as HSL phosphorylation by several fold in r.p. and iWAT explants, but responses from OVX and OVX E2 rats were comparable to SHAMs. To assess whether E2 can directly affect lipolysis, r.p. and iWAT tissue was treated with E2, CL or E2 + CL for 2, 4 or 8 hours using adipose tissue organ culture. CL stimulated FFA release (p<0.001), but was unaffected by E2. Overall, our results indicate that E2 does not directly regulate adipose tissue lipolysis.

Divergent functions of endotrophin on different cell populations in adipose tissue.

Endotrophin is a cleavage product of collagen 6 (Col6) in adipose tissue (AT). Previously, we demonstrated that endotrophin serves as a costimulator to trigger fibrosis and inflammation within the unhealthy AT milieu. However, how endotrophin affects lipid storage and breakdown in AT and how different cell types in AT respond to endotrophin stimulation remain unknown. In the current study, by using a doxycycline-inducible mouse model, we observed significant upregulation of adipogenic genes in the white AT (WAT) of endotrophin transgenic mice. We further showed that the mice exhibited inhibited lipolysis and accelerated hypertrophy and hyperplasia in WAT. To investigate the effects of endotrophin in vitro, we incubated different cell types from AT with conditioned medium from endotrophin-overexpressing 293T cells. We found that endotrophin activated multiple pathological pathways in different cell types. Particularly in 3T3-L1 adipocytes, endotrophin triggered a fibrotic program by upregulating collagen genes and promoted abnormal lipid accumulation by downregulating hormone-sensitive lipolysis gene and decreasing HSL phosphorylation levels. In macrophages isolated from WAT, endotrophin stimulated higher expression of the collagen-linking enzyme lysyl oxidase and M1 proinflammatory marker genes. In the stromal vascular fraction isolated from WAT, endotrophin induced upregulation of both profibrotic and proinflammatory genes. In conclusion, our study provides a new perspective on the effect of endotrophin in abnormal lipid accumulation and a mechanistic insight into the roles played by adipocytes and a variety of other cell types in AT in shaping the unhealthy microenvironment upon endotrophin treatment.

Decreased Whole-Body Fat Mass Produced by Chronic Alcohol Consumption is Associated with Activation of S6K1-Mediated Protein Synthesis and Increased Autophagy in Epididymal White Adipose Tissue.

Chronic alcohol consumption leads to a loss of white adipose tissue (WAT) but the underlying mechanisms for this lipodystrophy are not fully elucidated. This study tested the hypothesis that the reduction in WAT mass in chronic alcohol-fed mice is associated with a decreased protein synthesis specifically related to impaired function of mammalian target of rapamycin (mTOR). Adult male mice were provided an alcohol-containing liquid diet for 24weeks or an isonitrogenous isocaloric control diet. In vivo protein synthesis was determined at this time and thereafter epididymal WAT (eWAT) was excised for analysis of signal transduction pathways central to controling protein synthesis and degradation. While chronic alcohol feeding decreased whole-body and eWAT mass, this was associated with a discordant increase in protein synthesis in eWAT. This increase was not associated with a change in mTOR, 4E-BP1, Akt, or PRAS40 phosphorylation. Instead, a selective increase in phosphorylation of S6K1 and its downstream substrates, S6 and eIF4B was detected in alcohol-fed mice. Alcohol also increased eEF2K phosphorylation and decreased eEF2 phosphorylation consistent with increased translation elongation. Alcohol increased Atg12-5, LC3B-I and -II, and ULK1 S555 phosphorylation, suggesting increased autophagy, while markers of apoptosis (cleaved caspase-3 and -9, and PARP) were unchanged. Lipolytic enzymes (ATGL and HSL phosphorylation) were increased and lipogenic regulators (PPARγ and C/EBPα) were decreased in eWAT by alcohol. Although alcohol increased TNF-α, IL-6, and IL-1β mRNA, no change in key components of the NLRP3 inflammasome (NLRP3, ACS, and cleaved caspase-1) was detected suggesting alcohol did not increase pyroptosis. Plasma insulin did not differ between groups. These results demonstrate that the alcohol-induced decrease in whole-body fat mass resulted in part from activation of autophagy in eWAT as protein synthesis was increased and mediated by the specific increase in the activity of S6K1.

Fasting upregulates adipose triglyceride lipase and hormone-sensitive lipase levels and phosphorylation in mouse kidney.

Circulating non-esterified fatty acids (NEFA) rise during fasting and are taken up by the kidneys, either directly from the plasma or during re-uptake of albumin from glomerular filtrate, and are stored as triacylglycerol (TAG). Subsequent utilization of stored fatty acids requires their hydrolytic release from cellular lipid droplets, but relatively little is known about renal lipolysis. We found that total [(3)H]triolein hydrolase activity of kidney lysates was significantly increased by 15% in the fasted state. Adipose triglyceride lipase (Atgl) and hormone-sensitive lipase (Hsl) mRNA expression was time-dependently increased by fasting, along with other fatty acid metabolism genes (Pparα, Cd36, and Aox). ATGL and HSL protein levels were also significantly induced (by 239 ± 7% and 322 ± 8%, respectively). Concomitant with changes in total protein levels, there was an increase in ATGL phosphorylation at the AMPK-regulated serine 406 site in the 14-3-3 binding motif, and an increase in HSL phosphorylation at serines 565 and 660 that are regulated by AMPK and PKA, respectively. Using immunofluorescence, we further demonstrate nearly ubiquitous expression of ATGL in the renal cortex with a concentration on the apical/lumenal surface of some cortical tubules. Our findings suggest a role for ATGL and HSL in kidney lipolysis.

Adipose triglyceride lipase deletion from adipocytes, but not skeletal myocytes, impairs acute exercise performance in mice.

Adipose triglyceride lipase (ATGL) is the rate-limiting enzyme mediating triacylglycerol hydrolysis in virtually all cells, including adipocytes and skeletal myocytes, and hence, plays a critical role in mobilizing fatty acids. Global ATGL deficiency promotes skeletal myopathy and exercise intolerance in mice and humans, and yet the tissue-specific contributions to these phenotypes remain unknown. The goal of this study was to determine the relative contribution of ATGL-mediated triacylglycerol hydrolysis in adipocytes vs. skeletal myocytes to acute exercise performance. To achieve this goal, we generated murine models with adipocyte- and skeletal myocyte-specific targeted deletion of ATGL. We then subjected untrained mice to acute peak and submaximal exercise interventions and assessed exercise performance and energy substrate metabolism. Impaired ATGL-mediated lipolysis within adipocytes reduced peak and submaximal exercise performance, reduced peripheral energy substrate availability, shifted energy substrate preference toward carbohydrate oxidation, and decreased HSL Ser(660) phosphorylation and mitochondrial respiration within skeletal muscle. In contrast, impaired ATGL-mediated lipolysis within skeletal myocytes was not sufficient to reduce peak and submaximal exercise performance or peripheral energy substrate availability and instead tended to enhance metabolic flexibility during peak exercise. Furthermore, the expanded intramyocellular triacylglycerol pool in these mice was reduced following exercise in association with preserved HSL phosphorylation, suggesting that HSL may compensate for impaired ATGL action in skeletal muscle during exercise. These data suggest that adipocyte rather than skeletal myocyte ATGL-mediated lipolysis plays a greater role during acute exercise in part because of compensatory mechanisms that maintain lipolysis in muscle, but not adipose tissue, when ATGL is absent.

Skeletal muscle mitochondrial uncoupling drives endocrine cross-talk through the induction of FGF21 as a myokine

UCP1-Tg mice with ectopic expression of uncoupling protein 1 (UCP1) in skeletal muscle (SM) are a model of improved substrate metabolism and increased longevity. Analysis of myokine expression showed an induction of fibroblast growth factor 21 (FGF21) in SM, resulting in approximately fivefold elevated circulating FGF21 in UCP1-Tg mice. Despite a reduced muscle mass, UCP1-Tg mice showed no evidence for a myopathy or muscle autophagy deficiency but an activation of integrated stress response (ISR; eIF2α/ATF4) in SM. Targeting mitochondrial function in vitro by treating C2C12 myoblasts with the uncoupler FCCP resulted in a dose-dependent activation of ISR, which was associated with increased expression of FGF21, which was also observed by treatment with respiratory chain inhibitors antimycin A and myxothiazol. The cofactor required for FGF21 action, β-klotho, was expressed in white adipose tissue (WAT) of UCP1-Tg mice, which showed an increased browning of WAT similar to what occurred in altered adipocyte morphology, increased brown adipocyte markers (UCP1, CIDEA), lipolysis (HSL phosphorylation), and respiratory capacity. Importantly, treatment of primary white adipocytes with serum of transgenic mice resulted in increased UCP1 expression. Additionally, UCP1-Tg mice showed reduced body length through the suppressed IGF-I-GH axis and decreased bone mass. We conclude that the induction of FGF21 as a myokine is coupled to disturbance of mitochondrial function and ISR activation in SM. FGF21 released from SM has endocrine effects leading to increased browning of WAT and can explain the healthy metabolic phenotype of UCP1-Tg mice. These results confirm muscle as an important endocrine regulator of whole body metabolism.

Lipocalin 2 is an important regulator of lipid metabolism and energy utilization

We have recently identified lipocalin 2 (LCN2) as an adipose-derived cytokine playing a critical role in thermoregulation. In this study, we further explored the efficiency of energy utilization in response to acute cold exposure in Lcn2−/− mice. We found that after exposed to 4°C for 5hs, Lcn2−/− mice developed significantly lower blood glucose levels compared with wild type (WT) mice. The liver and muscle glycogen contents were markedly reduced, while serum fatty acid and lactate levels were significantly higher in Lcn2−/− mice compared to WT mice after 5h cold exposure. The gene expression patterns in brown adipose tissue and muscle showed that cold-induced expression of PGC1α and UCP1 was not different between WT and Lcn2−/− mice. Compared to WT, Lcn2−/− mice had significantly lower mRNA expression of mitochondrial function-related genes Nrf1, EERα, COXiv, and ATP5β in BAT and muscle. Furthermore, Lcn2−/− WAT had higher levels of cold-induced HSL phosphorylation than WT WAT indicating an increased lipolysis in Lcn2−/− mice. TZD treatment completely rescued cold intolerance and reversed serum parameters and mitochondrial gene expression. Our results suggest that LCN2 deficiency impairs mitochondrial function and lipid utilization leading to decreased energy production and cold intolerance. Thus, we conclude that LCN2 has a key role in lipid metabolism and energy utilization.

17β‐estradiol supplementation attenuates ovariectomy‐induced increases in ATGL signaling and reduced perilipin expression in visceral adipose tissue

Adipocytes from post-menopausal females have higher basal lipolytic rates than pre-menopausal females, which contributes to increased risk of developing dyslipidemia following menopause. The purpose of this study was to delineate cellular mechanisms affecting adipose tissue function in the ovariectomized (OVX) mouse and also determine if physical activity or estrogen supplementation alter any detected changes. Female C57/Bl6 mice were placed into SHAM, OVX sedentary (OVX), OVX exercise (OVX-Ex), and OVX sedentary + 17beta-estradiol (OVX + E(2)) groups. Visceral fat mass, glycerol, and NEFA levels were significantly higher in OVX mice compared to SHAM animals, but were not elevated in the E(2)-treated animals. Voluntary running failed to change circulating levels of glycerol or NEFA in OVX mice, but did partially attenuate the increase in visceral fat mass. Adipose triglyceride lipase (ATGL) protein content was significantly elevated in visceral fat from OVX and OVX-Ex groups compared to SHAM, while ATGL-CGI-58 interaction was significantly higher in OVX than SHAM and OVX + E(2) mice. No significant differences in HSL phosphorylation were detected between groups, however, ERK1/2 phosphorylation was significantly elevated in the OVX mice. To determine if ERK1/2 function was critical for the increased glycerol levels, visceral fat was treated with MEK inhibitor PD98059, with no differences in glycerol release detected. Perilipin protein content was decreased significantly in OVX and OVX-Ex mice compared to SHAM. Thus, these data suggest that increased ATGL signaling and reduced perilipin protein content may contribute to increased NEFA and glycerol levels in OVX mice, which are attenuated with E(2) treatment, but not by exercise.

Impaired Skeletal Muscle β-Adrenergic Activation and Lipolysis Are Associated with Whole-Body Insulin Resistance in Rats Bred for Low Intrinsic Exercise Capacity

Rats selectively bred for high endurance running capacity (HCR) have higher insulin sensitivity and improved metabolic health compared with those bred for low endurance capacity (LCR). We investigated several skeletal muscle characteristics, in vitro and in vivo, that could contribute to the metabolic phenotypes observed in sedentary LCR and HCR rats. After 16 generations of selective breeding, HCR had approximately 400% higher running capacity (P < 0.001), improved insulin sensitivity (P < 0.001), and lower fasting plasma glucose and triglycerides (P < 0.05) compared with LCR. Skeletal muscle ceramide and diacylglycerol content, basal AMP-activated protein kinase (AMPK) activity, and basal lipolysis were similar between LCR and HCR. However, the stimulation of lipolysis in response to 10 mum isoproterenol was 70% higher in HCR (P = 0.004). Impaired isoproterenol sensitivity in LCR was associated with lower basal triacylglycerol lipase activity, Ser660 phosphorylation of HSL, and beta2-adrenergic receptor protein content in skeletal muscle. Expression of the orphan nuclear receptor Nur77, which is induced by beta-adrenergic signaling and is associated with insulin sensitivity, was lower in LCR (P < 0.05). Muscle protein content of Nur77 target genes, including uncoupling protein 3, fatty acid translocase/CD36, and the AMPK gamma3 subunit were also lower in LCR (P < 0.05). Our investigation associates whole-body insulin resistance with impaired beta-adrenergic response and reduced expression of genes that are critical regulators of glucose and lipid metabolism in skeletal muscle. We identify impaired beta-adrenergic signal transduction as a potential mechanism for impaired metabolic health after artificial selection for low intrinsic exercise capacity.

Phosphorylation of hormone‐sensitive lipase by protein kinase A in vitro promotes an increase in its hydrophobic surface area

Hormone-sensitive lipase (EC 3.1.1.79; HSL) is a key enzyme in the mobilization of fatty acids from stored triacylglycerols. HSL activity is controlled by phosphorylation of at least four serines. In rat HSL, Ser563, Ser659 and Ser660 are phosphorylated by protein kinase A (PKA) in vitro as well as in vivo, and Ser660 and Ser659 have been shown to be the activity-controlling sites in vitro. The exact molecular events of PKA-mediated activation of HSL in vitro are yet to be determined, but increases in both Vmax and S0.5 seem to be involved, as recently shown for human HSL. In this study, the hydrophobic fluorescent probe 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) was found to inhibit the hydrolysis of triolein by purified recombinant rat adipocyte HSL, with a decrease in the effect of bis-ANS upon PKA phosphorylation of HSL. The interaction of HSL with bis-ANS was found to have a Kd of 1 microM in binding assays. Upon PKA phosphorylation, the interactions of HSL with both bis-ANS and the alternative probe SYPRO Orange were increased. By negative stain transmission electron microscopy, phosphorylated HSL was found to have a closer interaction with phospholipid vesicles than unphosphorylated HSL. Taken together, our results show that HSL increases its hydrophobic nature upon phosphorylation by PKA. This suggests that PKA phosphorylation induces a conformational change that increases the exposed hydrophobic surface and thereby facilitates binding of HSL to the lipid substrate.

Anti-lipolytic Action of AMP-activated Protein Kinase in Rodent Adipocytes

Despite its importance in terms of energy homeostasis, the role of AMP-activated protein kinase in adipose tissue remains controversial. Initial studies have described an anti-lipolytic role for AMP-activated protein kinase, whereas more recent studies have suggested the converse. Thus we have addressed the role of AMP-activated protein kinase in adipose tissue by modulating AMP-activated protein kinase activity in primary rodent adipocytes using pharmacological activators or by adenoviral expression of dominant negative or constitutively active forms of the kinase. We then studied the effects of AMP-activated protein kinase activity modulation on lipolytic mechanisms. Finally, we analyzed the consequences of a genetic deletion of AMP-activated protein kinase in mouse adipocytes. AMP-activated protein kinase activity in adipocytes is represented mainly by the alpha(1) isoform and is induced by all of the stimuli that increase cAMP in adipocytes, including fasting. When AMP-activated protein kinase activity is increased by 5-aminoimidazole-4-carboxamide-riboside, phenformin, or by the expression of a constitutively active form, isoproterenol-induced lipolysis is strongly reduced. Conversely, when AMP-activated protein kinase activity is decreased either by a dominant negative form or in AMP-activated protein kinase alpha(1) knock-out mice, lipolysis is increased. We present data suggesting that AMP-activated protein kinase acts on hormone-sensitive lipase by blocking its translocation to the lipid droplet. We conclude that, in mature adipocytes, AMP-activated protein kinase activation has a clear anti-lipolytic effect.

Mechanism of intracellular calcium ([Ca2+]i) inhibition of lipolysis in human adipocytes.

We investigated the mechanisms responsible for the anti-lipolytic effect of intracellular Ca2+ ([Ca2+]i) in human adipocytes. Increasing [Ca2+]i inhibited lipolysis induced by b-adrenergic receptor activation, A1 adenosine receptor inhibition, adenylate cyclase activation, and phosphodiesterase (PDE) inhibition, as well as by a hydrolyzable cAMP analog, but not by a nonhydrolyzable cAMP analog. This finding indicates that the anti-lipolytic effect of [Ca2+]i may be mediated by the activation of adipocyte PDE. Consistent with this theory, [Ca2+]i inhibition of isoproterenol-stimulated lipolysis was reversed completely by the nonselective PDE inhibitor isobutyl methylxanthine and also by the selective PDE 3B inhibitor cilostamide, but not by selective PDE 1 and 4 inhibitors. In addition, phosphatidylinositol-3 kinase inhibition with wortmannin completely prevented insulin's anti-lipolytic effect but only minimally blocked [Ca2+]i's effect, which suggests that [Ca2+]i and insulin may activate PDE 3B via different mechanisms. In contrast, the antilipolytic effect of [Ca2+]i was not affected by inhibitors of calmodulin, Ca2+/calmodulin-dependent kinase, protein phosphatase 2B, and protein kinase C. Finally, [Ca2+]i inhibited significantly isoproterenol-stimulated increases in cAMP levels and hormone-sensitive lipase phosphorylation in human adipocytes. In conclusion, increasing [Ca2+]i exerts an antilipolytic effect mainly by activation of PDE, leading to a decrease in cAMP and HSL phosphorylation and, consequently, inhibition of lipolysis.

On the control of lipolysis in adipocytes.

The lipolytic reaction in adipocytes is one of the most important reactions in the management of bodily energy reserves, and dysregulation of this reaction may contribute to the symptoms of Type 2 diabetes mellitus. Yet, progress on resolving the molecular details of this reaction has been relatively slow. However, recent developments at the molecular level begin to paint a clearer picture of lipolysis and point to a number of unanswered questions. While HSL has long been known to be the rate-limiting enzyme of lipolysis, the mechanism by which HSL attacks the droplet lipids is not yet firmly established. Certainly, the immunocytochemical evidence showing the movement of HSL to the lipid droplet upon stimulation leaves little doubt that this translocation is a key aspect of the lipolytic reaction, but whether or not HSL phosphorylation contributes to the translocation, and at which site(s), is as yet unresolved. It will be important to establish whether there is an activation step in addition to the translocation reaction. The participation of perilipin A is indicated by the findings that this protein can protect neutral lipids within droplets from hydrolysis, but active participation in the lipolytic reaction is yet to be proved. Again, it will be important to determine whether mutations of serine residues of PKA phosphorylation sites of perilipins prevent lipolysis, and whether such modifications abolish the physical changes in the droplet surfaces that accompany lipolysis.

Mechanism of hormone-stimulated lipolysis in adipocytes: translocation of hormone-sensitive lipase to the lipid storage droplet.

Hormone-sensitive lipase activity (HSL), which is found in the supernatant of centrifuged homogenates of lipolytically quiet isolated rat adipocytes, was greatly reduced in or absent from the supernatant of lipolytically stimulated cells. The lipase was purified 100- to 250-fold from the supernatant of lipolytically quiet cells to 10-20% purity by a single passage over phenyl-Sepharose resin with high (greater than 70%) activity yields. Western blotting of adipocyte homogenate fractions with polyclonal antiserum raised against HSL showed that the enzyme shifted quantitatively from the supernatant of control cells to the floating "fat cake" of lipolytically stimulated cells. A similar shift to the fat cake was observed when cells were disrupted by hypotonic lysis and centrifugation rather than by homogenization. We propose that upon lipolytic activation of adipocytes and phosphorylation of HSL by cAMP-dependent protein kinase, the critical event is not an increase in catalytic activity (i.e., turnover number) but a translocation of the lipase to its substrate at the surface of the lipid storage droplet.