FFA‐Induced Adipocyte Inflammation and Insulin Resistance: Involvement of ER Stress and IKKβ Pathways

Free‐fatty acids (FFAs) are well‐characterized factor for causing production of inflammatory factors and insulin resistance in adipocytes. Using cultured adipocytes, we demonstrate that FFAs can activate endoplasmic reticulum (ER) stress pathway by examination of ER stress sensor activation and marker gene expression. Chemical chaperone tauroursodeoxycholic acid (TUDCA) can reduce FFA‐induced adipocyte inflammation and improve insulin signaling whereas overexpression of spliced X‐box protein 1 (XBP‐1s) only attenuates FFA‐induced inflammation. PKR‐like eukaryotic initiation factor 2α kinase (PERK) is one of the three major ER stress sensor proteins and deficiency of PERK alleviates FFA‐induced inflammation and insulin resistance. The key downstream target of FFA‐induced ER stress is IκB kinase β (IKKβ), a master kinase for regulating expression of inflammatory genes. Deficiency of PERK attenuates FFA‐induced activation of IKKβ and deficiency of IKKβ alleviates FFA‐induced inflammation and insulin resistance. Consistently, overexpression of IKKβ in 3T3‐L1 CAR adipocytes causes inflammation and insulin resistance. In addition, IKKβ overexpression has profound effect on adipocyte lipid metabolism, including inhibition of lipogenesis and promotion of lipolysis. Furthermore, increased endogenous IKKβ expression and activation is also observed in isolated primary adipocytes from mice injected with lipids or fed on high‐fat diet (HFD) acutely. These results indicate that ER stress pathway is a key mediator for FFA‐induced inflammation and insulin resistance in adipocytes with PERK and IKKβ as the critical signaling components.     

Regulated localization of Rab18 to lipid droplets: effects of lipolytic stimulation and inhibition of lipid droplet catabolism

Rab GTPases are crucial regulators of membrane traffic. Here we have examined a possible association of Rab proteins with lipid droplets (LDs), neutral lipid-containing organelles surrounded by a phospholipid monolayer, also known as lipid bodies, which have been traditionally considered relatively inert storage organelles. Although we found close apposition between LDs and endosomal compartments labeled by expressed Rab5, Rab7, or Rab11 constructs, there was no detectable labeling of the LD surface itself by these Rab proteins. In contrast, GFP-Rab18 localized to LDs and immunoelectron microscopy showed direct association with the monolayer surface. Green fluorescent protein (GFP)-Rab18-labeled LDs underwent oscillatory movements in a localized area as well as sporadic, rapid, saltatory movements both in the periphery of the cell and toward the perinuclear region. In both adipocytes and non-adipocyte cell lines Rab18 localized to a subset of LDs. To gain insights into this specific localization, Rab18 was co-expressed with Cav3DGV, a truncation mutant of caveolin-3 shown to inhibit the catabolism and motility of lipid droplets. GFP-Rab18 and mRFP-Cav3DGV labeled mutually exclusive subpopulations of LDs. Moreover, in 3T3-L1 adipocytes, stimulation of lipolysis increased the localization of Rab18 to LDs, an effect reversed by beta-adrenergic antagonists. These results show that a Rab protein localizes directly to the monolayer surface of LDs. In addition, association with the LD surface was increased following stimulation of lipolysis and inhibited by a caveolin mutant suggesting that recruitment of Rab18 is regulated by the metabolic state of individual LDs.     

Regulation of lipogenesis by glucocorticoids and insulin in human adipose tissue

Patients with glucocorticoid (GC) excess, Cushing's syndrome, develop a classic phenotype characterized by central obesity and insulin resistance. GCs are known to increase the release of fatty acids from adipose, by stimulating lipolysis, however, the impact of GCs on the processes that regulate lipid accumulation has not been explored. Intracellular levels of active GC are dependent upon the activity of 11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) and we have hypothesized that 11β-HSD1 activity can regulate lipid homeostasis in human adipose tissue (Chub-S7 cell line and primary cultures of human subcutaneous (sc) and omental (om) adipocytes. Across adipocyte differentiation, lipogenesis increased whilst β-oxidation decreased. GC treatment decreased lipogenesis but did not alter rates of β-oxidation in Chub-S7 cells, whilst insulin increased lipogenesis in all adipocyte cell models. Low dose Dexamethasone pre-treatment (5 nM) of Chub-S7 cells augmented the ability of insulin to stimulate lipogenesis and there was no evidence of adipose tissue insulin resistance in primary sc cells. Both cortisol and cortisone decreased lipogenesis; selective 11β-HSD1 inhibition completely abolished cortisone-mediated repression of lipogenesis. GCs have potent actions upon lipid homeostasis and these effects are dependent upon interactions with insulin. These in vitro data suggest that manipulation of GC availability through selective 11β-HSD1 inhibition modifies lipid homeostasis in human adipocytes.     

Postlipolytic insulin-dependent remodeling of micro lipid droplets in adipocytes

Despite the lipolysis-lipogenesis cycle being a fundamental process in adipocyte biology, very little is known about the morphological changes that occur during this process. The remodeling of lipid droplets to form micro lipid droplets (mLDs) is a striking feature of lipolysis in adipocytes, but once lipolysis ceases, the cell must regain its basal morphology. We characterized mLD formation in cultured adipocytes, and in primary adipocytes isolated from mouse epididymal fat pads, in response to acute activation of lipolysis. Using real-time quantitative imaging and electron tomography, we show that formation of mLDs in cultured adipocytes occurs throughout the cell to increase total LD surface area by ~30% but does not involve detectable fission from large LDs. Peripheral mLDs are monolayered structures with a neutral lipid core and are sites of active lipolysis. Electron tomography reveals preferential association of mLDs with the endoplasmic reticulum. Treatment with insulin and fatty acids results in the reformation of macroLDs and return to the basal state. Insulin-dependent reformation of large LDs involves two distinct processes: microtubule-dependent homotypic fusion of mLDs and expansion of individual mLDs. We identify a physiologically important role for LD fusion that is involved in a reversible lipolytic cycle in adipocytes.     

Adipose triglyceride lipase regulates basal lipolysis and lipid droplet size in adipocytes

In adipocytes, lipid droplet (LD) size reflects a balance of triglyceride synthesis (lipogenesis) and hydrolysis (lipolysis). Perilipin A (Peri A) is the most abundant phosphoprotein on the surface of adipocyte LDs and has a crucial role in lipid storage and lipolysis. Adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) are the major rate-determining enzymes for lipolysis in adipocytes. Each of these proteins (Peri A, ATGL, and HSL) has been demonstrated to regulate lipid storage and release in the adipocyte. However, in the absence of protein kinase A (PKA) stimulation (basal state), the lipases (ATGL and HSL) are located mainly in the cytoplasm, and their contribution to basal rates of lipolysis and influence on LD size are poorly understood. In this study, we utilize an adenoviral system to knockdown or overexpress ATGL and HSL in an engineered model system of adipocytes in the presence or absence of Peri A. We are able to demonstrate in our experimental model system that in the basal state, LD size, triglyceride storage, and fatty acid release are mainly influenced by the expression of ATGL. These results demonstrate for the first time the relative contributions of ATGL, HSL, and Peri A on determination of LD size in the absence of PKA stimulation.     

Role of the sulfonylurea receptor in regulating human adipocyte metabolism.

A regulatory role for intracellular Ca2+ ([Ca2+]i) in adipocyte lipogenesis, lipolysis and triglyceride accumulation has been demonstrated. Compounds acting on the pancreatic sulfonylurea receptor (SUR) to increase (e.g., glibenclamide) or decrease (e.g., diazoxide) [Ca2+]i cause corresponding increases and decreases in weight gain. However, these weight gain and loss effects have been attributed to insulin release rather than to the primary effects of these compounds on the adipocyte SUR and its associated K(ATP) channel. Accordingly, we have evaluated the direct role of the human adipocyte SUR in regulating adipocyte metabolism. We used RT-PCR with primers for a highly conserved region of SUR1 to demonstrate that human adipocytes express SUR1. The PCR product was confirmed by sequence analysis and used as a probe to demonstrate adipocyte SUR1 expression by Northern blot analysis. Adipocytes exhibited glibenclamide dose-responsive (0-20 microM) increases in [Ca2+]i (P<0.05). Similarly, glibenclamide (10 microM) caused a 67% increase in adipocyte fatty acid synthase activity (P<0.001), a 48% increase in glycerol-3-phosphate dehydrogenase activity (P<0.01) and a 68% inhibition in lipolysis (P<0.01), whereas diazoxide (10 microM) completely prevented each of these effects. These data demonstrate that human adipocytes express a SUR that regulates [Ca2+]i and, consequently, exerts coordinate control over lipogenesis and lipolysis. Accordingly, the adipocyte SUR1 may represent an important target for the development of therapeutic interventions in obesity.     

Hexosamine Biosynthesis Is a Possible Mechanism Underlying Hypoxia’s Effects on Lipid Metabolism in Human Adipocytes

Introduction

Hypoxia regulates adipocyte metabolism. Hexosamine biosynthesis is implicated in murine 3T3L1 adipocyte differentiation and is a possible underlying mechanism for hypoxia’s effects on adipocyte metabolism.

Methods

Lipid metabolism was studied in human visceral and subcutaneous adipocytes in in vitro hypoxic culture with adipophilic staining, glycerol release, and palmitate oxidation assays. Gene expression and hexosamine biosynthesis activation was studied with QRTPCR, immunofluorescence microscopy, and Western blotting.

Results

Hypoxia inhibits lipogenesis and induces basal lipolysis in visceral and subcutaneous human adipocytes. Hypoxia induces fatty acid oxidation in visceral adipocytes but had no effect on fatty acid oxidation in subcutaneous adipocytes. Hypoxia inhibits hexosamine biosynthesis in adipocytes. Inhibition of hexosamine biosynthesis with azaserine attenuates lipogenesis and induces lipolysis in adipocytes in normoxic conditions, while promotion of hexosamine biosynthesis with glucosamine in hypoxic conditions slightly increases lipogenesis.

Conclusions

Hypoxia’s net effect on human adipocyte lipid metabolism would be expected to impair adipocyte buffering capacity and contribute to systemic lipotoxicity. Our data suggest that hypoxia may mediate its effects on lipogenesis and lipolysis through inhibition of hexosamine biosynthesis. Hexosamine biosynthesis represents a target for manipulation of adipocyte metabolism.     

Lipid metabolism and adipokine levels in fatty acid-binding protein null and transgenic mice

Fatty acid-binding proteins (FABPs) facilitate the diffusion of fatty acids within cellular cytoplasm. Compared with C57Bl/6J mice maintained on a high-fat diet, adipose-FABP (A-FABP) null mice exhibit increased fat mass, decreased lipolysis, increased muscle glucose oxidation, and attenuated insulin resistance, whereas overexpression of epithelial-FABP (E-FABP) in adipose tissue results in decreased fat mass, increased lipolysis, and potentiated insulin resistance. To identify the mechanisms that underlie these processes, real-time PCR analyses indicate that the expression of hormone-sensitive lipase is reduced, while perilipin A is increased in A-FABP/aP2 null mice relative to E-FABP overexpressing mice. In contrast, de novo lipogenesis and expression of genes encoding lipoprotein lipase, CD36, long-chain acyl-CoA synthetase 5, and diacylglycerol acyltransferase are increased in A-FABP/aP2 null mice relative to E-FABP transgenic animals. Consistent with an increase in de novo lipogenesis, there was an increase in adipose C16:0 and C16:1 acyl-CoA pools. There were no changes in serum free fatty acids between genotypes. Serum levels of resistin were decreased in the E-FABP transgenic mice, whereas serum and tissue adiponectin were increased in A-FABP/aP2 null mice and decreased in E-FABP transgenic animals; leptin expression was unaffected. These results suggest that the balance between lipolysis and lipogenesis in adipocytes is remodeled in the FABP null and transgenic mice and is accompanied by the reprogramming of adipokine expression in fat cells and overall changes in plasma adipokines.     

Dynamic changes in lipid droplet-associated proteins in the “browning” of white adipose tissues

The morphological and functional differences between lipid droplets (LDs) in brown (BAT) and white (WAT) adipose tissues will largely be determined by their associated proteins. Analysing mRNA expression in mice fat depots we have found that most LD protein genes are expressed at higher levels in BAT, with the greatest differences observed for Cidea and Plin5. Prolonged cold exposure, which induces the appearance of brown-like adipocytes in mice WAT depots, was accompanied with the potentiation of the lipolytic machinery, with changes in ATGL, CGI-58 and G0S2 gene expression. However the major change detected in WAT was the enhancement of Cidea mRNA. Together with the increase in Cidec, it indicates that LD enlargement through LD–LD transference of fat is an important process during WAT browning. To study the dynamics of this phenotypic change, we have applied 4D confocal microscopy in differentiated 3T3-L1 cells under sustained β-adrenergic stimulation. Under these conditions the cells experienced a LD remodelling cycle, with progressive reduction on the LD size by lipolysis, followed by the formation of new LDs, which were subjected to an enlargement process, likely to be CIDE-triggered, until the cell returned to the basal state. This transformation would be triggered by the activation of a thermogenic futile cycle of lipolysis/lipogenesis and could facilitate the molecular mechanism for the unilocular to multilocular transformation during WAT browning. This article is part of a Special Issue entitled Brown and White Fat: From Signaling to Disease.     

Stimulation of lipogenesis in rat adipocytes by ATP, a ligand for P2-receptors

The activation of P2-receptors has a wide range of diverse effects in many tissues. Here we show that extracellular ATP stimulates lipogenesis in adipocytes derived from the epididymal fat pads of male Wistar rats. The lipogenic effect of ATP is not susceptible to treatment of adipocytes with adenosine deaminase or an adenosine receptor antagonist. Degradation of ATP in adipocyte suspension by ectonucleotidases is slow and remaining ATP concentrations are sufficient to activate P2-receptors. ATP does not affect basal or insulin stimulated glucose transport, or basal or isoproterenol stimulated lipolysis, respectively. The lipogenic effect of ATP is mimicked by the adenine compounds, ADP, AMP, and beta,gamma-methylene-ATP, but not by other nucleotides (UTP, UDP, CTP, GTP, ITP, and diadenosine tetraphosphate), indicating that extracellular nucleotides stimulate lipogenesis via a P2-receptor. ATP and its receptor may define a signalling system in adipocytes, which regulates fat stores independently from established hormones.     

Creb-Pgc1α pathway modulates the interaction between lipid droplets and mitochondria and influences high fat diet-induced changes of lipid metabolism in the liver and isolated hepatocytes of yellow catfish

Although the crucial role of lipid droplets (LDs), mitochondria (MT) and their interactions in regulating lipid metabolism are well accepted, the mechanism of LDs-MT interactions in high fat diet (HFD)-induced changes of lipid metabolism remains unknown. Thus, this study was conducted to determine the mechanism of LDs-MT interactions in HFD-induced changes of lipid accumulation. We found that HFD not only up-regulated the expression of key proteins linked with TAG biosynthesis, but also increased the expression of proteins involved in lipolysis and fatty acid (FA) oxidation in LDs, including Rab32 (the only Rab protein associated with the MT). FA-induced LDs accumulation coincided with increased mitochondrial biogenesis, suggesting the potential LDs-MT interaction in hepatocytes after FA incubation. Also, FA incubation markedly increased the localization of Rab32 into LDs and MT, which confirmed the LDs-MT interaction and indicated the involvement of Rab32 in LDs-MT interaction following FA incubation. Inhibitors of Creb-Pgc1α pathway significantly blocked the localization of Rab32 into LDs and MT, and significantly reduced FA-induced LDs lipolysis by targeting Atgl and Plin5. Meanwhile, the FA-enhanced LDs accumulation, and mitochondrial biogenesis, fusion and oxidation were also significantly repressed. These indicated the regulatory role of Creb-Pgc1α in Rab32-mediated LDs-MT interactions and lipolysis after FA incubation. Taken together, these results revealed a novel mechanism of HFD- and FA-induced LDs-MT interactions in regulating hepatic LDs lipolysis, which provided new insight into the crosstalk between LDs-MT interaction and their potential role in HFD-induced hepatic steatosis.     

Effects of dietary fat and adipose tissue location on insulin action in young boar adipocytes

1. Regulation of lipogenesis and lipolysis by insulin was studied on adipocytes isolated from 100 kg Large white male pigs. Two adipose tissues were studied: subcutaneous and perirenal. Animals were fed either a control low fat diet or a diet containing 14.7% sunflower seed oil. 2. The cell diameter was higher in the group fed the sunflower diet. 3. De novo lipogenesis was decreased for each adipose tissue in the group fed the sunflower diet. The perirenal site had a higher lipogenic activity than subcutaneous site whatever the diet. 4. Insulin did not significantly stimulate lipogenesis but had an important antilipolytic effect on stimulated lipolysis by isoproterenol. 5. The antilipolytic action of insulin was higher in perirenal adipocytes with the control diet. With the sunflower diet, the decrease was about 54.4% for subcutaneous adipocytes, whereas the inhibition was decreased in perirenal adipocytes. Addition of theophylline reversed the antilipolytic action of insulin. 6. Insulin binding was not affected neither by the dietary fat nor by the adipose tissue location. 7. Absence of de novo lipogenesis stimulation by insulin was not due to an impairment in insulin binding. 8. The different effects of dietary fat and adipose tissue location on the antilipolytic action of insulin could not be explained by a modification of insulin binding but rather by a latter event, probably at a post-insulin binding stage.     

Partial Inhibition of Adipose Tissue Lipolysis Improves Glucose Metabolism and Insulin Sensitivity Without Alteration of Fat Mass

When energy is needed, white adipose tissue (WAT) provides fatty acids (FAs) for use in peripheral tissues via stimulation of fat cell lipolysis. FAs have been postulated to play a critical role in the development of obesity-induced insulin resistance, a major risk factor for diabetes and cardiovascular disease. However, whether and how chronic inhibition of fat mobilization from WAT modulates insulin sensitivity remains elusive. Hormone-sensitive lipase (HSL) participates in the breakdown of WAT triacylglycerol into FAs. HSL haploinsufficiency and treatment with a HSL inhibitor resulted in improvement of insulin tolerance without impact on body weight, fat mass, and WAT inflammation in high-fat-diet–fed mice. In vivo palmitate turnover analysis revealed that blunted lipolytic capacity is associated with diminution in FA uptake and storage in peripheral tissues of obese HSL haploinsufficient mice. The reduction in FA turnover was accompanied by an improvement of glucose metabolism with a shift in respiratory quotient, increase of glucose uptake in WAT and skeletal muscle, and enhancement of de novo lipogenesis and insulin signalling in liver. In human adipocytes, HSL gene silencing led to improved insulin-stimulated glucose uptake, resulting in increased de novo lipogenesis and activation of cognate gene expression. In clinical studies, WAT lipolytic rate was positively and negatively correlated with indexes of insulin resistance and WAT de novo lipogenesis gene expression, respectively. In obese individuals, chronic inhibition of lipolysis resulted in induction of WAT de novo lipogenesis gene expression. Thus, reduction in WAT lipolysis reshapes FA fluxes without increase of fat mass and improves glucose metabolism through cell-autonomous induction of fat cell de novo lipogenesis, which contributes to improved insulin sensitivity.     

Role for TBC1D20 and Rab1 in hepatitis C virus replication via interaction with lipid droplet-bound nonstructural protein 5A

Replication and assembly of hepatitis C virus (HCV) depend on the host's secretory and lipid-biosynthetic machinery. Viral replication occurs on endoplasmic reticulum (ER)-derived modified membranes, while viral assembly is thought to occur on lipid droplets (LDs). A physical association and coordination between the viral replication and assembly complexes are prerequisites for efficient viral production. Nonstructural protein 5A (NS5A), which localizes both to the ER and LDs, is an ideal candidate for this function. Here, the interaction of NS5A with host cell membranes and binding partners was characterized in living cells. The binding of NS5A to LDs is apparently irreversible, both in HCV-infected cells and when ectopically expressed. In HCV-infected cells, NS5A fluorescence was observed around the LDs and in perinuclear structures that were incorporated into a highly immobile platform superimposed over the ER membrane. Moreover, TBC1D20 and its cognate GTPase Rab1 are recruited by NS5A to LDs. The NS5A-TBC1D20 interaction was shown to be essential for the viral life cycle. In cells, expression of the Rab1 dominant negative (Rab1DN) GTPase mutant abolished steady-state LDs. In infected cells, Rab1DN induced the elimination of NS5A from viral replication sites. Our results demonstrate the significance of the localization of NS5A to LDs and support a model whereby its interaction with TBC1D20 and Rab1 affects lipid droplet metabolism to promote the viral life cycle.     

Regulation of lipolysis in isolated adipocytes of rainbow trout (Oncorhynchus mykiss): the role of insulin and glucagon

In the present study, we have examined the effects of insulin and glucagon on the lipolysis of rainbow trout (Oncorhynchus mykiss). To this end, adipocytes were isolated from mesenteric fat and incubated in the absence (basal lipolysis) or presence of different concentrations of insulin and glucagon. In addition, to further elucidate the effects of these hormones in vivo on adipocyte lipolysis, both fasting and intraperitoneal glucagon injection experiments were performed. Basal lipolysis, measured as the glycerol released in the adipocyte medium, increased proportionally with cell concentration and incubation time. Cell viability was verified by measuring the release of lactate dehydrogenase (LDH) activity in the medium. Insulin (at doses of 35 and 350 nM) decreased lipolysis in isolated adipocytes of rainbow trout in vitro, while glucagon was clearly lipolytic at concentrations of 10 and 100 nM. Furthermore, hypoinsulinemia induced by fasting, as well as glucagon injection, significantly increased lipolysis in isolated adipocytes approximately 1.5- and 1.4-fold, respectively, when compared with adipocytes from control fish. Our data demonstrate that lipolysis, as measured in isolated adipocytes of rainbow trout, can be regulated by both insulin and glucagon. These results not only indicate that insulin is an important hormone in lipid deposition via its anti-lipolytic effects on rainbow trout adipocytes, but also reveal glucagon as a lipolytic hormone, as shown by both in vitro and in vivo experiments.     

The Role of PDE3B Phosphorylation in the Inhibition of Lipolysis by Insulin

Inhibition of adipocyte lipolysis by insulin is important for whole-body energy homeostasis; its disruption has been implicated as contributing to the development of insulin resistance and type 2 diabetes mellitus. The main target of the antilipolytic action of insulin is believed to be phosphodiesterase 3B (PDE3B), whose phosphorylation by Akt leads to accelerated degradation of the prolipolytic second messenger cyclic AMP (cAMP). To test this hypothesis genetically, brown adipocytes lacking PDE3B were examined for their regulation of lipolysis. In Pde3b knockout (KO) adipocytes, insulin was unable to suppress β-adrenergic receptor-stimulated glycerol release. Reexpressing wild-type PDE3B in KO adipocytes fully rescued the action of insulin against lipolysis. Surprisingly, a mutant form of PDE3B that ablates the major Akt phosphorylation site, murine S273, also restored the ability of insulin to suppress lipolysis. Taken together, these data suggest that phosphorylation of PDE3B by Akt is not required for insulin to suppress adipocyte lipolysis.     

Regulation of adipocyte differentiation and insulin action with rapamycin

Here, we demonstrated that inhibition of mTOR with rapamycin has negative effects on adipocyte differentiation and insulin signaling. Rapamycin significantly reduced expression of most adipocyte marker genes including PPARgamma, adipsin, aP2, ADD1/SREBP1c, and FAS, and decreased intracellular lipid accumulation in 3T3-L1 and 3T3-F442A cells, suggesting that rapamycin would affect both lipogenesis and adipogenesis. Contrary to the previous report that suppressive effect of rapamycin on adipogenesis is limited to the clonal expansion, we revealed that its inhibitory effect persisted throughout the process of adipocyte differentiation. Thus, it is likely that constitutive activation of mTOR might be required for the execution of adipogenic programming. In differentiated 3T3-L1 adipocytes, chronic treatment of rapamycin blunted the phosphorylation of AKT and GSK, which is stimulated by insulin, and reduced insulin-dependent glucose uptake activity. Taken together, these results suggest that rapamycin not only prevents adipocyte differentiation by decrease of adipogenesis and lipogenesis but also downregulates insulin action in adipocytes, implying that mTOR would play important roles in adipogenesis and insulin action.     

Short photoperiod exposure increases adipocyte sensitivity to noradrenergic stimulation in Siberian hamsters

Siberian hamsters (Phodopus sungorus) exhibit a naturally occurring, reversible seasonal obesity with body fat peaking in long "summerlike" days (LDs) and reaching a nadir in short "winterlike" days (SDs). These SD-induced decreases in adiposity are mediated largely via sympathetic nervous system (SNS) innervation of white adipose tissue (WAT), as indicated by increased WAT norepinephrine (NE) turnover. We examined whether SDs also increase sensitivity to NE-stimulated lipolysis. This was accomplished by measuring NE- and beta3-adrenoceptor (beta3-AR) agonist (BRL-37344)-induced lipolysis (glycerol release) as well as NE-induced cAMP accumulation by inguinal, epididymal, and retroperitoneal WAT (IWAT, EWAT, and RWAT) in isolated adipocytes of LD- and SD-housed hamsters. SDs increased potency/efficacy of NE-triggered lipolysis in a temporally and fat pad-specific manner. Thus when WAT pad mass decreased most rapidly (5 wk of SDs), potency (sensitivity/EC50) and efficacy (maximal response asymptote) of NE-stimulated lipolysis were increased for all WAT pads and also at 10 wk for IWAT compared with their LD counterparts. SD enhancement of lipolysis was similar for NE and BRL-37344 in IWAT adipocytes. These results, coupled with our previous demonstration that SDs upregulate WAT beta3-AR mRNA expression, suggest that increased beta3-ARs mediated the SD-induced increased NE sensitivity. NE-stimulated adipocyte accumulation of cAMP was greater after 5 wk of SDs for IWAT and EWAT and after 10 wk of SDs for IWAT compared with LDs, with no photoperiod effect for RWAT. Therefore, the SD-induced increase in SNS drive to WAT and increased sensitivity to this drive may work together to increase lipolysis in SDs.