Lipolysis Signaling Research Articles

Epinephrine promotes breast cancer metastasis through a ubiquitin-specific peptidase 22-mediated lipolysis circuit.

Chronic stress-induced epinephrine (EPI) accelerates breast cancer progression and metastasis, but the molecular mechanisms remain unclear. Herein, we found a strong positive correlation between circulating EPI levels and the tumoral expression of ubiquitin-specific peptidase 22 (USP22) in patients with breast cancer. USP22 facilitated EPI-induced breast cancer progression and metastasis by enhancing adipose triglyceride lipase (ATGL)-mediated lipolysis. Targeted USP22 deletion decreased ATGL expression and lipolysis, subsequently inhibiting EPI-mediated breast cancer lung metastasis. USP22 acts as a bona fide deubiquitinase for the Atgl gene transcription factor FOXO1, and EPI architects a lipolysis signaling pathway to stabilize USP22 through AKT-mediated phosphorylation. Notably, USP22 phosphorylation levels are positively associated with EPI and with downstream pathways involving both FOXO1 and ATGL in breast cancers. Pharmacological USP22 inhibition synergized with β-blockers in treating preclinical xenograft breast cancer models. This study reveals a molecular pathway behind EPI's tumor-promoting effects and provides a strong rationale for combining USP22 inhibition with β-blockers to treat aggressive breast cancer.

NADPH oxidase-derived reactive oxygen species regulates adipose tissue lipolysis in humans

There is mounting evidence suggesting that reactive oxygen species (ROS) generated by NADPH oxidase (NOX) regulate physiological processes, as NOX has been shown to promote increases in lipolysis in cultured adipocytes. PURPOSE: To determine the mechanisms by which NOX stimulates adipose tissue lipolysis. METHODS: Young healthy females (n = 17; mean ± SD; age: 21.7 ± 5 years; body mass index: 22.2 ± 3.8 kg/m2) participated in this study. Participants underwent microdialysis and hyperinsulinemic-euglycemic clamp (clamp) procedures. Three microdialysis probes were inserted into subcutaneous abdominal adipose tissue to monitor local adipose tissue ROS (H2O2) production and lipolysis (as indicated by dialysate glycerol). To determine whether NOX stimulates lipolysis via distinct signaling pathways, microdialysis probes were perfused with either isoproterenol (Iso; β-adrenergic agonist to stimulate canonical lipolysis signaling) or atrial natriuretic peptide (ANP to stimulate non-canonical lipolysis signaling), then perfused with a local NOX inhibitor (apocynin). A control probe was perfused with apocynin but without any agonists of lipolysis. The microdialysis procedures were then repeated under hyperinsulinemic conditions during the same study visit. RESULTS: In the fasted state, dialysate H2O2 concentrations were lower in the control and Iso probes upon apocynin perfusion (control probe without apocynin: 1.06 ± 0.5 μM, with apocynin: 0.62 ± 0.2 μM; p = 0.005; Iso probe without apocynin: 1.01 ± 0.4 μM, with apocynin: 0.66 ± 0.2 μM; p = 0.003). Apocynin perfusion did not alter dialysate H2O2 concentrations in the ANP probe under the fasted state (p = 0.11). Dialysate glycerol concentrations did not change upon apocynin perfusion under any of the microdialysis probe conditions in the fasted state (p > 0.05). During the clamp, apocynin perfusion resulted in lower dialysate H2O2 concentrations in the three conditions (control probe without apocynin: 1.22 ± 0.6 μM, with apocynin: 0.75 ± 0.3 μM; p = 0.03; Iso probe without apocynin: 1.55 ± 0.8 μM, with apocynin: 0.79 ± 0.3 μM; p = 0.01; ANP probe without apocynin: 1.55 ± 0.6 μM, with apocynin: 0.92 ± 0.3 μM; p = 0.03). Dialysate glycerol concentrations were lower in the Iso probe upon apocynin perfusion during the clamp (Iso probe without apocynin: 124.7 ± 38 μM, with apocynin: 90.9 ± 25 μM; p = 0.003). CONCLUSIONS: This study presents adipose tissue NOX as a source of in vivo ROS production in human participants. In addition, we reveal a novel mechanism of NOX in adipose tissue function whereby NOX exerts increases in β-adrenergic-mediated lipolysis during hyperinsulinemia. Our findings highlight the roles of ROS as signaling mediators and suggest that an imbalance between NOX activity and insulin action on adipose tissue could negatively affect systemic fatty acid metabolism. Porter Pre-doctoral Fellowship from the American Physiological Society and an NIH National Research Service Award (F31HL154642). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Transcriptome analysis of norepinephrine-induced lipolysis in differentiated adipocytes of Bama pig

Sympathetic innervation of white adipose tissue (WAT) plays a key role in the regulation of lipid metabolism. Sympathetic activation promotes release of norepinephrine (NE), which binds to adrenergic receptors on adipocytes, promoting adipocyte lipolysis and enhanced oxidative metabolism. However, the mechanism by which sympathetic nerves regulate lipid metabolism in pig adipose tissue remains unclear. We used NE to simulate the process of sympathetic driving in pig adipocytes. RNA sequencing (RNA-seq) was used to determine the gene expression profile of pig adipocytes responding to NE stimulation. Our data suggests that the lipolytic signaling pathway is activated in pig adipocytes upon acute stimulation of NE, resulting in enhanced lipid metabolism and lipolysis, consistent with the phenomena found in humans and mice. Specifically, differentially expressed protein coding genes (PCGs) (SIRT4, SLC27A1) are mainly associated with functions that inhibit fatty acid oxidation and promote lipid synthesis. Similarly, we investigated the changes in regulatory transcripts such as long non-coding RNAs (lncRNAs) and transcripts of uncertain coding potential (TUCP) in response to NE and found that differentially expressed lncRNAs (lncG47338, lncG30660, lncG29516, lncG3790) and TUCP (TUCP_G38001) were co-expressed with target genes related to the promotion of fatty acid β-oxidation, lipolysis and oxidative metabolism, thus acting as regulators. These results indicate a broad suite of gene expression alterations in response to NE stimulation and promote the understanding of the molecular mechanisms by which NE regulates lipid metabolism in pigs.

TP53/p53 Facilitates Stress-Induced Exosome and Protein Secretion by Adipocytes.

Besides the secretion of fatty acids, lipolytic stimulation of adipocytes results in the secretion of triglyceride-rich extracellular vesicle (AdEVs) and some free proteins (e.g., Fatty Acid Binding Protein 4) that in sum affect adipose homeostasis as well as the development of metabolic disease. At the mechanistic level, lipolytic signals activate p53 in an ATGL-dependent manner and pharmacologic inhibition of p53 attenuates AdEV protein and FABP4 secretion. Mass spectrometry analyses of the lipolytic secretome identified proteins involved in glucose and fatty acid metabolism, translation, chaperone activities as well as redox control. Consistent with a role for p53 in adipocyte protein secretion, activation of p53 by the MDM2 antagonist nutlin potentiated AdEV particles and non-AdEV protein secretion from cultured 3T3-L1 or OP9 adipocytes while the levels of FABP4 and AdEV proteins were significantly reduced in serum from p53-/- mice compared to wild-type controls. The genotoxin doxorubicin increased AdEV protein and FABP4 secretion in a p53-dependent manner and DNA repair-depleted ERCC1-/Δ haploinsufficient mice expressed elevated p53 in adipose depots, along with significantly increased serum FABP4. In sum, these data suggest that lipolytic signals, and cellular stressors such as DNA damage, facilitate AdEV protein and FABP4 secretion by adipocytes in a p53-dependent manner.

Increased Aquaporin-7 Expression Is Associated with Changes in Rat Brown Adipose Tissue Whitening in Obesity: Impact of Cold Exposure and Bariatric Surgery

Glycerol is a key metabolite for lipid accumulation in insulin-sensitive tissues. We examined the role of aquaporin-7 (AQP7), the main glycerol channel in adipocytes, in the improvement of brown adipose tissue (BAT) whitening, a process whereby brown adipocytes differentiate into white-like unilocular cells, after cold exposure or bariatric surgery in male Wistar rats with diet-induced obesity (DIO) (n = 229). DIO promoted BAT whitening, evidenced by increased BAT hypertrophy, steatosis and upregulation of the lipogenic factors Pparg2, Mogat2 and Dgat1. AQP7 was detected in BAT capillary endothelial cells and brown adipocytes, and its expression was upregulated by DIO. Interestingly, AQP7 gene and protein expressions were downregulated after cold exposure (4 °C) for 1 week or one month after sleeve gastrectomy in parallel to the improvement of BAT whitening. Moreover, Aqp7 mRNA expression was positively associated with transcripts of the lipogenic factors Pparg2, Mogat2 and Dgat1 and regulated by lipogenic (ghrelin) and lipolytic (isoproterenol and leptin) signals. Together, the upregulation of AQP7 in DIO might contribute to glycerol influx used for triacylglycerol synthesis in brown adipocytes, and hence, BAT whitening. This process is reversible by cold exposure and bariatric surgery, thereby suggesting the potential of targeting BAT AQP7 as an anti-obesity therapy.

OPA1 Regulates Lipid Metabolism and Cold-Induced Browning of White Adipose Tissue in Mice.

Mitochondria play a vital role in white adipose tissue (WAT) homeostasis including adipogenesis, fatty acid synthesis, and lipolysis. We recently reported that the mitochondrial fusion protein optic atrophy 1 (OPA1) is required for induction of fatty acid oxidation and thermogenic activation in brown adipocytes. In the current study we investigated the role of OPA1 in WAT function in vivo. We generated mice with constitutive or inducible knockout of OPA1 selectively in adipocytes. Studies were conducted under baseline conditions, at thermoneutrality, following high-fat feeding or during cold exposure. OPA1 deficiency reduced mitochondrial respiratory capacity in white adipocytes, impaired lipolytic signaling, repressed expression of de novo lipogenesis and triglyceride synthesis pathways, and promoted adipose tissue senescence and inflammation. Reduced WAT mass was associated with hepatic triglycerides accumulation and glucose intolerance. Moreover, mice deficient for OPA1 in adipocytes had impaired adaptive thermogenesis and reduced cold-induced browning of subcutaneous WAT and were completely resistant to diet-induced obesity. In conclusion, OPA1 expression and function in adipocytes are essential for adipose tissue expansion, lipid biosynthesis, and fatty acid mobilization of WAT and brown adipocytes and for thermogenic activation of brown and beige adipocytes.

Cytokine-Mediated STAT3 Transcription Supports ATGL/CGI-58-Dependent Adipocyte Lipolysis in Cancer Cachexia

Adipose tissue inflammation is observed in multiple metabolically-altered states including cancer-associated cachexia and obesity. Although cachexia is a syndrome of adipose loss and obesity is a disease of adipose excess, both pathologies demonstrate increases in circulating levels of IL-6 family cytokines, β-adrenergic signaling, and adipocyte lipolysis. While β-adrenergic-stimulated adipocyte lipolysis is well described, there is limited mechanistic insight into how cancer cachexia-associated inflammatory cytokines contribute to adipocyte lipolysis under pathologic conditions. Here, we set out to compare adipocyte lipolysis signaling by cancer cachexia-associated IL-6 family cytokines (IL-6 and LIF) to that of the β-adrenergic agonist isoproterenol. Unlike isoproterenol, the IL-6 family of cytokines required JAK/STAT3-dependent transcriptional changes to induce adipocyte lipolysis. Furthermore, cachexia-associated cytokines that used STAT3 to induce lipolysis were primarily dependent on the lipase ATGL and its cofactor CGI-58 rather than lipases HSL and MAGL. Finally, administration of JAK but not β-adrenergic inhibitors suppressed adipose STAT3 phosphorylation and associated adipose wasting in a murine model of cancer cachexia characterized by increased systemic IL-6 family cytokine levels. Combined, our results demonstrate how the IL-6 family of cytokines diverge from β-adrenergic signals by employing JAK/STAT3-driven transcriptional changes to promote adipocyte ATGL/CGI-58-dependent lipolysis contributing to adipose wasting in cancer cachexia.

Characterizing the Effects of High Fat and High Carbohydrate Diets on Feeding Behavior and Energy Expenditure in Mice

ObjectiveWe sought to characterize how different compositions of the diet can alter feeding behavior and energy expenditure in mice, ultimately driven by changes in sympathetic activation and thermogenesis.HypothesisMice on a high fat diet (HFD) will consume more calories/day compared to high carbohydrate diet (HCD) and standard chow fed mice. Mice on HFD will have decreased expression of UCP1 in both brown and white adipose tissue. There will be decreased expression for several lipolytic enzymes in the white adipose of HFD mice.MethodsA total of 21 mice were divided into three ad libitum fed diet groups: HFD, HCD, and standard chow. Each group was fed their respective diet for 7 weeks. Changes in weight and percent body fat were recorded by NMR. At week 5, glucose tolerance tests were performed on all mice. Both a 24‐hour heat production and respiratory exchange ratio were collected on each group using a metabolic cage system. Mice were sacrificed at the conclusion of week 7 and organs were collected for molecular analysis.ResultsWe show that mice on both ad libitum HFD and HCD consume significantly more calories/day when compared to standard chow. Both HFD and HCD fed mice have a significantly increased body weight and percent body fat when compared to standard chow; however, the HFD mice had a far greater increase in weight compared to HCD fed mice despite no difference in calorie consumption. Mice on a HFD had a lower 24‐hour average heat production per body mass when compared to standard chow and HCD mice and a significantly impaired glucose tolerance test. Furthermore, UCP1 mRNA expression is significantly elevated in brown adipose tissue (BAT) of HCD mice compared to that of HFD and standard chow mice. There were no differences in UCP1 expression in subcutaneous or visceral white adipose tissue (WAT) among all groups. There were also no differences in tyrosine hydroxylase (TH) expression in BAT between all mice. Three markers of lipolysis were decreased in visceral WAT of HFD mice compared to HCD and standard chow counterparts.DiscussionMice consumed significantly more calories when fed a HFD or HCD. These mice gain significantly more weight compared to chow with HFD mice gaining weight far more than the HCD group. This major difference in weight gain despite no significant difference in calorie consumption between HFD and HCD suggests differences may be determined by energy expenditure. This evidence is further supported by the fact that HFD fed mice had a significantly lower daily heat production compared to HCD and standard chow mice. We also show that HCD increases thermogenesis in mouse BAT which may be protective against weight gain. Finally, lipolysis signaling seems to be impaired in HFD mice, but no data supports that this is due to differences in sympathetic activation. Further work will include analyzing the same tissues in isocaloric, restricted diet fed mice.

Lipolysis drives expression of the constitutively active receptor GPR3 to induce adipose thermogenesis

SummaryThermogenic adipocytes possess a therapeutically appealing, energy-expending capacity, which is canonically cold-induced by ligand-dependent activation of β-adrenergic G protein-coupled receptors (GPCRs). Here, we uncover an alternate paradigm of GPCR-mediated adipose thermogenesis through the constitutively active receptor, GPR3. We show that the N terminus of GPR3 confers intrinsic signaling activity, resulting in continuous Gs-coupling and cAMP production without an exogenous ligand. Thus, transcriptional induction of Gpr3 represents the regulatory parallel to ligand-binding of conventional GPCRs. Consequently, increasing Gpr3 expression in thermogenic adipocytes is alone sufficient to drive energy expenditure and counteract metabolic disease in mice. Gpr3 transcription is cold-stimulated by a lipolytic signal, and dietary fat potentiates GPR3-dependent thermogenesis to amplify the response to caloric excess. Moreover, we find GPR3 to be an essential, adrenergic-independent regulator of human brown adipocytes. Taken together, our findings reveal a noncanonical mechanism of GPCR control and thermogenic activation through the lipolysis-induced expression of constitutively active GPR3.

ATGL activity regulates GLUT1-mediated glucose uptake and lactate production via TXNIP stability in adipocytes

Traditionally, lipolysis has been regarded as an enzymatic activity that liberates fatty acids as metabolic fuel. However, recent work has shown that novel substrates, including a variety of lipid compounds such as fatty acids and their derivatives, release lipolysis products that act as signaling molecules and transcriptional modulators. While these studies have expanded the role of lipolysis, the mechanisms underpinning lipolysis signaling are not fully defined. Here, we uncover a new mechanism regulating glucose uptake, whereby activation of lipolysis, in response to elevated cAMP, leads to the stimulation of thioredoxin-interacting protein (TXNIP) degradation. This, in turn, selectively induces glucose transporter 1 surface localization and glucose uptake in 3T3-L1 adipocytes and increases lactate production. Interestingly, cAMP-induced glucose uptake via degradation of TXNIP is largely dependent upon adipose triglyceride lipase (ATGL) and not hormone-sensitive lipase or monoacylglycerol lipase. Pharmacological inhibition or knockdown of ATGL alone prevents cAMP-dependent TXNIP degradation and thus significantly decreases glucose uptake and lactate secretion. Conversely, overexpression of ATGL amplifies the cAMP response, yielding increased glucose uptake and lactate production. Similarly, knockdown of TXNIP elicits enhanced basal glucose uptake and lactate secretion, and increased cAMP further amplifies this phenotype. Overexpression of TXNIP reduces basal and cAMP-stimulated glucose uptake and lactate secretion. As a proof of concept, we replicated these findings in human primary adipocytes and observed TXNIP degradation and increased glucose uptake and lactate secretion upon elevated cAMP signaling. Taken together, our results suggest a crosstalk between ATGL-mediated lipolysis and glucose uptake.

Pathophysiological role of fatty acid-binding protein 4 in Asian patients with heart failure and preserved ejection fraction.

AimsSystemic metabolic impairment is the key pathophysiology of heart failure (HF) with preserved ejection fraction (HFpEF). Fatty acid‐binding protein 4 (FABP4) is highly expressed in adipocytes and secreted in response to lipolytic signals. We hypothesized that circulating FABP4 levels would be elevated in patients with HFpEF, would correlate with cardiac structural and functional abnormalities, and could predict clinical outcomes.Methods and resultsSerum FABP4 measurements and echocardiography were performed in patients with HFpEF (n = 92) and those with coronary artery disease free of HF (n = 20). Patients were prospectively followed‐up for a composite endpoint of all‐cause mortality or HF hospitalization. Compared with patients with coronary artery disease, those with HFpEF had higher FABP4 levels [12.5 (9.1–21.0) vs. 43.5 (24.6–77.4) ng/mL, P < 0.0001]. FABP4 levels were associated with cardiac remodelling (left ventricular mass index: r = 0.29, P = 0.002; left atrial volume index: r = 0.40, P < 0.0001), left ventricular systolic and diastolic dysfunction (global longitudinal strain: r = −0.24, P = 0.01; E/e′ ratio: r = 0.29, P = 0.002; and N‐terminal pro‐B‐type natriuretic peptide: r = 0.62, P < 0.0001), and right ventricular dysfunction (tricuspid annular plane systolic excursion: r = −0.43, P < 0.0001). During a median follow‐up of 9.1 months, there were 28 primary endpoints in the HFpEF cohort. Event‐free survival was significantly decreased in patients with FABP4 levels ≥43.5 ng/mL than in those with FABP4 levels <43.5 ng/mL (P = 0.003).ConclusionsSerum FABP4 levels were increased in HFpEF and were associated with cardiac remodelling and dysfunction, and poor outcomes. Thus, FABP4 could be a potential biomarker in the complex pathophysiology of HFpEF.

Moderate aerobic exercise-induced cytokines changes are disturbed in PPARα knockout mice

The nuclear transcriptional factor peroxisome proliferator activated receptor alpha (PPARα) plays a role in regulating genes involved in lipid metabolism, adipogenesis and inflammation. We aimed to assess the role of PPARα on exercise-mediated locally produced cytokines in adipose fat deposits and skeletal muscle. C57BL/6 (WT) and PPARα knockout (PPARα−/−) mice were examined. Each genotype was randomly subdivided into three groups: non-exercised, and euthanized 2 or 24 h after a moderate aerobic exercise session (run on a treadmill at 60% of maximum speed for 1 h). Fat content in gastrocnemius muscle and lipolytic activity in isolated adipose tissue from mesenteric (MEAT) and retroperitoneal (RPAT) adipose tissue were evaluated. In addition, Interleukin 6 (IL-6), interleukin 10 (IL-10), tumor necrosis factor α (TNF-α) and monocyte chemoattractant protein 1 (MCP-1) content were evaluated by ELISA. WT mice showed a maximum lipolysis rate, as well as higher IL-6, IL-10, and IL10/TNF-α ratio values 2 h post-exercise (RPAT only) compared with PPARα−/− mice. Taken together, our data suggests that PPARα knockout mice exhibited reduced lipolysis and anti-inflammatory response in adipose tissue following exercise, PPARα appears to play an important role in immunomodulatory and lipolysis signaling after acute moderate exercise.

Activation of cardiac AMPK-FGF21 feed-forward loop in acute myocardial infarction: Role of adrenergic overdrive and lipolysis byproducts

Fibroblast growth factor 21 (FGF21) is a metabolic hormone having anti-oxidative and anti-hypertrophic effects. However, the regulation of FGF21 expression during acute myocardial infarction (AMI) remains unclear. We tested blood samples from 50 patients with AMI and 43 patients with stable angina pectoris (sAP) for FGF21, fatty acid binding protein 4 (FABP4), a protein secreted from adipocytes in response to adrenergic lipolytic signal, and total and individual fatty acids. Compared with sAP patients, AMI patients had higher serum FGF21 levels on admission, which were significantly correlated with peak FABP4 and saturated fatty acids (SFAs) but not with peak levels of cardiac troponin T. In mice, myocardial ischemia rapidly induced FGF21 production by the heart, which accompanied activation of AMP-activated protein kinase (AMPK)-dependent pathway. Like AICAR, an activator of AMPK, catecholamines (norepinephrine and isoproterenol) and SFAs (palmitate and stearate) significantly increased FGF21 production and release by cardiac myocytes via AMPK activation. Recombinant FGF21 induced its own expression as well as members of down-stream targets of AMPK involved in metabolic homeostasis and mitochondrial biogenesis in cardiac myocytes. These findings suggest that adrenergic overdrive and resultant adipose tissue lipolysis induce cardiac AMPK-FGF21 feed-forward loop that potentially provides cardioprotection against ischemic damage.

304-LB: Connection between Dynamics of Lipid Droplets and Energy Expenditure in Adipose Tissue

Lipid droplets (LDs), which largely found in adipocytes, are major organelles for energy storage and burndown. It is well known that ß-adrenergic signaling pathway liberates fatty acid from LDs to activate and fuel the thermogenesis program in mitochondria. However, the detailed events on the surface of LDs remain unclear. Here, we identified a novel hydrolase named Carboxylesterase 3 (Ces3) that targets LDs upon cold exposure by LC-MS/MS. We further found that translocation of Ces3 to LDs is cAMP-PKA pathway dependent. To investigate the function of Ces3 in β-adrenergic-activated adipocytes, we suppressed Ces3 with WWL229 (a specific Ces3 inhibitor) and small interfering RNA (siRNA) in 3T3-L1 and BAC (immortalized brown adipocytes) cells. We found that blockage of Ces3 by both methods dramatically attenuates ISO-induced lipolysis in cells. Furthermore, Ces3 inhibition leads to impaired mitochondrial respiratory function and attenuates ISO-induced thermogenic program in adipocytes by down-regulating Ucp1 and Pgc1α genes via PPARγ. Importantly, we further confirmed our findings by in vivo assays in mice. Specifically, our data showed that the thermogenic program in adipose tissue upon cold exposure is downregulated in WWL229 treated wild type mice and adipose tissue-specific Ces3 knockout mice. As the result, the mice exhibit dramatically impaired cold tolerance. Furthermore, we found that serum collected from cold-exposed wild type mice massively increase UCP1 levels in BAC cells while serum from Ces3 deficient mice fails to do so. In conclusion, our study highlighted the Ces3-mediated lipolytic signaling as a unique process to regulate thermogenesis in adipose tissue. Disclosure L. Yang: None. X. Li: None. K. Sun: None. Funding National Institutes of Health

Aerobic exercise training prevents obesity and insulin resistance independent of the renin angiotensin system modulation in the subcutaneous white adipose tissue.

We investigate the effects of aerobic exercise training (AET) on the thermogenic response, substrate metabolism and renin angiotensin system (RAS) in the subcutaneous white adipose tissue (SC-WAT) of mice fed cafeteria diet (CAF). Male C57BL/6J mice were assigned into groups CHOW-SED (chow diet, sedentary; n = 10), CHOW-TR (chow diet, trained; n = 10), CAF-SED (CAF, sedentary; n = 10) and CAF-TR (CAF, trained; n = 10). AET consisted in running sessions of 60 min at 60% of maximal speed, five days per week for eight weeks. The CAF-SED group showed higher body weight and adiposity, glucose intolerance and insulin resistance (IR), while AET prevented such damages in CAF-TR group. AET reduced the p-AKT/t-AKT ratio and increased ATGL expression in CHOW-TR and CAF-TR groups and increased t-HSL and p-HSL/t-HSL ratio in CAF-TR. AET prevented adipocyte hypertrophy in CAF-TR group and increased UCP-1 protein expression only in CHOW-TR. Serum ACE2 increased in CHOW-TR and CAF-TR groups, and Ang (1–7) increased in the CHOW-TR group. In the SC-WAT, CAF-TR group increased the expression of AT1, AT2 and Mas receptors, whereas CHOW-TR increased Ang (1–7) and Ang (1–7)/Ang II ratio in SC-WAT. No changes were observed in ACE and Ang II. Positive correlations were observed between UCP-1 and kITT (r = 0.6), between UCP-1 and Ang (1–7) concentration (r = 0.6), and between UCP-1 and Ang (1–7)/Ang II ratio (r = 0.7). In conclusion, the AET prevented obesity and IR, reduced insulin signaling proteins and increased lipolysis signaling proteins in the SC-WAT. In addition, the CAF diet precludes the AET-induced thermogenic response and the partial modulation of the RAS suggests that the protective effect of AET against obesity and IR could not be associated with SC-WAT RAS.

A Unique Role of Carboxylesterase 3 (Ces3) in β-Adrenergic Signaling-Stimulated Thermogenesis.

Carboxylesterase 3 (Ces3) is a hydrolase with a wide range of activities in liver and adipose tissue. In this study, we identified Ces3 as a major lipid droplet surface-targeting protein in adipose tissue upon cold exposure by liquid chromatography-tandem mass spectrometry. To investigate the function of Ces3 in the β-adrenergic signaling-activated adipocytes, we applied WWL229, a specific Ces3 inhibitor, or genetic inhibition by siRNA to Ces3 on isoproterenol (ISO)-treated 3T3-L1 and brown adipocyte cells. We found that blockage of Ces3 by WWL229 or siRNA dramatically attenuated the ISO-induced lipolytic effect in the cells. Furthermore, Ces3 inhibition led to impaired mitochondrial function measured by Seahorse. Interestingly, Ces3 inhibition attenuated an ISO-induced thermogenic program in adipocytes by downregulating Ucp1 and Pgc1α genes via peroxisome proliferator-activated receptor γ. We further confirmed the effects of Ces3 inhibition in vivo by showing that the thermogenesis in adipose tissues was significantly attenuated in WWL229-treated or adipose tissue-specific Ces3 heterozygous knockout (Adn-Cre-Ces3flx/wt) mice. As a result, the mice exhibited dramatically impaired ability to defend their body temperature in coldness. In conclusion, our study highlights a lipolytic signaling induced by Ces3 as a unique process to regulate thermogenesis in adipose tissue.

EHD2 regulates adipocyte function and is enriched at cell surface-associated lipid droplets in primary human adipocytes.

Adipocytes play a central role in energy balance, and dysfunctional adipose tissue severely affects systemic energy homeostasis. The ATPase EH domain–containing 2 (EHD2) has previously been shown to regulate caveolae, plasma membrane-specific domains that are involved in lipid uptake and signal transduction. Here, we investigated the role of EHD2 in adipocyte function. We demonstrate that EHD2 protein expression is highly up-regulated at the onset of triglyceride accumulation during adipocyte differentiation. Small interfering RNA–mediated EHD2 silencing affected the differentiation process and impaired insulin sensitivity, lipid storage capacity, and lipolysis. Fluorescence imaging revealed localization of EHD2 to caveolae, close to cell surface–associated lipid droplets in primary human adipocytes. These lipid droplets stained positive for glycerol transporter aquaporin 7 and phosphorylated perilipin-1 following adrenergic stimulation. Further, EHD2 overexpression in human adipocytes increased the lipolytic signaling and suppressed the activity of transcription factor PPARγ. Overall, these data suggest that EHD2 plays a key role for adipocyte function.

Abstract 2398: Tumor cell-adipocyte gap junctions activate lipolysis in breast cancer

Abstract During mammary tumorigenesis, a cell-cell interface exists between adipocytes and cancer cells. Several studies have demonstrated that breast tumor cells can secrete cytokines that induce lipolysis in adjacent adipocytes. However, evidence of tumor-adjacent lipolysis in clinical samples has been lacking. We therefore assayed for lipolysis in normal tissue adjacent to breast tumors (NAT) using (1) the three-component breast composition measure, a radiographic imaging method derived from dual-energy mammography that allows lipid content of a tissue to be quantified, on breast tumors and NAT from 46 patients, (2) a publically available dataset of gene expression in primary breast tumors and NAT from 9 patients, (3) laser capture microdissection and proteomics on primary breast tumors, stroma and NAT from 75 patients, and (4) immunoblot analysis of NAT from several patient-derived and transgenic mouse models of breast cancer. We found strong evidence in all cases that lipolysis is activated in breast cancer-adjacent adipose tissue. We next set out to model the breast cancer-adipocyte interface and determine the contribution of cell-cell contact to induced lipolysis. Gap junctions are cell-cell junctions formed by proteins called connexins, which are known to transport a variety of small molecules (&amp;lt;1kD) including cAMP, a critical pro-lipolytic signaling molecule. Using established dye transfer assays, we determined that gap junctions form between breast cancer cells, and between breast cancer cells and adipocytes. Using biochemical assays, we demonstrated that cAMP is a substrate of breast cancer cell gap junctions, that transfer of cAMP from breast cancer cells to adipocytes occurs, and that breast cancer cells activate lipolytic signaling, all in a gap junction-dependent manner. Finally, we found that gap junction communication in this context is dependent upon connexin 31 (Cx31), and we establish the importance of Cx31 for breast tumor growth and activation of lipolysis in tumor-adjacent adipose tissue in vivo. Citation Format: Roman Camarda, Jeremy Williams, Serghei Malkov, Lisa J. Zimmerman, Suzanne Manning, Dvir Aran, Andrew Beardsley, Daniel Van de Mark, Jeffrey van Haren, Yong Chen, Charles Berdan, Sharon Louie, Celine Mahieu, Juliane Winkler, Elizabeth Willey, John D. Gagnon, Kosaku Shinoda, K. Mark Ansel, Zena Werb, Daniel C. Nomura, Shingo Kajimura, Torsten Wittmann, Atul J. Butte, Melinda E. Sanders, Daniel C. Liebler, Gregor Krings, John A. Shepherd, Andrei Goga. Tumor cell-adipocyte gap junctions activate lipolysis in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2398.

GLP-1 receptor agonist ameliorates obesity-induced chronic kidney injury via restoring renal metabolism homeostasis.

Increasing evidence indicates that obesity is highly associated with chronic kidney disease (CKD). GLP-1 receptor (GLP-1R) agonist has shown benefits on kidney diseases, but its direct role on kidney metabolism in obesity is still not clear. This study aims to investigate the protection and metabolic modulation role of liraglutide (Lira) on kidney of obesity. Rats were induced obese by high-fat diet (HFD), and renal function and metabolism changes were evaluated by metabolomic, biological and histological methods. HFD rats exhibited systemic metabolic disorders such as obesity, hyperlipidemia and impaired glucose tolerance, as well as renal histological and function damages, while Lira significantly ameliorated these adverse effects in HFD rats. Metabolomic data showed that Lira directly reduced renal lipids including fatty acid residues, cholesterol, phospholipids and triglycerides, and improved mitochondria metabolites such as succinate, citrate, taurine, fumarate and nicotinamide adenine dinucleotide (NAD+) in the kidney of HFD rats. Furthermore, we revealed that Lira inhibited renal lipid accumulation by coordinating lipogenic and lipolytic signals, and partly rescued renal mitochondria function via Sirt1/AMPK/PGC1α pathways in HFD rats. This study suggested that Lira alleviated HFD-induced kidney injury at least partly via directly restoring renal metabolism, thus GLP-1R agonist is a promising therapy for obesity-associated CKD.

Effects of prenatal exposure to triclosan on the liver transcriptome in chicken embryos

Triclosan (TCS), a commonly used antimicrobial compound, has recently been detected in the eggs of wild avian species. Exposure to TCS in rodents is known to interfere with thyroid hormone (TH), disrupt immune responses and cause liver disease. However, no attempt has been made to clarify the effects of TCS in avian species. The aim of this study is therefore to evaluate the toxic effects of in ovo exposure to TCS and explore the molecular mechanism by transcriptome analysis in the embryonic liver of a model avian species, chicken (Gallus gallus). Embryos were treated with graded concentration of TCS (0.1, 1 and 10 μg/g egg) at Hamburger Hamilton Stage (HHS) 1 (1st day), followed by 20 days of incubation to HHS 46. At the administration of 10 μg TCS/g egg, embryo mortality increased from 20% in control to 37% accompanied with 8% attenuation in tarsus length. While liver somatic index (LSI) in TCS treatments was enhanced, statistical difference was only observed at the treatment of 0.1 μg TCS/g egg in females. The up-regulation of several crucial differentially expressed genes (DEGs) in transcriptome analysis suggested that TCS induced xenobiotic metabolism (e.g. CYP2C23a, CYP2C45 and CYP3A37 in males; CYP2C45 in females) and activated the thyroid hormone receptor (THR) - mediated downstream signaling (e.g. THRSPB and DIO2 in males; THRSPB in females). In females, TCS may further activate the lipogenesis signaling (e.g. ACSL5, ELOVL2) and repress the lipolysis signaling (e.g. ABHD5, ACAT2). A battery of enriched transcription factors in relation to these TCS-induced signaling and phenotypes were found, including activated SREBF1, PPARa, LXRa, and LXRb in males and activated GLI2 in females; COUP-TFII was predicted to be suppressed in both genders. Finally, we developed adverse outcome pathways (AOPs) that provide insights into the molecular mechanisms underlying the alteration of phenotypes.