HSL Phosphorylation Research Articles

GPER/PKA-Dependent Enhancement of Hormone-Sensitive Lipase Phosphorylation in 3T3-L1 Adipocytes by Piceatannol.

We previously reported that piceatannol (PIC) had an anti-obesity effect only in ovariectomized (OVX) postmenopausal obesity mice. PIC was found to induce the phosphorylation of hormone-sensitive lipase (pHSL) in OVX mice. To elucidate the mechanism by which PIC activates HSL, we investigated the effect of PIC using 3T3-L1 adipocytes. PIC induced HSL phosphorylation at Ser563 in 3T3-L1 cells, as in vivo experiments showed. pHSL (Ser563) is believed to be activated through the β-adrenergic receptor (β-AR) and protein kinase A (PKA) pathways; however, the addition of a selective inhibitor of β-AR did not inhibit the effect of PIC. The addition of a PKA inhibitor with PIC blocked pHSL (Ser563), suggesting that the effects are mediated by PKA in a different pathway than β-AR. The addition of G15, a selective inhibitor of the G protein-coupled estrogen receptor (GPER), reduced the activation of HSL by PIC. Furthermore, PIC inhibited insulin signaling and did not induce pHSL (Ser565), which represents its inactive form. These results suggest that PIC acts as a phytoestrogen and phosphorylates HSL through a novel pathway that activates GPER and its downstream PKA, which may be one of the inhibitory actions of PIC on fat accumulation in estrogen deficiency.

1502-P: Adipose Tissue Protein Response and Insulin Sensitivity in Obesity

Dysregulation of adipose tissue (AT) lipolysis, a hallmark of obesity and type 2 diabetes, results in elevated postabsorptive and postprandial plasma free fatty acid (FFA) concentrations. This is thought to be due to adipose insulin resistance with regards to anti-lipolysis effects. Alterations in intracellular adipocyte pathways and/or proteins that mediate this phenomenon in humans are poorly understood. To test the hypothesis that impaired insulin signaling is associated with adipose insulin resistance measured whole body insulin sensitivity (the insulin concentration that suppresses palmitate flux by 50% (IC50 FFA)) in 12 lean, 7 lower body obese and 15 upper body obese (n=15) volunteers using a single step hyperinsulinemic-euglycemic clamp. We also measured adipose pAkt/Akt (a measure of insulin signaling) and hormone sensitive lipase (pHSL/HSL) (a measure of lipolysis protein regulation) in biopsies of abdominal and thigh adipose obtained before and during the insulin clamp were measured. Contrary to our hypothesis, there was a positive relationship between IC50 FFA and insulin clamp fold change of pAkt/Akt in thigh samples (ρ = 0.38, p <0.05) and no relationship for abdominal adipose samples. There was good correlation between abdominal and thigh adipose samples in the fractional change in pAkt/Akt in response to insulin clamp (ρ= 0.49, p < 0.01). IC50 FFA was correlated to fractional decrease in HSL phosphorylation in both abdominal (r = 0.36, p< 0.05) and thigh (r = 0.41, p <0.05) Surprisingly, fold changes of pAKT/AKT in response to insulin clamp were positively correlated to fold change of pHSL/HSL in abdominal adipose tissue (ρ= 0.41, p < 0.05) but not thigh samples. In conclusion we found that insulin resistance in adipose is not related to canonical insulin signaling responses but is related to lipase responses. Disclosure K.Lytle: None. M.D.Jensen: Other Relationship; Novo Nordisk, Elsevier. Funding National Institute of Diabetes and Digestive and Kidney Diseases (DK40484)

Impaired white adipose tissue fatty acid metabolism in mice fed a high-fat diet worsened by arsenic exposure, primarily affecting retroperitoneal adipose tissue

Fatty acid (FA) metabolism dysfunction of white adipose tissue (WAT) underlies obesity and insulin resistance in response to high calorie intake and/or endocrine-disrupting chemicals (EDCs), among other factors. Arsenic is an EDC that has been associated with metabolic syndrome and diabetes. However, the combined effect of a high-fat diet (HFD) and arsenic exposure on WAT FA metabolism has been little studied.FA metabolism was evaluated in visceral (epididymal and retroperitoneal) and subcutaneous WAT of C57BL/6 male mice fed control or HFD (12 and 40% kcal fat, respectively) for 16 weeks together with an environmentally relevant chronic arsenic exposure through drinking water (100 μg/L) during the second half of the study.In mice fed HFD, arsenic potentiated the increase of serum markers of selective insulin resistance in WAT and fatty acid re-esterification and the decrease of the lipolysis index. Retroperitoneal was the WAT most affected, where the combination of arsenic and HFD in contrast to HFD, generated higher adipose weight, larger adipocytes, increased triglyceride content, and decreased fasting stimulated lipolysis evidenced by lower phosphorylation of HSL and perilipin. At the transcriptional level, arsenic in mice fed either diet downregulated genes involved in fatty acid uptake (LPL, CD36), oxidation (PPARα, CPT1), lipolysis (ADRß3) and glycerol transport (AQP7 and AQP9). Additionally, arsenic potentiated hyperinsulinemia induced by HFD, despite a slight increase in weight gain and food efficiency.Thus, the second hit of arsenic in sensitized mice by HFD worsens fatty acid metabolism impairment in WAT, mainly retroperitoneal, along with an exacerbated insulin resistance phenotype.

PLK1 promotes cholesterol efflux and alleviates atherosclerosis by up-regulating ABCA1 and ABCG1 expression via the AMPK/PPARγ/LXRα pathway

Polo-like kinase 1 (PLK1) is a serine/threonine kinase involving lipid metabolism and cardiovascular disease. However, its role in atherogenesis has yet to be determined. The aim of this study was to observe the impact of PLK1 on macrophage lipid accumulation and atherosclerosis development and to explore the underlying mechanisms. We found a significant reduction of PLK1 expression in lipid-loaded macrophages and atherosclerosis model mice. Lentivirus-mediated overexpression of PLK1 promoted cholesterol efflux and inhibited lipid accumulation in THP-1 macrophage-derived foam cells. Mechanistic analysis revealed that PLK1 stimulated the phosphorylation of AMP-activated protein kinase (AMPK), leading to activation of the peroxisome proliferator-activated receptor γ (PPARγ)/liver X receptor α (LXRα) pathway and up-regulation of ATP binding cassette transporter A1 (ABCA1) and ABCG1 expression. Injection of lentiviral vector expressing PLK1 increased reverse cholesterol transport, improved plasma lipid profiles and decreased atherosclerotic lesion area in apoE-deficient mice fed a Western diet. PLK1 overexpression also facilitated AMPK and HSL phosphorylation and enhanced the expression of PPARγ, LXRα, ABCA1, ABCG1 and LPL in the aorta. In summary, these data suggest that PLK1 inhibits macrophage lipid accumulation and mitigates atherosclerosis by promoting ABCA1- and ABCG1-dependent cholesterol efflux via the AMPK/PPARγ/LXRα pathway.

Exogenous ANP Treatment Ameliorates Myocardial Insulin Resistance and Protects against Ischemia-Reperfusion Injury in Diet-Induced Obesity.

Increasing evidence suggests natriuretic peptides (NPs) coordinate interorgan metabolic crosstalk. We recently reported exogenous ANP treatment ameliorated systemic insulin resistance by inducing adipose tissue browning and attenuating hepatic steatosis in diet-induced obesity (DIO). We herein investigated whether ANP treatment also ameliorates myocardial insulin resistance, leading to cardioprotection during ischemia–reperfusion injury (IRI) in DIO. Mice fed a high-fat diet (HFD) or normal-fat diet for 13 weeks were treated with or without ANP infusion subcutaneously for another 3 weeks. Left ventricular BNP expression was substantially reduced in HFD hearts. Intraperitoneal-insulin-administration-induced Akt phosphorylation was impaired in HFD hearts, which was restored by ANP treatment, suggesting that ANP treatment ameliorated myocardial insulin resistance. After ischemia–reperfusion using the Langendorff model, HFD impaired cardiac functional recovery with a corresponding increased infarct size. However, ANP treatment improved functional recovery and reduced injury while restoring impaired IRI-induced Akt phosphorylation in HFD hearts. Myocardial ultrastructural analyses showed increased peri-mitochondrial lipid droplets with concomitantly decreased ATGL and HSL phosphorylation levels in ANP-treated HFD, suggesting that ANP protects mitochondria from lipid overload by trapping lipids. Accordingly, ANP treatment attenuated mitochondria cristae disruption after IRI in HFD hearts. In summary, exogenous ANP treatment ameliorates myocardial insulin resistance and protects against IRI associated with mitochondrial ultrastructure modifications in DIO. Replenishing biologically active NPs substantially affects HFD hearts in which endogenous NP production is impaired.

Tauroursodeoxycholic acid improves glucose tolerance and reduces adiposity in normal protein and malnourished mice fed a high-fat diet

Early childhood malnutrition may facilitate the onset of obesity and diabetes mellitus in adulthood which, when established, makes it more resistant to therapeutic interventions. The beneficial effects of tauroursodeoxycholic acid (TUDCA) in glucose homeostasis and body fat accumulation were analyzed in protein-restricted mice fed a high-fat diet (HFD). C57BL/6 mice were fed a control (14% protein [C]) or a protein‐restricted (6% protein [R]) diet for 6 weeks. Afterward, mice received an HFD or not for 12 weeks (C mice fed an HFD [CH] and R mice fed an HFD [RH]). In the last 15 days of this period, half of the mice fed a HFD received i.p. PBS (groups CH and RH) or 300 mg/kg TUDCA (groups CHT and RHT). RH mice developed obesity, as demonstrated by the increase in fat accumulation, liver steatosis, and metabolic inflexibility. Additionally, showed glucose intolerance and insulin hypersecretion. TUDCA reduced adiposity and improve metabolic flexibility through increased HSL phosphorylation and CPT1 expression in eWAT and BAT, and reduced ectopic fat deposition by activating the AMPK/HSL pathway in the liver. Also, improved glucose tolerance and insulin sensitivity, normalizing insulin secretion by reducing GDH expression and increasing insulin peripheral sensitivity by greater expression of the IRβ in muscle and adipose tissue and reducing PEPCK liver expression. Our data indicate that TUDCA reduces global adiposity and improves glucose tolerance and insulin sensitivity in protein malnourished mice fed a HFD. Therefore, this is a possible strategy to reverse metabolic disorders in individuals with the double burden of malnutrition.

Pre-Weaning Exposure to Maternal High-Fat Diet Is a Critical Developmental Window for Programming the Metabolic System of Offspring in Mice.

BackgroundMaternal high-fat diet (HFD) during pregnancy and lactation exerts long-term effects on the health of offspring. However, the critical developmental window for metabolic programming of maternal exposure to HFD on pathogenesis of obesity in offspring needs further clarification.Materials & MethodsFemale ICR mice were fed low-fat diet (LFD) or HFD for 8 weeks until delivery. During lactation, half of LFD dams received HFD while the other half of LFD dams and HFD dams maintained the previous diet. Male offspring were weaned at postnatal day 21 (P21) and fed LFD or HFD for 7 weeks. Metabolic parameters, biochemical, and histological indicators of thermogenesis, rectal temperature, and sympathetic nerve tone were detected at P21 and 10 weeks old.ResultsAt P21, LH (maternal LFD before delivery but HFD during lactation) and HH (maternal HFD before delivery and during lactation) offspring gained more body weight and showed higher serum glucose and triglyceride levels as compared with LL (maternal LFD before delivery and during lactation), and the metabolic characters were maintained until 10 weeks age when fed with LFD after weaning. However, LH offspring exhibited a greater degree of metabolic abnormalities compared to HH offspring, with increased body weight, as well as lower norepinephrine (NE)-stimulated rectal temperature rise when fed with HFD after weaning. The lower UCP1 levels and HSL phosphorylation in LH offspring further suggested that brown adipose tissue (BAT) thermogenic function was impaired.ConclusionExposure to maternal HFD feeding during pre-weaning period alone showed similar detrimental effects on programming metabolic system of offspring as those of both prenatal and early postnatal HFD feeding. Early postnatal stage is a critical time window for metabolic programming and has profound and long-lasting effects on BAT development and function through sympathetic nerve-mediated thermogenesis.

Pharmacological agents targeting autophagy and their effects on lipolysis in human adipocytes

Adipose tissue of metabolically compromised humans with obesity is often characterized by impaired regulation of autophagy pathway. However, data on the role of autophagy in human adipocyte lipid catabolism is scarce. Therefore, we investigated the effect of pharmacological agents (including 3-methyladenine (3MA), bafilomycin A1 (BAF), chloroquine (CQ) and lalistat-2 (L-stat), that target different stages of the autophagy pathway on lipid hydrolysis in differentiated human multipotent adipose-derived stem cells (hMADs). Glycerol and fatty acid release were measured as marker of lipid hydrolysis following starvation and β-adrenergic stimulation. Microtubule-associated protein light chain 3 ratio (LC3II/LC3I) and HSL phosphorylation (pHSL) were analyzed by Western blot. Our data indicate that pharmacological inhibition of the autophagy pathway reduced lipid hydrolysis in human adipocytes, although to a limited extent (10-15%). However, further research is needed to reveal the exact mechanism of action of these pharmacological agents and their interplay with cytosolic lipid breakdown in human adipocytes.

Cold-induction of afadin in brown fat supports its thermogenic capacity

The profound energy-expending nature of brown adipose tissue (BAT) thermogenesis makes it an attractive target tissue to combat obesity-associated metabolic disorders. While cold exposure is the strongest inducer of BAT activity, the temporal mechanisms tuning BAT adaptation during this activation process are incompletely understood. Here we show that the scaffold protein Afadin is dynamically regulated by cold in BAT, and participates in cold acclimation. Cold exposure acutely increases Afadin protein levels and its phosphorylation in BAT. Knockdown of Afadin in brown pre-adipocytes does not alter adipogenesis but restricts β3-adrenegic induction of thermogenic genes expression and HSL phosphorylation in mature brown adipocytes. Consistent with a defect in thermogenesis, an impaired cold tolerance was observed in fat-specific Afadin knockout mice. However, while Afadin depletion led to reduced Ucp1 mRNA induction by cold, stimulation of Ucp1 protein was conserved. Transcriptomic analysis revealed that fat-specific ablation of Afadin led to decreased functional enrichment of gene sets controlling essential metabolic functions at thermoneutrality in BAT, whereas it led to an altered reprogramming in response to cold, with enhanced enrichment of different pathways related to metabolism and remodeling. Collectively, we demonstrate a role for Afadin in supporting the adrenergic response in brown adipocytes and BAT function.

Adipocyte PHLPP2 inhibition prevents obesity-induced fatty liver

Increased adiposity confers risk for systemic insulin resistance and type 2 diabetes (T2D), but mechanisms underlying this pathogenic inter-organ crosstalk are incompletely understood. We find PHLPP2 (PH domain and leucine rich repeat protein phosphatase 2), recently identified as the Akt Ser473 phosphatase, to be increased in adipocytes from obese mice. To identify the functional consequence of increased adipocyte PHLPP2 in obese mice, we generated adipocyte-specific PHLPP2 knockout (A-PHLPP2) mice. A-PHLPP2 mice show normal adiposity and glucose metabolism when fed a normal chow diet, but reduced adiposity and improved whole-body glucose tolerance as compared to Cre- controls with high-fat diet (HFD) feeding. Notably, HFD-fed A-PHLPP2 mice show increased HSL phosphorylation, leading to increased lipolysis in vitro and in vivo. Mobilized adipocyte fatty acids are oxidized, leading to increased peroxisome proliferator-activated receptor alpha (PPARα)-dependent adiponectin secretion, which in turn increases hepatic fatty acid oxidation to ameliorate obesity-induced fatty liver. Consistently, adipose PHLPP2 expression is negatively correlated with serum adiponectin levels in obese humans. Overall, these data implicate an adipocyte PHLPP2-HSL-PPARα signaling axis to regulate systemic glucose and lipid homeostasis, and suggest that excess adipocyte PHLPP2 explains decreased adiponectin secretion and downstream metabolic consequence in obesity.

PKA and AMPK Signaling Pathways Differentially Regulate Luteal Steroidogenesis.

Luteinizing hormone (LH) via protein kinase A (PKA) triggers ovulation and formation of the corpus luteum, which arises from the differentiation of follicular granulosa and theca cells into large and small luteal cells, respectively. The small and large luteal cells produce progesterone, a steroid hormone required for establishment and maintenance of pregnancy. We recently reported on the importance of hormone-sensitive lipase (HSL, also known as LIPE) and lipid droplets for appropriate secretory function of the corpus luteum. These lipid-rich intracellular organelles store cholesteryl esters, which can be hydrolyzed by HSL to provide cholesterol, the main substrate necessary for progesterone synthesis. In the present study, we analyzed dynamic posttranslational modifications of HSL mediated by PKA and AMP-activated protein kinase (AMPK) as well as their effects on steroidogenesis in luteal cells. Our results revealed that AMPK acutely inhibits the stimulatory effects of LH/PKA on progesterone production without reducing levels of STAR, CYP11A1, and HSD3B proteins. Exogenous cholesterol reversed the negative effects of AMPK on LH-stimulated steroidogenesis, suggesting that AMPK regulates cholesterol availability in luteal cells. AMPK evoked inhibitory phosphorylation of HSL (Ser565). In contrast, LH/PKA decreased phosphorylation of AMPK at Thr172, a residue required for its activation. Additionally, LH/PKA increased phosphorylation of HSL at Ser563, which is crucial for enzyme activation, and decreased inhibitory phosphorylation of HSL at Ser565. The findings indicate that LH and AMPK exert opposite posttranslational modifications of HSL, presumptively regulating cholesterol availability for steroidogenesis.

Cardiac Autophagy Deficiency Attenuates ANP Production and Disrupts Myocardial-Adipose Cross Talk, Leading to Increased Fat Accumulation and Metabolic Dysfunction.

Increased myocardial autophagy has been established as an important stress-induced cardioprotective response. Three weeks after generating cardiomyocyte-specific autophagy deficiency, via inducible deletion of autophagy-related protein 7 (Atg7), we found that these mice (AKO) had increased body weight and fat mass without altered food intake. Glucose and insulin tolerance tests indicated reduced insulin sensitivity in AKO mice. Metabolic cage analysis showed reduced ambulatory activity and oxygen consumption with a trend of elevated respiratory exchange ratio in AKO mice. Direct analysis of metabolism in subcutaneous and visceral adipocytes showed increased glucose oxidation and reduced ATGL expression and HSL phosphorylation with no change in lipid synthesis or fatty acid oxidation. Importantly, we found AKO mice had reduced myocardial and circulating levels of atrial natriuretic peptide (ANP), an established mediator of myocardial-adipose cross talk. When normal ANP levels were restored to AKO mice with use of osmotic pump, the metabolic dysfunction evident in AKO mice was corrected. We conclude that cardiac autophagy deficiency alters myocardial-adipose cross talk via decreased ANP levels with adverse metabolic consequences.

Anti-adipogenic effect of Lactobacillus fermentum MG4231 and MG4244 through AMPK pathway in 3T3-L1 preadipocytes.

This study evaluated the anti-adipogenic effects and mechanisms underlying the action of Lactobacillus fermentum MG4231 and MG4244 strains on adipogenesis and lipid accumulation in 3T3-L1 preadipocytes. Treatment with cell-free extracts (CFEs) from the two strains reduced lipid accumulation and intracellular triglyceride production in 3T3-L1 adipocytes by more than 50%. The inhibitory effects of L. fermentum on lipid accumulation were mediated by the downregulation of FAS and aP2 resulting from the inhibition of PPARγ and C/EBPα gene expression. Moreover, AMPK and HSL phosphorylation was upregulated by CFE treatment. These results indicated that the anti-adipogenic and lipolysis activities of L. fermentum strains were caused by increased AMPK and HSL phosphorylation. Both strains displayed high leucine arylamidase and β-galactosidase enzymatic activity, with excellent adhesion to epithelial cells. Therefore, we identified L. fermentum as potential new probiotics for the prevention of obesity.

Breast cancer mammospheres secrete Adrenomedullin to induce lipolysis and browning of adjacent adipocytes

BackgroundCancer cells cooperate with cells that compose their environment to promote tumor growth and invasion. Among them, adipocytes provide lipids used as a source of energy by cancer cells and adipokines that contribute to tumor expansion. Mechanisms supporting the dynamic interactions between cancer cells and stromal adipocytes, however, remain unclear.MethodsWe set-up a co-culture model with breast cancer cells grown in 3D as mammospheres and human adipocytes to accurately recapitulate intrinsic features of tumors, such as hypoxia and cancer cell–adipocytes interactions.ResultsHerein, we observed that the lipid droplets’ size was reduced in adipocytes adjacent to the mammospheres, mimicking adipocyte morphology on histological sections. We showed that the uncoupling protein UCP1 was expressed in adipocytes close to tumor cells on breast cancer histological sections as well as in adipocytes in contact with the mammospheres.Mammospheres produced adrenomedullin (ADM), a multifactorial hypoxia-inducible peptide while ADM receptors were detected in adipocytes. Stimulation of adipocytes with ADM promoted UCP1 expression and increased HSL phosphorylation, which activated lipolysis. Invalidation of ADM in breast cancer cells dramatically reduced UCP1 expression in adipocytes.ConclusionsBreast tumor cells secreted ADM that modified cancer–associated adipocytes through paracrine signaling, leading to metabolic changes and delipidation. Hence, ADM appears to be crucial in controlling the interactions between cancer cells and adipocytes and represents an excellent target to hinder them.

Protein Arginine Methyltransferase 4 Regulates Adipose Tissue Lipolysis in Type 1 Diabetic Mice.

PurposeHypertriglyceridemia is considered to be driven by increased lipolysis in type 1 diabetes mellitus (T1DM). However, information regarding the transcriptional circuitry that governs lipolysis remains incomplete in T1DM. Protein arginine methyltransferase 4 (PRMT4), a transcriptional coactivation factor, promotes autophagy and may play an important role in lipolysis. We wonder whether activated lipolysis in T1DM is regulated by PRMT4.Materials and MethodsRecombinant adeno-associated virus was adopted to overexpress PRMT4 in adipose tissue of mice. Streptozotocin (150 mg/kg) was injected intraperitoneally into mice to induce T1DM. Plasma insulin, triglycerides, free fatty acids (FFAs) levels were determined using commercial assay kits. Differentiated adipocytes were applied to verify the regulation of PRMT4 on lipolysis.ResultsElevated serum triglycerides and FFAs were observed in PRMT4-overexpressed T1DM mice. We also observed that PRMT4 over-expression induced the decrease of fat pads weights and adipocyte sizes. Moreover, expression levels of lipolysis-related molecules, including ATGL, HSL, and MAGL, and HSL phosphorylation levels were increased in PRMT4-overexpressed mice when compared to those of control mice. In vitro, PRMT4 promoted FFAs release and activated HSL phosphorylation, whereas PRMT4 knockdown inhibited these processes.ConclusionPRMT4 promotes lipolysis and increases serum triglyceride in T1DM.

L-Carnitine Is Involved in Hyperbaric Oxygen-Mediated Therapeutic Effects in High Fat Diet-Induced Lipid Metabolism Dysfunction.

Lipid metabolism dysfunction and obesity are serious health issues to human beings. The current study investigated the effects of hyperbaric oxygen (HBO) against high fat diet (HFD)-induced lipid metabolism dysfunction and the roles of L-carnitine. C57/B6 mice were fed with HFD or normal chew diet, with or without HBO treatment. Histopathological methods were used to assess the adipose tissues, serum free fatty acid (FFA) levels were assessed with enzymatic methods, and the endogenous circulation and skeletal muscle L-carnitine levels were assessed with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Additionally, western blotting was used to assess the expression levels of PPARα, CPT1b, pHSL/HSL, and UCP1. HFD treatment increased body/adipose tissue weight, serum FFA levels, circulation L-carnitines and decreased skeletal muscle L-carnitine levels, while HBO treatment alleviated such changes. Moreover, HFD treatment increased fatty acid deposition in adipose tissues and decreased the expression of HSL, while HBO treatment alleviated such changes. Additionally, HFD treatment decreased the expression levels of PPARα and increased those of CPT1b in skeletal muscle, while HBO treatment effectively reverted such changes as well. In brown adipose tissues, HFD increased the expression of UCP1 and the phosphorylation of HSL, which was abolished by HBO treatment as well. In summary, HBO treatment may alleviate HFD-induced fatty acid metabolism dysfunction in C57/B6 mice, which seems to be associated with circulation and skeletal muscle L-carnitine levels and PPARα expression.

Melatonin ameliorates SGLT2 inhibitor-induced diabetic ketoacidosis by inhibiting lipolysis and hepatic ketogenesis in type 2 diabetic mice.

Sodium-glucose cotransporter-2 inhibitors (SGLT2i) are effective hypoglycemic agents that can induce glycosuria. However, there are increasing concerns that they might induce diabetic ketoacidosis. This study investigated the effect of melatonin on SGTL2i-induced ketoacidosis in insulin-deficient type 2 diabetic (T2D) mice. The SGLT2i dapagliflozin reduced blood glucose level and plasma insulin concentrations in T2D mice, but induced increases in the concentrations of plasma β-hydroxybutyrate, acetoacetate, and free fatty acid and a decrease in the concentration of plasma bicarbonate, resulting in ketoacidosis. Melatonin inhibited dapagliflozin-induced ketoacidosis without inducing any change in blood glucose level or plasma insulin concentration. In white adipose tissue, melatonin inhibited lipolysis and downregulated phosphorylation of PKA, HSL, and perilipin-1. In liver tissue, melatonin suppressed cellular cyclic AMP levels and downregulated phosphorylation of PKA, AMPK, and acetyl-CoA carboxylase (ACC). In addition, melatonin increased hepatic ACC activity, but decreased hepatic CPT1a activity and acetyl-CoA content. These effects of melatonin on lipolysis and hepatic ketogenesis were blocked by pretreatment with melatonin receptor antagonist or PKA activator. Collectively, these results suggest that melatonin can ameliorate SGLT2i-induced ketoacidosis by inhibiting lipolysis and hepatic ketogenesis though cyclic AMP/PKA signaling pathways in T2D mice. Thus, melatonin treatment may offer protection against SGLT2i-induced ketoacidosis.

High-fat diet feeding and palmitic acid increase CRC growth in \u03b22AR-dependent manner

Epidemiology studies indicate that consumption of high-fat diet (HFD) is directly associated with the development of colorectal cancer (CRC). However, the exact component in HFD and the mechanism underlying its effect on CRC growth remained unclear. Our study shows that HFD feeding increases β2AR expression in the xenograft tissues of CRC-bearing mouse model; the elevated β2AR expression is reduced when HFD is replaced by control diet, which strongly suggests an association between HFD feeding and β2AR expression in CRC. HFD feeding increases palmitic acid and stearic acid levels in CRC; however, only palmitic acid increases β2AR expression, which is dependent upon Sp1. β2AR plays the dominant role in promoting CRC cell proliferation among all the β-AR subtypes. More importantly, knockout of β2AR or knockdown of Sp1 abolishes the palmitic acid increased CRC cell proliferation, suggesting palmitic acid increases CRC cell proliferation in β2AR-dependent manner. HFD or palmitic acid-rich diet (PAD) also fails to increase the tumor growth in xenograft mouse models bearing β2AR-knockout CRC cells. β2AR promotes CRC growth by increasing the phosphorylation of HSL at the residue S552. The phosphorylated and activated HSL (S552) changes the metabolic phenotype of CRC and increases energy production, which promotes CRC growth. Our study has revealed the unique tumorigenic properties of palmitic acid in promoting CRC growth, and have delineated the underlying mechanism of action. We are also the first to report the linkage between HFD feeding and β-adrenergic signaling pathway in relation to CRC growth.

1782-P: Mechanism of Lipid-Induced White Adipose Tissue Insulin Resistance

White adipose tissue (WAT), as an energy storage organ, plays a major role in regulating peripheral insulin resistance and hepatic gluconeogenesis, but the mechanism by which lipid induces insulin resistance in WAT is poorly understood. To examine the potential role of the diacylglycerol (DAG) - protein kinase Cε (PKCε) - insulin receptor (INSR) pathway in mediating lipid-induced WAT insulin resistance, we assessed insulin action in liver, muscle and WAT in 7-day high fat diet (HFD) and regular chow (RC) fed rats by hyperinsulinemic-euglycemic clamp (HEC) combined with [3-3H] glucose, [14C] deoxyglucose and [2H5]glycerol infusions. Using this approach, we found that HFD induced WAT insulin resistance as reflected by impaired suppression (∼20%, P<0.05 vs. RC) of WAT lipolysis during the HEC. WAT insulin resistance was associated with increased PKCε translocation (P<0.05 vs. RC) and increased phosphorylation of perilipin, HSL and ATGL (all P<0.05 vs. RC). To investigate if PKCε induced Thr1150phosphorylation mediates lipid-induced WAT insulin resistance by inhibiting INSR kinase activity, we studied mice with a threonine-to-alanine knockin mutation at INSR Thr1150(InsrT1150Amice). InsrT1150Amice were protected from HFD-induced WAT insulin resistance as reflected by normalization of insulin’s ability to suppress WAT lipolysis and normalization of insulin’s ability to stimulate INSR and Akt2 phosphorylation in WAT, as well as a restoration of insulin’s ability to suppress phosphorylation of perilipin, HSL and ATGL (all P<0.05 vs. WT). Conclusion: The DAG-PKCε-INSR Thr1150pathway plays a major role in mediating lipid-induced WAT insulin resistance and represents a potential therapeutic target to improve insulin sensitivity in WAT. Disclosure D. Zhang: None. K. Lyu: None. J.D. Song: None. X. Li: None. R.J. Perry: Research Support; Self; AstraZeneca. V. Samuel: None. G.I. Shulman: Advisory Panel; Self; AstraZeneca, Janssen Research & Development, Merck & Co., Inc. Advisory Panel; Spouse/Partner; Merck & Co., Inc. Board Member; Self; Novo Nordisk A/S. Consultant; Self; Aegerion Pharmaceuticals, IMetabolic BioPharma Corporation, Longitude Capital, Nimbus Discovery, Inc., Staten Biotechnology B.V. Funding National Institutes of Health (R01DK116774)

High Intensity Interval Training Activates Specific Site HSL Phosphorylation and Reduces Visceral Fat

High Intensity Interval Training Activates Specific Site HSL Phosphorylation and Reduces Visceral Fat