White Adipose Tissue Of Mice Research Articles

Effects of metformin on metabolism of white and brown adipose tissue in obese C57BL/6J mice

BackgroundTo investigate effects of metformin on the regulation of proteins of white adipose tissue (WAT) and brown adipose tissue (BAT) in obesity and explore the underlying mechanisms on energy metabolism.MethodsC57BL/6J mice were fed with normal diet (ND, n = 6) or high-fat diet (HFD, n = 12) for 22 weeks. HFD-induced obese mice were treated with metformin (MET, n = 6). After treatment for 8 weeks, oral glucose tolerance test (OGTT) and hyperinsulinemic–euglycemic clamp were performed to evaluate the improvement of glucose tolerance and insulin sensitivity. Protein expressions of WAT and BAT in mice among ND, HFD, and MET group were identified and quantified with isobaric tag for relative and absolute quantification (iTRAQ) coupled with 2D LC–MS/MS. The results were analyzed by MASCOT, Scaffold and IPA.ResultsThe glucose infusion rate in MET group was increased significantly compared with HFD group. We identified 4388 and 3486 proteins in WAT and BAT, respectively. As compared MET to HFD, differential expressed proteins in WAT and BAT were mainly assigned to the pathways of EIF2 signaling and mitochondrial dysfunction, respectively. In the pathways, CPT1a in WAT, CPT1b and CPT2 in BAT were down-regulated by metformin significantly.ConclusionsMetformin improved the body weight and insulin sensitivity of obese mice. Meanwhile, metformin might ameliorate endoplasmic reticulum stress in WAT, and affect fatty acid metabolism in WAT and BAT. CPT1 might be a potential target of metformin in WAT and BAT.

Uncoupling protein 1 and the capacity for nonshivering thermogenesis are components of the glucose homeostatic system.

Uncoupling protein 1 (Ucp1) provides nonshivering thermogenesis (NST) fueled by the dissipation of energy from macronutrients in brown and brite adipocytes. The availability of thermogenic fuels is facilitated by the uptake of extracellular glucose. This conjunction renders thermogenic adipocytes in brown and white adipose tissue (WAT) a potential target against obesity and glucose intolerance. We employed wild-type (WT) and Ucp1-ablated mice to elucidate this relationship. In three experiments of similar setup, Ucp1-ablated mice fed a high-fat diet (HFD) had either reduced or similar body mass gain, food intake, and metabolic efficiency compared with WT mice, challenging the hypothesized role of this protein in the development of diet-induced obesity. Despite the absence of increased body mass, oral glucose tolerance was robustly impaired in Ucp1-ablated mice in response to HFD. Postprandial glucose uptake was attenuated in brown adipose tissue but enhanced in subcutaneous WAT of Ucp1-ablated mice. These differences were explainable by expression of the insulin-responsive member 4 of the facilitated glucose transporter family and fully in line with the capacity for NST in these very tissues. Thus, the postprandial glucose uptake of adipose tissues serves as a surrogate measure for Ucp1-dependent and independent capacity for NST. Collectively, our findings corroborate Ucp1 as a modulator of adipose tissue glucose uptake and systemic glucose homeostasis but challenge its hypothesized causal effect on the development of obesity.

Effects of psychological stress on xanthine oxidase expression, activity and related markers in adipose tissue of mice

To investigate the effects of psychological stress on xanthine oxidase (XO) expression, activity and related markers in adipose tissue of mice. Twenty male Kunming mice were randomly divided into two groups (10 in each group), stress group and control group (10 in each group). Stress group were restrained in self-made restraint device for 2 hours per day for 14 days, then blood samples and white adipose tissues(WAT) were collected. The expression levels of XO and NADPH oxidase-4 (Nox-4) in WAT were detected by immunohistochemistry. The expression of XO, Nox-4, antioxidant proteins (manganese superoxide dismutase (Mn SOD), glutathione peroxidase (GSH-Px), and catalase (CAT)), adipocytokines (adiponectin (ADPN), monocyte chemotactic protein 1 (MCP-1), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α)) in WAT were further detected by quantitative PCR. Relative expressions of glucose metabolism (insulin receptor substrate-1(IRS-1) and glucose transporter type 4(GLUT-4)) and thrombin(tissue factor (TF) and plasminogen activator inhibitor 1 (PAI-1))were measured. XO activity and serum concentrations of (triglyceride (TG), total cholesterol (T-Cho), free fatty acid, (FFA), and uric acid (UA)) were detected by ELISA. XO expressed in stress mice inguinal WAT was deeper and more abundant than that of control group, mainly expressed in adipocytes. RT-PCR and ELISA results showed that the levels of XO mRNA, serum XO concentration and the activities of XO enzyme in WAT of stress group were significantly higher than those of control group(P ＜0.01). Compared with control group the concentrations of free fatty acid (FFA) and uric acid (UA) in stress group were increased significantly than in control group (P＜ 0.01). Nox-4 positive cells mainly expressed in adipocytes. The expression of Nox-4 in WAT of stress group was significantly higher(P ＜0.01); The levels of antioxidant proteins (Mn-SOD, GSH-Px, Catalase) in WAT of stress group were significantly lower (P＜ 0.01). WAT in stress group showed a large number of infiltration reactions and inflammatory changes of monocytes, neutrophils, eosinophils and plasma cells. Stress significantly decreased the expression of adiponectin in WAT, and significantly increased the expressions of MCP-1, IL-6 and TNF-α (P＜0.01). The levels of IRS-1 and GLUT-4 in WAT of stress mice were increased significantly (P＜ 0.01). The expressions of TF and PAI-1 in WAT of stress mice and blood concentrations were significantly higher (P＜0.01). Stress can induce excessive expressions of XO in adipose tissue, which eventaully can lead to adipose inflammation, glycometabolism and abnormal prothrombin.

Timeliness of β3 adrenergic receptor agonist-induced browning of white adipose tissues in mice

To characterize the timeliness of β3 adrenergic receptor agonist CL316,243-induced browning of white adipose tissues in mice. Methods: Male C57BL/6J mice at 10 weeks of age were housed in conventional cages and given sterile saline for the control group or CL316,243 (1 μg/g) for the experimental group via intraperitoneal injection for 1, 3, and 5 days. Food intake and body weight were measured daily. Interscapular brown adipose tissue (iBAT), inguinal subcutaneous white adipose (sWAT) and epididymal white adipose tissue (eWAT) were harvested for histological and gene expression analysis. Results: Compared with the control group, intraperitoneal injection of CL316,243 reduced the weight of eWAT on the first day. Meanwhile, CL316,243 continuously promoted the mRNA and protein expression of uncoupling protein-1 (UCP-1) in sWAT and eWAT. Furthermore, CL316,243 injection significantly decreased the food intake and weight gain of the mice, and reduced the diameter of adipocyte and accumulation of small lipid droplets in adipose tissues. Conclusion: CL316,243 can induce the brown-like remodeling in adipose tissues of mice in vivo, which show different time-dependent manners in different adipose tissues.

The CCR4-NOT Deadenylase Complex Maintains Adipocyte Identity.

Shortening of poly(A) tails triggers mRNA degradation; hence, mRNA deadenylation regulates many biological events. In the present study, we generated mice lacking the Cnot1 gene, which encodes an essential scaffold subunit of the CCR4–NOT deadenylase complex in adipose tissues (Cnot1-AKO mice) and we examined the role of CCR4–NOT in adipocyte function. Cnot1-AKO mice showed reduced masses of white adipose tissue (WAT) and brown adipose tissue (BAT), indicating abnormal organization and function of those tissues. Indeed, Cnot1-AKO mice showed hyperinsulinemia, hyperglycemia, insulin resistance, and glucose intolerance and they could not maintain a normal body temperature during cold exposure. Muscle-like fibrous material appeared in both WAT and BAT of Cnot1-AKO mice, suggesting the acquisition of non-adipose tissue characteristics. Gene expression analysis using RNA-sequencing (RNA-seq) showed that the levels of adipose tissue-related mRNAs, including those of metabolic genes, decreased, whereas the levels of inflammatory response-related mRNAs increased. These data suggest that the CCR4–NOT complex ensures proper adipose tissue function by maintaining adipocyte-specific mRNAs at appropriate levels and by simultaneously suppressing mRNAs that would impair adipocyte function if overexpressed.

Fat-Specific Knockout of Mecp2 Upregulates Slpi to Reduce Obesity by Enhancing Browning.

Abnormalities of methyl-CpG binding protein 2 (Mecp2) cause neurological disorders with metabolic dysfunction; however, its role in adipose tissues remains unclear. Here, we report upregulated Mecp2 in white adipose tissues (WAT) of obese humans, as well as in obese mice and during in vitro adipogenesis. Normal chow-fed adipocyte-specific Mecp2 knockout mice (Mecp2 Adi KO mice) showed a lean phenotype, with downregulated lipogenic genes and upregulated thermogenic genes that were identified using RNA sequencing. Consistently, the deficiency of Mecp2 in adipocytes protected mice from high-fat diet (HFD)-induced obesity and inhibited in vitro adipogenesis. Furthermore, Mecp2 Adi KO mice showed increased browning under different stimuli, including cold treatment. Mechanistically, Mecp2 bound to the promoter of secretory leukocyte protease inhibitor (Slpi) and negatively regulated its expression. Knockdown of Slpi in inguinal WAT of Mecp2 Adi KO mice prevented cold-induced browning. Moreover, recombinant SLPI treatment reduced the HFD-induced obesity via enhancing browning. Together, our results suggest a novel non-central nervous system function of Mecp2 in obesity by suppressing browning, at least partially, through regulating adipokine Slpi.

Shared Autonomic Pathways Connect Bone Marrow and Peripheral Adipose Tissues Across the Central Neuraxis.

Bone marrow adipose tissue (BMAT) is increased in both obesity and anorexia. This is unique relative to white adipose tissue (WAT), which is generally more attuned to metabolic demand. It suggests that there may be regulatory pathways that are common to both BMAT and WAT and also those that are specific to BMAT alone. The central nervous system (CNS) is a key mediator of adipose tissue function through sympathetic adrenergic neurons. Thus, we hypothesized that central autonomic pathways may be involved in BMAT regulation. To test this, we first quantified the innervation of BMAT by tyrosine hydroxylase (TH) positive nerves within the metaphysis and diaphysis of the tibia of B6 and C3H mice. We found that many of the TH+ axons were concentrated around central blood vessels in the bone marrow. However, there were also areas of free nerve endings which terminated in regions of BMAT adipocytes. Overall, the proportion of nerve-associated BMAT adipocytes increased from proximal to distal along the length of the tibia (from ~3–5 to ~14–24%), regardless of mouse strain. To identify the central pathways involved in BMAT innervation and compare to peripheral WAT, we then performed retrograde viral tract tracing with an attenuated pseudorabies virus (PRV) to infect efferent nerves from the tibial metaphysis (inclusive of BMAT) and inguinal WAT (iWAT) of C3H mice. PRV positive neurons were identified consistently from both injection sites in the intermediolateral horn of the spinal cord, reticular formation, rostroventral medulla, solitary tract, periaqueductal gray, locus coeruleus, subcoeruleus, Barrington's nucleus, and hypothalamus. We also observed dual-PRV infected neurons within the majority of these regions. Similar tracings were observed in pons, midbrain, and hypothalamic regions from B6 femur and tibia, demonstrating that these results persist across mouse strains and between skeletal sites. Altogether, this is the first quantitative report of BMAT autonomic innervation and reveals common central neuroanatomic pathways, including putative “command” neurons, involved in coordinating multiple aspects of sympathetic output and facilitation of parallel processing between bone marrow/BMAT and peripheral adipose tissue.

Supplementation of the Flavonoid Myricitrin Attenuates the Adverse Metabolic Effects of Long-Term Consumption of a High-Fat Diet in Mice.

The flavonoid myricitrin exhibits various pharmacological and physiological effects. However, studies on the effects of myricitrin on obesity are limited. We hypothesized that dietary myricitrin would attenuate the adiposity and metabolic dysfunction that occur in obesity. To test this hypothesis, mice were randomly fed a high-fat diet (HFD) or HFD supplemented with myricitrin for 16 weeks. Myricitrin significantly reduced white adipose tissue (WAT) mass, adipocyte size, and plasma leptin levels, and also attenuated dyslipidemia. These changes appeared to result from increased energy expenditure and activation of the carnitine acyltransferase (CPT) and β-oxidation in WAT. Expressions of the proinflammatory genes NF-κB, TLR2, MCP1, and TNF-α were also lower in the WAT of myricitrin-supplemented mice. Moreover, myricitrin markedly reduced hepatic triglyceride accumulation and plasma aspartate transaminase levels by increasing CPT activity and reducing fatty acid synthase activity in the liver. Myricitrin-supplemented mice also showed improved glucose tolerance, insulin sensitivity, and decreased hyperinsulinemia, along with decreased levels of circulating resistin. In conclusion, long-term consumption of a myricitrin-supplemented diet may effectively protect against HFD-induced obesity and related metabolic disorders.

Co-Administration of Curcumin and Artepillin C Induces Development of Brown-Like Adipocytes in Association with Local Norepinephrine Production by Alternatively Activated Macrophages in Mice.

Classical brown adipocytes, characterized by interscapular depots, have multilocular fat depots and are known to release excess energy. Recent studies have shown that induction of brown-like adipocytes, also referred to as beige or brite cells, in white adipose tissue (WAT) results in the release of excess energy through mitochondrial heat production via uncoupling protein 1. This has potential a therapeutic strategy for obesity and related diseases as well as classical brown adipocytes. In our previous studies, we found that artepillin C (ArtC, 10 mg/kg body weight), a characteristic constituent of Brazilian propolis, significantly induced the development of brown-like adipocytes in inguinal WAT (iWAT) of mice. Furthermore, we recently demonstrated that curcumin (Cur, 4.5 mg/kg) also significantly induced the development of brown-like adipocytes in mice. The combined administration of several food-derived factors can enhance their bioactivity and reduce their required functional doses. In this study, we showed that co-administration of Cur and ArtC at lower doses (Cur, 1.5 mg/kg; ArtC, 5 mg/kg) additively induce brown-like adipocyte development in mouse iWAT. Moreover, this induction is associated with the localized production of norepinephrine following accumulation of alternatively activated macrophages in iWAT. These findings suggest that co-administration of Cur and ArtC is significantly effective to reduce the dose and enhance the formation of brown-like adipocyte via a unique molecular mechanism.

Metformin prevents the pathological browning of subcutaneous white adipose tissue

ObjectiveBrowning, the conversion of white adipose tissue (WAT) to a beige phenotype, has gained interest as a strategy to induce weight loss and improve insulin resistance in metabolic disorders. However, for hypermetabolic conditions stemming from burn trauma or cancer cachexia, browning is thought to contribute to energy wasting and supraphysiological nutritional requirements. Metformin's impact on this phenomenon and underlying mechanisms have not been explored. MethodsWe used both a murine burn model and human ex vivo adipose explants to assess metformin and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR)'s effects on the development of subcutaneous beige adipose. Enzymes involved in fat homeostasis and browning, as well as mitochondrial dynamics, were assessed to determine metformin's effects. ResultsTreatment with the biguanide metformin lowers lipolysis in beige fat by inducing protein phosphatase 2A (PP2A) independently of adenosine monophosphate kinase (AMPK) activation. Increased PP2A activity catalyzes the dephosphorylation of acetyl-CoA carboxylase (Ser 79) and hormone sensitive lipase (Ser 660), thus promoting fat storage and the “whitening” of otherwise lipolytic beige adipocytes. Moreover, co-incubation of metformin with the PP2A inhibitor okadaic acid countered the anti-lipolytic effects of this biguanide in human adipose. Additionally, we show that metformin does not activate this pathway in the WAT of control mice and that AICAR sustains the browning of white adipose, offering further evidence that metformin acts independently of this cellular energy sensor. ConclusionsThis work provides novel insights into the mechanistic underpinnings of metformin's therapeutic benefits and potential as an agent to reduce the lipotoxicity associated with hypermetabolism and adipose browning.

Analysis of Tks4 Knockout Mice Suggests a Role for Tks4 in Adipose Tissue Homeostasis in the Context of Beigeing.

Obesity and adipocyte malfunction are related to and arise as consequences of disturbances in signaling pathways. Tyrosine kinase substrate with four Src homology 3 domains (Tks4) is a scaffold protein that establishes a platform for signaling cascade molecules during podosome formation and epidermal growth factor receptor (EGFR) signaling. Several lines of evidence have also suggested that Tks4 has a role in adipocyte biology; however, its roles in the various types of adipocytes at the cellular level and in transcriptional regulation have not been studied. Therefore, we hypothesized that Tks4 functions as an organizing molecule in signaling networks that regulate adipocyte homeostasis. Our aims were to study the white and brown adipose depots of Tks4 knockout (KO) mice using immunohistology and western blotting and to analyze gene expression changes regulated by the white, brown, and beige adipocyte-related transcription factors via a PCR array. Based on morphological differences in the Tks4-KO adipocytes and increased uncoupling protein 1 (UCP1) expression in the white adipose tissue (WAT) of Tks4-KO mice, we concluded that the beigeing process was more robust in the WAT of Tks4-KO mice compared to the wild-type animals. Furthermore, in the Tks4-KO WAT, the expression profile of peroxisome proliferator-activated receptor gamma (PPARγ)-regulated adipogenesis-related genes was shifted in favor of the appearance of beige-like cells. These results suggest that Tks4 and its downstream signaling partners are novel regulators of adipocyte functions and PPARγ-directed white to beige adipose tissue conversion.

Comprehensive map and functional annotation of the mouse white adipose tissue proteome.

White adipose tissue (WAT) plays a significant role in energy metabolism and the obesity epidemic. In this study, we sought to (1) profile the mouse WAT proteome with advanced 2DLC/MS/MS approach, (2) provide insight into WAT function based on protein functional annotation, and (3) predict potentially secreted proteins. A label-free 2DLC/MS/MS proteomic approach was used to identify the WAT proteome from female mouse WAT. A total of 6,039 proteins in WAT were identified, among which 5,160 were quantified (spanning a magnitude of 106) using an intensity-based absolute quantification algorithm, and 3,117 proteins were reported by proteomics technology for the first time in WAT. To comprehensively analyze the function of WAT, the proteins were divided into three quantiles based on abundance and we found that proteins of different abundance performed different functions. High-abundance proteins (the top 90%, 1,219 proteins) were involved in energy metabolism; middle-abundance proteins (90–99%, 2,273 proteins) were involved in the regulation of protein synthesis; and low-abundance proteins (99–100%, 1,668 proteins) were associated with lipid metabolism and WAT beiging. Furthermore, 800 proteins were predicted by SignalP4.0 to have signal peptides, 265 proteins had never been reported, and five have been reported as adipokines. The above results provide a large dataset of the normal mouse WAT proteome, which might be useful for WAT function research.

CircNrxn2 Promoted WAT Browning via Sponging miR-103 to Relieve Its Inhibition of FGF10 in HFD Mice

The accumulation of excess white adipose tissue (WAT) has harmful consequences on metabolic health. WAT browning confers beneficial effects on adiposity, insulin resistance, and hyperlipidemia. In this study, it was found out that circNrxn2 sponged miR-103 and enhanced FGF10 levels in HFD mice WAT. We discovered that circNrxn2 promoted WAT browning and mitochondria functions. Furthermore, circNrxn2 also increased M2 macrophage polarization in HFD mouse adipose tissue, and the PPARγ signaling pathway participated in these biological processes. Moreover, eliminating adipose tissue macrophages (ATMs) by clodronate-crippled circNrxn2 promoted WAT browning, and the simulation co-culture of macrophages and adipocytes results suggested that circNrxn2 promoted WAT browning through increasing M2 macrophage polarization. Our finding revealed that circNrxn2 acted as an endogenous miR-103 sponge, blocked miR-103 effects, and relieved its inhibition of FGF10 expression to promote WAT browning through increasing M2 macrophage polarization. This study provides a good therapeutic strategy for treating obesity and improving obesity-related metabolic disorders.

Chrysanthemum Leaf Ethanol Extract Prevents Obesity and Metabolic Disease in Diet-Induced Obese Mice via Lipid Mobilization in White Adipose Tissue.

This study aimed to elucidate the molecular mechanism of Chrysanthemum morifolium Ramat. against obesity and diabetes, by comparing the transcriptional changes in epididymal white adipose tissue (eWAT) with those of the bioactive compound in C. morifolium, luteolin (LU). Male C57BL/6J mice were fed a normal diet, high-fat diet (HFD), and HFD supplemented with 1.5% w/w chrysanthemum leaf ethanol extract (CLE) for 16 weeks. Supplementation with CLE and LU significantly decreased the body weight gain and eWAT weight by stimulating mRNA expressions for thermogenesis and energy expenditure in eWAT via lipid mobilization, which may be linked to the attenuation of dyslipidemia. Furthermore, CLE and LU increased uncoupling protein-1 protein expression in brown adipose tissue, leading to energy expenditure. Of note, CLE and LU supplements enhanced the balance between lipid storage and mobilization in white adipose tissue (WAT), in turn, inhibiting adipocyte inflammation and lipotoxicity of peripheral tissues. Moreover, CLE and LU attenuated hepatic steatosis by suppressing hepatic lipogenesis, thereby ameliorating insulin resistance and dyslipidemia. Our data suggest that CLE helps inhibit obesity and its comorbidities via the complex interplay between liver and WAT in diet-induced obese mice.

2000-P: Intracellular Metabolism-Dependent PDGF-B Induction in Inflammatory Macrophages Contributes to Adipose Tissue Expansion with Obesity

White adipose tissue (WAT) expands with angiogenesis. We have demonstrated that adipose-tissue-macrophages (ATMs) highly express platelet-derived growth factor (PDGF)-B in obesity, and exposure of PDGF-B stimulates detachment of pericytes (PCs) from vessels. Since vessel-attached PCs inhibit endothelial cell growth, the detachment restarts angiogenesis. In the present study, we aimed to clarify the significance of ATMs on adipose angiogenesis. Intraperitoneal injection of liposome-encapsulated clodronate (Clod) for 6 weeks achieved depletion of macrophages and decreased expression of Pdgfb mRNA in WAT of HFD-fed mice. Clod-treated mice showed smaller WAT with vessels well covered with PCs compared to control mice. We next investigated underlying mechanism of Pdgfb induction. LPS but not IL-4 stimulated Pdgfb expression in peritoneal macrophages. In RAW 264.7 macrophages, Pdgfb expression induced by high glucose plus LPS stimulation was completely blocked by pretreatment with 2-deoxyglucose (2-DG), a hexokinase inhibitor or heptelidic acid, a GAPDH inhibitor, whereas it was not affected by treatment with 6-aminonicotinamide, an inhibitor of glucose-6-phosphate dehydrogenase acting as the rate-limiting enzyme of the pentose phosphate pathway. In addition, high glucose plus LPS induced phosphorylations of S6 kinase, ERK, and JNK but not p38 MAP kinase. These phosphorylations were significantly attenuated by treatment with 2DG. Importantly, enhanced expression of Pdgfb by high glucose plus LPS was effectively suppressed by inhibitor or knockdown of Erk but not by rapamycin. The expression was also attenuated by inhibition of NFκb pathway. In conclusion, hyperglycemia and chronic inflammation reprogram metabolic pathways toward glycolysis, and subsequent activation of NFκB plays a pivotal role in the production of PDGF-B in ATMs during obesity development. Disclosure T. Wada: None. Y. Onogi: None. A. Okekawa: None. T. Matsuzawa: None. E. Watanabe: None. G. Komatsu: None. H. Tsuneki: None. T. Sasaoka: None. Funding Japan Society for the Promotion of Science (JP18K08469)

Protective Effects of Curcumin in High Fat Diet (HFD)-Induced Obesity Include Anti-Inflammatory Effects in Adipose Tissue and Changes in Gut Microbiome (P06-075-19)

ObjectivesCurcumin, a traditionally used spice in Asia has several health-protecting effects. However, its role on gut microbiota and obesity-associated inflammation is still poorly understood. The objective of this study was to determine whether the protective effects of curcumin in high fat diet (HFD)-induced obesity are mediated by reduced white adipose tissue (WAT) inflammation and changes in gut bacteria. MethodsMale B6 mice were fed a HFD (45% kcal fat) or HFD supplemented with 0.4% (w/w) curcumin (HFC) for thirteen weeks. Body weight, adiposity, glucose and, insulin tolerances, and serum triglycerides, insulin, leptin, resistin levels were measured. Gut microbiome composition was determined by 16S RNA metagenomics sequencing. Expression of inflammation-related genes in WAT was measured by qRT-PCR. Macrophage contents in WAT were evaluated by galectin-3 immunohistochemical staining. ResultsPro-inflammatory transcription factor nuclear factor NF-kappa-B p65 subunit (p65) and toll-like receptor-4 (TLR-4) gene expression was downregulated in HFC group compared to HFD mice. Furthermore, curcumin reduced total macrophage infiltration in WAT in HFC mice compared to HFD group. Expression of both M1 (CD80, CD38) and M2 (Arginase-1) associated genes was decreased. The relative abundance of bacteria representing the Clostridium genus, which contains numerous short-chain fatty acid (SCFA) producing species, was increased by the curcumin supplement. ConclusionsCurcumin exerts protective effects in dietary obesity, in part through downregulation of adipose tissue inflammation which may be due to the production of SCFA and, possibly other curcumin metabolites by gut microflora. Funding SourcesStartup funds and Come N Go award from the College of Human Sciences at Texas Tech University.

The Anti-obesity Effects of Direct Delivery of Resveratrol-encapsulated Liposomes to Inguinal White Adipose Tissue in Male APOE*3Leiden.CETP Transgenic Mice (P21-006-19)

ObjectivesBrowning of white adipose tissue (WAT) is a promising strategy for preventing and treating obesity. Trans-resveratrol (R) has the potential to increase the energy expenditure via inducing WAT browning, but its application in obesity is limited by a low level of aqueous solubility, a high level of hepatic metabolism and toxicity. We have successfully synthesized biocompatible and biodegradable R encapsulated liposomes (Rlipo). The objective of this project is to deliver Rlipo directly to the inguinal WAT (iWAT) in mice with the goal of maximizing anti-obesity efficacy while minimizing side effects and toxicity. MethodsRlipo were prepared using R and soy phosphatidylcholine (soy-PC) dissolved in ethanol. After mixing and drying them under nitrogen, deionized water was added followed by sonication. Unencapsulated R was removed by ultrafiltration. The void liposomes (Vlipo) were prepared using only soy-PC.The particle size and polydispersity index (PI) of Rlipo and Vlipo were measured using a Brookhaven BI-MAS particle size analyzer. R’s encapsulation efficiency and solubility were measured using a liquid chromatography-mass spectrometry.Male APOE*3Leiden.CETP mice having human-like lipoprotein metabolism were fed with a high-fat diet (45% of calorie from fat) for 10 weeks. After treating the high-fat diet for 4 weeks, mice received iWAT injection of Rlipo, Vlipo, free R and saline (control) once a week. R concentration was 17.5 mg/kg body weight/week. Body weight and food intake were measured weekly. Body composition of mice was measured using an EchoMRITM every other week. Core body temperatures of mice were determined using a Type T thermometer. Glucose tolerance test (GTT) was performed at the 4th week of treatment. ResultsRlipo increased the aqueous solubility of R by more than 100 times. The size of Rlipo and Vlipo was 170 ± 6 nm and 140 ± 7 nm, respectively. Their PI values were less than 0.2. The encapsulation efficiency of Rlipo was 99.8%. The post-treatment core body temperatures for control, free R, Vlipo and Rlipo groups were 36.2°C, 36.2°C, 36.6°C, and 37.0°C, respectively. Food intake was similar among 4 groups. Compared to other groups, the Rlipo-treated mice had the lowest body weight, body fat mass, and body fat percentage. The Rlipo group had the lowest area under the curve in the GTT among 4 groups. ConclusionsNanoencapsulation increased R’s solubility and enhanced its anti-obesity efficacy. Funding SourcesNIH/NCCIH (Grant R15AT008733).

272-LB: The Micropeptide ORF60 Modulates Insulin Sensitivity

Although long non-coding RNAs (lncRNAs) are non-protein-coding transcripts by definition, recent studies have shown that a number of putative small open reading frames within lncRNAs are translated. The biological significance of these hidden polypeptides is still unclear, although studies have demonstrated functional significance for some polypeptides. Recently, our lab discovered a novel micropeptide ORF60 encoded by a lncRNA. The micropeptide is conserved across species. Topology prediction indicates that the micropeptide is likely transmembrane. RNA transcript expression is nearly ubiquitous across tissues. In mice, ORF60 expression was found to be increased in the setting of high fat diet (HFD). Using CRISPR/Cas9 technology we developed ORF60 KO mice. In both mice feeding on normal chow diet and HFD, we observed impaired glucose tolerance in KO mice compared to wild type mice. ORF60 KO mice also demonstrated impaired response to insulin compared to WT mice on insulin tolerance testing (ITT), suggesting insulin resistance. Mouse tissue harvested under the condition of insulin infusion demonstrated insulin signaling attenuation in white adipose tissue (WAT) and brown adipose tissue (BAT). We are currently dissecting the molecular mechanism underlying ORF60’s influence on insulin signaling pathways. In summary, our results demonstrate that the conserved micropeptide ORF60 regulates insulin sensitivity in WAT and BAT in mice. Micropeptides involved in diabetes may serve as novel drug targets. Disclosure N. Gupta: None. Y. Shi: None. P. Li: None. H. Cao: None.

Browning is activated in the subcutaneous white adipose tissue of mice metabolically challenged with a high-fructose diet submitted to high-intensity interval training

Fructose may induce an endocrine dysfunction in adipose tissue in rodents. Browning is identified by deposits of beige adipocytes in subcutaneous white adipose tissue (sWAT). We study the effects of the high-intensity interval training (HIIT) on the formation of beige adipocytes in the sWAT of mice fed a high-fructose diet. Sixty male mice (3 months old; C57BL/6) were fed two diets for 18 weeks (n=30 each): control diet (C) or high-fructose diet (F). At the 10th week, for an additional 8-week period, the groups were (n=15 each) nontrained (NT) or trained (HIIT): C-NT, C-HIIT, F-NT and F-HIIT. We evaluated body mass, energy expenditure and molecular analyses for browning and thermogenic markers in sWAT. The HIIT groups showed significantly lower body mass and increased energy expenditure. The consumption of fructose was linked with an increased sWAT mass. However, HIIT caused a reduction of sWAT mass compared to the NT groups. Energy intake was parallel in the groups, regardless of the diet type and HIIT. Fructose was related to higher glucose and insulin levels and hypertrophied sWAT adipocytes, but HIIT decreased both glucose and insulin levels and led to the appearance of brown fat-like adipocytes dispersed in sWAT with higher expression of browning markers. Also, fructose reduced the sWAT markers of mitochondrial biogenesis and beta-oxidation, which were enhanced by HIIT. In conclusion, HIIT might stimulate the sWAT browning in mice fed a high-fructose diet associated with beneficial changes in mitochondrial biogenesis and beta-oxidation markers, contributing to a whole-body metabolic improvement.

Bromodomain-containing protein 2 promotes lipolysis via ERK/HSL signalling pathway in white adipose tissue of mice

White adipose tissue (WAT) dysfunction is prevalent among patients with type 2 diabetes mellitus (T2DM). Uncontrolled free fatty acid (FFA) release from WAT stores has detrimental effects on lipid metabolism, leading to insulin resistance. Bromodomain-containing protein 2 (Brd2) has emerged as a central transcriptional regulator of adipocyte differentiation and pancreatic β-cell bioactivity. A recent study shows that Brd2 overexpression leads to insulin resistance in mice. However, the mechanisms underlying these effects have not been fully elucidated. This study provides the first evidence that adenoviral-mediated Brd2 overexpression in the WAT of mice increases lipolysis-related gene expression in addition to significantly reducing WAT size and promoting plasma FFA release. Brd2 overexpression in adipocytes also inhibits fat synthesis-related gene expression, while activating hormone-sensitive lipase (HSL) expression and ERK-dependent perilipin 1 inhibition as well as promoting glycerol release, which are all involved in lipolysis. Collectively, these results indicate that Brd2 triggers insulin resistance via lipolysis-mediated FFA release.