TM4SF19-mediated control of lysosomal activity in macrophages contributes to obesity-induced inflammation and metabolic dysfunction.

Adipose tissue (AT) adapts to overnutrition in a complex process, wherein specialized immune cells remove and replace dysfunctional and stressed adipocytes with new fat cells. Among immune cells recruited to AT, lipid-associated macrophages (LAMs) have emerged as key players in obesity and in diseases involving lipid stress and inflammation. Here, we show that LAMs selectively express transmembrane 4 L six family member 19 (TM4SF19), a lysosomal protein that represses acidification through its interaction with Vacuolar-ATPase. Inactivation of TM4SF19 elevates lysosomal acidification and accelerates the clearance of dying/dead adipocytes in vitro and in vivo. TM4SF19 deletion reduces the LAM accumulation and increases the proportion of restorative macrophages in AT of male mice fed a high-fat diet. Importantly, male mice lacking TM4SF19 adapt to high-fat feeding through adipocyte hyperplasia, rather than hypertrophy. This adaptation significantly improves local and systemic insulin sensitivity, and energy expenditure, offering a potential avenue to combat obesity-related metabolic dysfunction.

A Combination of Soy Isoflavone and L-Carnitine Improves Running Endurance in Mice.

The present study aimed to investigate the effect of APIC, a mixture containing soy isoflavone and L-carnitine on running endurance. Male C57BL/6 mice were orally administered APIC for 8 weeks. The APIC group exhibited a significant increase in treadmill running time until exhaustion compared to the control group. The respiratory exchange ratio in the APIC group was lower, indicating an enhancement in fatty acid oxidative metabolism. Furthermore, APIC supplementation increased the proportion of oxidative myofibers. Biochemical parameters associated with endurance capacity were also affected by APIC, as evidenced by increased muscle ATP levels and decreased levels of muscle triglycerides and blood lactate. qPCR and immunoblot analysis of C2C12 myotubes and gastrocnemius muscles indicated that APIC treatment stimulated AMPK signaling, mitochondrial biogenesis, and fatty acid metabolism. Additionally, treatment with APIC led to an increased oxygen consumption rate in C2C12 myotubes. Collectively, these findings suggest that APIC supplementation enhances mitochondrial biogenesis, promotes a switch from glycolytic to oxidative fiber types, and improves fatty acid metabolism through the activation of the AMPK signaling pathway in murine skeletal muscle. Ultimately, these effects contribute to the enhancement of running endurance.

Use of CIDEA Reporter Mouse Model for Screening Thermogenic Fat-Activating Drugs.

Excessive fat accumulation is a risk factor for metabolic diseases. Activating non-shivering thermogenesis in adipose tissue increases energy expenditure and potentially reverses obesity-related metabolic dysfunctions. While brown/beige adipocytes specialize in non-shivering thermogenesis and catabolic lipid metabolism, thermogenic stimuli and pharmacological intervention can induce the recruitment and metabolic activation of these cell types in adipose tissue. Thus, these adipocytes are attractive therapeutic targets to combat obesity, and there is an increasing need for efficient screening strategies for thermogenic drugs. Cell death-inducing DNA fragmentation factor-like effector A (CIDEA) is a well-known marker of the thermogenic capacity of brown and beige adipocytes. We recently developed a CIDEA reporter mouse model that expresses multicistronic mRNAs encoding CIDEA, luciferase 2, and tdTomato proteins under endogenous Cidea promoter control. Here, we introduce the CIDEA reporter model system as a tool for in vitro and in vivo screening of drug candidate molecules with thermogenic effects and provide a detailed protocol to monitor CIDEA reporter expression.

Green tea extract exhibits antidiabetic effects partly through regulating dipeptidyl peptidase-4 expression in adipose tissue.

The antidiabetic effects of green tea have been demonstrated in clinical trials and epidemiological studies. This study investigated the antidiabetic effects of green tea extract (GTE) and its underlying molecular mechanisms using a leptin receptor-deficient db/db mouse model (Leprdb/db). Treatment with GTE for 2 weeks improved glucose tolerance and insulin sensitivity in Leprdb/db mice. In addition, GTE treatment reduced the body weight and adiposity of Leprdb/db mice. Furthermore, GTE treatment reduced pro-inflammatory gene expression, including nuclear factor kappa B (NF-κB) in white adipose tissue (WAT), and also reduced dipeptidyl peptidase-4 (DPP4) expression levels in WAT as well as in the serum. The promoter region of Dpp4 contains the NF-κB binding site, and DPP4 was found to be a direct target of NF-κB. Consistently, in vitro treatment of cells with GTE or its main constituent epigallocatechin gallate reduced lipopolysaccharide-induced NF-κB/DPP4 expression in 3T3-L1 adipocytes and RAW264.7 cells. Overall, our data demonstrated that GTE exerts an anti-diabetic effect by regulating the expression levels of NF-κB and DPP4 in WAT.

Macrophage-Specific Connexin 43 Knockout Protects Mice from Obesity-Induced Inflammation and Metabolic Dysfunction.

Adipose tissue macrophages are a major immune cell type contributing to homeostatic maintenance and pathological adipose tissue remodeling. However, the mechanisms underlying macrophage recruitment and polarization in adipose tissue during obesity remain poorly understood. Previous studies have suggested that the gap junctional protein, connexin 43 (Cx43), plays a critical role in macrophage activation and phagocytosis. Herein, we investigated the macrophage-specific roles of Cx43 in high fat diet (HFD)-induced pathological remodeling of adipose tissue. Expression levels of Cx43 were upregulated in macrophages co-cultured with dying adipocytes in vitro, as well as in macrophages associated with dying adipocytes in the adipose tissue of HFD-fed mice. Cx43 knockdown reduced lipopolysaccharide (LPS)-induced ATP release from macrophages and decreased inflammatory responses of macrophages co-cultured with dying adipocytes. Based on global gene expression profiling, macrophage-specific Cx43-knockout (Cx43-MKO) mice were resistant to HFD-induced inflammatory responses in adipose tissue, potentially via P2X7-mediated signaling pathways. Cx43-MKO mice exhibited reduced HFD-induced macrophage recruitment in adipose tissue. Moreover, Cx43-MKO mice showed reduced inflammasome activation in adipose tissues and improved glucose tolerance. Collectively, these findings demonstrate that Cx43 expression in macrophages facilitates inflammasome activation, which, in turn, contributes to HFD-induced metabolic dysfunction.

Adipocyte lysoplasmalogenase TMEM86A regulates plasmalogen homeostasis and protein kinase A-dependent energy metabolism.

Dysregulation of adipose tissue plasmalogen metabolism is associated with obesity-related metabolic diseases. We report that feeding mice a high-fat diet reduces adipose tissue lysoplasmalogen levels and increases transmembrane protein 86 A (TMEM86A), a putative lysoplasmalogenase. Untargeted lipidomic analysis demonstrates that adipocyte-specific TMEM86A-knockout (AKO) increases lysoplasmalogen content in adipose tissue, including plasmenyl lysophosphatidylethanolamine 18:0 (LPE P-18:0). Surprisingly, TMEM86A AKO increases protein kinase A signalling pathways owing to inhibition of phosphodiesterase 3B and elevation of cyclic adenosine monophosphate. TMEM86A AKO upregulates mitochondrial oxidative metabolism, elevates energy expenditure, and protects mice from metabolic dysfunction induced by high-fat feeding. Importantly, the effects of TMEM86A AKO are largely reproduced in vitro and in vivo by LPE P-18:0 supplementation. LPE P-18:0 levels are significantly lower in adipose tissue of human patients with obesity, suggesting that TMEM86A inhibition or lysoplasmalogen supplementation might be therapeutic approaches for preventing or treating obesity-related metabolic diseases.

REEP6 knockout leads to defective ß-adrenergic signaling in adipocytes and promotes obesity-related metabolic dysfunction.

INTRODUCTION: The mobilization and catabolism of lipid energy is a central function of adipocytes that is under the control of the ß-adrenergic signaling pathway, and defects in ß-adrenergic signaling in adipocytes have been linked to obesity and obesity-related metabolic diseases. Receptor expression-enhancing proteins (REEPs) are endoplasmic reticulum (ER) proteins that play critical roles in subcellular targeting of receptor signaling complexes. Examination of gene expression profiles indicates that, among REEPs expressed in adipocytes, REEP6 expression is uniquely upregulated by sympathetic nervous system activation, suggesting involvement in regulating adrenergic signal transduction. OBJECTIVE: The aim of this study was to assess the contribution of REEP6 to the thermogenic activation of adipocytes and characterize the metabolic consequences of REEP6 deficiency in vivo. METHODS: Expression levels of Reep6 in adipose tissue were examined by using public transcriptomic data and validated by Western blot and qPCR analyses. Adipocyte-specific regulatory roles of REEP6 were investigated in vitro in C3H10T1/2 adipocytes and in primary adipocytes obtained from REEP6 KO mice. Effects of in vivo REEP6 deficiency on energy expenditure were measured by indirect calorimetry. Mitochondrial content in adipose tissue was accessed by immunoblot, mitochondrial DNA analysis, and confocal and electron microscopy. Effects of REEP6 KO on obesity-induced metabolic dysfunction were tested in a high-fat diet-induced obesity mouse model by glucose tolerance test, Western blot, and histological analyses. RESULTS: REEP6 expression is highly enriched in murine adipocytes and is sharply upregulated upon adipocyte differentiation and by cold exposure. Inactivation of REEP6 in mice increased adiposity, and reduced energy expenditure and cold tolerance. REEP6 KO severely reduced protein kinase A-mediated signaling in BAT and greatly reduced mitochondrial mass. The effect of REEP6 inactivation on diminished ß-adrenergic signaling was reproduced in cultured adipocytes, indicating that this effect is cell-autonomous. REEP6 KO also suppressed expression of adenylate cyclase 3 (Adcy3) in brown adipose tissue and knockdown of REEP6 in adipocytes reduced targeting of ADCY3 to the plasma membrane. Lastly, REEP6 KO exacerbated high-fat diet-induced insulin resistance and inflammation in adipose tissue. CONCLUSIONS: This study indicates that REEP6 plays an important role in ß-adrenergic signal transduction in adipocytes involving the expression and trafficking of Adcy3. Genetic inactivation of REEP6 reduces energy expenditure, increases adiposity, and the susceptibility to obesity-related metabolic dysfunction.

Development of CIDEA reporter mouse model and its application for screening thermogenic drugs.

Cell death-inducing DNA fragmentation factor-like effector A (CIDEA) is a lipid droplet-associated protein and is a known marker of the thermogenic capacity of brown/beige adipocytes. To monitor the expression of CIDEA in live mice in a non-invasive manner, we generated CIDEA reporter mice expressing multicistronic mRNAs encoding CIDEA, luciferase 2, and tdTomato proteins under the control of the Cidea promoter. The expression level of endogenous CIDEA protein in adipose tissue was not affected by the expression of polycistronic reporters. The two CIDEA reporters, luciferase 2 and tdTomato, correctly reflected CIDEA protein levels. Importantly, luciferase activity was induced by cold exposure and the treatment with ß3-adrenergic receptor agonist CL316,243 in interscapular and inguinal adipose tissue, which was detectable by in vivo bioluminescence imaging. We further evaluated the effects of candidate brown adipogenic agents using this CIDEA reporter system and demonstrated a positive correlation between drug-induced luciferase activity and thermogenic gene expression levels both in vitro and in vivo. Collectively, we established a dual CIDEA reporter mouse model in which fluorescence and luminescence signals correctly reflect CIDEA expression, and therefore, suggested that this reporter system can be used to evaluate the thermogenic efficacy of candidate molecules.

TM4SF5 Knockout Protects Mice From Diet-Induced Obesity Partly by Regulating Autophagy in Adipose Tissue.

Transmembrane 4 L six family member 5 (TM4SF5) functions as a sensor for lysosomal arginine levels and activates the mammalian target of rapamycin complex 1 (mTORC1). While the mTORC1 signaling pathway plays a key role in adipose tissue metabolism, the regulatory function of TM4SF5 in adipocytes remains unclear. In this study we aimed to establish a TM4SF5 knockout (KO) mouse model and investigated the effects of TM4SF5 KO on mTORC1 signaling-mediated autophagy and mitochondrial metabolism in adipose tissue. TM4SF5 expression was higher in inguinal white adipose tissue (iWAT) than in brown adipose tissue and significantly upregulated by a high-fat diet (HFD). TM4SF5 KO reduced mTORC1 activation and enhanced autophagy and lipolysis in adipocytes. RNA sequencing analysis of TM4SF5 KO mouse iWAT showed that the expression of genes involved in peroxisome proliferator-activated receptor α signaling pathways and mitochondrial oxidative metabolism was upregulated. Consequently, TM4SF5 KO reduced adiposity and increased energy expenditure and mitochondrial oxidative metabolism. TM4SF5 KO prevented HFD-induced glucose intolerance and inflammation in adipose tissue. Collectively, the results of our study demonstrate that TM4SF5 regulates autophagy and lipid catabolism in adipose tissue and suggest that TM4SF5 could be therapeutically targeted for the treatment of obesity-related metabolic diseases.

STK3/STK4 signalling in adipocytes regulates mitophagy and energy expenditure.

Obesity reduces adipocyte mitochondrial function, and expanding adipocyte oxidative capacity is an emerging strategy to improve systemic metabolism. Here, we report that serine/threonine-protein kinase 3 (STK3) and STK4 are key physiological suppressors of mitochondrial capacity in brown, beige and white adipose tissues. Levels of STK3 and STK4, kinases in the Hippo signalling pathway, are greater in white than brown adipose tissues, and levels in brown adipose tissue are suppressed by cold exposure and greatly elevated by surgical denervation. Genetic inactivation of Stk3 and Stk4 increases mitochondrial mass and function, stabilizes uncoupling protein 1 in beige adipose tissue and confers resistance to metabolic dysfunction induced by high-fat diet feeding. Mechanistically, STK3 and STK4 increase adipocyte mitophagy in part by regulating the phosphorylation and dimerization status of the mitophagy receptor BNIP3. STK3 and STK4 expression levels are elevated in human obesity, and pharmacological inhibition improves metabolic profiles in a mouse model of obesity, suggesting STK3 and STK4 as potential targets for treating obesity-related diseases.

Anti-Obesity Effects of Soybean Embryo Extract and Enzymatically-Modified Isoquercitrin.

Soy isoflavones are bioactive phytoestrogens with known health benefits. Soybean embryo extract (SEE) has been consumed as a source of isoflavones, mainly daidzein, glycitein, and genistein. While previous studies have reported the anti-obesity effects of SEE, this study investigates their molecular mechanisms and the synergistic effects of co-treatment with SEE and enzymatically modified isoquercitrin (EMIQ). SEE upregulated genes involved in lipolysis and brown adipocyte markers and increased mitochondrial content in differentiated C3H10T1/2 adipocytes in vitro. Next, we use a high-fat diet-induced obesity mouse model to determine the anti-obesity effect of SEE. Two weeks of single or combined treatment with SEE and EMIQ significantly reduced body weight gain and improved glucose tolerance. Mechanistically, SEE treatment increased mitochondrial content and upregulated genes involved in lipolysis in adipose tissue through the cAMP/PKA-dependent signaling pathway. These effects required a cytosolic lipase adipose triglyceride lipase (ATGL) expression, confirmed by an adipocyte-specific ATGL knockout mouse study. Collectively, this study demonstrates that SEE exerts anti-obesity effects through the activation of adipose tissue metabolism and exhibits a synergistic effect of co-treatment with EMIQ. These results improve our understanding of the mechanisms underlying the anti-obesity effects of SEE related to adipose tissue metabolism.

Epigallocatechin-3-Gallate Reduces Visceral Adiposity Partly through the Regulation of Beclin1-Dependent Autophagy in White Adipose Tissues.

Epigallocatechin-3-gallate (EGCG) is a primary bioactive phytochemical in green tea. Its therapeutic potential in metabolic diseases has been reported; however, the molecular mechanisms of the anti-obesity effect of EGCG have not been fully elucidated. In this study, we examined the effects of EGCG on lipid metabolism and autophagy in adipose tissue. After 8 weeks of high-fat diet feeding, mice were treated with EGCG (20 mg/kg/day) for 2 weeks to test in vivo anti-obesity effects of EGCG. EGCG treatment improved glucose tolerance and caused body weight loss. Interestingly, reduced adipose tissue mass was more prominent in visceral compared to subcutaneous white adipose tissue. Mechanistically, EGCG treatment increased autophagic flux in white adipose tissue through the AMP-activated protein kinase-mediated signaling pathway. Adipocyte-specific knockout of Beclin1 mitigated the effects of EGCG on visceral adipose tissue mass and glucose tolerance, indicating that the anti-obesity effect of EGCG requires Beclin1-dependent autophagy. Collectively, our data demonstrated that EGCG has anti-obesity effects through the upregulation of Beclin1-dependent autophagy and lipid catabolism in white adipose tissue (WAT).

Adipocyte-specific Beclin1 deletion impairs lipolysis and mitochondrial integrity in adipose tissue.

OBJECTIVE: Beclin1 is a core molecule of the macroautophagy machinery. Although dysregulation of macroautophagy is known to be involved in metabolic disorders, the function of Beclin1 in adipocyte metabolism has not been investigated. In the present study, we aimed to study the role of Beclin1 in lipolysis and mitochondrial homeostasis of adipocytes. METHODS: Autophagic flux during lipolysis was examined in adipocytes cultured in vitro and in the adipose tissue of mice. Adipocyte-specific Beclin1 knockout (KO) mice were used to investigate the activities of Beclin1 in adipose tissues. RESULTS: cAMP/PKA signaling increased the autophagic flux in adipocytes differentiated from C3H10T1/2 cells. In vivo autophagic flux was higher in the brown adipose tissue (BAT) than that in the white adipose tissue and was further increased by the ß3 adrenergic receptor agonist CL316243. In addition, surgical denervation of BAT greatly reduced autophagic flux, indicating that sympathetic nerve activity is a major regulator of tissue autophagy. Adipocyte-specific KO of Beclin1 led to a hypertrophic enlargement of lipid droplets in BAT and impaired CL316243-induced lipolysis/lipid mobilization and energy expenditure. While short-term effects of Beclin1 deletion were characterized by an increase in mitochondrial proteins, long-term Beclin1 deletion led to severe disruption of autophagy, resulting in mitochondrial loss, and dramatically reduced the expression of genes involved in lipid metabolism. Consequently, adipose tissue underwent increased activation of cell death signaling pathways, macrophage recruitment, and inflammation, particularly in BAT. CONCLUSIONS: The present study demonstrates the critical roles of Beclin1 in the maintenance of lipid metabolism and mitochondrial homeostasis in adipose tissues.