In vitro studies of manganese transport and homeostasis.

Manganese (Mn) is an essential micronutrient required for fundamental cell functions and vital physiological processes. More than a dozen putative Mn transporters have been described over the last two decades, but few have been thoroughly evaluated. Recent genetic studies have revealed vital roles for solute carrier family 39, member 8 (SLC39A8) in Mn homeostasis. SLC39A8 can mediate the cellular uptake of the essential metals zinc, iron, and Mn, as well as the non-essential metal cadmium. However, loss-of-function mutations in SLC39A8 have been found in patients with severe Mn deficiency in the blood without affecting other metals. An in vitro study from our laboratory showed that SLC39A8 is a cell-surface transporter that strongly stimulates 54Mn incorporation into cells (Choi, Nguyen, Gupta, Iwase, & Seo, 2018). By contrast, the disease-associated mutations completely abrogated the cellular uptake of 54Mn (Choi et al., 2018), thereby providing a causal link between SLC39A8 deficiency and Mn deficiency. The importance of SLC39A8 is now increasingly recognized in multiple disease processes, and SLC39A8 has emerged as a critical regulator of Mn homeostasis. Thus, exploring the function of SLC39A8 in cellular Mn homeostasis is of significant research interest. This chapter describes the advanced methods used in our laboratory to examine Mn homeostasis and transport. Specifically, genetic and molecular approaches are described in HeLa cells overexpressing SLC39A8 and disease-associated SLC39A8 mutants. These methods are useful for characterizing the roles of Mn in diverse cellular events.

Phosphorylation and stabilization of EZH2 by DCAF1/VprBP trigger aberrant gene silencing in colon cancer.

Our recent work has shown that DCAF1 (also known as VprBP) is overexpressed in colon cancer and phosphorylates histone H2AT120 to drive epigenetic gene inactivation and oncogenic transformation. We have extended these observations by investigating whether DCAF1 also phosphorylates non-histone proteins as an additional mechanism linking its kinase activity to colon cancer development. We now demonstrate that DCAF1 phosphorylates EZH2 at T367 to augment its nuclear stabilization and enzymatic activity in colon cancer cells. Consistent with this mechanistic role, DCAF1-mediated EZH2 phosphorylation leads to elevated levels of H3K27me3 and altered expression of growth regulatory genes in cancer cells. Furthermore, our preclinical studies using organoid and xenograft models revealed that EZH2 requires phosphorylation for its oncogenic function, which may have therapeutic implications for gene reactivation in colon cancer cells. Together, our data define a mechanism underlying DCAF1-driven colonic tumorigenesis by linking DCAF1-mediated EZH2 phosphorylation to EZH2 stability that is crucial for establishing H3K27me3 and gene silencing program.

Multiplexed Ultrasound Imaging Using Spectral Analysis on Gas Vesicles.

Current advances in ultrasound imaging techniques combined with the next generation contrast agents such as gas vesicles (GV) revolutionize the visualization of biological tissues with spatiotemporal precision. In optics, fluorescent proteins enable understanding of molecular and cellular functions in biological systems due to their multiplexed imaging capability. Here, a panel of GVs is investigated using mid-band fit (MBF) spectral imaging to realize multiplexed ultrasound imaging to uniquely visualize locations of different types of stationary GVs. The MBF spectral imaging technique demonstrates that stationary clustered GVs are efficiently localized and distinguished from unclustered GVs in agarose gel phantom and 3D vessel structures are visualized in ex vivo mouse liver specimens. Mouse macrophages serve as carriers of clustered and unclustered GVs and multiplexing beacons to report cells' spatial locations by emitting distinct spectral signals. 2D MBF spectral images are reconstructed, and pixels in these images are classified depending on MBF values by comparing predetermined filters that predict the existence of cells with clustered and unclustered GVs. This pseudo-coloring scheme clearly distinguishes the locations of two classes of cells like pseudo-color images in fluorescence microscopy.

VprBP directs epigenetic gene silencing through histone H2A phosphorylation in colon cancer.

Histone modification is aberrantly regulated in cancer and generates an unbalanced state of gene transcription. VprBP, a recently identified kinase, phosphorylates histone H2A on threonine 120 (T120) and is involved in oncogenic transcriptional dysregulation; however, its specific role in colon cancer is undefined. Here, we show that VprBP is overexpressed in colon cancer and directly contributes to epigenetic gene silencing and cancer pathogenesis. Mechanistically, the observed function of VprBP is mediated through H2AT120 phosphorylation (H2AT120p)-driven transcriptional repression of growth regulatory genes, resulting in a significantly higher proliferative capacity of colon cancer cells. Our preclinical studies using organoid and xenograft models demonstrate that treatment with the VprBP inhibitor B32B3 impairs colonic tumor growth by blocking H2AT120p and reactivating a transcriptional program resembling that of normal cells. Collectively, our work describes VprBP as a master kinase contributing to the development and progression of colon cancer, making it a new molecular target for novel therapeutic strategies.

Measurements of acoustic radiation force of ultrahigh frequency ultrasonic transducers using model-based approach.

Even though ultrahigh frequency ultrasonic transducers over 60 MHz have been used for single-cell-level manipulation such as intracellular delivery, acoustic tweezers, and stimulation to investigate cell phenotype and cell mechanics, no techniques have been available to measure the actual acoustic radiation force (ARF) applied to target cells. Therefore, we have developed an approach to measure the ARF of ultrahigh frequency ultrasonic transducers using a theoretical model of the dynamics of a solid sphere in a gelatin phantom. To estimate ARF at the focus of a 130 MHz transducer, we matched measured maximum displacements of a solid sphere with theoretical calculations. We selected appropriate ranges of input voltages and pulse durations for single-cell applications, and the estimated ARF was in the range of tens of µN. To gauge the influence of pulse duration, an impulse of different pulse durations was estimated. Fluorescence resonance energy transfer live cell imaging was demonstrated to visualize calcium transport between cells after a target single cell was stimulated by the developed ultrasonic transducer.

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.

Antiobesity effects of coumestrol through expansion and activation of brown adipose tissue metabolism.

Coumestrol is a dietary phytoestrogen with estrogen-mimicking characteristics. This study investigated the molecular mechanisms of antiobesity effects of coumestrol. Two weeks of coumestrol treatment reduced body weight and improved glucose tolerance of high-fat diet (HFD)-fed mice. Notably, coumestrol treatment reduced adiposity but expanded brown adipose tissue mass. In addition, coumestrol treatment induced up-regulation of brown adipocyte markers and lipolytic gene expression in adipose tissue. Mechanistically, coumestrol induced an increase in mitochondrial contents of brown adipose tissue, which was associated with up-regulation of adenosine monophosphate-activated protein kinase and sirtuin 1. In vitro knockdown of estrogen receptor 1 inhibited the effect of coumestrol on brown adipogenic marker expression, increase in mitochondrial contents and oxygen consumption rate in brown adipocytes. Furthermore, lineage tracing of platelet-derived growth factor receptor A-positive (PDGFRA+) adipocyte progenitors confirmed increased levels of de novo brown adipogenesis from PDGFRA+ cells by coumestrol treatment. In conclusion, our results indicate that coumestrol has antiobesity effects through the expansion and activation of brown adipose tissue metabolism.

MicroRNA-10a-5p regulates macrophage polarization and promotes therapeutic adipose tissue remodeling.

OBJECTIVE: This study investigated the role of microRNAs generated from adipose tissue macrophages (ATMs) during adipose tissue remodeling induced by pharmacological and nutritional stimuli. METHODS: Macrophage-specific Dicer knockout (KO) mice were used to determine the roles of microRNA generated in macrophages in adipose tissue remodeling induced by the ß3-adrenergic receptor agonist CL316,243 (CL). RNA-seq was performed to characterize microRNA and mRNA expression profiles in isolated macrophages and PDGFRα+ adipocyte stem cells (ASCs). The role of miR-10a-5p was further investigated in cell culture, and in adipose tissue remodeling induced by CL treatment and high fat feeding. RESULTS: Macrophage-specific deletion of Dicer elevated pro-inflammatory gene expression and prevented CL-induced de novo beige adipogenesis in gonadal white adipose tissue (gWAT). Co-culture of ASCs with ATMs of wild type mice promoted brown adipocyte gene expression upon differentiation, but co-culture with ATMs of Dicer KO mice did not. Bioinformatic analysis of RNA expression profiles identified miR-10a-5p as a potential regulator of inflammation and differentiation in ATMs and ASCs, respectively. CL treatment increased levels of miR-10a-5p in ATMs and ASCs in gWAT. Interestingly, CL treatment elevated levels of pre-mir-10a in ATMs but not in ASCs, suggesting possible transfer from ATMs to ASCs. Elevating miR-10a-5p levels inhibited proinflammatory gene expression in cultured RAW 264.7 macrophages and promoted the differentiation of C3H10T1/2 cells into brown adipocytes. Furthermore, treatment with a miR-10a-5p mimic in vivo rescued CL-induced beige adipogenesis in Dicer KO mice. High fat feeding reduced miR-10a-5p levels in ATMs of gWAT, and treatment of mice with a miR-10a-5p mimic suppressed pro-inflammatory responses, promoted the appearance of new white adipocytes in gWAT, and improved systemic glucose tolerance. CONCLUSIONS: These results demonstrate an important role of macrophage-generated microRNAs in adipogenic niches and identify miR-10a-5p as a key regulator that reduces adipose tissue inflammation and promotes therapeutic adipogenesis.

Myricetin Exerts Anti-Obesity Effects through Upregulation of SIRT3 in Adipose Tissue.

Myricetin is a biologically active natural polyphenol with beneficial effects on metabolic health. This study aimed to examine the effects of myricetin on the expression levels of genes involved in lipolysis and mitochondrial respiration in adipocytes and the anti-obesity potential of myricetin. The results indicated that myricetin reduced triglyceride (TG) content and increased mitochondrial content and oxygen consumption rate (OCR) in adipocytes in vitro. To determine anti-obesity effect of myricetin, C57BL6/J mice were fed a high-fat diet (HFD) for eight weeks and then treated with myricetin (10 mg/kg) for 2 weeks. The in vivo treatment of myricetin reduced body weight by 11%. Furthermore, it improved the glucose tolerance, and increased fatty acid consumption of HFD-fed mice. Myricetin treatment increased Sirt3 expression and reduced the acetylation of mitochondrial proteins in adipose tissue. Finally, the knockdown of Sirt3 in adipocytes reduced the myricetin-induced increase in mitochondrial oxygen consumption rate by about 27% compared to controls. Our results indicated that myricetin exerted anti-obesity effects through the upregulation of Sirt3 expression and mitochondrial metabolism in adipose tissue.

Anti-inflammatory role of 15-lipoxygenase contributes to the maintenance of skin integrity in mice.

15-lipoxygenase is involved in the generation of specialized pro-resolving lipid mediators that play essential roles in resolution and inflammatory responses. Here, we investigated anti-inflammatory role of Alox15 in skin homeostasis. We demonstrated that knockout (KO) of Alox15 led to hair loss and disrupted the structural integrity of the dorsal skin. Alox15 KO resulted in loss of hair follicle stem cells and abnormal transition of dermal adipocytes into fibroblasts. Alox15 deficiency increased infiltration of proinflammatory macrophages and upregulated proinflammatory and necroptotic signaling in dermal adipose tissue in the dorsal skin. Lipidomic analysis revealed severe loss of resolvin D2 in the dorsal skin of Alox15 KO mice compared to wild type controls. Treatment with resolvin D2 reduced skin inflammation in Alox15 KO mice. Collectively, these results indicate that Alox15-mediated production of resolvin D2 is required to maintain skin integrity by suppressing dermal inflammation.

Connexin 43 is required for the maintenance of mitochondrial integrity in brown adipose tissue.

We investigated the role of connexin 43 (Cx43) in maintaining the integrity of mitochondria in brown adipose tissue (BAT). The functional effects of Cx43 were evaluated using inducible, adipocyte-specific Cx43 knockout in mice (Gja1 adipoq KO) and by overexpression and knockdown of Cx43 in cultured adipocytes. Mitochondrial morphology was evaluated by electron microscopy and mitochondrial function and autophagy were assessed by immunoblotting, immunohistochemistry, and qPCR. The metabolic effects of adipocyte-specific knockout of Cx43 were assessed during cold stress and following high fat diet feeding. Cx43 expression was higher in BAT compared to white adipose tissue. Treatment with the ß3-adrenergic receptor agonist CL316,243 increased Cx43 expression and mitochondrial localization. Gja1 adipoq KO mice reduced mitochondrial density and increased the presence of damaged mitochondria in BAT. Moreover, metabolic activation with CL316,243 further reduced mitochondrial integrity and upregulated autophagy in the BAT of Gja1 adipoq KO mice. Inhibition of Cx43 in cultured adipocytes increased the generation of reactive oxygen species and induction of autophagy during ß-adrenergic stimulation. Gja1 adipoq KO mice were cold intolerant, expended less energy in response to ß3-adrenergic receptor activation, and were more insulin resistant after a high-fat diet challenge. Collectively, our data demonstrate that Cx43 is required for maintaining the mitochondrial integrity and metabolic activity of BAT.

Effects of Epigallocatechin-3-Gallate on Autophagic Lipolysis in Adipocytes.

Previous studies demonstrated effects of green tea on weight loss; however, green tea-induced modulation of adipocyte function is not fully understood. Here, we investigated effects of the major green tea phytochemical, epigallocatechin-3-gallate (EGCG) on triglyceride contents, lipolysis, mitochondrial function, and autophagy, in adipocytes differentiated from C3H10T1/2 cells and immortalized pre-adipocytes in vitro. EGCG reduced the triglycerol content significantly in adipocytes by 25%, comparable to the nutrient starvation state. EGCG did not affect protein kinase A signaling or brown adipocyte marker expression in adipocytes; however, EGCG increased autophagy, as measured by autophagy flux analysis and immunoblot analysis of LC3B, ATG7, and Beclin1. EGCG treatment reduced mitochondrial membrane potential by 56.8% and intracellular ATP levels by 49.1% compared to controls. Although mammalian target of rapamycin signaling was not upregulated by EGCG treatment, EGCG treatment induced AMP-activated protein kinase phosphorylation, indicating an energy-depleted state. In addition, EGCG increased the association between RAB7 and lipid droplets, suggesting that lipophagy was activated. Finally, knockdown of Rab7 attenuated the EGCG-dependent reduction in lipid contents. Collectively, these results indicated that EGCG upregulated autophagic lipolysis in adipocytes, supporting the therapeutic potential of EGCG as a caloric restriction mimetic to prevent obesity and obesity-related metabolic diseases.

Phenolics and neolignans isolated from the fruits of Juglans mandshurica Maxim. and their effects on lipolysis in adipocytes.

Juglans mandshurica Maxim. (Juglandaceae) is a traditional folk medicine used for treatment of dermatosis and to relieve aches in Korea and China. In this study, eight compounds, along with six known compounds, were isolated from the fruit of J. mandshurica. Among the six known compounds, the absolute configuration of two compounds were determined. The structures of compounds were determined on the basis of extensive spectroscopic methods, including 1D and 2D NMR and CD spectroscopic data. All isolated compounds were tested for their lipolytic activities in differentiated adipocytes using C3H10T1/2 mouse embryonic fibroblasts. Among them, 2-(4-formyl-2-methoxyphenoxy)-propan-1,3-diol and 2-[4-(3-hydroxypropyl)-2-methoxyphenoxy]-1,3-propanediol exhibited the most potent lipolytic activities.

Metabolic heterogeneity of activated beige/brite adipocytes in inguinal adipose tissue.

Sustained ß3 adrenergic receptor (ADRB3) activation simultaneously upregulates fatty acid synthesis and oxidation in mouse brown, beige, and white adipose tissues; however, the cellular basis of this dual regulation is not known. Treatment of mice with the ADRB3 agonist CL316,243 (CL) increased expression of fatty acid synthase (FASN) and medium chain acyl-CoA dehydrogenase (MCAD) protein within the same cells in classic brown and white adipose tissues. Surprisingly, in inguinal adipose tissue, CL-upregulated FASN and MCAD in distinct cell populations: high MCAD expression occurred in multilocular adipocytes that co-expressed UCP1+, whereas high FASN expression occurred in paucilocular adipocytes lacking detectable UCP1. Genetic tracing with UCP1-cre, however, indicated nearly half of adipocytes with a history of UCP1 expression expressed high levels of FASN without current expression of UCP1. Global transcriptomic analysis of FACS-isolated adipocytes confirmed the presence of distinct anabolic and catabolic phenotypes, and identified differential expression of transcriptional pathways known to regulate lipid synthesis and oxidation. Surprisingly, paternally-expressed genes of the non-classical gene imprinted network were strikingly enriched in anabolic phenotypes, suggesting possible involvement in maintaining the balance of metabolic phenotypes. The results indicate that metabolic heterogeneity is a distinct property of activated beige/brite adipocytes that might be under epigenetic control.

Sex differences in sympathetic innervation and browning of white adipose tissue of mice.

BACKGROUND: The higher prevalence of obesity-related metabolic disease in males suggests that female sex hormones provide protective mechanisms against the pathogenesis of metabolic syndrome. Because browning of white adipose tissue (WAT) is protective against obesity-related metabolic disease, we examined sex differences in ß3-adrenergic remodeling of WAT in mice. METHODS: Effects of the ß3-adrenergic receptor agonist CL316,243 (CL) on browning of white adipose tissue were investigated in male and female C57BL mice. The role of ovarian hormones in female-specific browning was studied in control female C57BL mice and mice with ovarian failure induced by 4-vinylcyclohexene diepoxide treatment for 15 days. RESULTS: We found that treatment with CL-induced upregulation of brown adipocyte markers and mitochondrial respiratory chain proteins in gonadal WAT (gWAT) of female mice, but was without effect in males. In contrast, CL treatment was equally effective in males and females in inducing brown adipocyte phenotypes in inguinal WAT. The tissue- and sex-specific differences in brown adipocyte recruitment were correlated with differences in sympathetic innervation, as determined by tyrosine hydroxylase immunostaining and western blotting. Levels of the neurotrophins NGF and BDNF were significantly higher in gWAT of female mice. CL treatment significantly increased NGF levels in gWAT of female mice but did not affect BDNF expression. In contrast, estradiol treatment doubled BDNF expression in female adipocytes differentiated in vitro. Ovarian failure induced by 4-vinylcyclohexene diepoxide treatment dramatically reduced BDNF and TH expression in gWAT, eliminated induction of UCP1 by CL, and reduced tissue metabolic rate. CONCLUSIONS: Collectively, these data demonstrate that female mice are more responsive than males to the recruitment of brown adipocytes in gonadal WAT and this difference corresponds to greater levels of estrogen-dependent sympathetic innervation.

Sex-specific metabolic interactions between liver and adipose tissue in MCD diet-induced non-alcoholic fatty liver disease.

Higher susceptibility to metabolic disease in male exemplifies the importance of sexual dimorphism in pathogenesis. We hypothesized that the higher incidence of non-alcoholic fatty liver disease in males involves sex-specific metabolic interactions between liver and adipose tissue. In the present study, we used a methionine-choline deficient (MCD) diet-induced fatty liver mouse model to investigate sex differences in the metabolic response of the liver and adipose tissue. After 2 weeks on an MCD-diet, fatty liver was induced in a sex-specific manner, affecting male mice more severely than females. The MCD-diet increased lipolytic enzymes in the gonadal white adipose tissue (gWAT) of male mice, whereas it increased expression of uncoupling protein 1 and other brown adipocyte markers in the gWAT of female mice. Moreover, gWAT from female mice demonstrated higher levels of oxygen consumption and mitochondrial content compared to gWAT from male mice. FGF21 expression was increased in liver tissue by the MCD diet, and the degree of upregulation was significantly higher in the livers of female mice. The endocrine effect of FGF21 was responsible, in part, for the sex-specific browning of gonadal white adipose tissue. Collectively, these data demonstrated that distinctively female-specific browning of white adipose tissue aids in protecting female mice against MCD diet-induced fatty liver disease.

Adipogenic role of alternatively activated macrophages in ß-adrenergic remodeling of white adipose tissue.

De novo brown adipogenesis involves the proliferation and differentiation of progenitors, yet the mechanisms that guide these events in vivo are poorly understood. We previously demonstrated that treatment with a ß3-adrenergic receptor (ADRB3) agonist triggers brown/beige adipogenesis in gonadal white adipose tissue following adipocyte death and clearance by tissue macrophages. The close physical relationship between adipocyte progenitors and tissue macrophages suggested that the macrophages that clear dying adipocytes might generate proadipogenic factors. Flow cytometric analysis of macrophages from mice treated with CL 316,243 identified a subpopulation that contained elevated lipid and expressed CD44. Lipidomic analysis of fluorescence-activated cell sorting-isolated macrophages demonstrated that CD44+ macrophages contained four- to five-fold higher levels of the endogenous peroxisome-proliferator activated receptor gamma (PPARγ) ligands 9-hydroxyoctadecadienoic acid (HODE), and 13-HODE compared with CD44- macrophages. Gene expression profiling and immunohistochemistry demonstrated that ADRB3 agonist treatment upregulated expression of ALOX15, the lipoxygenase responsible for generating 9-HODE and 13-HODE. Using an in vitro model of adipocyte efferocytosis, we found that IL-4-primed tissue macrophages accumulated lipid from dying fat cells and upregulated expression of Alox15. Furthermore, treatment of differentiating adipocytes with 9-HODE and 13-HODE potentiated brown/beige adipogenesis. Collectively, these data indicate that noninflammatory removal of adipocyte remnants and coordinated generation of PPARγ ligands by M2 macrophages provides localized adipogenic signals to support de novo brown/beige adipogenesis.