Subcutaneous Transplantation of White Adipose Tissue.

In the research setting, white adipose tissue (WAT) transplantation, also known as fat transplantation, is often used to understand the physiological function of adipocytes or associated stromal vascular cells such as macrophages in the context of local and systemic metabolism. The mouse is the most common animal model used where WAT from a donor is transferred either to a subcutaneous site of the same organism or to a subcutaneous region of a recipient. Here, we describe in detail the procedure for heterologous fat transplantation, and, given the need for survival surgery, peri- and postoperative care and subsequent histological confirmation of fat grafts will also be discussed.

Profilin-1 regulates DNA replication forks in a context-dependent fashion by interacting with SNF2H and BOD1L.

DNA replication forks are tightly controlled by a large protein network consisting of well-known core regulators and many accessory factors which remain functionally undefined. In this study, we report previously unknown nuclear functions of the actin-binding factor profilin-1 (PFN1) in DNA replication, which occur in a context-dependent fashion and require its binding to poly-L-proline (PLP)-containing proteins instead of actin. In unperturbed cells, PFN1 increases DNA replication initiation and accelerates fork progression by binding and stimulating the PLP-containing nucleosome remodeler SNF2H. Under replication stress, PFN1/SNF2H increases fork stalling and functionally collaborates with fork reversal enzymes to enable the over-resection of unprotected forks. In addition, PFN1 binds and functionally attenuates the PLP-containing fork protector BODL1 to increase the resection of a subset of stressed forks. Accordingly, raising nuclear PFN1 level decreases genome stability and cell survival during replication stress. Thus, PFN1 is a multi-functional regulator of DNA replication with exploitable anticancer potential.

Metabolomics reveals inosine 5'-monophosphate is increased during mice adipocyte browning.

Adipocyte browning is one of the potential strategies for the prevention of obesity-related metabolic syndromes, but it is a complex process. Although previous studies make it increasingly clear that several transcription factors and enzymes are essential to induce browning, it is unclear what dynamic and metabolic changes occur in induction of browning. Here, we analyzed the effect of a beta-adrenergic receptor agonist (CL316243, accelerator of browning) on metabolic change in mice adipose tissue and plasma using metabolome analysis and speculated that browning is regulated partly by inosine 5'-monophosphate (IMP) metabolism. To test this hypothesis, we investigated whether Ucp-1, a functional marker of browning, mRNA expression is influenced by IMP metabolism using immortalized adipocytes. Our study showed that mycophenolic acid, an IMP dehydrogenase inhibitor, increases the mRNA expression of Ucp-1 in immortalized adipocytes. Furthermore, we performed a single administration of mycophenolate mofetil, a prodrug of mycophenolic acid, to mice and demonstrated that mycophenolate mofetil induces adipocyte browning and miniaturization of adipocyte size, leading to adipose tissue weight loss. These findings showed that IMP metabolism has a significant effect on adipocyte browning, suggesting that the regulator of IMP metabolism has the potential to prevent obesity.

Effects of Persimmon Fruit Polyphenols on Postprandial Plasma Glucose Elevation in Rats and Humans.

Persimmon is a fruit rich in polyphenols (proanthocyanidins or condensed tannins). Using rats and humans, the effects of Kaki-tannin (Nara-type), persimmon polyphenols prepared using a new method, on postprandial plasma glucose levels were investigated in this study. Kaki-tannin (Nara-type) comprised mainly proanthocyanidins, composed of epicatechin : epicatechin gallate : epigallocatechin : epigallocatechin gallate in a ratio of 1 : 1 : 2 : 2 with a molecular weight of approximately 8,000 Da, with epicatechin gallate as a terminal unit. These polyphenols inhibited amylolytic enzymes, such as α-amylase, maltase, sucrase, and α-glucosidase in vitro, and sodium-dependent glucose transporter 1 in Caco-2 cells. These results suggested that the polyphenols suppressed digestion and absorption in the intestinal tract. The ingestion of 250 mg/kg body weight of the polyphenols significantly suppressed increased blood glucose levels after carbohydrate (2 g/kg body weight of glucose or maltose) loading in rats. In a human trial, 1.88 g of Kaki-tannin (Nara-type) significantly delayed increased plasma glucose levels after carbohydrate (150 kcal of maltooligosaccharides) loading. Thus, Kaki-tannin (Nara-type) holds promise to be developed as a food material that potentially improve blood glucose elevation after meals.

Comparative Analysis of the Preventive Effects of Canagliflozin, a Sodium-Glucose Co-Transporter-2 Inhibitor, on Body Weight Gain Between Oral Gavage and Dietary Administration by Focusing on Fatty Acid Metabolism.

PURPOSE: Sodium-glucose co-transporter-2 (SGLT2) inhibitors have various pleiotropic effects, including body weight reduction, and therefore have the potential to be used in various applications. However, such effects have not been fully investigated; thus, non-clinical studies using animal models are needed. In animal experiments, SGLT2 inhibitors are usually administered by oral or dietary methods. However, the detailed characteristics of these dosing methods, especially to induce their pleiotropic effects, have not been reported. Therefore, we compared the preventive effects of canagliflozin, an SGLT2 inhibitor, on body weight gain following oral gavage and dietary administration methods in a mouse model of diet-induced obesity. METHODS: Canagliflozin was dosed by oral gavage or dietary administration for 9 weeks to 6-week-old C57BL/6N mice fed a high-fat diet, and parameters related to obesity were evaluated. RESULTS: The suppression of body weight gain, fat mass, and hepatic lipid content was observed following both dosing methods, whereas the effect on body weight tended to be stronger in the dietary administration group. In adipose tissue, fatty acid synthase expression was significantly decreased in the dietary administration group, and its expression was significantly correlated with fat mass. However, the expression of genes related to fatty acid oxidation was unchanged, indicating that the preventive effect on body weight gain was mediated mainly through the suppression of lipid synthesis rather than the promotion of lipid oxidation. CONCLUSION: Canagliflozin prevented body weight gain through the suppression of lipid synthesis via both dosing methods, although there were some differences in the efficacy. The findings of our study can help to identify new mechanisms of action of SGLT2 inhibitors and potential applications.

Glycerol kinase stimulates uncoupling protein 1 expression by regulating fatty acid metabolism in beige adipocytes.

Browning of adipose tissue is induced by specific stimuli such as cold exposure and consists of up-regulation of thermogenesis in white adipose tissue. Recently, it has emerged as an attractive target for managing obesity in humans. Here, we performed a comprehensive analysis to identify genes associated with browning in murine adipose tissue. We focused on glycerol kinase (GYK) because its mRNA expression pattern is highly correlated with that of uncoupling protein 1 (UCP1), which regulates the thermogenic capacity of adipocytes. Cold exposure-induced Ucp1 up-regulation in inguinal white adipose tissue (iWAT) was partially abolished by Gyk knockdown (KD) in vivo Consistently, the Gyk KD inhibited Ucp1 expression induced by treatment with the ß-adrenergic receptors (ßAR) agonist isoproterenol (Iso) in vitro and resulted in impaired uncoupled respiration. Gyk KD also suppressed Iso- and adenylate cyclase activator-induced transcriptional activation and phosphorylation of the cAMP response element-binding protein (CREB). However, we did not observe these effects with a cAMP analog. Therefore Gyk KD related to Iso-induced cAMP products. In Iso-treated Gyk KD adipocytes, stearoyl-CoA desaturase 1 (SCD1) was up-regulated, and monounsaturated fatty acids such as palmitoleic acid (POA) accumulated. Moreover, a SCD1 inhibitor treatment recovered the Gyk KD-induced Ucp1 down-regulation and POA treatment down-regulated Iso-activated Ucp1 Our findings suggest that Gyk stimulates Ucp1 expression via a mechanism that partially depends on the ßAR-cAMP-CREB pathway and Gyk-mediated regulation of fatty acid metabolism.

Long non-coding RNA 2310069B03Rik functions as a suppressor of Ucp1 expression under prolonged cold exposure in murine beige adipocytes.

Specific conditions, such as exposure to cold, can induce the production of brown-like adipocytes in white adipose tissue. These adipocytes express high levels of uncoupling protein 1 (UCP1) and energy expended by generating heat. Thus, these are a potential target for the prevention or treatment of obesity. The present study involved a comprehensive analysis of the adipose tissue to understand the relationship between long non-coding RNA (lncRNA) 2310069B03Rik and UCP1. Cold exposure increased both lncRNA 2310069B03Rik and Ucp1 expression in inguinal white adipose tissue (iWAT). However, overexpression of lncRNA 2310069B03Rik suppressed the Ucp1 mRNA expression and the promoter activity of UCP1 in the iWAT primary adipocytes. In addition, compared to the early induction of Ucp1 expression by cold stimulation, the induction of lncRNA 2310069B03Rik expression was later. These results suggest that lncRNA 2310069B03Rik functions as a suppression factor of Ucp1 expression.

ß-Cryptoxanthin Induces UCP-1 Expression via a RAR Pathway in Adipose Tissue.

While ß-cryptoxanthin is hypothesized to have a preventive effect on lifestyle-related diseases, its underlying mechanisms are unknown. We investigated the effect of ß-cryptoxanthin on energy metabolism in adipose tissues and its underlying mechanism. C57BL/6J mice were fed a high-fat diet (60% kcal fat) containing 0 or 0.05% ß-cryptoxanthin for 12 weeks. ß-cryptoxanthin treatment was found to reduce body fat gain and plasma glucose level, while increasing energy expenditure. The expression of uncoupling protein (UCP) 1 was elevated in adipose tissues in the treatment group. Furthermore, the in vivo assays showed that the Ucp1 mRNA expression was higher in the ß-cryptoxanthin treatment group, an effect that disappeared upon cotreatment with a retinoic acid receptor (RAR) antagonist. In conclusion, we report that ß-cryptoxanthin reduces body fat and body weight gain and that ß-cryptoxanthin increases the expression of UCP1 via the RAR pathway.

The Mevalonate Pathway Is Indispensable for Adipocyte Survival.

The mevalonate pathway is essential for the synthesis of isoprenoids and cholesterol. Adipose tissue is known as a major site for cholesterol storage; however, the role of the local mevalonate pathway and its synthesized isoprenoids remains unclear. In this study, adipose-specific mevalonate pathway-disrupted (aKO) mice were generated through knockout of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase (HMGCR). aKO mice showed serious lipodystrophy accompanied with glucose and lipid metabolic disorders and hepatomegaly. These metabolic variations in aKO mice were dramatically reversed after fat transplantation. In addition, HMGCR-disrupted adipocytes exhibited loss of lipid accumulation and an increase of cell death, which were ameliorated by the supplementation of mevalonate and geranylgeranyl pyrophosphate but not farnesyl pyrophosphate and squalene. Finally, we found that apoptosis may be involved in adipocyte death induced by HMGCR down-regulation. Our findings indicate that the mevalonate pathway is essential for adipocytes and further suggest that this pathway is an important regulator of adipocyte turnover.

A Phytol-Enriched Diet Activates PPAR-α in the Liver and Brown Adipose Tissue to Ameliorate Obesity-Induced Metabolic Abnormalities.

SCOPE: Peroxisome proliferator-activated receptor alpha (PPAR-α) is a ligand-activated transcription factor that regulates lipid and carbohydrate metabolism. We investigate the effects of naturally occurring PPAR-α agonists, phytol, and its metabolite phytanic acid, on obesity-induced metabolic disorders using a mouse model. METHODS AND RESULTS: A luciferase reporter assay shows that phytanic acid potently activates PPAR-α among PPAR subtypes. In high-fat-diet-induced, severely obese mice, a phytol-enriched diet increases phytanic acid levels in the liver and adipose tissue, where PPAR-α is abundantly expressed. A phytol-enriched diet ameliorates severe obesity and the related metabolic abnormalities of white adipose tissue. Moreover, the expression of PPAR-α target genes in the liver and brown adipose tissue is enhanced by a phytol-enriched diet, suggesting that phytol and phytanic acid activate PPAR-α in these organs. We confirm that phytanic acid treatment induced PPAR-α target gene expression in both primary hepatocytes and brown adipocytes from wild-type mice, but not in these cells from PPAR-α-deficient mice. CONCLUSION: A phytol-enriched diet may increase phytanic acid levels in the liver and brown adipocytes, thereby activating PPAR-α in these organs and ameliorating obesity-induced metabolic diseases.

α-Linolenic acid-derived metabolites from gut lactic acid bacteria induce differentiation of anti-inflammatory M2 macrophages through G protein-coupled receptor 40.

Among dietary fatty acids with immunologic effects, ω-3 polyunsaturated fatty acids, such as α-linolenic acid (ALA), have been considered as factors that contribute to the differentiation of M2-type macrophages (M2 macrophages). In this study, we examined the effect of ALA and its gut lactic acid bacteria metabolites 13-hydroxy-9(Z),15(Z)-octadecadienoic acid (13-OH) and 13-oxo-9(Z),15(Z)-octadecadienoic acid (13-oxo) on the differentiation of M2 macrophages from bone marrow-derived cells (BMDCs) and investigated the underlying mechanisms. BMDCs were stimulated with ALA, 13-OH, or 13-oxo in the presence of IL-4 or IL-13 for 24 h, and significant increases in M2 macrophage markers CD206 and Arginase-1 (Arg1) were observed. In addition, M2 macrophage phenotypes were less prevalent following cotreatment with GPCR40 antagonists or inhibitors of PLC-ß and MEK under these conditions, suggesting that GPCR40 signaling is involved in the regulation of M2 macrophage differentiation. In further experiments, remarkable M2 macrophage accumulation was observed in the lamina propria of the small intestine of C57BL/6 mice after intragastric treatments with ALA, 13-OH, or 13-oxo at 1 g/kg of body weight per day for 3 d. These findings suggest a novel mechanism of M2 macrophage differentiation involving fatty acids from gut lactic acid bacteria and GPCR40 signaling.-Ohue-Kitano, R., Yasuoka, Y., Goto, T., Kitamura, N., Park, S.-B., Kishino, S., Kimura, I., Kasubuchi, M., Takahashi, H., Li, Y., Yeh, Y.-S., Jheng, H.-F., Iwase, M., Tanaka, M., Masuda, S., Inoue, T., Yamakage, H., Kusakabe, T., Tani, F., Shimatsu, A., Takahashi, N., Ogawa, J., Satoh-Asahara, N., Kawada, T. α-Linolenic acid-derived metabolites from gut lactic acid bacteria induce differentiation of anti-inflammatory M2 macrophages through G protein-coupled receptor 40.

Suksdorfin Promotes Adipocyte Differentiation and Improves Abnormalities in Glucose Metabolism via PPARγ Activation.

Although the Apiaceae herb family has been traditionally used for the management of type 2 diabetes, its molecular mechanism has not been clarified. Coumarin derivatives, which are abundant in plants of the Apiaceae family, were evaluated for their effects on adipogenesis. We found that suksdorfin significantly promoted adipocyte differentiation and enhanced production of adiponectin, an anti-diabetic adipokine. We also demonstrated that suksdorfin activates peroxisome proliferator-activated receptor gamma (PPARγ), a master regulator of adipogenesis. Furthermore, we showed metabolic disorders in obese diabetic KK-Ay mice were attenuated by suksdorfin feeding. Suksdorfin intake induced adipocyte miniaturization and increased expression levels of PPARγ target genes related to adipocyte differentiation. These results indicated that suksdorfin induces adipogenesis in white adipose tissue (WAT) via the activation of PPARγ, leading to improvement of obesity-induced metabolic disorders. Therefore, suksdorfin-mediated amelioration of WAT dysfunctions might be responsible for the anti-diabetic effects of traditional herbal medicine therapy with Apiaceae.

The hepatokine FGF21 is crucial for peroxisome proliferator-activated receptor-α agonist-induced amelioration of metabolic disorders in obese mice.

Obesity causes excess fat accumulation in white adipose tissues (WAT) and also in other insulin-responsive organs such as the skeletal muscle, increasing the risk for insulin resistance, which can lead to obesity-related metabolic disorders. Peroxisome proliferator-activated receptor-α (PPARα) is a master regulator of fatty acid oxidation whose activator is known to improve hyperlipidemia. However, the molecular mechanisms underlying PPARα activator-mediated reduction in adiposity and improvement of metabolic disorders are largely unknown. In this study we investigated the effects of PPARα agonist (fenofibrate) on glucose metabolism dysfunction in obese mice. Fenofibrate treatment reduced adiposity and attenuated obesity-induced dysfunctions of glucose metabolism in obese mice fed a high-fat diet. However, fenofibrate treatment did not improve glucose metabolism in lipodystrophic A-Zip/F1 mice, suggesting that adipose tissue is important for the fenofibrate-mediated amelioration of glucose metabolism, although skeletal muscle actions could not be completely excluded. Moreover, we investigated the role of the hepatokine fibroblast growth factor 21 (FGF21), which regulates energy metabolism in adipose tissue. In WAT of WT mice, but not of FGF21-deficient mice, fenofibrate enhanced the expression of genes related to brown adipocyte functions, such as Ucp1, Pgc1a, and Cpt1b Fenofibrate increased energy expenditure and attenuated obesity, whole body insulin resistance, and adipocyte dysfunctions in WAT in high-fat-diet-fed WT mice but not in FGF21-deficient mice. These findings indicate that FGF21 is crucial for the fenofibrate-mediated improvement of whole body glucose metabolism in obese mice via the amelioration of WAT dysfunctions.