Ursodeoxycholic Acid Regulates Hepatic Energy Homeostasis and White Adipose Tissue Macrophages Polarization in Leptin-Deficiency Obese Mice

Obesity has been shown to play a role in the pathogenesis of several forms of metabolic syndrome, including non-alcoholic fatty liver disease (NAFLD) and type 2 diabetes. Ursodeoxycholic acid (UDCA) has been shown to possess antioxidant and anti-inflammatory properties and prevents mitochondrial dysfunction in the progression of obesity-associated diseases. The aim of the study was to evaluate the mechanisms of UDCA during obesity-linked hepatic mitochondrial dysfunction and obesity-associated adipose tissue macrophage-induced inflammation in obese mice. UDCA significantly decreased lipid droplets, reduced free fatty acids (FFA) and triglycerides (TG), improved mitochondrial function, and enhanced white adipose tissue browning in ob/ob mice. This is associated with increased hepatic energy expenditure, mitochondria biogenesis, and incorporation of bile acid metabolism (Abca1, Abcg1 mRNA and BSEP, FGFR4, and TGR5 protein). In addition, UDCA downregulated NF-κB and STAT3 phosphorylation by negative regulation of the expression of SOCS1 and SOCS3 signaling. These changes were accompanied by decreased angiogenesis, as shown by the downregulation of VEGF, VCAM, and TGF-βRII expression. Importantly, UDCA is equally effective in reducing whole body adiposity. This is associated with decreased adipose tissue expression of macrophage infiltration (CD11b, CD163, and CD206) and lipogenic capacity markers (lipofuscin, SREBP-1, and CD36). Furthermore, UDCA significantly upregulated adipose browning in association with upregulation of SIRT-1-PGC1-α signaling in epididymis adipose tissue (EWAT). These results suggest that multi-targeted therapies modulate glucose and lipid biosynthesis fluxes, inflammatory response, angiogenesis, and macrophage differentiation. Therefore, it may be suggested that UDCA treatment may be a novel therapeutic agent for obesity.


Introduction
Obesity-induced non-alcoholic fatty liver disease (NAFLD) is a major risk factor for type 2 diabetes, hyperlipidemia, and some types of cancer [1,2]. Although the mechanism by which obesity causes NAFLD is unclear, inflammation has been linked to the development of local and systemic metabolic syndrome, especially, the inflammatory signaling occurs in liver and white adipose tissue (WAT) [3,4].

Animals
Male C57BL/6J (normal) mice and leptin-deficient C57BL/6J (also named ob/ob) mice were obtained from the National Laboratory Animal Center (Taiwan). All animals were fed standard chow and water ad libitum and housed under conditions of control temperature (26 • C) and illumination (12-h light/dark cycle, light off at 7 pm). Twenty-week-old mice were randomized into three groups (n = 5 per group): (i) normal mice, (ii) ob/ob mice, and (iii) ob/ob mice were fed with UDCA (50 mg/kg/mouse, stock solution in 33% ethanol, 33% dimethyl sulfoxide, 33% Tween-80, finally diluted 1:50 in 0.5 M NaCl; UDCA from Sigma-Aldrich, U5127) once a day orally for 14 consecutive days. Each experimental group consisted of five animals and all mice remained on their assigned diet until they were sacrificed by CO 2 . All protocols were conducted in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the Chang Gung University Animal Care and Use Committee.

Oil Red O Stain
Cells were incubated in 6-well plates with UDCA or DMSO control in 1 mM HFFA DMEM/F12 medium for 24 h. Cultured AML12 cells were fixed in buffered formalin. For the quantitative analysis of lipids, cells were incubated with Oil Red O (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Fresh liver tissues were embedded carefully in an optimal cutting temperature compound (OCT) in a plastic mold, followed by freezing at −80 • C. Cells and liver tissue sections (10 µm thick) were stained with Oil Red O working solution (w/v, 60% isopropyl alcohol and 40% water) for 15 min. Cells and liver tissue sections were rinsed with 50% isopropanol and counterstained with hematoxylin for the nucleus. Fat droplets in the liver were stained red.

Auto-Fluorescence Detection of Lipofuscin
The liver and EWAT sections were deparaffinized, hydrated, and mounted into 40% glycerol/TBS mounting medium. Lipofuscin auto-fluorescence was then evidenced by excitation at 450-490 nm, using a dichromatic mirror at 510 nm and a long-pass filter at 515 nm [25]. In addition, DAPI was used for nuclear staining (Thermo Fisher Scientific).

Plasma Aminotransferase (ALT) Analysis
ALT measurements were used in the diagnosis and treatment of certain liver diseases. Plasma ALT was determined with the ALT assay kit (RANDOX, Antrim, United Kingdom).

Plasma and Hepatic Triglyceride (TG) Measurement
The plasma sample was used directly. We extracted the lipids from liver samples using chloroform/methanol (2:1) for 1 h at 25 • C, as described previously [26], and then analyzed hepatic TG with TG kit (RANDOX).

RNA Isolation and Real-Time PCR Analysis
Total RNA was isolated from tissues using TRzol and RNeasy kit (Thermo Fisher Scientific). Equal amounts of RNA were retrotranscribed to cDNA using High-Capacity cDNA Reverse transcription kit (Thermo Fisher Scientific). qRT-PCR (real-time reverse transcription-PCR) was run in 10 µL reactions in a Roche real-time PCR system machine from Applied Biosystems using SYBR Green PCR master mix (Roche, Schweizerische) according to the manufacturer's instructions. Standard and melting curves were run in every plate for every gene to ensure efficiency and specificity of the reaction. The sequences of primers used for RT-qPCR are listed in Table 2.

Statistical Analysis
Data were presented as the means ± standard error of the mean (SEM). The statistical analyses were performed using Student's t-test and one-way analysis of variance followed by the Student Newman-Keuls multiple-range test. A value of P < 0.05 was considered to indicate a statistically significant difference.

UDCA Attenuates Hepatosteatosis by Reducing
Lipogenesis and β-Oxidation in ob/ob Mice ob/ob mice showed a high body weight compared with normal mice; we determined that UDCA significantly reduced body weight and improved the ratio of liver weight to body weight (Table 3). Plasma ALT, cholesterol, plasma and hepatic TG, and FFA levels were significantly decreased in ob/ob mice treated with UDCA (Table 3), suggesting UDCA ameliorated obesity and altered lipid metabolism. To further investigate the effects of UDCA on hepatosteatosis in ob/ob mice, we measured lipid deposits and the pathway of lipid metabolism in ob/ob mice. Histological sections of ob/ob liver showed large lipid-filled vacuoles, whereas ob/ob mice treatment UDCA showed a significantly smaller lipid-filled vacuole ( Figure 2A). Immunohistochemical analysis showed the high CD36 and SREBP-1c protein levels in ob/ob mice, and high Srebp-1c, Fas, and Scd-1 mRNA expression were found in liver tissue. We showed that UDCA significantly reduced the lipogenesis pathway in the liver (Figure 2A-C). UDCA attenuated fatty acid transporter protein (Fatp), Cd36, carnitine palmitoyltransferase 1 (Cpt-1), acyl-CoA oxidase (Aco), and decreased peroxisome proliferator-activated receptor (Ppar) α, Pparγ, PPARγ coactivator 1-beta (Ppargc1b), hormone-sensitive lipase (Hsl), diacylglycerol acyltransferase (Dgat), and long-chain Acyl-CoA dehydrogenase (Lcad) mRNA expression in ob/ob mice ( Figure 2D-F).

UDCA Ameliorates Hepatic Glucose Metabolism Disorder and Notch1 Signaling in ob/ob Mice
To determine the dynamics of recovery from obesity-associated glucose dysmetabolism, we identified the mechanisms of hepatic glucose metabolism in mice. Protein levels of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) were increased in ob/ob mice, and the mRNA expression of pyruvate dehydrogenase lipoamide kinase isozyme 4 (Pdk4) was suppressed in ob/ob mice compared with normal mice ( Figure 3A,B). UDCA significantly reduced protein levels and mRNA expression of PEPCK and G6Pase and had little effect on the mRNA expression of Pdk4 in ob/ob mice ( Figure 3A,B), suggesting UDCA reduces gluconeogenesis in ob/ob mice. We next detected that ob/ob mice had higher mRNA expression of hepatic pyruvate dehydrogenase alpha 1 (Pdha1), glycogen synthase (Gys) 1 and Gys2, and lower mRNA expression of phosphatidylinositol 3-kinase regulatory subunit 1 (Pik3r1) than normal mice; these were significantly improved in ob/ob mice treated with UDCA, but had no effect on Gys2 ( Figure 3C). We focused on the molecular mechanisms by which UDCA affect glucose metabolism in the liver.

UDCA Ameliorates Hepatic Glucose Metabolism Disorder and Notch1 Signaling in ob/ob Mice
To determine the dynamics of recovery from obesity-associated glucose dysmetabolism, we identified the mechanisms of hepatic glucose metabolism in mice.
Protein levels of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) were increased in ob/ob mice, and the mRNA expression of pyruvate dehydrogenase lipoamide kinase isozyme 4 (Pdk4) was suppressed in ob/ob mice compared with normal mice ( Figure 3A,B). UDCA significantly reduced protein levels and mRNA expression of PEPCK and G6Pase and had little effect on the mRNA expression of Pdk4 in ob/ob mice ( Figure 3A,B), suggesting UDCA reduces gluconeogenesis in ob/ob mice. We next detected that ob/ob mice had higher mRNA expression of hepatic pyruvate dehydrogenase alpha 1 (Pdha1), glycogen synthase (Gys) 1 and Gys2, and lower mRNA expression of phosphatidylinositol 3-kinase regulatory subunit 1 (Pik3r1) than normal mice; these were significantly improved in ob/ob mice treated with UDCA, but had no effect on Gys2 ( Figure 3C). We focused on the molecular mechanisms by which UDCA affect glucose metabolism in the liver.
Moreover, analysis of Notch signaling pathways by Western blotting and qPCR revealed that Notch signaling-related genes were upregulated in liver obtained from ob/ob mice compared with both normal and ob/ob mice treated with UDCA ( Figure 3D,F). We detected both Notch ligands and jagged 1 (Jag1); deltas like canonical Notch ligand 1 (Dll1) and Dll3 were induced in ob/ob mouse liver, resulting in increased neurogenic locus notch homolog protein 1 (Notch1) protein levels ( Figure 3D,E). UDCA decreased the protein level of the Notch intracellular domain (NICD) and Notch1 in liver ( Figure 3D). Notch receptor-ligand interactions trigger the downstream responses, including the Notch transcriptional targets, such as the recombining binding protein suppressor of hairless (Rbpj), hairy and enhancer of split (Hes) 1 and Hes 5 in ob/ob mice ( Figure 3F). Consistently, UDCA significantly downregulated the Notch target genes, Rbpj, Hes1, Hes5, G6pase, and Pepck in the liver of ob/ob mice. UDCA blockade of hepatic Notch signaling resulted in the inhibition of hepatic glucose; the metabolism was associated with downregulation of gluconeogenic mRNA expression ( Figure 3). both normal and ob/ob mice treated with UDCA ( Figure 3D,F). We detected both Notch ligands and jagged 1 (Jag1); deltas like canonical Notch ligand 1 (Dll1) and Dll3 were induced in ob/ob mouse liver, resulting in increased neurogenic locus notch homolog protein 1 (Notch1) protein levels ( Figure  3D,E). UDCA decreased the protein level of the Notch intracellular domain (NICD) and Notch1 in liver ( Figure 3D). Notch receptor-ligand interactions trigger the downstream responses, including the Notch transcriptional targets, such as the recombining binding protein suppressor of hairless (Rbpj), hairy and enhancer of split (Hes) 1 and Hes 5 in ob/ob mice ( Figure 3F). Consistently, UDCA significantly downregulated the Notch target genes, Rbpj, Hes1, Hes5, G6pase, and Pepck in the liver of ob/ob mice. UDCA blockade of hepatic Notch signaling resulted in the inhibition of hepatic glucose; the metabolism was associated with downregulation of gluconeogenic mRNA expression ( Figure 3).

UDCA Improves Hepatic Mitochondrial Dysfunction and Biogenesis in ob/ob Mice
Next, we aimed at understanding the mechanistic underpinnings of the obesity-induced hepatic mitochondrial dysfunction observed in liver tissue of ob/ob mice under UDCA treatment. Compared to normal mice, ob/ob mice had lower hepatic mitochondrial biogenesis regulators, NAD-dependent deacetylase sirtuin-1 (SIRT1), PPARγ Coactivator-1 (PGC-1α) protein levels ( Figure 4A,B), and mitochondrial transcription factor A (Tfam) mRNA expression ( Figure 4C).  Further, we showed that ob/ob mice had lower basal hepatic mitochondrial Complex I, II, III, IV, and V mRNA expression than normal mice ( Figure 4D). Moreover, treatment with UDCA increased mitochondrial biogenesis regulators; factors; and the mitochondrial Complex I, II and V, but no effect on mitochondrial Complex III and IV mRNA expression in ob/ob mice ( Figure 4D). To identify mechanisms of hepatic mitochondrial dysfunction, we assessed the ROS generation key regulator in obese states. Accordingly, ob/ob mice had higher p22phox, p47phox, NADPH oxidase (Nox) 2, and Nox4 mRNA expression than normal mice, and UDCA treatment completely inhibited the effects ( Figure  4E), suggesting that UDCA ameliorates hepatic mitochondrial dysfunction and inflammation in ob/ob mice.

Effects of UDCA on Regulates Bile Acid Metabolism and Alters Transporters Expression in ob/ob Mice
Based on the observation of increased bile acids levels in plasma and liver of ob/ob mice (Table  3), we investigated whether bile acid regulator and transporters are altered in liver from ob/ob mice treatment of UDCA. Indeed, a reduced level of FXR and an increased level of cholesterol 7 alphahydroxylase (CYP7A1) protein in liver and UDCA reverses these changes in ob/ob mice ( Figure 5A, B), but there is no effect of plasma and the hepatic concentrations of bile acid (Table 3). To explore the mechanisms whereby disrupting hepatic CYP7A1 normalizes bile acids homeostasis, we assessed , and Nox4 mRNA expression in liver. Relative mRNA expression was normalized to Gapdh and then normalized to the controls. In all panels, results are expressed as the mean ± S.E.M. of five independent experiments, and statistical significance of differences between means was assessed using an unpaired Student's t-test (* p ≤ 0.05; normal vs. ob/ob. # p ≤ 0.05; ob/ob vs. ob/ob + UDCA). SIRT1, nicotinamide adenine dinucleotide (NAD)-dependent protein deacetylase sirtuin-1; PGC-1α, peroxisome proliferator-activated receptor gamma coactivator 1-alpha; Tfam, mitochondrial transcription factors A; Nox, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase.
Further, we showed that ob/ob mice had lower basal hepatic mitochondrial Complex I, II, III, IV, and V mRNA expression than normal mice ( Figure 4D). Moreover, treatment with UDCA increased mitochondrial biogenesis regulators; factors; and the mitochondrial Complex I, II and V, but no effect on mitochondrial Complex III and IV mRNA expression in ob/ob mice ( Figure 4D). To identify mechanisms of hepatic mitochondrial dysfunction, we assessed the ROS generation key regulator in obese states. Accordingly, ob/ob mice had higher p22phox, p47phox, NADPH oxidase (Nox) 2, and Nox4 mRNA expression than normal mice, and UDCA treatment completely inhibited the effects ( Figure 4E), suggesting that UDCA ameliorates hepatic mitochondrial dysfunction and inflammation in ob/ob mice.

Effects of UDCA on Regulates Bile Acid Metabolism and Alters Transporters Expression in ob/ob Mice
Based on the observation of increased bile acids levels in plasma and liver of ob/ob mice (Table 3), we investigated whether bile acid regulator and transporters are altered in liver from ob/ob mice treatment of UDCA. Indeed, a reduced level of FXR and an increased level of cholesterol 7 alpha-hydroxylase (CYP7A1) protein in liver and UDCA reverses these changes in ob/ob mice ( Figure 5A, B), but there is no effect of plasma and the hepatic concentrations of bile acid (Table 3). To explore the mechanisms whereby disrupting hepatic CYP7A1 normalizes bile acids homeostasis, we assessed plasma and hepatic cholesterol and cholesterol transporters in ob/ob mice (Table 3, Figure 5C). Levels of plasma cholesterol and mRNA expression of hepatic ATP-binding cassette transporter A1 (Abca1), 5-hydroxy-3-methylglutaryl-coenzyme A synthase (HMG-CoA S), and HMG-CoA reductase (R) were higher, and mRNA expression of hepatic ATP-binding cassette sub-family G member 1 (Abcg1) was lower in ob/ob mice compared with normal mice. UDCA improved plasma cholesterol, hepatic Abca1 and Abcg1, and increased HMG-CoA R, but had no effect on HMG-CoA S mRNA expression in ob/ob mice ( Figure 5C). Indeed, detoxification-related enzyme, sulfotransferase family 2A member 1 (Sult2a1), and hepatic bile acid transporters, organic anion-transporting polypeptide (OATP1, Slco1a1), Na + -taurocholate cotransporting polypeptide (NTCP, SLC10A1), organic solute transporters (OSTβ), multidrug resistance-associated protein 3 (MRP3, Abcc3), and bile salt export pump (BSEP) protein levels were significantly increased in ob/ob mice, and UDCA reduced hepatic bile acid transporters in liver ( Figure 5D,E).  (Table 3, Figure 5C). Levels of plasma cholesterol and mRNA expression of hepatic ATP-binding cassette transporter A1 (Abca1), 5-hydroxy-3-methylglutaryl-coenzyme A synthase (HMG-CoA S), and HMG-CoA reductase (R) were higher, and mRNA expression of hepatic ATP-binding cassette sub-family G member 1 (Abcg1) was lower in ob/ob mice compared with normal mice. UDCA improved plasma cholesterol, hepatic Abca1 and Abcg1, and increased HMG-CoA R, but had no effect on HMG-CoA S mRNA expression in ob/ob mice ( Figure 5C). Indeed, detoxification-related enzyme, sulfotransferase family 2A member 1 (Sult2a1), and hepatic bile acid transporters, organic anion-transporting polypeptide (OATP1, Slco1a1), Na + -taurocholate cotransporting polypeptide (NTCP, SLC10A1), organic solute transporters (OSTβ), multidrug resistance-associated protein 3 (MRP3, Abcc3), and bile salt export pump (BSEP) protein levels were significantly increased in ob/ob mice, and UDCA reduced hepatic bile acid transporters in liver ( Figure 5D and E). Accordingly, G-protein-coupled bile acid receptor 1 (GPBAR1, TGR5) and fibroblast growth factor receptor 4 (FGFR4) were higher in obese mice, and UDCA significantly reduced FGFR4 but did not significantly reduce TGR5 in obese mice ( Figure 5E), suggesting UDCA may have remodified bile acid metabolism by modifying the function of bile acid transporters.

UDCA Attenuates Hepatic Inflammation and Alternative Macrophage Activation in ob/ob Mice
To determine the extent of inflammation in the livers of obese mice, we investigated the possible involvement of Toll-like receptor (TLR) 4, NF-κB ( Figure 6A,B), and phospho-signal transducer and activator of transcription (p-STAT) 3 in the obese models switching effect of UDCA ( Figure 6B). These transcription factors are known to play a crucial role in insensitivity or resistance to mitochondrial-mediated inflammation. UDCA significantly attenuated TLR4, NF-κB, p-STAT3 protein level, and increased Socs1 and Socs3 mRNA expression in ob/ob mice ( Figure 6A-C). Moreover, UDCA significantly reduced hepatic proinflammatory cytokine interleukin (Il)-10, M1 markers Il-6, and tumor necrosis factor (Tnf) α mRNA expression in ob/ob mice ( Figure 6D). To further estimate the impact of ob/ob on liver tissue inflammation, we assessed the protein levels of neutrophil and macrophage M1 and M2 markers. Accordingly, G-protein-coupled bile acid receptor 1 (GPBAR1, TGR5) and fibroblast growth factor receptor 4 (FGFR4) were higher in obese mice, and UDCA significantly reduced FGFR4 but did not significantly reduce TGR5 in obese mice ( Figure 5E), suggesting UDCA may have remodified bile acid metabolism by modifying the function of bile acid transporters.

UDCA Attenuates Hepatic Inflammation and Alternative Macrophage Activation in ob/ob Mice
To determine the extent of inflammation in the livers of obese mice, we investigated the possible involvement of Toll-like receptor (TLR) 4, NF-κB ( Figure 6A,B), and phospho-signal transducer and activator of transcription (p-STAT) 3 in the obese models switching effect of UDCA ( Figure 6B). These transcription factors are known to play a crucial role in insensitivity or resistance to mitochondrialmediated inflammation. UDCA significantly attenuated TLR4, NF-κB, p-STAT3 protein level, and increased Socs1 and Socs3 mRNA expression in ob/ob mice ( Figure 6A-C). Moreover, UDCA significantly reduced hepatic proinflammatory cytokine interleukin (Il)-10, M1 markers Il-6, and tumor necrosis factor (Tnf) α mRNA expression in ob/ob mice ( Figure 6D). To further estimate the impact of ob/ob on liver tissue inflammation, we assessed the protein levels of neutrophil and macrophage M1 and M2 markers. Here, the ob/ob mice showed a significant up-regulation of neutrophil, F4/80, CD11b, and classical M1 polarization markers (CCR7 and CD11c), and down-regulation of M2 polarization markers (CD163 and CD206) in ob/ob mice ( Figure 7A-D). UDCA significantly attenuated M1 Here, the ob/ob mice showed a significant up-regulation of neutrophil, F4/80, CD11b, and classical M1 polarization markers (CCR7 and CD11c), and down-regulation of M2 polarization markers (CD163 and CD206) in ob/ob mice ( Figure 7A-D). UDCA significantly attenuated M1 markers, increased M2 macrophages markers, and attenuated the ratio of M1 to M2 macrophages (CCR7/CD163 and CD11c/CD206) in ob/ob mice ( Figure 7C,D). The beneficial effects of UDCA on M2 activation are associated with improved inflammatory conditions in obesity-induced diseases. markers, increased M2 macrophages markers, and attenuated the ratio of M1 to M2 macrophages (CCR7/CD163 and CD11c/CD206) in ob/ob mice ( Figure 7C,D). The beneficial effects of UDCA on M2 activation are associated with improved inflammatory conditions in obesity-induced diseases.

UDCA Partially Ameliorates Inflammation-Induced Hepatic Angiogenesis in ob/ob Mice
Next, we aimed at understanding the mechanistic underpinning of the inflammation and oxidative stress-driven angiogenesis observed in ob/ob mice. ob/ob mice had higher levels of lipofuscin, alpha-smooth muscle actin (α-SMA), transforming growth factor beta receptor II (TGFβ RII), vascular endothelial growth factor (VEGF), VEGF receptor I (VEGF RI), vascular cell adhesion molecule (VCAM), c-Myc and β-catenin. UDCA significantly reduced liver angiogenesis expression in ob/ob mice ( Figure 8A,B). These data suggest that UDCA may be important in the regulation of inflammation-induced hepatic angiogenesis in ob/ob mice. (D) Western blot analysis of CCR7, CD11c, CD163, and CD206 protein levels in liver. In all panels, results are expressed as the mean ± S.E.M. of five independent experiments, and statistical significance of differences between means was assessed using an unpaired Student's t-test (* p ≤ 0.05; normal vs. ob/ob. # p ≤ 0.05; ob/ob vs. ob/ob + UDCA). CD, cluster of differentiation; CCR7, C-C chemokine receptor type 7.

UDCA Partially Ameliorates Inflammation-Induced Hepatic Angiogenesis in ob/ob Mice
Next, we aimed at understanding the mechanistic underpinning of the inflammation and oxidative stress-driven angiogenesis observed in ob/ob mice. ob/ob mice had higher levels of lipofuscin, alpha-smooth muscle actin (α-SMA), transforming growth factor beta receptor II (TGFβ RII), vascular endothelial growth factor (VEGF), VEGF receptor I (VEGF RI), vascular cell adhesion molecule (VCAM), c-Myc and β-catenin. UDCA significantly reduced liver angiogenesis expression in ob/ob mice ( Figure 8A,B). These data suggest that UDCA may be important in the regulation of inflammation-induced hepatic angiogenesis in ob/ob mice. In all panels, results are expressed as the mean ± S.E.M. of five independent experiments, and statistical significance of differences between means was assessed using an unpaired Student's t-test (* p ≤ 0.05; normal vs. ob/ob. # p ≤ 0.05; ob/ob vs. ob/ob + UDCA). α-SMA, alpha-smooth muscle actin; TGFβ RII, transforming growth factor beta receptor II; VEGF, vascular endothelial growth factor; VEGFRI, VEGF receptor I; VCAM, vascular cell adhesion molecule.

UDCA Alters EWAT M1 and M2 Macrophage Polarization and Ameliorates Initial Angiogenesis in ob/ob Mice
In order to analyze whether UDCA is able to affect adipose tissue macrophages activation, protein expression analyses were performed in EWAT of ob/ob mice, as defined by macrophage markers. We found that CD11b, F4/80, CCR7, and CD11c protein levels were significantly increased in ob/ob mice as compared with normal mice (Figure 9A,B). We also observed that alternatively activated M2 macrophage markers (CD206 and CD163) were downregulated in ob/ob mice ( Figure  9A,B). UDCA significantly reduced M1 markers, increased M2 markers, and improved the ratio of M1 to M2 (CCR7/CD163 and CD11c/CD206) macrophages in ob/ob mice ( Figure 9A,B). and β-catenin were detected by Western blot analysis in the liver. In all panels, results are expressed as the mean ± S.E.M. of five independent experiments, and statistical significance of differences between means was assessed using an unpaired Student's t-test (* p ≤ 0.05; normal vs. ob/ob. # p ≤ 0.05; ob/ob vs. ob/ob + UDCA). α-SMA, alpha-smooth muscle actin; TGFβ RII, transforming growth factor beta receptor II; VEGF, vascular endothelial growth factor; VEGFRI, VEGF receptor I; VCAM, vascular cell adhesion molecule.

Discussion
The main important findings on the properties of UDCA arose from the present study: Firstly, the biochemical mechanisms of UDCA in modulating the cross-talk between liver and white adipose tissue regarding bile acid metabolism and fatty acid partitioning in obese mice. Secondly, UDCA activated the SIRT1-PGC1-α signaling pathway and reduced Sterol regulatory element-binding protein-1 (SREBP-1), a key transcription factor that regulates genes in de novo lipogenesis and glycolysis pathways. These findings suggest that UDCA increases the gene expression associated with lipogenesis, and gluconeogenesis induces the down-regulation of intracellular ROS levels and is more effective at reducing body weight gain in obesity mice. Thirdly, UDCA blockade of the Notch1 inflammatory response improves macrophage infiltration and is directly involved in the crosstalk of inflammatory and metabolic pathways. Finally, UDCA is sufficient to induce fat differentiation, including mitochondrial biogenesis and respiratory uncoupling (PPARγ, UCP1, and PGC-1α).
UDCA is currently used as a 'panacea' for pharmacological treatment of a wide range of hepatobiliary disorders. Dysregulated bile acid (BA) metabolism is an important indicator in the pathology of NAFLD, which could progress into more severe forms of liver injury, including lipid, glucose metabolism, immune response, and mitochondrial dysfunction [27,28].
Previous studies showed a considerable increase in cholesterol and BA synthesis in obesity [29]. We found that UDCA changes BA and cholesterol synthesis via induction of several BA and cholesterol synthetic markers and enzymes such as FXR and CYP7A1 and hepatic SREBP1 and TGR5 in obese mice. Since diminished cholesterol synthesis in turn decreased BA formation, no net effect on hepatic cholesterol levels was observed. UDCA stimulates CYP7A1 via suppressed hepatic FXR and SREBP-1 signaling pathways. Therefore, UDCA, which constitutes the amount of BAs, then has only a low affinity for and hence no agonistic activity on FXR. Moreover, our data suggest the adaptive changes in bile acid transporters expression in liver provide alternative excretory pathways for biliary constituents during UDCA treatment and may thus attenuate liver injury in ob/ob mice. Changes in bile acid transporters constitute a central element in the pathogenesis and clinical manifestation of cholestasis, and several clinically effective drugs modulate transporter expression via regulatory nuclear receptors (NRs), translating into a reduction of the hepatocellular bile acid burden, thus ameliorating tissue injury [30]. Interestingly, we note that UDCA reduced cholesterol synthesis and bile acid transporter levels, but did not reduce the plasma bile acid level, suggesting that UDCA may induce slightly different programs, highlighting the complexity of bile acid signaling.
SIRT1 is an NAD-dependent deacetylase and has been reported to have many beneficial effects for controlled glucose homeostasis, lipid metabolism, and insulin resistance in the liver tissue of obese mice [31]. PGC1-α is regulated by SIRT-1 according to cellular energy requirements and works as a regulator of mitochondrial biogenesis [32]. SIRT1-deficient mice lack AMPK activity, but SREBP-1c expression is promoted in mice with induced obesity and hepatic steatosis compared to wild-type HFD-induced obese mice [33]. Our study has found that UDCA can activate SIRT-1 and reduce hepatic steatosis by increasing fatty acid β-oxidation and reducing SREBP-1c expression. Therefore, sirt1-PGC1-SREBP pathway activity potentially inhibits lipid accumulation in hepatocytes and improves hepatic steatosis. Notably, our study showed that UDCA elevates hepatic and adipose tissue levels of SIRT1, PGC1-α, and simultaneously decreases SREBP-1 in fat mice. Interestingly, accelerating lipid decomposition is an important strategy for improving NAFLD. In the liver, excessive lipid biosynthesis is mainly a phenomenon of excessive energy accumulation. Notably, excess TGs in the liver are broken down, and released free fatty acids must be decomposed to produce energy via fatty acid β-oxidation and reduce the damage to liver and adipose tissue. To our knowledge, this is the first study of UDCA which improves mitochondria function in liver and adipose tissue. UDCA appears to induce adaptive thermogenesis via the SIRT-1-PGC-1α signaling pathway in WAT when mice are challenged with fat accumulation. This adaptive thermogenesis does act through the same signaling pathway in hepatic steatosis. In addition, adaptive thermogenesis in adipose tissue occurs through an increase in mitochondrial mass to accommodate increased metabolic demands for respiratory uncoupling. Importantly, UDCA induces a marked elevation of SIRT-1-PGC-1α in adipose tissues and may indicate that UDCA improves WAT and liver mitochondrial capacity in obese mice.
We have provided compelling genetic, physiological, metabolic, histological, cellular, and molecular evidence to demonstrate that blockage of Notch signaling promotes browning of WAT. White to brown fat transition involves a series of cellular processes, including increased UCP1 expression and mitochondria production, lipolysis, and β-oxidation. We found that obesity increased Notch activity in liver and WAT. These results indicate that the activation of Notch signaling is linked to the initiation and development of obesity. Treating obese mice with UDCA reduced body weight and ameliorated glucose metabolism-such a drastic effect may be elicited not only by browning of white adipose tissue, but also through targeting other important metabolic organs, including liver, which directly regulates lipid metabolism and nutrient absorption respectively. Previous studies have demonstrated that Notch gain-of-function causes fatty liver, and is correlated with an increasing rate of SREBP-1c-mediated lipogenesis and increases hepatic glucose production by co-activating Foxo1 at the G6Pase and PEPCK, and it is regulated in response to metabolic stimuli in the liver [34]. Moreover, liver-specific REPJ deletion protects from diet-induced steatosis, reduces de novo lipogenesis, improves glucose tolerance, and reduces hepatocyte glucose production in mice [34,35]. Here, UDCA led to reducing lipogenesis, gluconeogenesis, and Notch 1 signaling proteins in ob/ob mice, suggesting that UDCA plays an important role in lipid, glucose metabolism, and energy storage capacity in those with obesity.
Moreover, Notch could reprogram for proinflammatory macrophage activation [36]; mitochondrial abnormalities and abnormal morphologic changes associated with NAFLD include decreased activity of respiratory chain complexes, impaired mitochondrial β-oxidation ultrastructural lesions, as well as depletion of mtDNA that have been observed in patients and animal models with NASH [37,38]. The mitochondrial dehydrogenase activities related to complex I (NADH-dehydrogenase) and complex II (succinate dehydrogenase) were decreased in the diabetic rats that were normalized in the UDCA-treated animals [39]. In this report, we have shown that UDCA reverses obesity-reduced SIRT1, PGC1α, and Tfam expression, that also increased mitochondria complex I, II, V mRNA expression in ob/ob mice, suggesting UDCA is a potential agent to promote mitochondrial biogenesis and improved mitochondrial function. Mitochondria are a target for ROS produced by NADPH oxidase (NOX) and their associated regulatory subunits such as p22phox and p47phox, which are a significant source of ROS, which under certain conditions may stimulate NADPH oxidases [40,41]. Our study reveales that UDCA significantly improved obesity-induced mitochondrial dysfunction by suppressing NOX family enzymes mRNA expression and Notch-mediated lipogenesis, gluconeogenesis in ob/ob mice.
Fatty acids released from hypertrophied adipocytes themselves serve as ligands for TLR 4, which is expressed on adipocytes and macrophages and can also provoke inflammation in liver and adipose tissue [42]. Importantly, downregulation of TLR4, NF-κB, and STAT3 signals in ob/ob mice after treat with UDCA. Our study showed that the expression of M2 macrophage [43] markers (CD206 and CD163) were down-regulated in the liver and EWAT biopsy samples of the obese subjects. Recent studies also showed that CCR7(-/-) mice were protected from fatty liver and dyslipidemia and exhibited increased thermogenesis on high-fat feeding [44]. By analyzing the expression of genes and the angiocrine factor secretion profiles of different subsets of macrophages, Spiller et al. showed that M1 macrophages express genes involved in the initiation of angiogenesis, including VEGF-A and FGF2 [45]. Thereby, considering that obesity induces a phenotypic switch from an anti-inflammatory M2 polarized state to a proinflammatory M1 state, increased M1 macrophages along with pro-inflammatory cytokines and chemokines contribute to chronic inflammation [46]. Thus, our findings may support the notion that the M2 macrophage polarization plays a prominent role in ob/ob mice treat with UDCA.
In accordance with previous observations, adipose tissue has a key role in peripheral energy homeostasis, as obesity is also associated with dysfunctional adipose tissue and fatty liver [47,48]. In our study, we observed that UDCA treatment induced important changes at the level of SIRT-1-PGC-1 and SREBP-1 signaling involved in lipid metabolism and energy in adipose tissue. These data accord with results that UDCA affects energy balance by promoting a profound remodeling in lipid-specific adipocyte functions. These changes were associated with enhanced mitochondrial function and increased M2 macrophage activation in liver and EWAT. Thus, in agreement with previously published evidence [49], UDCA has the effect of reducing the lipid storage capacity of liver and WAT. The reduced energy storage capacity was linked to decreased body weight in ob/ob mice. Moreover, protein levels on analysis confirmed upregulation of thermogenic genes and mitochondrial biogenesis in EWAT, suggesting the browning of this fat depot in ob/ob mice treatment with UDCA. Furthermore, adipose-derived stem/stromal/progenitor cells (ASCs), CD34, and CD90 were markedly enhanced in the ob/ob mice treatment with UDCA. Altogether, UDCA was able to increase the number of multilocular UCP1 and Tmem26-positive adipocytes in EWAT, therefore inducing EWAT thermogenesis in ob/ob mice. UDCA promotes profound energy metabolism, not only of liver, but also of EWAT, including alternatively activated macrophages that work concomitantly with mitochondrial function effects to play a pivotal role in increased stem cells activation and browning EWAT as well as a thermogenic program. In contrast, our current experimental approaches cannot directly estimate the contribution of alternatively activated macrophages in comparison with UDCA to the enhancement of mitochondrial function observed in ob/ob mice, or determine whether the M2 macrophages are local sources of sufficient mitochondrial concentrations with physiological relevance. In summary, we conclude that UDCA in liver and EWAT leads to an increased mitochondrial function and M2 macrophage abundance, both of which play critical roles in the emergence of the ob/ob mice. This compelling evidence illustrates the crosstalk between adipocytes and immune cells in concert with the liver and EWAT, in which UDCA occupies a key regulatory function.