MicroRNA-29a Suppresses CD36 to Ameliorate High Fat Diet-Induced Steatohepatitis and Liver Fibrosis in Mice

MicroRNA-29 (miR-29) has been shown to play a critical role in reducing inflammation and fibrosis following liver injury. Non-alcoholic fatty liver disease (NAFLD) occurs when fat is deposited (steatosis) in the liver due to causes other than excessive alcohol use and is associated with liver fibrosis. In this study, we asked whether miR-29a could reduce experimental high fat diet (HFD)-induced obesity and liver fibrosis in mice. We performed systematical expression analyses of miR-29a transgenic mice (miR-29aTg mice) and wild-type littermates subjected to HFD-induced NAFLD. The results demonstrated that increased miR-29a not only alleviated HFD-induced body weight gain but also subcutaneous, visceral, and intestinal fat accumulation and hepatocellular steatosis in mice. Furthermore, hepatic tissue in the miR-29aTg mice displayed a weak fibrotic matrix concomitant with low fibrotic collagen1α1 expression within the affected tissues compared to the wild-type (WT) mice fed the HFD diet. Increased miR-29a signaling also resulted in the downregulation of expression of the epithelial mesenchymal transition-executing transcription factor snail, mesenchymal markers vimentin, and such pro-inflammation markers as il6 and mcp1 within the liver tissue. Meanwhile, miR-29aTg-HFD mice exhibited significantly lower levels of peroxisome proliferator-activated receptor γ (PPARγ), mitochondrial transcription factor A TFAM, and mitochondria DNA content in the liver than the WT-HFD mice. An in vitro luciferase reporter assay further confirmed that miR-29a mimic transfection reduced fatty acid translocase CD36 expression in HepG2 cells. Conclusion: Our data provide new insights that miR-29a can improve HDF-induced obesity, hepatocellular steatosis, and fibrosis, as well as highlight the role of miR-29a in regulation of NAFLD.

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CD44 contributes to hyaluronan-mediated insulin resistance in skeletal muscle of high-fat-fed C57BL/6 mice

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00215.2019 ◽

2019 ◽

Vol 317 (6) ◽

pp. E973-E983 ◽

Cited By ~ 3

Author(s):

Annie Hasib ◽

Chandani K. Hennayake ◽

Deanna P. Bracy ◽

Aimée R. Bugler-Lamb ◽

Louise Lantier ◽

...

Keyword(s):

Insulin Resistance ◽

Skeletal Muscle ◽

High Fat Diet ◽

Critical Role ◽

Chow Diet ◽

Wild Type ◽

High Fat ◽

Insulin Resistant ◽

Beneficial Effects

Extracellular matrix hyaluronan is increased in skeletal muscle of high-fat-fed insulin-resistant mice, and reduction of hyaluronan by PEGPH20 hyaluronidase ameliorates diet-induced insulin resistance (IR). CD44, the main hyaluronan receptor, is positively correlated with type 2 diabetes. This study determines the role of CD44 in skeletal muscle IR. Global CD44-deficient ( cd44−/−) mice and wild-type littermates ( cd44+/+) were fed a chow diet or 60% high-fat diet for 16 wk. High-fat-fed cd44−/− mice were also treated with PEGPH20 to evaluate its CD44-dependent action. Insulin sensitivity was measured by hyperinsulinemic-euglycemic clamp (ICv). High-fat feeding increased muscle CD44 protein expression. In the absence of differences in body weight and composition, despite lower clamp insulin during ICv, the cd44−/− mice had sustained glucose infusion rate (GIR) regardless of diet. High-fat diet-induced muscle IR as evidenced by decreased muscle glucose uptake (Rg) was exhibited in cd44+/+ mice but absent in cd44−/− mice. Moreover, gastrocnemius Rg remained unchanged between genotypes on chow diet but was increased in high-fat-fed cd44−/− compared with cd44+/+ when normalized to clamp insulin concentrations. Ameliorated muscle IR in high-fat-fed cd44−/− mice was associated with increased vascularization. In contrast to previously observed increases in wild-type mice, PEGPH20 treatment in high-fat-fed cd44−/− mice did not change GIR or muscle Rg during ICv, suggesting a CD44-dependent action. In conclusion, genetic CD44 deletion improves muscle IR, and the beneficial effects of PEGPH20 are CD44-dependent. These results suggest a critical role of CD44 in promoting hyaluronan-mediated muscle IR, therefore representing a potential therapeutic target for diabetes.

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A Novel TWIST2-ID2 Axis That Modulates The Growth and Imatinib Sensitivity Of CML CD34+ Cells

Blood ◽

10.1182/blood.v122.21.511.511 ◽

2013 ◽

Vol 122 (21) ◽

pp. 511-511

Author(s):

Yun Zhao ◽

Wenjuan Ma ◽

Xiuyan Zhang ◽

Jiangxia Cui ◽

Ivan Sloma ◽

...

Keyword(s):

Dna Binding ◽

Progenitor Cells ◽

Lentiviral Vector ◽

Critical Role ◽

K562 Cells ◽

Luciferase Reporter ◽

Control Group ◽

Wild Type ◽

Mesenchymal Transition ◽

Cd34 Cells

Abstract TWIST is a basic helix-loop-helix transcription factor that specifies Drosophila mesoderm development. In mammals there are 2 members, TWIST1 and TWIST2. TWIST2 is a regulator of osteoblasts and muscle development and plays a critical role in the epithelial-mesenchymal transition process, as well as in cancer initiation and metastasis. Twist2-deficient mice develop a myeloproliferative disease. These findings led us to query a potential role of TWIST2 in normal and leukemic (CML) human hematopoietic cells. RT-PCR and immuno-fluorescence analysis of CD34+ bone marrow (BM) cells obtained from healthy donors demonstrated their expression of TWIST2 transcripts and protein. Lentiviral vector-mediated knockdown of TWIST2 with 2 independent shRNA sequences enhanced the erythroid and granulopoietic colony-forming activity of transduced normal BM cells ∼2-fold compared with control transduced cells (n=3, p<0.05). Interestingly, ChIP studies showed that TWIST2 can bind directly to the DNA promoter for ID2 in CD34-enriched BM cells and knockdown of TWIST2 reduced ID2 expression by 50%. In lin-CD34+ cells from 14 chronic myeloid leukemia (CML) patients, we found both TWIST2 and ID2 transcripts to be 5 and 6 fold lower than those from 6 healthy BM donors (p<0.05), with similar findings for TWIST2 and ID2 protein in the same cells. BCR-ABL1-transduced Baf/3 cells also showed a reduction in Twist2 expression. Conversely, TWIST2 expression became elevated when K562 cells were treated with Imatinib mesylate (IM). We then generated a lentiviral vector encoding TWIST2 which proved capable of inhibiting the growth of K562 and MEG-01 cells as well as CFC production from CML CD34+ cells (n=11, p<0.05). Overexpression of TWIST2 in MEG-01 cells also reduced their tumorigenic ability in subcutaneously injected nude mice (0/8 for TWIST2 group, 7/8 for control group). In addition, increased TWIST2 sensitized the IM response of K562 cells and IM-resistant CD34+ cells from CML patients (2 in chronic phase and 2 in blast crisis). Correspondingly, knockdown of TWIST2 in K562 cells enhanced their cloning efficiency by 15% and made them IM-resistant. To obtain further insight into these biological effects of TWIST2, we generated several TWIST2 mutant cDNAs, including ones with a N-terminal truncation (ΔN), a C-terminal truncation (ΔC), a F86P dimerization mutant and a b- DNA binding mutant. Analysis of the effects of these mutants when overexpressed in CML cells and cell lines showed TWIST2 dimer formation was critical for the effects obtained with wild-type TWIST2, whereas the DNA binding domain could modulate these effects but was not essential, and the N-terminal and C-terminal domains were dispensable. We also found that overexpression of TWIST2 enhanced ID2 expression in CML CD34+ cells (n=3), as well as K562 and MEG-01 cells, and ChIP analyses confirmed the binding of TWIST2 to ID2 promoter DNA from K562 and MEG-01 cells. Using ID2 promoter-driven luciferase reporter and a mutant derivative (with only the E-box sequence altered), we found that TWIST2 could activate the wild-type promoter but not the mutated one in both K562 and MEG-01 cells. Finally, we co-transduced CML cells from 3 patients with TWIST2 and shRNA against ID2 and found that this reversed the suppressed production of CFC obtained with TWIST2 alone. Similarly in K562 cells this treatment partially restored their growth rate and IM resistance. Taken together, we report a novel TWIST2-ID2 regulatory axis in normal hematopoietic progenitor cells, which can also modulate the growth and IM response of CML progenitor cells. These findings provide a baseline for the future development of more effective therapy of CML. Disclosures: No relevant conflicts of interest to declare.

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High fat diet induces dysregulation of hepatic oxygen gradients and mitochondrial function in vivo

Biochemical Journal ◽

10.1042/bj20080868 ◽

2008 ◽

Vol 417 (1) ◽

pp. 183-193 ◽

Cited By ~ 174

Author(s):

Sudheer K. Mantena ◽

Denty Paul Vaughn ◽

Kelly K. Andringa ◽

Heather B. Eccleston ◽

Adrienne L. King ◽

...

Keyword(s):

Nitric Oxide ◽

High Fat Diet ◽

Protein Modification ◽

Control Diet ◽

Critical Role ◽

Liver Mitochondria ◽

Mitochondrial Proteins ◽

High Fat ◽

Alcoholic Fatty Liver

NAFLD (non-alcoholic fatty liver disease), associated with obesity and the cardiometabolic syndrome, is an important medical problem affecting up to 20% of western populations. Evidence indicates that mitochondrial dysfunction plays a critical role in NAFLD initiation and progression to the more serious condition of NASH (non-alcoholic steatohepatitis). Herein we hypothesize that mitochondrial defects induced by exposure to a HFD (high fat diet) contribute to a hypoxic state in liver and this is associated with increased protein modification by RNS (reactive nitrogen species). To test this concept, C57BL/6 mice were pair-fed a control diet and HFD containing 35% and 71% total calories (1 cal≈4.184 J) from fat respectively, for 8 or 16 weeks and liver hypoxia, mitochondrial bioenergetics, NO (nitric oxide)-dependent control of respiration, and 3-NT (3-nitrotyrosine), a marker of protein modification by RNS, were examined. Feeding a HFD for 16 weeks induced NASH-like pathology accompanied by elevated triacylglycerols, increased CYP2E1 (cytochrome P450 2E1) and iNOS (inducible nitric oxide synthase) protein, and significantly enhanced hypoxia in the pericentral region of the liver. Mitochondria from the HFD group showed increased sensitivity to NO-dependent inhibition of respiration compared with controls. In addition, accumulation of 3-NT paralleled the hypoxia gradient in vivo and 3-NT levels were increased in mitochondrial proteins. Liver mitochondria from mice fed the HFD for 16 weeks exhibited depressed state 3 respiration, uncoupled respiration, cytochrome c oxidase activity, and mitochondrial membrane potential. These findings indicate that chronic exposure to a HFD negatively affects the bioenergetics of liver mitochondria and this probably contributes to hypoxic stress and deleterious NO-dependent modification of mitochondrial proteins.

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Fenofibrate Alleviates Insulin Resistance and Hepatic Steatosis via Modulation of let-7/ SERCA2b Axis

10.21203/rs.3.rs-83279/v1 ◽

2020 ◽

Author(s):

Dan Zhang ◽

Shan-zhuang Niu ◽

Yi-cheng Ma ◽

Bo Zhou ◽

Yi Deng ◽

...

Keyword(s):

Insulin Resistance ◽

Endoplasmic Reticulum ◽

Er Stress ◽

Hepatic Steatosis ◽

High Fat Diet ◽

Luciferase Reporter ◽

Control Group ◽

Chow Diet ◽

High Fat ◽

Alcoholic Fatty Liver

Abstract Background: Fenofibrate is a peroxisome proliferator-activated receptor alpha agonist, which is widely used in clinical practice to effectively ameliorates the development of NAFLD. However, the molecular mechanism remains largely unknown, the present study aimed to investigate the role and specific mechanism of fenofibrate on lipid metabolism disorders associated diseases.Methods: The male C57BL6/J mice were divided into 3 groups, the mice in control group (n=10) were fed with normal chow diet, and the mice in HFD-fed group (n =10) were fed with a high fat diet (HFD) for 14 weeks. For the fenofibrate +HFD-fed group (n =10), the mice fed HFD were orally gavaged with fenofibrate (40 mg/kg) daily for the last 4 weeks. Body weight and hip width were measured. Macrosteatosis and fat deposition in the liver were measured by H&E staining and Oil red O staining individually. The levels of serum and hepatic triglyceride were measured, and HOMA-IR, HOMA-ISI were analyzed. The levels of SCD-1, Bip, CHOP and SERCA2b were measured by western blotting. The expression of let-7 were analyzed by qPCR, and the complementarity between the 3′-UTR of SERCA2b gene and let-7 was measured by luciferase reporter assay.Results: Fenofibrate reduces hepatic steatosis and insulin resistance in HFD-fed mice. Fnofibrate alleviates endoplasmic reticulum stress (ER stress) of mice fed a high fat diet (HFD). Fenofibrate increases the levels of Sarco-endoplasmic reticulum Ca2+-ATPase 2b (SERCA2b) which serves as a regulator of ER stress. Further, the levels of let-7 microRNA is also regulated by fenofibrate, and let-7 directly targets 3’-UTR of SERCA2b. Conclusion: The present data suggests that fenofibrate alleviates ER stress through the let-7/SERCA2b axis to protect against excessive lipid accumulation in the liver of Non-alcoholic fatty liver disease (NAFLD) mice.

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Group 3 Innate Lymphoid Cells Protect Steatohepatitis From High-Fat Diet Induced Toxicity

Frontiers in Immunology ◽

10.3389/fimmu.2021.648754 ◽

2021 ◽

Vol 12 ◽

Author(s):

Masahide Hamaguchi ◽

Takuro Okamura ◽

Takuya Fukuda ◽

Kensuke Nishida ◽

Yuta Yoshimura ◽

...

Keyword(s):

Liver Fibrosis ◽

Fatty Liver ◽

High Fat Diet ◽

Innate Lymphoid Cells ◽

Lymphoid Cells ◽

High Fat ◽

Primary Hepatocytes ◽

M1 Macrophages ◽

Alcoholic Fatty Liver ◽

Group 3

Background and Aims: Emerging evidence has revealed that innate lymphoid cells (ILCs) play a key role in regulating metabolic disorders. Here, we investigated the role of group 3 ILCs (ILC3s) in the modulation of Non-alcoholic fatty liver disease (NAFLD).Methods: RORγ gfp/gfp (RORgt KI/KI) and Rag2−/− mice with the administration of A213, RORgt antagonist, fed with a high-fat-diet (HFD) for 12 weeks, were used. We performed flow cytometry, real time PCR, and lipidomics analysis of serum and liver, and used RAW264.7 cells and murine primary hepatocytes in vitro.Results: HFD increased ILC3s and M1 macrophages in the liver, and RORgt KI/KI mice deficient in ILC3 showed significant fatty liver, liver fibrosis and significantly increased palmitic acid levels in serum and liver. In addition, administration of A213 to Rag2−/− mice caused significant fatty liver, liver fibrosis, and a significant increase in serum and liver palmitate concentrations, as in RORgt KI/KI mice. Addition of palmitc acid stimulated IL-23 production in cell experiments using RAW264.7. IL-22 produced by ILC3s inhibited the palmitate-induced apoptosis of primary hepatocytes.Conclusions: HFD stimulates IL-23 production by M1 macrophages, thus promoting ILC3 proliferation, whereas IL-22 secreted by ILC3s contributes to the upregulation of hepatic lipid metabolism and has anti-apoptosis activity.

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Phytosterol esters attenuate hepatic steatosis in rats with non-alcoholic fatty liver disease rats fed a high-fat diet

Scientific Reports ◽

10.1038/srep41604 ◽

2017 ◽

Vol 7 (1) ◽

Cited By ~ 23

Author(s):

Lihua Song ◽

Dan Qu ◽

Qing Zhang ◽

Jing jiang ◽

Haiyue Zhou ◽

...

Keyword(s):

High Fat Diet ◽

Hepatic Tissue ◽

High Dose ◽

Chow Diet ◽

High Fat ◽

Alcoholic Fatty Liver ◽

Inflammatory Stress ◽

Beneficial Effects ◽

Ppar Γ ◽

Phytosterol Esters

Abstract Given the adverse effects of drugs used for NAFLD treatment, identifying novel and effective natural compound to prevent NAFLD is urgently needed. In the present study, the effects of phytosterol esters (PSEs) on NAFLD were explored. Adult SD rats were randomized into five groups: normal chow diet (NC), high-fat diet (HF), low-, medium- and high-dose PSE treatment plus high-fat diet groups (PSEL, PSEM, and PSEH). Our results showed that the levels of LDL-C in the PSEL group and hepatic TG, TC, and FFA in the three PSEs groups were significantly decreased. Notably, the uric acid (UA) level was significantly decreased by PSEs intervention. The hepatic inflammatory stress was ameliorated via the inhibition of the cytokines, including TGF-β, IL-6, IL-10 and CRP in the PSEs intervention groups. Further, the oxidative status was improved by PSE treatment through adjusting the enzyme activity (SOD and XOD) and decreasing the MDA level. These beneficial effects of PSE may have been partly due to its regulation on the expression of TGF-β1, TGF-β2, TNF-α, UCP-2, PPAR-α and PPAR-γ in hepatic tissue at both mRNA and protein level. The results of this study suggest that PSEs may be used as therapeutic agents for the prevention and progression of NAFLD and that hyperuricemia is induced by high-fat diet consumption.

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Pathophysiological importance of bile cholesterol reabsorption: hepatic NPC1L1-exacerbated steatosis and decreasing VLDL-TG secretion in mice fed a high-fat diet

Lipids in Health and Disease ◽

10.1186/s12944-019-1179-0 ◽

2019 ◽

Vol 18 (1) ◽

Author(s):

Yu Toyoda ◽

Tappei Takada ◽

Yoshihide Yamanashi ◽

Hiroshi Suzuki

Keyword(s):

High Fat Diet ◽

Early Stage ◽

Lipid Homeostasis ◽

Mrna Levels ◽

Hepatic Cholesterol ◽

Wild Type ◽

High Fat ◽

Wild Type Littermate ◽

Alcoholic Fatty Liver ◽

Hepatic Expression

Abstract Background Non-alcoholic fatty liver disease (NAFLD) is one of the most common liver diseases worldwide, although its pathogenesis remains to be elucidated. A recent study revealed that hepatic Niemann-Pick C1-Like 1 (NPC1L1), a cholesterol re-absorber from bile to the liver expressed on the bile canalicular membrane, is an exacerbation factor of NAFLD. Indeed, transgenic mice with hepatic expression of human NPC1L1 under a liver-specific promoter (L1-Tg mice) developed steatosis with a high-fat diet (HFD) containing cholesterol within a few weeks. However, the mechanism underlying diet-induced hepatic NPC1L1-mediated lipid accumulation is poorly defined. Methods To achieve a deeper understanding of steatosis development in L1-Tg mice, the biochemical features of hepatic NPC1L1-mediated steatosis were investigated. Hemizygous L1-Tg mice and wild-type littermate controls fed a HFD or control-fat diet were used. At the indicated time points, the livers were evaluated for cholesterol and triglyceride (TG) contents as well as mRNA levels of hepatic genes involved in the maintenance of lipid homeostasis. The hepatic ability to secrete very low-density lipoprotein (VLDL)-TG was also investigated. Results Unlike the livers of wild-type mice that have little expression of hepatic Npc1l1, the livers of L1-Tg mice displayed time-dependent changes that indicated steatosis formation. In steatosis, there were three different stages of development: mild accumulation of hepatic cholesterol and TG (early stage), acceleration of hepatic TG accumulation (middle stage), and further accumulation of hepatic cholesterol (late stage). In the early stage, between WT and L1-Tg mice fed a HFD for 2 weeks, there were no significant differences in the hepatic expression of Pparα, Acox1, Fat/Cd36, Srebf1, and Srebf2; however, the hepatic ability to secrete VLDL-TG decreased in L1-Tg mice (P < 0.05). Furthermore, this decrease was completely prevented by administration of ezetimibe, an NPC1L1-selective inhibitor. Conclusion Hepatic NPC1L1 exacerbates diet-induced steatosis, which was accompanied by decreased hepatic ability of VLDL-TG secretion. The obtained results provide a deeper understanding of L1-Tg mice as a promising NAFLD animal model that is able to re-absorb biliary-secreted cholesterol similar to humans. Furthermore, this work supports further studies of the pathophysiological impact of re-absorbed biliary cholesterol on the regulation of hepatic lipid homeostasis.

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Fatty Acids and a High-Fat Diet Induce Epithelial–Mesenchymal Transition by Activating TGFβ and β-Catenin in Liver Cells

International Journal of Molecular Sciences ◽

10.3390/ijms22031272 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1272

Author(s):

Oliwia Kwapisz ◽

Judyta Górka ◽

Agata Korlatowicz ◽

Jerzy Kotlinowski ◽

Agnieszka Waligórska ◽

...

Keyword(s):

Fatty Acids ◽

Liver Fibrosis ◽

Epithelial Cells ◽

High Fat Diet ◽

Transforming Growth Factor ◽

Epithelial Mesenchymal Transition ◽

Negative Regulator ◽

Prolonged Treatment ◽

High Fat ◽

Mesenchymal Transition

Nonalcoholic fatty liver disease is defined as the accumulation of excessive fat in the liver in the absence of excessive alcohol consumption or any secondary cause. Although the disease generally remains asymptomatic, chronic liver inflammation leads to fibrosis, liver cirrhosis, and even to the development of hepatocellular carcinoma (HCC). Fibrosis results from epithelial–mesenchymal transition (EMT), which leads to dedifferentiation of epithelial cells into cells with a mesenchymal-like phenotype. During EMT, epithelial cells with high expression of E-cadherin, influenced by growth factors, cytokines, and inflammatory processes, undergo morphological changes via enhanced expression of, e.g., vimentin, fibronectin, and N-cadherin. An inducer of EMT and, consequently, of fibrosis development is transforming growth factor beta (TGFβ), a pleiotropic cytokine associated with the progression of hepatocarcinogenesis. However, the understanding of the molecular events that direct the development of steatosis into steatohepatitis and liver fibrosis remains incomplete. Our study revealed that both prolonged exposure of hepatocarcinoma cells to fatty acids in vitro and high-fat diet in mice (20 weeks) result in inflammation. Prolonged treatment with fatty acids increased the levels of TGFβ, MMP9, and β-catenin, important EMT inducers. Moreover, the livers of mice fed a high-fat diet exhibited features of liver fibrosis with increased TGFβ and IL-1 levels. Increased expression of IL-1 correlated with a decrease in monocyte chemoattractant protein-induced protein 1 (MCPIP1), a negative regulator of the inflammatory response that regulates the stability of proinflammatory transcripts encoding IL-1. Our study showed that a high-fat diet induced EMT by increasing the levels of EMT-activating transcription factors, including Zeb1, Zeb2, and Snail and changed the protein profile to a profile characteristic of the mesenchymal phenotype.

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