Macrophage infiltration in adipose tissue is strongly correlated with obesity. The exact role of macrophage in the development of obesity, however, has not been fully understood. In this study, using intraperitoneal injection of clodronate liposomes, we tissue-specifically depleted visceral adipose tissue macrophages (VATMs) and explored their roles in initiation and progression of obesity. Two sets of experiments were conducted, using mice on a high-fat diet as the animal model. Mice were injected with clodronate liposomes at the beginning of high-fat diet feeding to investigate the role of VATMs in the initiation of obesity. Treatment starting on week 5 was designed to explore the function of VATMs in the progression of weight gain. The results show that intraperitoneal injection of clodronate liposomes effectively depleted VATMs, which blocked high-fat diet-induced weight gain, fat accumulation, insulin resistance, and hepatic steatosis. Similarly, clodronate liposomes suppressed progression of weight gain in mice after being fed with a high-fat diet for 4 weeks and improved insulin sensitivity. Gene expression analysis showed that depletion of VATMs was associated with downregulation of the expression of genes involved in lipogenesis and gluconeogenesis including acc1, fas, scd1, and pepck, decreased expression of genes involved in chronic inflammation including mcp1 and tnfα, and suppressed expression of macrophage specific marker genes of f4/80 and cd11c in adipose tissue. Depletion of VATMs was associated with prevention of the formation of crown-like structures in white adipose tissue and the maintenance of a low level of blood TNF-α. Collectively, these data demonstrate that VATMs appeared to play a crucial role in the development of obesity and obesity-associated diseases and suggest that adipose tissue macrophages could be regarded as a potential target for drug development in prevention and therapy of obesity and obesity-associated complications.
high-fat diet-induced obesity; inflammation; insulin resistance; liposomes; visceral adipose tissue macrophage
The nuclear receptor farnesoid X receptor (FXR), an endogenous sensor for bile acids, plays an important role in cholesterol, lipid and carbohydrate metabolism. The objective of this study is to examine the effect of FXR activation on diet-induced obesity and hepatic steatosis. Activation of FXR by its synthetic agonist, 3-[2-[2-Chloro-4-[[3-(2,6-dichlorophenyl)-5-(1-methylethyl)-4-isoxazolyl]methoxy]phenyl]ethenyl]benzoic acid (GW4064), suppressed weight gain in C57BL/6 mice fed with either a high-fat diet (HFD) or high-fat, high-cholesterol diet. GW4064 treatment of mice on HFD significantly repressed diet-induced hepatic steatosis as evidenced by lower triglyceride and free fatty acid level in the liver. Analysis of genes involved in lipid metabolism showed GW4064 markedly reduced lipid transporter CD36 expression without affecting expression of genes that are directly involved in lipogenesis. GW4064 treatment attenuated hepatic inflammation while having no effect on white adipose tissue. In addition, activation of FXR by GW4064 avoided diet-induced hyperinsulinemia and hyperglycemia through decreasing the transcript levels of phosphoenolpyruvate carboxykinase (Pepck) and glucose-6-phosphatase (G6pase), two key enzymes in gluconeogenesis. These results verify the important function of FXR in diet-induced obesity and suggest that FXR agonists are promising therapeutic agents for obesity-associated metabolic disorders.
Obesity; farnesoid X receptor; hepatic steatosis; glucose homeostasis
Liver X receptor (LXR) has been identified as a potential target for treatment of atherosclerosis and diabetes. Activation of LXR, however, is associated with increased lipogenesis and fat accumulation in the liver. The objective of the current study was to examine the effect of resveratrol on LXR activator-induced fat accumulation in liver using mice as an animal model. Three groups of C57BL/6 mice were studied. Animals in group 1 were treated with T0901317, a potent activator of LXR in mice. Animals in group 2 served as the control and were treated with carrier solution and those in group 3 were treated with T0901317/resveratrol combination. Using histochemical and biochemical methods, we demonstrate that resveratrol treatment significantly suppressed fat accumulation in the liver induced by T0901317. In addition, resveratrol completely blocked elevation of blood levels of triglyceride and cholesterol and reduced blood glucose level. Quantitative PCR analysis revealed that resveratrol treatment did not change the mRNA levels of abca1, abcg1, cyp7a1, srebp-1c, chrebp, and acc genes compared to that of animals treated with T0901317 alone but reduced pepck and g6p gene expressions. Immunohistochemistry and Western blot analyses show resveratrol treatment activated AMP-activated protein kinase (AMPK) and increased phosphorylation of acetyl-CoA carboxylase. Treatment with T0901317 on hepatocytes increased intracellular fat accumulation and this increase was suppressed by resveratrol; the suppressive effect of resveratrol was greatly repressed by Compound C which is an inhibitor of AMPK. Collectively, these data suggest that resveratrol blocks T0901317-induced lipid accumulation in the liver and can be considered for inclusion into the treatment of diseases involving activation of liver X receptor.
AMPK; fatty liver; gluconeogenesis; lipogenesis; liver X receptor; resveratrol; T0901317
Adiponectin and its receptors are inversely related to the degree of obesity and have been identified as potential therapeutic targets for the treatment of obesity. In this study, we evaluated the effect of hydrodynamic delivery of adiponectin and/or its receptor 2 (adipoR2) genes on controlling the development of obesity and insulin resistance in AKR/J mice fed a high-fat diet. An increase in adiponectin and adipoR2 gene expression by hydrodynamic gene delivery prevented diet-induced weight gain, reduced fat accumulation in liver and adipose tissue, and improved insulin sensitivity. Beneficial effects were seen with reduced gluconeogenesis in the liver and lipogenesis in the liver, white adipose tissue and skeletal muscle. Real-time PCR analysis demonstrated overexpression of adiponectin and adipoR2 significantly suppressed transcription of phosphoenolpyruvate carboxykinase (pepck), glucose-6-phosphatase (g6pase), stearoyl CoA desaturase 1 (scd-1), and fatty acid synthase (fas) gene. Inhibition effects were mediated by activating the AMP-activated protein kinase (AMPK). These results prove that elevation of adiponectin and/or adipoR2 expression via gene transfer is an effective approach in managing obesity epidemics.
Obesity; insulin resistance; adiponectin; adiponectin receptor; gene therapy
The effect of activation of liver X receptor by N-(2,2,2-trifluoroethyl)-N-[4-[2,2,2-trifluoro-1-hydroxy-1(trifluoromethyl)ethyl]phenyl] benzenesulfonamide (T0901317) on high fat diet (HFD)-induced obesity and insulin resistance was examined in C57BL/6 mice. When on HFD continuously for 10 weeks, C57BL/6 mice became obese with an average body weight of 42 g, insulin resistant, and glucose intolerant. Twice weekly intraperitoneal injections of T0901317 at 50 mg/kg in animals on the same diet completely blocked obesity development, obesity-associated insulin resistance, and glucose intolerance. Quantitative real-time PCR analysis showed that T0901317-treated animals had significantly higher mRNA levels of genes involved in energy metabolism, including Ucp-1, Pgc1a, Pgc1b, Cpt1a, Cpt1b, Acadm, Acadl, Aox, and Ehhadh. Transcription activation of Cyp7a1, Srebp-1c, Fas, Scd-1, and Acc-1 genes was also seen in T0901317-treated animals. T0901317 treatment induced reversible aggregation of lipids in the liver. These results suggest that liver X receptor could be a potential target for prevention of obesity and obesity-associated insulin resistance.
Electronic supplementary material
The online version of this article (doi:10.1208/s12248-012-9429-3) contains supplementary material, which is available to authorized users.
diabetes; high fat diet-induced obesity; liver X receptor; nuclear receptor; T0901317
Development of a safe and effective method for gene delivery to hepatocytes is a critical step toward gene therapy for liver diseases. Here, we assessed the parameters for gene delivery to the livers of large animals (pigs, 40–65 kg) using an image-guided hydrodynamics-based procedure that involves image-guided catheter insertion into the lobular hepatic vein and hydrodynamic injection of reporter plasmids using a computer-controlled injector. We demonstrated that injection parameters (relative position of the catheter in the hepatic vasculature, intravascular pressure upon injection, and injection volume) are directly related to the safety and efficiency of the procedure. By optimizing these parameters, we explored for the first time, the advantage of the procedure for sequential injections to multiple lobes in human-sized pigs. The optimized procedure resulted in sustained expression of the human α-1 antitrypsin gene in livers for more than 2 months after gene delivery. In addition, repeated hydrodynamic gene delivery was safely conducted and no adverse events were seen in the entire period of the study. Our results support the clinical applicability of the image-guided hydrodynamic gene delivery method for the treatment of liver diseases.
gene therapy; human α-1 antitrypsin; hydrodynamic gene delivery; image-guided gene delivery; non-viral vector
Liver X receptor (LXR) activation improves glucose homeostasis in obesity. This improvement, however, is associated with several side effects including hyperlipidemia and hepatic steatosis. Activation of peroxisome proliferator-activated receptor alpha (PPARα), on the other hand, increases fatty acid oxidation, leading to a reduction of hyperlipidemia. The objective of this study was to investigate whether concurrent activation of LXR/PPARα can produce synergistic benefits in treating obesity-associated metabolic disorders. Treatment of high fat diet-induced obese mice with T0901317, an LXR activator, or fenofibrate, the PPARα agonist, or in combination alleviated insulin resistance and improved glucose tolerance. The combined treatment dramatically exacerbated hepatic steatosis. Gene expression analysis in the liver showed that combined treatment increased the expression of genes involved in lipogenesis and fatty acid transport, including srebp-1c, chrebp, acc1, fas, scd1 and cd36. Histochemistry and ex vivo glycerol releasing assay showed that combined treatment accelerated lipid mobilization in adipose tissue. Combined treatment also increased the transcription of glut4, hsl, atgl and adiponectin, and decreased that of plin1, cd11c, ifnγ and leptin. Combined treatment markedly elevated the transcription of fgf21 in liver but not in adipose tissue. These results suggest that concurrent activation of LXR and PPARα as a strategy to control glucose and lipid metabolism in obesity is beneficial but could lead to elevation of lipid accumulation in the liver.
Human arylamine N-acetyltransferase 1, (HUMAN)NAT1, is a phase II xenobiotic-metabolizing enzyme that plays an important role in drug and carcinogen biotransformation and cancer development. Its gene expression has been shown to be regulated by environmental factors. The purpose of the current study is to determine the involvement of nuclear receptors in transcriptional regulation of (HUMAN)NAT1 gene. We show that among the nuclear receptors examined, including the glucocorticoid receptor, retinoid acid receptor-related orphan receptor alpha, constitutive androstane receptor, pregnane X receptor, aryl hydrocarbon receptor, and retinoic acid receptor, the glucocorticoid receptor plays a dominant role in regulating (HUMAN)NAT1 gene expression through distal promoter (P3). The involvement of the glucocorticoid receptor in transcription regulation of (HUMAN)NAT1 gene expression was demonstrated by dexamethasone treatment, reporter assay using plasmid-containing 3 kbp of 5′-end region of promoter 3, and treatment of anti-glucocorticoid RU486 in primary culture of human hepatocytes and transfected HepG2 cells. In addition, translation inhibition did not affect dexamethasone-induced gene expression through P3, suggesting that dexamethasone effect is directly mediated by glucocorticoid receptor activation. Furthermore, deletion analysis revealed the presence of multiple responsive elements within the 3 kbp fragment of P3. Transfection assays in mice using hydrodynamics-based procedure and reporter gene assay in a mouse cell line revealed that glucocorticoid-induced NAT gene expression is species dependent. Dexamethasone treatment of transfected mice and mouse cell line decreased (MOUSE)Nat2 gene expression, (HUMAN)NAT1 homologue. These results suggest that glucocorticoids serve as a modulator for (HUMAN)NAT1 gene expression via the P3-containing 5′-flanking region.
arylamine N-acetyltransferases; glucocorticoids; phase-II enzymes; promoter analysis; regulation of gene expression; transcriptional regulation
To mimic advanced stage of cancer development involving multi-organ metastasis, hydrodynamic delivery commonly used in gene transfer was explored for establishing concurrent tumors in the lung, liver and kidney using B16-F1 melanoma cells, 4T1 breast cells and Renca renal carcinoma cells, as a model. The procedure involves a rapid injection into a mouse tail-vein of serum-free medium, containing tumor cells in a volume equal to approximately 7–9% of body weight. Compared with the conventional tail vein injection of tumor cells resulting in tumor growth only in the lung, hydrodynamic injection is highly effective in establishing tumor growth in the liver, kidney and lung. All tumor cells examined including melanoma, breast metastatic and renal carcinoma cells showed significant tumor growth in these organs. These results suggest that the hydrodynamic delivery can be a valuable tool for modeling cancer in laboratory animals, especially in experimental mice.
hydrodynamic delivery; tumor models; cell delivery
Pregnane X receptor (PXR) is known to function as a xenobiotic sensor to regulate xenobiotic metabolism through selective transcription of genes responsible for maintaining physiological homeostasis. Here we report that the activation of PXR by pregnenolone 16α-carbonitrile (PCN) in AKR/J mice can prevent the development of high-fat diet-induced obesity and insulin resistance. The beneficial effects of PCN treatment are seen with reduced lipogenesis and gluconeogenesis in the liver, and lack of hepatic accumulation of lipid and lipid storage in the adipose tissues. RT-PCR analysis of genes involved in gluconeogenesis, lipid metabolism and energy homeostasis reveal that PCN treatment on high-fat diet-fed mice reduces expression in the liver of G6Pase, Pepck, Cyp7a1, Cd36, L-Fabp, Srebp, and Fas genes and slightly enhances expression of Cyp27a1 and Abca1 genes. RT-PCR analysis of genes involved in adipocyte differentiation and lipid metabolism in white adipose tissue show that PCN treatment reduces expression of Pparγ2, Acc1, Cd36, but increases expression of Cpt1b and Pparα genes in mice fed with high-fat diet. Similarly, PCN treatment of animals on high-fat diet increases expression in brown adipose tissue of Pparα, Hsl, Cpt1b, and Cd36 genes, but reduces expression of Acc1 and Scd-1 genes. PXR activation by PCN in high-fat diet fed mice also increases expression of genes involved in thermogenesis in brown adipose tissue including Dio2, Pgc-1α, Pgc-1β, Cidea, and Ucp-3. These results verify the important function of PXR in lipid and energy metabolism and suggest that PXR represents a novel therapeutic target for prevention and treatment of obesity and insulin resistance.
The transfer of genes into cells, both in vitro and in vivo, is critical for studying gene function and conducting gene therapy. Methods that utilize viral and nonviral vectors, as well as physical approaches, have been explored. Viral vector-mediated gene transfer employs replication-deficient viruses such as retro-virus, adenovirus, adeno-associated virus and herpes simplex virus. A major advantage of viral vectors is their high gene delivery efficiency. The nonviral vectors developed so far include cationic liposomes, cationic polymers, synthetic peptides and naturally occurring compounds. These nonviral vectors appear to be highly effective in gene delivery to cultured cells in vitro but are significantly less effective in vivo. Physical methods utilize mechanical pressure, electric shock or hydrodynamic force to transiently permeate the cell membrane to transfer DNA into target cells. They are simpler than viral- and nonviral-based systems and highly effective for localized gene delivery. The past decade has seen significant efforts to establish the most desirable method for safe, effective and target-specific gene delivery, and good progress has been made. The objectives of this review are to (i) explain the rationale for the design of viral, nonviral and physical methods for gene delivery; (ii) provide a summary on recent advances in gene transfer technology; (iii) discuss advantages and disadvantages of each of the most commonly used gene delivery methods; and (iv) provide future perspectives.
Numerous xenobiotic metabolizing enzymes are regulated by nuclear receptors at transcriptional level. The challenge we currently face is to understand how a given nuclear receptor interacts with its xenobiotics, migrates into nucleus, binds to the xenobiotic response element of a target gene, and regulates transcription. Toward this end, new methods have been developed to introduce the nuclear receptor gene into appropriate cells and study its activity in activating reporter gene expression under the control of a promoter containing xenobiotic response elements. The goal of this review is to critically examine the gene transfer methods currently available. We concentrate on the gene transfer mechanism, advantages and limitations of each method when employed for nuclear receptor-mediated gene regulation studies. It is our hope that the information provided highlights the importance of gene transfer in studying the mechanisms by which our body eliminates the potentially harmful substances and maintains the homeostasis.
Xenobiotics; nuclear receptor; transcription factor; drug metabolism; non-viral vector; gene delivery; cationic lipid; electroporation; hydrodynamic gene delivery
Hydrodynamic delivery has emerged as the simplest and most effective method for intracellular delivery of membrane impermeable substances in rodents. The system employs a physical force generated by a rapid injection of large volume of solution into a blood vessel to enhance the permeability of endothelium and the plasma membrane of the parenchyma cells to allow delivery of substance into cells. The procedure was initially established for gene delivery in mice and its applications have been extended to the delivery of proteins, oligo nucleotides, genomic DNA and RNA sequences, and small molecules. The focus of this review is on applications of hydrodynamic delivery in pharmaceutical research. Examples are provided to highlight the use of hydrodynamic delivery for study of transcriptional regulation of CYP enzymes, for establishment of animal model for viral infections, and for gene drug discovery and gene function analysis.
Hydrodynamic delivery; nonviral gene delivery; oligo nucleotides; gene therapy; siRNA; protein drug discovery
In this study, we examined the effect of various factors on gene delivery efficiency of tail vein injection of plasmid DNA into rats. We measured the level of reporter gene expression in the internal organs including the lung, heart, spleen, kidney, and liver as function of injection volume, injection time, and DNA dose. Persistency of reporter gene expression in transfected animals was also examined. We demonstrated that plasmid delivery to rats by the tail vein is effective as long as the volume of injected DNA solution is adjusted to 7–8% of body weight with an injection time of less than 10 s. With the exception of a short-term increase in serum concentration of alanine aminotransferase and transient irregularity in cardiac function during and soon after the injection, the procedure is well tolerated. Lac Z staining of the liver from transfected animals showed approximately 5–10% positive cells. Persistency test for transgene expression in animals using plasmid carrying cDNA of human alpha 1 antitrypsin gene driven by chicken beta actin gene promoter with CMV enhancers showed peak level of transgene product 1 day after the injection followed by a gradual decline with time. Peak level was regained by a second injection performed on day 38 after the first injection. These results show that tail vein injection is an effective means for introducing plasmid DNA into liver cells in rats. We believe that this procedure will be extremely useful for gene function studies in the context of whole animal in rats.
gene delivery; gene therapy; hydrodynamic gene delivery; nonviral vectors; siRNA delivery
Development of an effective, safe and convenient method for gene delivery to muscle is a critical step toward gene therapy for muscle-associated diseases. Toward this end, we have explored the possibility of combining the image-guided catheter insertion technique with the principle of hydrodynamic delivery to achieve muscle specific gene transfer in pigs. We demonstrate that gene transfer efficiency of the procedure is directly related to flow rate, injection pressure and injection volume. The optimal gene delivery was achieved at a flow rate of 15 ml/sec with injection pressure of 300 psi and injection volume equal to 1.5% of body weight. Under such a condition, hydrodynamic injection of saline containing pCMV-Luc (100 µg/ml) resulted in luciferase activity of 106 –107 relative light units (RLU)/mg of proteins extracted from the targeted muscle 5 days after hydrodynamic gene delivery. Result from immunohistochemical analysis revealed 70–90% transfection efficiency of muscle groups in the hind limb and persistent reporter gene expression for 2 months in transfected cells. With an exception of transient edema and elevation of creatine phosphokinase, no permanent tissue damage was observed. These results suggest that the image-guided, intravenous hydrodynamic delivery is an effective and safe method for gene delivery to skeletal muscle.
Hydrodynamic gene delivery; gene therapy; gene delivery; nonviral vectors; skeletal muscle
Image-guided, lobe-specific hydrodynamic gene delivery to liver was assessed in pigs. The procedure involved image-guided insertion of a balloon catheter to the hepatic vein of the selected lobe from the jugular vein and hydrodynamic injection of plasmid DNA using a newly developed computer-controlled injection device. We demonstrated that the impact of the procedure was regional with minimal effects on neighboring lobes. Level of gene expression resulted from the procedure was 107 RLU/mg in the targeted lobes and 102−105 RLU/mg in the non-targeted lobes 4 hr after hydrodynamic injection of pCMV-Luc plasmids. Occlusion of blood flow in the inferior vena cava or inferior vena cava plus portal vein was effective in elevating hydrodynamic pressure in the targeted vasculature but did not enhance gene delivery efficiency. Physiological examination on pigs with inferior vena cava occlusion revealed transient decreases of blood pressure and respiration rate. Removal of occlusion from inferior vena cava resulted in a rapid and transient increase in heart rate. Occlusion of the portal vein and hepatic vein showed no effect on physiological and cardiac activities. No major changes in serum composition were observed. These results suggest that: (1) image-guided, lobe-specific hydrodynamic procedure is safe and effective for regional gene delivery to liver; (2) blockade in inferior vena cava should be avoided for hydrodynamic gene delivery to the liver; and (3) clinic application of hydrodynamic gene delivery to liver is feasible.
Hydrodynamic gene delivery; gene therapy; gene delivery; nonviral vectors; hydrojector
The liver is a key organ for numerous metabolic pathways and involves many inherited diseases that, although being different in their pathology, are often caused by lack or overproduction of a critical gene product in the diseased cells. In principle, a straightforward method to fix such problem is to introduce into these cells with a gene-coding sequence to provide the missing gene product or with the nucleic acid sequence to inhibit production of the excessive gene product. Practically, however, success of nucleic acid-based pharmaceutics is dependent on the availability of a method capable of delivering nucleic acid sequence in the form of DNA or RNA to liver cells. In this review, we will summarize the progress toward the development of physical methods for nucleic acid delivery to the liver. Emphasis is placed on the mechanism of action, pros, and cons of each method developed so far. We hope the information provided will encourage new endeavor to improve the current methodologies or develop new strategies that will lead to safe and effective delivery of nucleic acids to the liver.
gene delivery; liver; nonviral vectors; physical method; transfection
Liver is a key organ for numerous metabolic pathways and involves many inherited diseases that, although being different in their pathology, are often caused by lack or overproduction of a critical gene product in the diseased cells. In principle, a straightforward method to fix such problem is to introduce into these cells with a gene-coding sequence to provide the missing gene product, or with the nucleic acid sequence to inhibit production of the excessive gene product. Practically, however, success of nucleic acid-based pharmaceutics is dependent on availability of a method capable of delivering nucleic acid sequence in the form of DNA or RNA to liver cells. In this review, we will summarize the progress toward development of physical methods for nucleic acid delivery to liver. Emphasis is placed on the mechanism of action, pros and cons of each method developed so far. We hope the information provided will encourage new endeavor to improve the current methodologies or develop new strategies that will lead to safe and effective delivery of nucleic acids to liver.
Gene delivery; non-viral vectors; physical method; liver; transfection
Gene delivery using nonviral approaches has been extensively studied as a basic tool for intracellular gene transfer and gene therapy. In the past, the primary focus has been on application of physical, chemical, and biological principles to development of a safe and efficient method that delivers a transgene into target cells for appropriate expression. This review summarizes the current status of the most commonly used nonviral methods, with an emphasis on their mechanism of action for gene delivery, and their advantages and limitations for gene therapy applications. The technical aspects of each delivery system are also reviewed, with a focus on how to achieve optimal delivery efficiency. A brief discussion of future development and further improvement of the current systems is intended to stimulate new ideas and encourage rapid advancement in this new and promising field.
Gene delivery; gene therapy; nonviral vectors; transfection