Role of non-coding RNAs on liver metabolism and NAFLD pathogenesis.

Obesity and type 2 diabetes are major contributors to the growing prevalence of non-alcoholic fatty liver disease (NAFLD), a chronic liver condition characterized by the accumulation of fat in individuals without a significant amount of alcohol intake. The NAFLD spectrum ranges from simple steatosis (early stages, known as NAFL) to non-alcoholic steatohepatitis, which can progress to fibrosis and cirrhosis or hepatocellular carcinoma. Obesity, type 2 diabetes and NAFLD are strongly associated with insulin resistance. In the liver, insulin resistance increases hepatic glucose output, lipogenesis and very-low-density lipoprotein secretion, leading to a combination of hyperglycemia and hypertriglyceridemia. Aberrant gene expression is a hallmark of insulin resistance. Non-coding RNAs (ncRNAs) have emerged as prominent regulators of gene expression that operate at the transcriptional, post-transcriptional and post-translational levels. In the last couple of decades, a wealth of studies have provided evidence that most processes of liver metabolism are orchestrated by ncRNAs. This review focuses on the role of microRNAs, long non-coding RNAs and circular RNAs as coordinators of hepatic function, as well as the current understanding on how their dysregulation contributes to abnormal metabolism and pathophysiology in animal models of insulin resistance and NAFLD. Moreover, ncRNAs are emerging as useful biomarkers that may be able to discriminate between the different stages of NAFLD. The potential of ncRNAs as therapeutic drugs for NAFLD treatment and as biomarkers is discussed.

Lack of liver glycogen causes hepatic insulin resistance and steatosis in mice.

Disruption of the Gys2 gene encoding the liver isoform of glycogen synthase generates a mouse strain (LGSKO) that almost completely lacks hepatic glycogen, has impaired glucose disposal, and is pre-disposed to entering the fasted state. This study investigated how the lack of liver glycogen increases fat accumulation and the development of liver insulin resistance. Insulin signaling in LGSKO mice was reduced in liver, but not muscle, suggesting an organ-specific defect. Phosphorylation of components of the hepatic insulin-signaling pathway, namely IRS1, Akt, and GSK3, was decreased in LGSKO mice. Moreover, insulin stimulation of their phosphorylation was significantly suppressed, both temporally and in an insulin dose response. Phosphorylation of the insulin-regulated transcription factor FoxO1 was somewhat reduced and insulin treatment did not elicit normal translocation of FoxO1 out of the nucleus. Fat overaccumulated in LGSKO livers, showing an aberrant distribution in the acinus, an increase not explained by a reduction in hepatic triglyceride export. Rather, when administered orally to fasted mice, glucose was directed toward hepatic lipogenesis as judged by the activity, protein levels, and expression of several fatty acid synthesis genes, namely, acetyl-CoA carboxylase, fatty acid synthase, SREBP1c, chREBP, glucokinase, and pyruvate kinase. Furthermore, using cultured primary hepatocytes, we found that lipogenesis was increased by 40% in LGSKO cells compared with controls. Of note, the hepatic insulin resistance was not associated with increased levels of pro-inflammatory markers. Our results suggest that loss of liver glycogen synthesis diverts glucose toward fat synthesis, correlating with impaired hepatic insulin signaling and glucose disposal.

Sterol regulatory element-binding protein-1 (SREBP-1) is required to regulate glycogen synthesis and gluconeogenic gene expression in mouse liver.

Sterol regulatory element-binding protein-1 (SREBP-1) is a key transcription factor that regulates genes in the de novo lipogenesis and glycolysis pathways. The levels of SREBP-1 are significantly elevated in obese patients and in animal models of obesity and type 2 diabetes, and a vast number of studies have implicated this transcription factor as a contributor to hepatic lipid accumulation and insulin resistance. However, its role in regulating carbohydrate metabolism is poorly understood. Here we have addressed whether SREBP-1 is needed for regulating glucose homeostasis. Using RNAi and a new generation of adenoviral vector, we have silenced hepatic SREBP-1 in normal and obese mice. In normal animals, SREBP-1 deficiency increased Pck1 and reduced glycogen deposition during fed conditions, providing evidence that SREBP-1 is necessary to regulate carbohydrate metabolism during the fed state. Knocking SREBP-1 down in db/db mice resulted in a significant reduction in triglyceride accumulation, as anticipated. However, mice remained hyperglycemic, which was associated with up-regulation of gluconeogenesis gene expression as well as decreased glycolysis and glycogen synthesis gene expression. Furthermore, glycogen synthase activity and glycogen accumulation were significantly reduced. In conclusion, silencing both isoforms of SREBP-1 leads to significant changes in carbohydrate metabolism and does not improve insulin resistance despite reducing steatosis in an animal model of obesity and type 2 diabetes.

Enhancing hepatic mitochondrial fatty acid oxidation stimulates eating in food-deprived mice.

Hepatic fatty acid oxidation (FAO) has long been implicated in the control of eating. Nevertheless, direct evidence for a causal relationship between changes in hepatic FAO and changes in food intake is still missing. Here we tested whether increasing hepatic FAO via adenovirus-mediated expression of a mutated form of the key regulatory enzyme of mitochondrial FAO carnitine palmitoyltransferase 1A (CPT1mt), which is active but insensitive to inhibition by malonyl-CoA, affects eating and metabolism in mice. CPT1mt expression increased hepatocellular CPT1 protein levels. This resulted in an increase in circulating ketone body levels in fasted CPT1mt-expressing mice, suggesting an increase in hepatic FAO. These mice did not show any significant changes in cumulative food intake, energy expenditure, or respiratory quotient after 4-h food deprivation. After 24-h food deprivation, however, the CPT1mt-expressing mice displayed increased food intake. Thus expression of CPT1mt in the liver increases hepatic FAO capacity, but does not inhibit eating. Rather, it may even stimulate eating after prolonged food deprivation. These data do not support the hypothesis that an increase in hepatic FAO decreases food intake.

Insights from a high-fat diet fed mouse model with a humanized liver.

Non-alcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disorder worldwide and is increasing at an alarming rate. NAFLD is strongly associated with obesity and insulin resistance. The use of animal models remains a vital aspect for investigating the molecular mechanisms contributing to metabolic dysregulation and facilitating novel drug target identification. However, some differences exist between mouse and human hepatocyte physiology. Recently, chimeric mice with human liver have been generated, representing a step forward in the development of animal models relevant to human disease. Here we explored the feasibility of using one of these models (cDNA-uPA/SCID) to recapitulate obesity, insulin resistance and NAFLD upon feeding a Western-style diet. Furthermore, given the importance of a proper control diet, we first evaluated whether there are differences between feeding a purified ingredient control diet that matches the composition of the high-fat diet and feeding a grain-based chow diet. We show that mice fed chow have a higher food intake and fed glucose levels than mice that received a low-fat purified ingredient diet, suggesting that the last one represents a better control diet. Upon feeding a high-fat or matched ingredient control diet for 12 weeks, cDNA-uPA/SCID chimeric mice developed extensive macrovesicular steatosis, a feature previously associated with reduced growth hormone action. However, mice were resistant to diet-induced obesity and remained glucose tolerant. Genetic background is fundamental for the development of obesity and insulin resistance. Our data suggests that using a background that favors the development of these traits, such as C57BL/6, may be necessary to establish a humanized mouse model of NAFLD exhibiting the metabolic dysfunction associated with obesity.

Aberrant gene expression induced by a high fat diet is linked to H3K9 acetylation in the promoter-proximal region.

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease, with an estimated global prevalence of 1 in 4 individuals. Aberrant transcriptional control of gene expression is central to the pathophysiology of metabolic diseases. However, the molecular mechanisms leading to gene dysregulation are not well understood. Histone modifications play important roles in the control of transcription. Acetylation of histone 3 at lysine 9 (H3K9ac) is associated with transcriptional activity and is implicated in transcript elongation by controlling RNA polymerase II (RNAPII) pause-release. Hence, changes in this histone modification may shed information on novel pathways linking transcription control and metabolic dysfunction. Here, we carried out genome-wide analysis of H3K9ac in the liver of mice fed a control or a high-fat diet (an animal model of NAFLD), and asked whether this histone mark associates with changes in gene expression. We found that over 70% of RNAPII peaks in promoter-proximal regions overlapped with H3K9ac, consistent with a role of H3K9ac in the regulation of transcription. When comparing high-fat with control diet, approximately 17% of the differentially expressed genes were associated with changes in H3K9ac in their promoters, showing a strong correlation between changes in H3K9ac signal and gene expression. Overall, our data indicate that in response to a high-fat diet, dysregulated gene expression of a subset of genes may be attributable to changes in transcription elongation driven by H3K9ac. Our results point at an added mechanism of gene regulation that may be important in the development of metabolic diseases.

Gene therapy for inherited retinal and optic nerve degenerations.

INTRODUCTION: The eye is a target for investigational gene therapy due to the monogenic nature of many inherited retinal and optic nerve degenerations (IRD), its accessibility, tight blood-ocular barrier, the ability to non-invasively monitor for functional and anatomic outcomes, as well as its relative immune privileged state.Vectors currently used in IRD clinical trials include adeno-associated virus (AAV), small single-stranded DNA viruses, and lentivirus, RNA viruses of the retrovirus family. Both can transduce non-dividing cells, but AAV are non-integrating, while lentivirus integrate into the host cell genome, and have a larger transgene capacity. AREAS COVERED: This review covers Leber's congenital amaurosis, choroideremia, retinitis pigmentosa, Usher syndrome, Stargardt disease, Leber's hereditary optic neuropathy, Achromatopsia, and X-linked retinoschisis. EXPERT OPINION: Despite great potential, gene therapy for IRD raises many questions, including the potential for less invasive intravitreal versus subretinal delivery, efficacy, safety, and longevity of response, as well as acceptance of novel study endpoints by regulatory bodies, patients, clinicians, and payers. Also, ultimate adoption of gene therapy for IRD will require widespread genetic screening to identify and diagnose patients based on genotype instead of phenotype.

Impact of silencing hepatic SREBP-1 on insulin signaling.

Sterol Regulatory Element Binding Protein-1 (SREBP-1) is a conserved transcription factor of the basic helix-loop-helix leucine zipper family (bHLH-Zip) that plays a central role in regulating expression of genes of carbohydrate and fatty acid metabolism in the liver. SREBP-1 activity is essential for the control of insulin-induced anabolic processes during the fed state. In addition, SREBP-1 regulates expression of key molecules in the insulin signaling pathway, including insulin receptor substrate 2 (IRS2) and a subunit of the phosphatidylinositol 3-kinase (PI3K) complex, PIK3R3, suggesting that feedback mechanisms exist between SREBP-1 and this pathway. Nevertheless, the overall contribution of SREBP-1 activity to maintain insulin signal transduction is unknown. Furthermore, Akt is a known activator of mTORC1, a sensor of energy availability that plays a fundamental role in metabolism, cellular growth and survival. We have silenced SREBP-1 and explored the impact on insulin signaling and mTOR in mice under fed, fasted and refed conditions. No alterations in circulating levels of insulin were observed. The studies revealed that depletion of SREBP-1 had no impact on IRS1Y612, AktS473, and downstream effectors GSK3αS21 and FoxO1S256 during the fed state. Nevertheless, reduced levels of these molecules were observed under fasting conditions. These effects were not associated with changes in phosphorylation of mTOR. Overall, our data indicate that the contribution of SREBP-1 to maintain insulin signal transduction in liver is modest.

Gene therapy for age-related macular degeneration.

INTRODUCTION: In neovascular age related macular degeneration (nAMD), gene therapy to chronically express anti-vascular endothelial growth factor (VEGF) proteins could ameliorate the treatment burden of chronic intravitreal therapy and improve limited visual outcomes associated with 'real world' undertreatment. Areas covered: In this review, the authors assess the evolution of gene therapy for AMD. Adeno-associated virus (AAV) vectors can transduce retinal pigment epithelium; one such early application was a phase I trial of AAV2-delivered pigment epithelium derived factor gene in advanced nAMD. Subsequently, gene therapy for AMD shifted to the investigation of soluble fms-like tyrosine kinase-1 (sFLT-1), an endogenously expressed VEGF inhibitor, binding and neutralizing VEGF-A. After some disappointing results, research has centered on novel vectors, including optimized AAV2, AAV8 and lentivirus, as well as genes encoding other anti-angiogenic proteins, including ranibizumab, aflibercept, angiostatin and endostatin. Also, gene therapy targeting the complement system is being investigated for geographic atrophy due to non-neovascular AMD. Expert opinion: The success of gene therapy for AMD will depend on the selection of the most appropriate therapeutic protein and its level of chronic expression. Future investigations will center on optimizing vector, promoter and delivery methods, and evaluating the risks of the chronic expression of anti-angiogenic or anti-complement proteins.

Gene targets of mouse miR-709: regulation of distinct pools.

MicroRNA (miRNA) are short non-coding RNA molecules that regulate multiple cellular processes, including development, cell differentiation, proliferation and death. Nevertheless, little is known on whether miRNA control the same gene networks in different tissues. miR-709 is an abundant miRNA expressed ubiquitously. Through transcriptome analysis, we have identified targets of miR-709 in hepatocytes. miR-709 represses genes implicated in cytoskeleton organization, extracellular matrix attachment, and fatty acid metabolism. Remarkably, none of the previously identified targets in non-hepatic tissues are silenced by miR-709 in hepatocytes, even though several of these genes are abundantly expressed in liver. In addition, miR-709 is upregulated in hepatocellular carcinoma, suggesting it participates in the genetic reprogramming that takes place during cell division, when cytoskeleton remodeling requires substantial changes in gene expression. In summary, the present study shows that miR-709 does not repress the same pool of genes in separate cell types. These results underscore the need for validating gene targets in every tissue a miRNA is expressed.

Basal insulin gene expression significantly improves conventional insulin therapy in type 1 diabetic rats.

Although a conventional insulin regimen for type 1 diabetes with twice-daily insulin injections is effective in preventing postprandial blood glucose excursions, this treatment is limited by its inadequate control of fasting hyperglycemia. Alternatively, sustained basal hepatic insulin gene expression has been shown to result in fasting normoglycemia in type 1 diabetic rats, although the treated animals still exhibited moderate postprandial hyperglycemia. To test the hypothesis that basal hepatic insulin production can be used as an auxiliary treatment to conventional insulin therapy for achieving better glycemic control, streptozotocin-induced diabetic rats were treated with twice-daily insulin injections, basal hepatic insulin production, or both in combination. Diabetic rats treated by conventional insulin therapy still suffered from fasting hyperglycemia, but when complemented with basal hepatic insulin production, near-normoglycemia under both fed and fasting conditions was achieved without fasting hypoglycemia. In addition, the combination-treated animals showed significantly enhanced glucose tolerance and markedly improved profiles in lipid metabolism. Furthermore, the combination treatment reduced the elevated fructosamine, glycated hemoglobin, and advanced glycation end products concentrations to normal. These results provide a proof of concept for basal hepatic insulin production as an adjuvant treatment to conventional insulin therapy in type 1 diabetes.

Novel targets and therapeutic strategies for type 2 diabetes.

The number of people diagnosed with type 2 diabetes mellitus (T2DM) is increasing at an alarming rate in western societies and has become a major health concern. During the past decade, studies using transgenic animals, gene transfer and pharmacological agents have yielded many data that have helped understand the molecular alterations characteristic of T2DM. This has opened the possibility for the development of potentially more-effective therapies, mainly focused on attenuating hepatic glucose production, enhancing glucose-dependent insulin secretion, enhancing the insulin signal transduction pathway, inhibiting lipolysis from the adipose tissue and promoting fatty acid oxidation.

Adenovirus-mediated expression of glucokinase in the liver as an adjuvant treatment for type 1 diabetes.

Glucokinase (GK) plays a crucial role in hepatic glucose disposal. Its activity is decreased in patients with maturity-onset diabetes of the young and in some animal models of diabetes. We investigated the feasibility of manipulating GK expression as an adjuvant treatment for type 1 diabetes, using an E1/E3-deleted adenoviral vector (Ad.EF1(alpha)GK) delivered to the liver of streptozotocin-induced type 1 diabetic rats. First, we studied the metabolic impact of constitutive glucokinase expression in the absence of insulin. Normal blood glucose levels were observed after gene transfer, and glucose tolerance was substantially enhanced compared with diabetic control animals, suggesting that hepatic GK expression is a feasible mechanism to enhance glucose disposal. In a second study we administered Ad.EF1(alpha)GK together with subcutaneous insulin injections to determine whether the combined action of insulin plus GK activity would provide better glucose homeostasis than insulin treatment alone. This combination approach resulted in constant, near-normal glucose values under fed conditions. Furthermore, the animals stayed in the normoglycemic range after an overnight fast, indicating that the risk to develop hypoglycemia is not increased by expression of GK. Alterations of other metabolic routes were observed, suggesting that insulin-regulated expression of GK may be necessary to use the strategy as a treatment of type 1 diabetes.

Lethal toxicity, severe endothelial injury, and a threshold effect with high doses of an adenoviral vector in baboons.

The effects of intravenous administration of a first-generation adenoviral vector expressing beta-galactosidase were compared in two baboons receiving a high dose or lower dose of vector, 1.2 x 10(13) or 1.2 x 10(12) particles/kg, respectively. The high-dose baboon developed acute symptoms, decreased platelet counts, and increased liver enzymes, and became moribund at 48 hr after injection, while the lower-dose baboon developed no symptoms. Expression of the beta-galactosidase transgene was prominent in liver, spleen, and endothelium of the arterial vasculature in the high-dose baboon, but was much more limited and spared the endothelium in the lower-dose baboon. Injury to the vascular endothelium was the most prominent abnormality in the high-dose baboon. Extensive histological studies provide a detailed picture of the pathology associated with a lethal dose of first-generation adenoviral vector in a primate.

Vector and helper genome rearrangements occur during production of helper-dependent adenoviral vectors.

Helper-dependent adenoviral vectors (HD Ad) hold extreme promise for gene therapy of human diseases. All viral genes are deleted in HD Ad vectors, and therefore, the presence of a helper virus is required for their production. Current methods to minimize helper contamination in large-scale preparations rely on the use of the Cre/loxP system. The inclusion of loxP sites flanking the packaging signal results in its excision in the presence of Cre recombinase, preventing helper genome encapsidation. It is well established that the level of Cre recombinase activity is important in determining the degree of helper contamination. However, there is little information on other mechanisms that could also play an important role. We have generated several HD Ad vectors containing a rapalog-inducible system to regulate transgene expression, or LacZ under the control of the elongation factor 1 α promoter. Large-scale production of these vectors resulted in abundant helper contamination. Viral DNA analysis revealed the presence of rearrangements between vector and helper genomes. The rearrangements involved a helper DNA molecule with a fragment of the left arm of the HD Ad vector, including its ITR, packaging signal, and some stuffer sequence. Overall, our data suggest that helper DNA molecules that accumulate after Cre recombinase activity are prone to rearrangements, resulting in helper genomes that have incorporated a packaging signal from the vector. Helper particles with rearranged genomes have a growth advantage. This study identifies a novel mechanism leading to helper contamination during helper-dependent adenoviral vector production.

shRNA-induced interferon-stimulated gene analysis.

RNA interference (RNAi) is a cellular mechanism to inhibit the expression of gene products in a highly specific manner. In recent years, RNAi has become the cornerstone of gene function studies, shortening the otherwise long process of target identification and validation. In addition, small interfering RNA (siRNA) and short-hairpin RNA (shRNA) therapies are being developed for the treatment of a variety of human diseases. Despite its huge potential for gene silencing, a hurdle to safe and effective RNAi is the activation of innate immune responses. Induction of innate immunity is dose- and sequence-dependent, and is also influenced by target tissue and delivery vehicle. Research on the molecular mechanisms mediating this response is helping to improve the design of the RNAi molecules. Nevertheless, appropriate testing for the presence of this undesired effect is needed prior to making conclusions on the outcome of the silencing treatment.

Comparative nucleic acid transfection efficacy in primary hepatocytes for gene silencing and functional studies.

BACKGROUND: Primary hepatocytes are the best resource for in vitro studies directed at understanding hepatic processes at the cellular and molecular levels, necessary for novel drug development to treat highly prevalent diseases such as non-alcoholic steatohepatitis, cardiovascular disease and type 2 diabetes. There is a need to identify simple methods to genetically manipulate primary hepatocytes and conduct functional studies with plasmids, small interfering RNA (siRNA) or microRNA (miRNA). New lipofection reagents are available that have the potential to yield higher levels of transfection with reduced toxicity. FINDINGS: We have tested several liposome-based transfection reagents used in molecular biology research. We show that transfection efficiency with one of the most recently developed formulations, Metafectene Pro, is high with plasmid DNA (>45% cells) as well as double stranded RNA (>90% with siRNA or microRNA). In addition, negligible cytotoxicity was present with all of these nucleic acids, even if cells were incubated with the DNA:lipid complex for 16 hours. To provide the proof of concept that these conditions can be used not only for overexpression of a gene of interest, but also in RNA interference applications, we targeted two liver expressed genes, Sterol Regulatory Element-Binding Protein-1 and Fatty Acid Binding Protein 5 using plasmid-mediated short hairpin RNA expression. In addition, similar transfection conditions were used to optimally deliver siRNA and microRNA. CONCLUSIONS: We have identified a lipid-based reagent for primary hepatocyte transfection of nucleic acids currently used in molecular biology laboratories. The conditions described here can be used to expedite a large variety of research applications, from gene function studies to microRNA target identification.

Constitutive expression of short hairpin RNA in vivo triggers buildup of mature hairpin molecules.

RNA interference (RNAi) has become the cornerstone technology for studying gene function in mammalian cells. In addition, it is a promising therapeutic treatment for multiple human diseases. Virus-mediated constitutive expression of short hairpin RNA (shRNA) has the potential to provide a permanent source of silencing molecules to tissues, and it is being devised as a strategy for the treatment of liver conditions such as hepatitis B and hepatitis C virus infection. Unintended interaction between silencing molecules and cellular components, leading to toxic effects, has been described in vitro. Despite the enormous interest in using the RNAi technology for in vivo applications, little is known about the safety of constitutively expressing shRNA for multiple weeks. Here we report the effects of in vivo shRNA expression, using helper-dependent adenoviral vectors. We show that gene-specific knockdown is maintained for at least 6 weeks after injection of 1 × 10(11) viral particles. Nonetheless, accumulation of mature shRNA molecules was observed up to weeks 3 and 4, and then declined gradually, suggesting the buildup of mature shRNA molecules induced cell death with concomitant loss of viral DNA and shRNA expression. No evidence of well-characterized innate immunity activation (such as interferon production) or saturation of the exportin-5 pathway was observed. Overall, our data suggest constitutive expression of shRNA results in accumulation of mature shRNA molecules, inducing cellular toxicity at late time points, despite the presence of gene silencing.

Accurate single-day titration of adenovirus vectors based on equivalence of protein VII nuclear dots and infectious particles.

Protein VII is an abundant component of adenovirus particles and is tightly associated with the viral DNA. It enters the nucleus along with the infecting viral genome and remains bound throughout early phase. Protein VII can be visualized by immunofluorescent staining as discrete dots in the infected cell nucleus. Comparison between protein VII staining and expression of the 72kDa DNA-binding protein revealed a one-to-one correspondence between protein VII dots and infectious viral genomes. A similar relationship was observed for a helper-dependent adenovirus vector expressing green fluorescent protein. This relationship allowed accurate titration of adenovirus preparations, including wild-type and helper-dependent vectors, using a 1-day immunofluorescence method. The method can be applied to any adenovirus vector and gives results equivalent to the standard plaque assay.

Robust hepatic gene silencing for functional studies using helper-dependent adenoviral vectors.

RNA interference is currently envisioned as the basis of gene function and drug target validation studies. This novel technology has the advantage of providing a remarkably faster tool for gene silencing than traditional transgenic animal methodologies. In vivo administration of short interfering RNA (siRNA) typically results in reduced target gene expression for approximately 1 week. Viral vectors offer the possibility to express constitutive levels of short hairpin RNA (shRNA) so that the effects of knocking down the target gene can be studied for a few weeks, rather than a few days. Helper-dependent vectors have a significant advantage over previous generations of adenoviral vectors because of their much higher cloning capacity, potential for long-term transgene expression, and enhanced safety profiles on administration in vivo. Therefore, this advanced type of vector is an excellent tool to carry out in vivo studies directed at constitutive expression of shRNA. Here we show it is possible to obtain more than 90% target gene knockdown in an animal model of type 2 diabetes for several weeks, thereby consolidating this technology as an alternative to generating liver-specific knockout animals.