Promoterless, Nuclease-Free Genome Editing Confers a Growth Advantage for Corrected Hepatocytes in Mice With Methylmalonic Acidemia.

BACKGROUND AND AIMS: Adeno-associated viral (AAV) gene therapy has shown great promise as an alternative treatment for metabolic disorders managed using liver transplantation, but remains limited by transgene loss and genotoxicity. Our study aims to test an AAV vector with a promoterless integrating cassette, designed to provide sustained hepatic transgene expression and reduced toxicity in comparison to canonical AAV therapy. APPROACH AND RESULTS: Our AAV vector was designed to insert a methylmalonyl-CoA mutase (MMUT) transgene into the 3' end of the albumin locus and tested in mouse models of methylmalonic acidemia (MMA). After neonatal delivery, we longitudinally evaluated hepatic transgene expression, plasma levels of methylmalonate, and the MMA biomarker, fibroblast growth factor 21 (Fgf21), as well as integration of MMUT in the albumin locus. At necropsy, we surveyed for AAV-related hepatocellular carcinoma (HCC) in all treated MMA mice and control littermates. AAV-mediated genome editing of MMUT into the albumin locus resulted in permanent hepatic correction in MMA mouse models, which was accompanied by decreased levels of methylmalonate and Fgf21, and improved survival without HCC. With time, levels of transgene expression increased and methylmalonate progressively decreased, whereas the number of albumin-MMUT integrations and corrected hepatocytes in MMA mice increased, but not in similarly treated wild-type animals. Additionally, expression of MMUT in the setting of MMA conferred a selective growth advantage upon edited cells, which potentiates the therapeutic response. CONCLUSIONS: In conclusion, our findings demonstrate that AAV-mediated, promoterless, nuclease-free genome editing at the albumin locus provides safe and durable therapeutic benefit in neonatally treated MMA mice.

Macrophages with a deletion of the phosphoenolpyruvate carboxykinase 1 (Pck1) gene have a more proinflammatory phenotype.

Phosphoenolpyruvate carboxykinase (Pck1) is a metabolic enzyme that is integral to the gluconeogenic and glyceroneogenic pathways. However, Pck1's role in macrophage metabolism and function is unknown. Using stable isotopomer MS analysis in a mouse model with a myeloid cell-specific Pck1 deletion, we show here that this deletion increases the proinflammatory phenotype in macrophages. Incubation of LPS-stimulated bone marrow-derived macrophages (BMDM) with [U-13C]glucose revealed reduced 13C labeling of citrate and malate and increased 13C labeling of lactate in Pck1-deleted bone marrow-derived macrophages. We also found that the Pck1 deletion in the myeloid cells increases reactive oxygen species (ROS). Of note, this altered macrophage metabolism increased expression of the M1 cytokines TNFα, IL-1ß, and IL-6. We therefore conclude that Pck1 contributes to M1 polarization in macrophages. Our findings provide important insights into the factors determining the macrophage inflammatory response and indicate that Pck1 activity contributes to metabolic reprogramming and polarization in macrophages.

Effect of mono- and diglycerides on the digestion and absorption of lutein in lymph fistula rats.

Breast milk lutein is better absorbed by infants than lutein delivered in infant formula. Therefore, we wanted to better understand the possible absorption differences of lutein in breast milk vs. that in infant formula by determining its bioavailability after gastric administration and whether the intestinal absorption of lutein can be improved by using new delivery vehicles. Study 1 compared the intestinal uptake,and the lymphatic and portal transport of lutein in conscious lymph fistula rats. Four groups of lymph- and portal vein-cannulated rats ( n = 8-10/group) were randomized to receive via gastric tube increasing doses (10, 20, 40, or 80 mg/kg) of 20% lutein in safflower oil (SO) suspension to assess whether there was a saturable level of lutein that could be absorbed and transported in lymph. Aliquots of hourly portal blood and lymph were taken for lutein and zeaxanthin analyses. The dose-response study showed that 20 mg/kg lutein was the saturable level of lymphatic lutein absorption with no lutein detected in portal circulation at any dosage level tested. Study 2 randomized five groups of lymph fistula rats ( n = 4-9/group) to receive 20 mg/kg lutein from either lutein in SO or lutein in four different mono- and diglyceride oils (MDGs). Gastric infusion of lutein suspended in MDG (20 mg/kg) significantly improved (71-211%, P < 0.05) lymphatic lutein output 2-6 h after lipid feeding vs. lutein in SO. Lymphatic zeaxanthin (10% of the lutein fed mixture) transport in both Study 1 and Study 2 followed that of lutein. We conclude that a mixture of MDGs helps solubilize lutein and facilitate gastrointestinal micelle formation, thus improving lymphatic lutein absorption compared with triglyceride oils. NEW & NOTEWORTHY This paper describes how lutein is digested and absorbed by the gastrointestinal tract by using the conscious lymph fistula rat model. Our dose-response study showed that absorption and lymphatic transport of lutein is a saturable process with no lutein detected in portal circulation at any dosage level tested. Our paper also provides insight into how this process can be improved by modifying the typical lipid mixtures carrying the lutein.

Zinc finger protein 407 overexpression upregulates PPAR target gene expression and improves glucose homeostasis in mice.

The peroxisome proliferator-activated receptor (PPAR) family of nuclear receptors is central to the pathophysiology and treatment of metabolic disease through the receptors' ability to regulate the expression of genes involved in glucose homeostasis, adipogenesis, and lipid metabolism. However, the mechanism by which PPAR is regulated remains incompletely understood. We generated a transgenic mouse strain (ZFP-TG) that overexpressed Zfp407 primarily in muscle and heart. Transcriptome analysis by RNA-Seq identified 1,300 differentially expressed genes in the muscle of ZFP-TG mice, among which PPAR target genes were significantly enriched. Among the physiologically important PPARγ target genes, Glucose transporter (Glut)-4 mRNA and protein levels were increased in heart and muscle. The increase in Glut4 and other transcriptional effects of Zfp407 overexpression together decreased body weight and lowered plasma glucose, insulin, and HOMA-IR scores relative to control littermates. When placed on high-fat diet, ZFP-TG mice remained more glucose tolerant than their wild-type counterparts. Cell-based assays demonstrated that Zfp407 synergistically increased the transcriptional activity of all PPAR subtypes, PPARα, PPARγ, and PPARδ. The increased PPAR activity was not associated with increased PPAR mRNA or protein levels, suggesting that Zfp407 posttranslationally regulates PPAR activity. Collectively, these results demonstrate that Zfp407 overexpression improved glucose homeostasis. Thus, Zfp407 represents a new drug target for treating metabolic disease.

Constitutive Activation of the Nlrc4 Inflammasome Prevents Hepatic Fibrosis and Promotes Hepatic Regeneration after Partial Hepatectomy.

TThe molecular mechanisms responsible for the development of hepatic fibrosis are not fully understood. The Nlrc4 inflammasome detects cytosolic presence of bacterial components, activating inflammatory cytokines to facilitate clearance of pathogens and infected cells. We hypothesized that low-grade constitutive activation of the Nlrc4 inflammasome may lead to induced hepatocyte proliferation and prevent the development of hepatic fibrosis. The gene of Nlrc4 contains two single nucleotide polymorphisms (SNPs), one located within the Nlrc4 promoter and one contained within exon 5. These SNPs regulate Nlrc4 gene transcription and activation as measured through gene reporter assays and IL-1ß secretion. The 17C-6 mice have increased IL-1ß in plasma after chronic carbon tetrachloride (CCl4) administration compared to B6 mice. After two-thirds partial hepatectomy (2/3PH) 17C-6 mice have earlier restoration of liver mass with greater cyclin D1 protein and BrdU incorporation compared to B6 mice at several time points. These data reveal mild constitutive activation of the Nlrc4 inflammasome as the results of two SNPs, which leads to the stimulation of hepatocyte proliferation. The increased liver regeneration induces rapid liver mass recovery after hepatectomy and may prevent the development of hepatotoxin-induced liver fibrosis.

Genetic resistance to liver fibrosis on A/J mouse chromosome 17.

BACKGROUND: Because the histological and biochemical progression of liver disease is similar in alcoholic steatohepatitis (ASH) and nonalcoholic steatohepatitis (NASH), we hypothesized that the genetic susceptibility to these liver diseases would be similar. To identify potential candidate genes that regulate the development of liver fibrosis, we studied a chromosome substitution strain (CSS-17) that contains chromosome 17 from the A/J inbred strain substituted for the corresponding chromosome on the C57BL/6J (B6) genetic background. Previously, we identified quantitative trait loci (QTLs) in CSS-17, namely obesity-resistant QTL 13 and QTL 15 (Obrq13 and Obrq15, respectively), that were associated with protection from diet-induced obesity and hepatic steatosis on a high-fat diet. METHODS: To test whether these or other CSS-17 QTLs conferred resistance to alcohol-induced liver injury and fibrosis, B6, A/J, CSS-17, and congenics 17C-1 and 17C-6 were either fed Lieber-DeCarli ethanol (EtOH)-containing diet or had carbon tetrachloride (CCl4 ) administered chronically. RESULTS: The congenic strain carrying Obrq15 showed resistance from alcohol-induced liver injury and liver fibrosis, whereas Obrq13 conferred susceptibility to liver fibrosis. From published deep sequencing data for chromosome 17 in the B6 and A/J strains, we identified candidate genes in Obrq13 and Obrq15 that contained single-nucleotide polymorphisms (SNPs) in the promoter region or within the gene itself. NADPH oxidase organizer 1 (Noxo1) and NLR family, CARD domain containing 4 (Nlrc4) showed altered hepatic gene expression in strains with the A/J allele at the end of the EtOH diet study and after CCl4 treatment. CONCLUSIONS: Aspects of the genetics for the progression of ASH are unique compared to NASH, suggesting that the molecular mechanisms for the progression of disease are at least partially distinct. Using these CSSs, we identified 2 candidate genes, Noxo1 and Nlrc4, which modulate genetic susceptibility in ASH.

Alcohol-induced liver injury is modulated by Nlrp3 and Nlrc4 inflammasomes in mice.

Alcoholic liver disease (ALD) is characterized by increased hepatic lipid accumulation (steatosis) and inflammation with increased expression of proinflammatory cytokines. Two of these cytokines, interleukin-1 ß (IL-1 ß ) and IL-18, require activation of caspase-1 via members of the NOD-like receptor (NLR) family. These NLRs form an inflammasome that is activated by pathogens and signals released through local tissue injury or death. NLR family pyrin domain containing 3 (Nlrp3) and NLR family CARD domain containing protein 4 (Nlrc4) have been studied minimally for their role in the development of ALD. Using mice with gene targeted deletions for Nlrp3 (Nlrp3(-/-)) and Nlrc4 (Nlrc4(-/-)), we analyzed the response to chronic alcohol consumption. We found that Nlrp3(-/-) mice have more severe liver injury with higher plasma alanine aminotransferase (ALT) levels, increased activation of IL-18, and reduced activation of IL-1B. In contrast, the Nlrc4(-/-) mice had similar alcohol-induced liver injury compared to C57BL/6J (B6) mice but had greatly reduced activation of IL-1 ß . This suggests that Nlrp3 and Nlrc4 inflammasomes activate IL-1 ß and IL-18 via caspase-1 in a differential manner. We conclude that the Nlrp3 inflammasome is protective during alcohol-induced liver injury.

Apolipoprotein A-IV-Deficient Mice in 129/SvJ Background Are Susceptible to Obesity and Glucose Intolerance.

Apolipoprotein A-IV (apoA-IV), synthesized by enterocytes, is potentially involved in regulating lipid absorption and metabolism, food intake, and glucose metabolism. In this study, we backcrossed apoA-IV knockout (apoA-IV-/-) mice onto the 129/SvJ background for eight generations. Compared to the wild-type (WT) mice, the 129/SvJ apoA-IV-/- mice gained more weight and exhibited delayed glucose clearance even on the chow diet. During a 16-week high-fat diet (20% by weight of fat) study, apoA-IV-/- mice were more obese than the WT mice, which was associated with their increased food intake as well as reduced energy expenditure and physical activity. In addition, apoA-IV-/- mice developed significant insulin resistance (indicated by HOMA-IR) with severe glucose intolerance even though their insulin levels were drastically higher than the WT mice. In conclusion, we have established a model of apoA-IV-/- mice onto the 129/SvJ background. Unlike in the C57BL/6J strain, apoA-IV-/- 129/SvJ mice become significantly more obese and insulin-resistant than WT mice. Our current investigations of apoA-IV in the 129/SvJ strain and our previous studies in the C57BL/6J strain underline the impact of genetic background on apoA-IV metabolic effects.

Deficiency of apoA-IV in Female 129X1/SvJ Mice Leads to Diet-Induced Obesity, Insulin Resistance, and Decreased Energy Expenditure.

Obesity is one of the main risk factors for cardiovascular diseases, type II diabetes, hypertension, and certain cancers. Obesity in women at the reproductive stage adversely affects contraception, fertility, maternal well-being, and the health of their offspring. Being a major protein component in chylomicrons and high-density lipoproteins, apolipoprotein A-IV (apoA-IV) is involved in lipid metabolism, food intake, glucose homeostasis, prevention against atherosclerosis, and platelet aggregation. The goal of the present study is to determine the impact of apoA-IV deficiency on metabolic functions in 129X1/SvJ female mouse strain. After chronic high-fat diet feeding, apoA-IV-/- mice gained more weight with a higher fat percentage than wild-type (WT) mice, as determined by measuring their body composition. Increased adiposity and adipose cell size were also observed with a microscope, particularly in periovarian fat pads. Based on plasma lipid and adipokine assays, we found that obesity in apoA-IV-/- mice was not associated with hyperlipidemia but with higher leptin levels. Compared to WT mice, apoA-IV deficiency displayed glucose intolerance and elevated insulin levels, according to the data of the glucose tolerance test, and increased HOMA-IR values at fasting, suggesting possible insulin resistance. Lastly, we found obesity in apoA-IV-/- mice resulting from reduced energy expenditure but not food intake. Together, we established a novel and excellent female mouse model for future mechanistic study of obesity and its associated comorbidities.

Palladium-catalyzed annulation of 2,2'-diiodobiphenyls with alkynes: synthesis and applications of phenanthrenes.

A range of phenanthrene derivatives were efficiently synthesized by the palladium-catalyzed annulation of 2,2'-diiodobiphenyls with alkynes. The scope, limitations and regioselectivity of the reaction were investigated. The described method was adopted to synthesize 9,10-dialkylphenanthrenes, sterically overcrowded 4,5-disubstituted phenanthrenes and phenanthrene-based alkaloids. Reactions of highly methoxy-substituted biphenyls with 2-(2-propynyl)pyrrolidine and 2-(2-propynyl)piperidine gave 2-(9-phenanthylmethyl)pyrrolidines and 2-(9-phenanthylmethyl)piperidines, respectively. The products were transformed to phenanthroindolizidine and phenanthroquinolizidine alkaloids by the Pictet-Spengler reaction.

Novel AAV-mediated genome editing therapy improves health and survival in a mouse model of methylmalonic acidemia.

Methylmalonic acidemia (MMA) is an inborn error of metabolism mostly caused by mutations in the mitochondrial methylmalonyl-CoA mutase gene (MMUT). MMA patients suffer from frequent episodes of metabolic decompensation, which can be life threatening. To mimic both the dietary restrictions and metabolic decompensation seen in MMA patients, we developed a novel protein-controlled diet regimen in a Mmut deficient mouse model of MMA and demonstrated the therapeutic benefit of mLB-001, a nuclease-free, promoterless recombinant AAV GeneRideTM vector designed to insert the mouse Mmut into the endogenous albumin locus via homologous recombination. A single intravenous administration of mLB-001 to neonatal or adult MMA mice prevented body weight loss and mortality when challenged with a high protein diet. The edited hepatocytes expressed functional MMUT protein and expanded over time in the Mmut deficient mice, suggesting a selective growth advantage over the diseased cells. In mice with a humanized liver, treatment with a human homolog of mLB-001 resulted in site-specific genome editing and transgene expression in the transplanted human hepatocytes. Taken together, these findings support the development of hLB-001 that is currently in clinical trials in pediatric patients with severe forms of MMA.

Regulation of intestinal lipid metabolism: current concepts and relevance to disease.

Lipids entering the gastrointestinal tract include dietary lipids (triacylglycerols, cholesteryl esters and phospholipids) and endogenous lipids from bile (phospholipids and cholesterol) and from shed intestinal epithelial cells (enterocytes). Here, we comprehensively review the digestion, uptake and intracellular re-synthesis of intestinal lipids as well as their packaging into pre-chylomicrons in the endoplasmic reticulum, their modification in the Golgi apparatus and the exocytosis of the chylomicrons into the lamina propria and subsequently to lymph. We also discuss other fates of intestinal lipids, including intestinal HDL and VLDL secretion, cytosolic lipid droplets and fatty acid oxidation. In addition, we highlight the applicability of these findings to human disease and the development of therapeutics targeting lipid metabolism. Finally, we explore the emerging role of the gut microbiota in modulating intestinal lipid metabolism and outline key questions for future research.

Apolipoprotein A-IV: A Multifunctional Protein Involved in Protection against Atherosclerosis and Diabetes.

Apolipoprotein A-IV (apoA-IV) is a lipid-binding protein, which is primarily synthesized in the small intestine, packaged into chylomicrons, and secreted into intestinal lymph during fat absorption. In the circulation, apoA-IV is present on chylomicron remnants, high-density lipoproteins, and also in lipid-free form. ApoA-IV is involved in a myriad of physiological processes such as lipid absorption and metabolism, anti-atherosclerosis, platelet aggregation and thrombosis, glucose homeostasis, and food intake. ApoA-IV deficiency is associated with atherosclerosis and diabetes, which renders it as a potential therapeutic target for treatment of these diseases. While much has been learned about the physiological functions of apoA-IV using rodent models, the action of apoA-IV at the cellular and molecular levels is less understood, let alone apoA-IV-interacting partners. In this review, we will summarize the findings on the molecular function of apoA-IV and apoA-IV-interacting proteins. The information will shed light on the discovery of apoA-IV receptors and the understanding of the molecular mechanism underlying its mode of action.

Use of Isotope Tracers to Assess Lipid Absorption in Conscious Lymph Fistula Mice.

This protocol provides a comprehensive reference for the evolution of the lymph fistula model, the mechanism of lipid absorption, the detailed procedure for studying lipid absorption using the lymph fistula model, the interpretation of the results, and consideration of the experimental design. The lymph fistula model is an approach to assess the concentration and rate of a range of molecules transported by the lymph by cannulating lymph duct in animals. In this protocol, mice first undergo surgery with the implantation of cannulae in the duodenum and mesenteric lymph duct and are allowed to recover overnight in Bollman restraining cages housed in a temperature-regulated environment. To study in vivo lipid absorption, a lipid emulsion is prepared with labeled tracers, including [3 H]-triolein and [14 C]-cholesterol. On the day of the experiment, mice are continuously infused with lipid emulsion via the duodenum for 6 hr, and lymph is usually collected hourly. At the end of the study, gastrointestinal segments and their luminal contents are collected separately for determination of the digestion, uptake, and transport of exogenous lipids. © 2019 by John Wiley & Sons, Inc.

Using Stable Isotopes in Bone Marrow Derived Macrophage to Analyze Metabolism.

Using gas chromatography mass spectrometry (GC-MS) to analyze the citric acid cycle (CAC) and related intermediates (such as glutamate, glutamine, GABA, and aspartate) is an analytical approach to identify unexpected correlations between apparently related and unrelated pathways of energy metabolism. Intermediates can be as expressed as their absolute concentrations or relative ratios by using known amounts of added reference standards to the sample. GC-MS can also distinguish between heavy labeled molecules (2H- or 13C-labeled) and the naturally occurring most abundant molecules. Applications using tracers can also assess the turnover of specific metabolic pools under various physiological and pathological conditions as well as for pathway discovery. The following protocol is a relatively simple method that is not only sensitive for small concentrations of metabolic intermediates but can also be used in vivo or in vitro to determine the integrity of various metabolic pathways, such as flux changes within specific metabolite pools. We used this protocol to determine the role of phosphoenolpyruvate carboxykinase 1 (Pck1) gene in mouse macrophage cells to determine the percent contribution from a precursor of 13C labeled glucose into specific CAC metabolite pools.

Apolipoprotein A-IV constrains HPA and behavioral stress responsivity in a strain-dependent manner.

There is a critical gap in our knowledge of the mechanisms that govern interactions between daily life experiences (e.g., stress) and metabolic diseases, despite evidence that stress can have profound effects on cardiometabolic health. Apolipoprotein A-IV (apoA-IV) is a protein found in chylomicrons (lipoprotein particles that transport lipids throughout the body) where it participates in lipid handling and the regulation of peripheral metabolism. Moreover, apoA-IV is expressed in brain regions that regulate energy balance including the arcuate nucleus. Given that both peripheral and central metabolic processes are important modulators of hypothalamic-pituitary-adrenocortical (HPA) axis activity, the present work tests the hypothesis that apoA-IV activity affects stress responses. As emerging data suggests that apoA-IV actions can vary with background strain, we also explore the strain-dependence of apoA-IV stress regulation. These studies assess HPA axis, metabolic (hyperglycemia), and anxiety-related behavioral responses to psychogenic stress in control (wildtype) and apoA-IV-deficient (KO) mice on either the C57Bl/6J (C57) or 129×1/SvJ (129) background strain. The results indicate that apoA-IV KO increases post-stress corticosterone and anxiety-related behavior specifically in the 129 strain, and increases stress-induced hyperglycemia exclusively in the C57 strain. These data support the hypothesis that apoA-IV is a novel factor that limits stress reactivity in a manner that depends on genetic background. An improved understanding of the complex relationship among lipid homeostasis, stress sensitivity, and genetics is needed to optimize the development of personalized treatments for stress- and metabolism-related diseases.