Practical and Statistical Considerations for the Long Term Follow-Up of Gene Therapy Trial Participants.

Study sponsors and market authorization holders are required by the US Food and Drug Administration (FDA) and European Medicines Agency (EMA) to enroll patients administered a gene therapy product, whether in a trial setting or post-licensure, in a long term follow-up safety study to continue the safety assessments of their product. These follow-up studies range between 5 and 15 years after dosing. This unprecedented duration of engagement with patients and caregivers raises logistical challenges that will require innovation and collaboration across sponsors and regulators. In this paper we delineate some of the key considerations for designing long term follow-up protocols in the gene therapy setting, with an eye toward platform and master protocol approaches, and offer guidance for innovative operational and statistical methods that can help assess the safety profile and durability of response for these novel therapeutics.

Topical Ocular TRPV1 Antagonist SAF312 (Libvatrep) for Postoperative Pain After Photorefractive Keratectomy.

Purpose: Evaluation of safety and efficacy of topical ocular SAF312 (Libvatrep) in post-photorefractive keratectomy (PRK) pain. Methods: In this placebo (vehicle)-controlled, participant- and investigator-masked study, 40 participants were randomized (1:1) to two treatment sequences in a bilateral PRK crossover design (SAF312 2.5% followed by vehicle [or vice versa], one eye drop, four times daily for 72 hours after PRK). Primary endpoints were visual analog scale (VAS) pain scores at 6 hours after first drop of study drug and average VAS scores over 0 to 12 hours postoperatively. Secondary endpoints included postoperative oral rescue medication (ORM) use and adverse events (AEs). Results: All 40 participants completed the study. Both primary endpoints were met; mean difference in VAS pain scores between SAF312- and vehicle-treated eyes was -11.13 (P = 0.005, -25%) at 6 hours postoperatively and -8.56 (P = 0.017, -22%) over 0 to 12 hours. Mean VAS pain scores with SAF312 were consistently lower than with vehicle from 1 hour postoperatively up to 30 hours (P ≤ 0.10 observed in 8/11 time points). Less ORM was taken with SAF312 up to 0 to 72 hours postoperatively, with a trend of fewer participants taking ORM at 0 to 24 hours postoperatively with SAF312 versus vehicle. No serious AEs were reported. All ocular AEs were mild and transient, and none were drug related. SAF312-treated eyes showed no delay in wound healing and had a lower grade 4 conjunctival hyperemia 24 hours postoperatively versus vehicle-treated eyes. Conclusions: SAF312 was well tolerated and effective in reducing ocular pain post-PRK. Translational Relevance: Topical SAF312 presents a new therapeutic option for patients undergoing PRK.

Navigating the Light-Sheet Image Analysis Software Landscape: Concepts for Driving Cohesion From Data Acquisition to Analysis.

From the combined perspective of biologists, microscope instrumentation developers, imaging core facility scientists, and high performance computing experts, we discuss the challenges faced when selecting imaging and analysis tools in the field of light-sheet microscopy. Our goal is to provide a contextual framework of basic computing concepts that cell and developmental biologists can refer to when mapping the peculiarities of different light-sheet data to specific existing computing environments and image analysis pipelines. We provide our perspective on efficient processes for tool selection and review current hardware and software commonly used in light-sheet image analysis, as well as discuss what ideal tools for the future may look like.

Digital endpoints for self-administered home-based functional assessment in pediatric Friedreich's ataxia.

BACKGROUND: Friedreich's ataxia is an inherited, progressive, neurodegenerative disease that typically begins in childhood. Disease severity is commonly assessed with rating scales, such as the modified Friedreich's Ataxia Rating Scale, which are usually administered in the clinic by a neurology specialist. OBJECTIVE: This study evaluated the utility of home-based, self-administered digital endpoints in children with Friedreich's ataxia and unaffected controls and their relationship to standard clinical rating scales. METHODS: In a cross-sectional study with 25 participants (13 with Friedreich's ataxia and 12 unaffected controls, aged 6-15 years), home-based digital endpoints that reflect activities of daily living were recorded over 1 week. Domains analyzed were hand motor function with a digitized drawing, automated analysis of speech with a recorded oral diadochokinesis test, and gait and balance with wearable sensors. RESULTS: Hand-drawing and speech tests were easy to conduct and generated high-quality data. The sensor-based gait and balance tests suffered from technical limitations in this study setup. Several parameters discriminated between groups or correlated strongly with modified Friedreich's Ataxia Rating Scale total score and activities of daily living total score in the Friedreich's ataxia group. Hand-drawing parameters also strongly correlated with standard 9-hole peg test scores. INTERPRETATION: Deploying digital endpoints in home settings is feasible in this population, results in meaningful and robust data collection, and may allow for frequent sampling over longer periods of time to track disease progression. Care must be taken when training participants, and investigators should consider the complexity of the tasks and equipment used.

Clinical Design and Analysis Strategies for the Development of Gene Therapies: Considerations for Quantitative Drug Development in the Age of Genetic Medicine.

Cell and gene therapies have shown enormous promise across a range of diseases in recent years. Numerous adoptive cell therapy modalities as well as systemic and direct-to-target tissue gene transfer administrations are currently in clinical development. The clinical trial design, development, reporting, and analysis of novel cell and gene therapies can differ significantly from established practices for small molecule drugs and biologics. Here, we discuss important quantitative considerations and key competencies for drug developers in preclinical requirements, trial design, and lifecycle planning for gene therapies. We argue that the unique development path of gene therapies requires practicing quantitative drug developers-statisticians, pharmacometricians, pharmacokineticists, epidemiologists, and medical and translational science leads-to exercise active collaboration and cross-functional learning across development stages.

High Glucose and Liver Fatty Acid Binding Protein Gene Ablation Differentially Impact Whole Body and Liver Phenotype in High-Fat Pair-Fed Mice.

Ad libitum-fed diets high in fat and carbohydrate (especially fructose) induce weight gain, obesity, and nonalcoholic fatty liver disease (NAFLD) in humans and animal models. However, interpretation is complicated since ad libitum feeding of such diets induces hyperphagia and upregulates expression of liver fatty acid binding protein (L-FABP)-a protein intimately involved in fatty acid and glucose regulation of lipid metabolism. Wild-type (WT) and L-fabp gene ablated (LKO) mice were pair-fed either high-fat diet (HFD) or high-fat/high-glucose diet (HFGD) wherein total carbohydrate was maintained constant but the proportion of glucose was increased at the expense of fructose. In LKO mice, the pair-fed HFD increased body weight and lean tissue mass (LTM) but had no effect on fat tissue mass (FTM) or hepatic fatty vacuolation as compared to pair-fed WT counterparts. These LKO mice exhibited upregulation of hepatic proteins in fatty acid uptake and cytosolic transport (caveolin and sterol carrier protein-2), but lower hepatic fatty acid oxidation (decreased serum ß-hydroxybutyrate). LKO mice pair-fed HFGD also exhibited increased body weight; however, these mice had increased FTM, not LTM, and increased hepatic fatty vacuolation as compared to pair-fed WT counterparts. These LKO mice also exhibited upregulation of hepatic proteins in fatty acid uptake and cytosolic transport (caveolin and acyl-CoA binding protein, but not sterol carrier protein-2), but there was no change in hepatic fatty acid oxidation (serum ß-hydroxybutyrate) as compared to pair-fed WT counterparts.

Partitioning the risk of tuberculosis transmission in household contact studies.

Household contact studies of tuberculosis (TB) are a common way to study disease transmission dynamics. However these studies lack a mechanism for accounting for community transmission, which is known to be significant, particularly in high burden settings. We illustrate a statistical approach for estimating both the correlates with transmission of TB in a household setting and the probability of community transmission using a modified Bayesian mixed-effects model. This is applied to two household contact studies in Vitória, Brazil from 2008-2013 and Kampala, Uganda from 1995-2004 that enrolled households with an individual that was recently diagnosed with pulmonary TB. We estimate the probability of community transmission to be higher in Uganda (ranging from 0.21 to 0.69, depending on HHC age and HIV status of the index case) than in Brazil (ranging from 0.13 for young children to 0.50 in adults). These estimates are consistent with a higher overall burden of disease in Uganda compared to Brazil. Our method also estimates an increasing risk of community-acquired TB with age of the household contact, consistent with existing literature. This approach is a useful way to integrate the role of the community in understanding TB disease transmission dynamics in household contact studies.

Sterol Carrier Protein-2/Sterol Carrier Protein-x/Fatty Acid Binding Protein-1 Ablation Impacts Response of Brain Endocannabinoid to High-Fat Diet.

Brain endocannabinoids (EC) such as arachidonoylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG) primarily originate from serum arachidonic acid (ARA), whose level is regulated in part by a cytosolic ARA-binding protein, that is, liver fatty acid binding protein-1 (FABP1), not expressed in the brain. Ablation of the Fabp1 gene (LKO) increases brain AEA and 2-AG by decreasing hepatic uptake of ARA to increase serum ARA, thereby increasing ARA availability for uptake by the brain. The brain also expresses sterol carrier protein-2 (SCP-2), which is also a cytosolic ARA-binding protein. To further resolve the role of SCP-2 independent of FABP1, mice ablated in the Scp-2/Scp-x gene (DKO) were crossed with mice ablated in the Fabp1 gene (LKO) mice to generate triple knock out (TKO) mice. TKO impaired the ability of LKO to increase brain AEA and 2-AG. While a high-fat diet (HFD) alone increased brain AEA, TKO impaired this effect. Overall, these TKO-induced blocks were not attributable to altered expression of brain proteins in ARA uptake, AEA/2-AG synthesis, or AEA/2-AG degrading enzymes. Instead, TKO reduced serum levels of free ARA and/or total ARA and thereby decreased ARA availability for uptake to the brain and downstream synthesis of AEA and 2-AG therein. In summary, Scp-2/Scp-x gene ablation in Fabp1 null (LKO) mice antagonized the impact of LKO and HFD on brain ARA and, subsequently, EC levels. Thus, both FABP1 and SCP-2 participate in regulating the EC system in the brain.

Effect of liver fatty acid binding protein (L-FABP) gene ablation on lipid metabolism in high glucose diet (HGD) pair-fed mice.

Liver fatty acid binding protein (L-FABP) is the major fatty acid binding/"chaperone" protein in hepatic cytosol. Although fatty acids can be derived from the breakdown of dietary fat and glucose, relatively little is known regarding the impact of L-FABP on phenotype in the context of high dietary glucose. Potential impact was examined in wild-type (WT) and Lfabp gene ablated (LKO) female mice fed either a control or pair-fed high glucose diet (HGD). WT mice fed HGD alone exhibited decreased whole body weight gain and weight gain/kcal food consumed-both as reduced lean tissue mass (LTM) and fat tissue mass (FTM). Conversely, LKO alone increased weight gain, lean tissue mass, and fat tissue mass while decreasing serum ß-hydroxybutyrate (indicative of hepatic fatty acid oxidation)-regardless of diet. Both LKO alone and HGD alone significantly altered the serum lipoprotein profile and increased triacylglycerol (TG), but in HGD mice the LKO did not further exacerbate serum TG content. HGD had little effect on hepatic lipid composition in WT mice, but prevented the LKO-induced selective increase in hepatic phospholipid, free-cholesterol and cholesteryl-ester. Taken together, these findings suggest that high glucose diet diminished the effects of LKO on the whole body and lipid phenotype of these mice.

Endothelial nitric oxide synthase protein distribution and nitric oxide production in endothelial cells along the coronary vascular tree.

OBJECTIVE: Freshly isolated endothelial cells from both conduit arteries and microvasculature were used to test the hypothesis that eNOS protein content and nitric oxide production in coronary endothelial cells increases with vessel radius. METHODS: Porcine hearts were obtained from a local abattoir. Large and small arteries as well as arterioles were dissected free of myocardium and homogenized as whole vessels. Additionally, endothelial cells were isolated from both conduit arteries and left ventricular myocardium by tissue digestion with collagenase, followed by endothelial cell isolation using biotinylated-anti-CD31 and streptavidin-coated paramagnetic beads. Purity of isolated endothelial cells was confirmed by immunofluorescence and immunoblot. RESULTS: In whole vessel lysate, immunoblot analysis revealed that protein content for eNOS was greater in arterioles compared to small and large arteries. Nitric oxide metabolites (nitrite plus nitrate; NOx) levels measured from whole vessel lysate decreased as vessel size increased, with both arterioles and small arteries displaying significantly greater NOx content than conduit. Consistent with our hypothesis, both eNOS protein level and NOx were significantly greater in endothelial cells isolated from conduit arteries compared with those from coronary microvasculature. Furthermore, confocal microscopy revealed that eNOS protein was present in all conduit and microvascular endothelial cells, although eNOS staining was less intense in microvascular cells than those of conduit artery. CONCLUSIONS: These findings demonstrate increased eNOS protein and NOx content in endothelial cells of conduit arteries compared with the microcirculation and underscore the importance of comparing endothelial-specific molecules in freshly isolated endothelial cells, rather than whole lysate of different sized vessels.

Impact of Fabp1 Gene Ablation on Uptake and Degradation of Endocannabinoids in Mouse Hepatocytes.

Liver fatty-acid-binding protein (FABP1, L-FABP) is the major cytosolic binding/chaperone protein for both precursor arachidonic acid (ARA) and the endocannabinoid (EC) products N-arachidonoylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG). Although FABP1 regulates hepatic uptake and metabolism of ARA, almost nothing is known regarding FABP1's impact on AEA and 2-AG uptake, intracellular distribution, and targeting of AEA and 2-AG to degradative hepatic enzymes. In vitro assays revealed that FABP1 considerably enhanced monoacylglycerol lipase hydrolysis of 2-AG but only modestly enhanced AEA hydrolysis by fatty-acid amide hydrolase. Conversely, liquid chromatography-mass spectrometry of lipids from Fabp1 gene-ablated (LKO) hepatocytes confirmed that loss of FABP1 markedly diminished hydrolysis of 2-AG. Furthermore, the real-time imaging of novel fluorescent NBD-labeled probes (NBD-AEA, NBD-2-AG, and NBD-ARA) resolved FABP1's impact on uptake vs intracellular targeting/hydrolysis. FABP1 bound NBD-ARA with 2:1 stoichiometry analogous to ARA, but bound NBD-2-AG and NBD-AEA with 1:1 stoichiometry-apparently at different sites in FABP1's binding cavity. All three probes were taken up, but NBD-2-AG and NBD-AEA were targeted to lipid droplets. LKO reduced the uptake of NBD-ARA as expected, significantly enhanced that of NBD-AEA, but had little effect on NBD-2-AG. These data indicated that FABP1 impacts hepatocyte EC levels by binding EC and differentially impacts their intracellular hydrolysis (2-AG) and uptake (AEA).

Structural and Functional Interaction of Δ9-Tetrahydrocannabinol with Liver Fatty Acid Binding Protein (FABP1).

Although serum Δ9-tetrahydrocannabinol (Δ9-THC) undergoes rapid hepatic clearance and metabolism, almost nothing is known regarding the mechanism(s) whereby this highly lipophilic phytocannabinoid is transported for metabolism/excretion. A novel NBD-arachidonoylethanolamide (NBD-AEA) fluorescence displacement assay showed that liver fatty acid binding protein (FABP1), the major hepatic endocannabinoid (EC) binding protein, binds the first major metabolite of Δ9-THC (Δ9-THC-OH) as well as Δ9-THC itself. Circular dichroism (CD) confirmed that not only Δ9-THC and Δ9-THC-OH but also downstream metabolites Δ9-THC-COOH and Δ9-THC-CO-glucuronide directly interact with FABP1. Δ9-THC and metabolite interaction differentially altered the FABP1 secondary structure, increasing total α-helix (all), decreasing total ß-sheet (Δ9-THC-COOH, Δ9-THC-CO-glucuronide), increasing turns (Δ9-THC-OH, Δ9-THC-COOH, Δ9-THC-CO-glucuronide), and decreasing unordered structure (Δ9-THC, Δ9-THC-OH). Cultured primary hepatocytes from wild-type (WT) mice took up and converted Δ9-THC to the above metabolites. Fabp1 gene ablation (LKO) dramatically increased hepatocyte accumulation of Δ9-THC and even more so its primary metabolites Δ9-THC-OH and Δ9-THC-COOH. Concomitantly, rtPCR and Western blotting indicated that LKO significantly increased Δ9-THC's ability to regulate downstream nuclear receptor transcription of genes important in both EC ( Napepld > Daglb > Dagla, Naaa, Cnr1) and lipid ( Cpt1A > Fasn, FATP4) metabolism. Taken together, the data indicated that FABP1 may play important roles in Δ9-THC uptake and elimination as well as Δ9-THC induction of genes regulating hepatic EC levels and downstream targets in lipid metabolism.

Using routinely collected laboratory data to identify high rifampicin-resistant tuberculosis burden communities in the Western Cape Province, South Africa: A retrospective spatiotemporal analysis.

BACKGROUND: South Africa has the highest tuberculosis incidence globally (781/100,000), with an estimated 4.3% of cases being rifampicin resistant (RR). Control and elimination strategies will require detailed spatial information to understand where drug-resistant tuberculosis exists and why it persists in those communities. We demonstrate a method to enable drug-resistant tuberculosis monitoring by identifying high-burden communities in the Western Cape Province using routinely collected laboratory data. METHODS AND FINDINGS: We retrospectively identified cases of microbiologically confirmed tuberculosis and RR-tuberculosis from all biological samples submitted for tuberculosis testing (n = 2,219,891) to the Western Cape National Health Laboratory Services (NHLS) between January 1, 2008, and June 30, 2013. Because the NHLS database lacks unique patient identifiers, we performed a series of record-linking processes to match specimen records to individual patients. We counted an individual as having a single disease episode if their positive samples came from within two years of each other. Cases were aggregated by clinic location (n = 302) to estimate the percentage of tuberculosis cases with rifampicin resistance per clinic. We used inverse distance weighting (IDW) to produce heatmaps of the RR-tuberculosis percentage across the province. Regression was used to estimate annual changes in the RR-tuberculosis percentage by clinic, and estimated average size and direction of change was mapped. We identified 799,779 individuals who had specimens submitted from mappable clinics for testing, of whom 222,735 (27.8%) had microbiologically confirmed tuberculosis. The study population was 43% female, the median age was 36 years (IQR 27-44), and 10,255 (4.6%, 95% CI: 4.6-4.7) cases had documented rifampicin resistance. Among individuals with microbiologically confirmed tuberculosis, 8,947 (4.0%) had more than one disease episode during the study period. The percentage of tuberculosis cases with rifampicin resistance documented among these individuals was 11.4% (95% CI: 10.7-12.0). Overall, the percentage of tuberculosis cases that were RR-tuberculosis was spatially heterogeneous, ranging from 0% to 25% across the province. Our maps reveal significant yearly fluctuations in RR-tuberculosis percentages at several locations. Additionally, the directions of change over time in RR-tuberculosis percentage were not uniform. The main limitation of this study is the lack of unique patient identifiers in the NHLS database, rendering findings to be estimates reliant on the accuracy of the person-matching algorithm. CONCLUSIONS: Our maps reveal striking spatial and temporal heterogeneity in RR-tuberculosis percentages across this province. We demonstrate the potential to monitor RR-tuberculosis spatially and temporally with routinely collected laboratory data, enabling improved resource targeting and more rapid locally appropriate interventions.

Scp-2/Scp-x ablation in Fabp1 null mice differentially impacts hepatic endocannabinoid level depending on dietary fat.

Dysregulation of the hepatic endocannabinoid (EC) system and high fat diet (HFD) are associated with non-alcoholic fatty liver disease. Liver cytosol contains high levels of two novel endocannabinoid binding proteins-liver fatty acid binding protein (FABP1) and sterol carrier protein-2 (SCP-2). While Fabp1 gene ablation significantly increases hepatic levels of arachidonic acid (ARA)-containing EC and sex-dependent response to pair-fed high fat diet (HFD), the presence of SCP-2 complicates interpretation. These issues were addressed by ablating Scp-2/Scp-x in Fabp1 null mice (TKO). In control-fed mice, TKO increased hepatic levels of arachidonoylethanolamide (AEA) in both sexes. HFD impacted hepatic EC levels by decreasing AEA in TKO females and decreasing 2-arachidonoyl glycerol (2-AG) in WT of both sexes. Only TKO males on HFD had increased hepatic 2-AG levels. Hepatic ARA levels were decreased in control-fed TKO of both sexes. Changes in hepatic AEA/2-AG levels were not associated with altered amounts of hepatic proteins involved in AEA/2-AG synthesis or degradation. These findings suggested that ablation of the Scp-2/Scp-x gene in Fabp1 null mice exacerbated hepatic EC accumulation and antagonized the impact of HFD on hepatic EC levels-suggesting both proteins play important roles in regulating the hepatic EC system.

Human Liver Fatty Acid Binding Protein-1 T94A Variant, Nonalcohol Fatty Liver Disease, and Hepatic Endocannabinoid System.

Hepatic endocannabinoids (EC) and their major binding/"chaperone" protein (i.e., liver fatty acid binding protein-1 [FABP1]) are associated with development of nonalcoholic fatty liver (NAFLD) in animal models and humans. Since expression of the highly prevalent human FABP1 T94A variant induces serum lipid accumulation, it is important to determine its impact on hepatic lipid accumulation and the EC system. This issue was addressed in livers from human subjects expressing only wild-type (WT) FABP1 T94T (TT genotype) or T94A variant (TC or CC genotype). WT FABP1 males had lower total lipids (both neutral cholesteryl esters, triacylglycerols) and phospholipids than females. WT FABP1 males' lower lipids correlated with lower levels of the N-acylethanolamide DHEA and 2-monoacylglycerols (2-MAG) (2-OG, 2-PG). T94A expression in males increased the hepatic total lipids (triacylglycerol, cholesteryl ester), which is consistent with their higher level of CB1-potentiating 2-OG and lower antagonistic EPEA. In contrast, in females, T94A expression did not alter the total lipids, neutral lipids, or phospholipids, which is attributable to the higher cannabinoid receptor-1 (CB1) agonist arachidonoylethanolamide (AEA) and its CB1-potentiator OEA being largely offset by reduced potentiating 2-OG and increased antagonistic EPEA. Taken together, these findings indicate that T94A-induced alterations in the hepatic EC system contribute at least in part to the hepatic accumulation of lipids associated with NAFLD, especially in males.

Δ9-Tetrahydrocannabinol induces endocannabinoid accumulation in mouse hepatocytes: antagonism by Fabp1 gene ablation.

Phytocannabinoids, such as Δ9-tetrahydrocannabinol (THC), bind and activate cannabinoid (CB) receptors, thereby "piggy-backing" on the same pathway's endogenous endocannabinoids (ECs). The recent discovery that liver fatty acid binding protein-1 (FABP1) is the major cytosolic "chaperone" protein with high affinity for both Δ9-THC and ECs suggests that Δ9-THC may alter hepatic EC levels. Therefore, the impact of Δ9-THC or EC treatment on the levels of endogenous ECs, such as N-arachidonoylethanolamide (AEA) and 2-arachidonoylglycerol (2-AG), was examined in cultured primary mouse hepatocytes from WT and Fabp1 gene-ablated (LKO) mice. Δ9-THC alone or 2-AG alone significantly increased AEA and especially 2-AG levels in WT hepatocytes. LKO alone markedly increased AEA and 2-AG levels. However, LKO blocked/diminished the ability of Δ9-THC to further increase both AEA and 2-AG. In contrast, LKO potentiated the ability of exogenous 2-AG to increase the hepatocyte level of AEA and 2-AG. These and other data suggest that Δ9-THC increases hepatocyte EC levels, at least in part, by upregulating endogenous AEA and 2-AG levels. This may arise from Δ9-THC competing with AEA and 2-AG binding to FABP1, thereby decreasing targeting of bound AEA and 2-AG to the degradative enzymes, fatty acid amide hydrolase and monoacylglyceride lipase, to decrease hydrolysis within hepatocytes.

Ablating both Fabp1 and Scp2/Scpx (TKO) induces hepatic phospholipid and cholesterol accumulation in high fat-fed mice.

Although singly ablating Fabp1 or Scp2/Scpx genes may exacerbate the impact of high fat diet (HFD) on whole body phenotype and non-alcoholic fatty liver disease (NAFLD), concomitant upregulation of the non-ablated gene, preference for ad libitum fed HFD, and sex differences complicate interpretation. Therefore, these issues were addressed in male and female mice ablated in both genes (Fabp1/Scp2/Scpx null or TKO) and pair-fed HFD. Wild-type (WT) males gained more body weight as fat tissue mass (FTM) and exhibited higher hepatic lipid accumulation than WT females. The greater hepatic lipid accumulation in WT males was associated with higher hepatic expression of enzymes in glyceride synthesis, higher hepatic bile acids, and upregulation of transporters involved in hepatic reuptake of serum bile acids. While TKO had little effect on whole body phenotype and hepatic bile acid accumulation in either sex, TKO increased hepatic accumulation of lipids in both, specifically phospholipid and cholesteryl esters in males and females and free cholesterol in females. TKO-induced increases in glycerides were attributed not only to complete loss of FABP1, SCP2 and SCPx, but also in part to sex-dependent upregulation of hepatic lipogenic enzymes. These data with WT and TKO mice pair-fed HFD indicate that: i) Sex significantly impacted the ability of HFD to increase body weight, induce hepatic lipid accumulation and increase hepatic bile acids; and ii) TKO exacerbated the HFD ability to induce hepatic lipid accumulation, regardless of sex, but did not significantly alter whole body phenotype in either sex.

Sex-dependent impact of Scp-2/Scp-x gene ablation on hepatic phytol metabolism.

While prior studies focusing on male mice suggest a role for sterol carrier protein-2/sterol carrier protein-x (SCP-2/SCP-x; DKO) on hepatic phytol metabolism, its role in females is unresolved. This issue was addressed using female and male wild-type (WT) and DKO mice fed a phytoestrogen-free diet without or with 0.5% phytol. GC/MS showed that hepatic: i) phytol was absent and its branched-chain fatty acid (BCFA) metabolites were barely detectable in WT control-fed mice; ii) accumulation of phytol as well as its peroxisomal metabolite BCFAs (phytanic acid ¼ pristanic and 2,3-pristenic acids) was increased by dietary phytol in WT females, but only slightly in WT males; iii) accumulation of phytol and BCFA was further increased by DKO in phytol-fed females, but much more markedly in males. Livers of phytol-fed WT female mice as well as phytol-fed DKO female and male mice also accumulated increased proportion of saturated straight-chain fatty acids (LCFA) at the expense of unsaturated LCFA. Liver phytol accumulation was not due to increased SCP-2 binding/transport of phytol since SCP-2 bound phytanic acid, but not its precursor phytol. Thus, the loss of Scp-2/Scp-x contributed to a sex-dependent hepatic accumulation of dietary phytol and BCFA.

Loss of fatty acid binding protein-1 alters the hepatic endocannabinoid system response to a high-fat diet.

Upregulation of the hepatic endocannabinoid (EC) receptor [cannabinoid receptor-1 (CB1)] and arachidonoylethanolamide (AEA) is associated with nonalcoholic fatty liver disease (NAFLD). Male mice fed high-fat diet (HFD) ad libitum also exhibit NAFLD, increased hepatic AEA, and obesity. But, preference for HFD complicates interpretation and almost nothing is known about these effects in females. These issues were addressed by pair-feeding HFD. Similarly to ad libitum-fed HFD, pair-fed HFD also increased WT male and female mouse fat tissue mass (FTM), but preferentially at the expense of lean tissue mass. In contrast, pair-fed HFD did not elicit NAFLD in WT mice regardless of sex. Concomitantly, pair-fed HFD oppositely impacted hepatic AEA, 2-arachidonoyl glycerol, and/or CB1 in WT males versus females. In pair-fed HFD mice, liver FA binding protein-1 (Fabp1) gene ablation (LKO): i) exacerbated FTM in both sexes; ii) did not elicit liver neutral lipid accumulation in males and only slightly in females; iii) increased liver AEA in males, but decreased it in females; and iv) decreased CB1 only in males. Thus, pair-fed HFD selectively impacted hepatic ECs more in females, but did not elicit NAFLD in either sex. These effects were modified by LKO consistent with FABP1's ability to impact EC and FA metabolism.