PNPLA3 is a triglyceride lipase that mobilizes polyunsaturated fatty acids to facilitate hepatic secretion of large-sized very low-density lipoprotein.

The I148M variant of PNPLA3 is closely associated with hepatic steatosis. Recent evidence indicates that the I148M mutant functions as an inhibitor of PNPLA2/ATGL-mediated lipolysis, leaving the role of wild-type PNPLA3 undefined. Despite showing a triglyceride hydrolase activity in vitro, PNPLA3 has yet to be established as a lipase in vivo. Here, we show that PNPLA3 preferentially hydrolyzes polyunsaturated triglycerides, mobilizing polyunsaturated fatty acids for phospholipid desaturation and enhancing hepatic secretion of triglyceride-rich lipoproteins. Under lipogenic conditions, mice with liver-specific knockout or acute knockdown of PNPLA3 exhibit aggravated liver steatosis and reduced plasma VLDL-triglyceride levels. Similarly, I148M-knockin mice show decreased hepatic triglyceride secretion during lipogenic stimulation. Our results highlight a specific context whereby the wild-type PNPLA3 facilitates the balance between hepatic triglyceride storage and secretion, and suggest the potential contribution of a loss-of-function by the I148M variant to the development of fatty liver disease in humans.

Mitochondrial amidoxime-reducing component 1 p.Ala165Thr increases protein degradation mediated by the proteasome.

OBJECTIVE: Metabolic dysfunction-associated steatotic liver disease (MASLD) is a global health concern with no effective and specific drug treatment available. The rs2642438 minor allele in mitochondrial amidoxime-reducing component 1 (MARC1) results in an aminoacidic substitution (p.Ala165Thr) and associates with protection against MASLD. However, the mechanisms behind this protective effect are unknown. In this study, we examined the consequences of this aminoacidic substitution on protein stability and subcellular localization. METHODS: We overexpressed the human MARC1 A165 (wild-type) or 165T (mutant) in vivo in mice and in vitro in human hepatoma cells (HepG2 and HuH-7), generated several mutants at position 165 by in situ mutagenesis and then examined protein levels. We also generated HepG2 cells stably overexpressing MARC1 A165 or 165T to test the effect of this substitution on MARC1 subcellular localization. RESULTS: MARC1 165T overexpression resulted in lower protein levels than A165 both in vivo and in vitro. Similarly, any mutant at position 165 showed lower protein levels compared to the wild-type protein. We showed that the 165T mutant protein is polyubiquitinated and its degradation is accelerated through lysine-48 ubiquitin-mediated proteasomal degradation. We also showed that the 165T substitution does not affect the MARC1 subcellular localization. CONCLUSIONS: This study shows that alanine at position 165 in MARC1 is crucial for protein stability, and the threonine substitution at this position leads to a hypomorphic protein variant due to lower protein levels. Our result supports the notion that lowering hepatic MARC1 protein level may be a successful therapeutic strategy for treating MASLD.

Substrate-Specific Function of PNPLA3 Facilitates Hepatic VLDL-Triglyceride Secretion During Stimulated Lipogenesis.

The I148M variant of PNPLA3 is strongly linked to hepatic steatosis. Evidence suggests a gain-of-function role for the I148M mutant as an ATGL inhibitor, leaving the physiological relevance of wild-type PNPLA3 undefined. Here we show that PNPLA3 selectively degrades triglycerides (TGs) enriched in polyunsaturated fatty acids (PUFAs) independently of ATGL in cultured cells and mice. Lipidomics and metabolite tracing analyses demonstrated that PNPLA3 mobilizes PUFAs from intracellular TGs for phospholipid desaturation, supporting hepatic secretion of TG-rich lipoproteins. Consequently, mice with liver-specific knockout or acute knockdown of PNPLA3 both exhibited aggravated liver steatosis and concomitant decreases in plasma VLDL-TG, phenotypes that manifest only under lipogenic conditions. I148M-knockin mice similarly displayed impaired hepatic TG secretion during lipogenic stimulation. Our results highlight a specific context whereby PNPLA3 facilitates the balance between hepatic TG storage and secretion and suggest the potential contributions of I148M variant loss-of-function to the development of hepatic steatosis in humans. Summary Statement: We define the physiological role of wild type PNPLA3 in maintaining hepatic VLDL-TG secretion.

Practical advice on variable selection and reporting using Akaike information criterion.

The various debates around model selection paradigms are important, but in lieu of a consensus, there is a demonstrable need for a deeper appreciation of existing approaches, at least among the end-users of statistics and model selection tools. In the ecological literature, the Akaike information criterion (AIC) dominates model selection practices, and while it is a relatively straightforward concept, there exists what we perceive to be some common misunderstandings around its application. Two specific questions arise with surprising regularity among colleagues and students when interpreting and reporting AIC model tables. The first is related to the issue of 'pretending' variables, and specifically a muddled understanding of what this means. The second is related to p-values and what constitutes statistical support when using AIC. There exists a wealth of technical literature describing AIC and the relationship between p-values and AIC differences. Here, we complement this technical treatment and use simulation to develop some intuition around these important concepts. In doing so we aim to promote better statistical practices when it comes to using, interpreting and reporting models selected when using AIC.

Staphylectomy in nonbrachycephalic dogs: A retrospective study of 27 cases.

Objective: To report the outcomes and complications associated with staphylectomy in nonbrachycephalic dogs. Animal: Twenty-seven nonbrachycephalic dogs with elongated soft palates and undergoing staphylectomy. Procedure: Retrospective study. Results: Increased upper airway noise (70.4%) and dyspnea (44.4%) were the most common presenting clinical signs. Concurrent upper airway abnormalities found in the study population included laryngeal collapse (25.9%) and laryngeal paralysis (14.8%). The most common staphylectomy technique used in this study was sharp excision (66.7%) with sutured oral and nasal mucosal apposition. The dogs in this study had an overall minor postoperative complication rate of 33.3%, with regurgitation/vomiting (11.1%) and coughing (11.1%) occurring most commonly. No dog required supplemental oxygen therapy or temporary tracheostomy. Conclusion: Staphylectomy was well-tolerated in nonbrachycephalic dogs and was associated with a relatively low rate of complications. Concurrent airway abnormalities were common among nonbrachycephalic dogs with elongated soft palates, similar to brachycephalic dogs. Clinical relevance: Clinicians should be aware that elongated soft palate can occur in nonbrachycephalic dogs, and surgical correction can be achieved with rare major or catastrophic complications.

Staphylectomie chez des chiens non-brachycéphales : une étude rétrospective de 27 cas. Objectif: Rapporter les résultats et les complications associés à la staphylectomie chez des chiens non-brachycéphales. Animal: Vingt-sept chiens non-brachycéphales au palais mou allongé et subissant une staphylectomie. Procédure: Étude rétrospective. Résultats: L'augmentation du bruit des voies respiratoires supérieures (70,4 %) et la dyspnée (44,4 %) étaient les signes cliniques les plus fréquents. Les anomalies concomitantes des voies respiratoires supérieures trouvées dans la population étudiée comprenaient un collapsus laryngé (25,9 %) et une paralysie laryngée (14,8 %). La technique de staphylectomie la plus couramment utilisée dans cette étude était l'exérèse fine (66,7 %) avec apposition suturée des muqueuses buccale et nasale. Les chiens de cette étude présentaient un taux global de complications postopératoires mineures de 33,3 %, les régurgitations/vomissements (11,1 %) et la toux (11,1 %) étant les plus fréquents. Aucun chien n'a eu besoin d'une oxygénothérapie supplémentaire ou d'une trachéotomie temporaire. Conclusion: La staphylectomie a été bien tolérée chez les chiens non-brachycéphales et a été associée à un taux relativement faible de complications. Les anomalies concomitantes des voies respiratoires étaient courantes chez les chiens nonbrachycéphales avec des palais mous allongés, semblables aux chiens brachycéphales. Pertinence clinique: Les cliniciens doivent être conscients qu'un palais mou allongé peut survenir chez les chiens non-brachycéphales et qu'une correction chirurgicale peut être obtenue avec de rares complications majeures ou catastrophiques.(Traduit par Dr Serge Messier).

Multiomics analysis of naturally efficacious lipid nanoparticle coronas reveals high-density lipoprotein is necessary for their function.

In terms of lipid nanoparticle (LNP) engineering, the relationship between particle composition, delivery efficacy, and the composition of the biocoronas that form around LNPs, is poorly understood. To explore this we analyze naturally efficacious biocorona compositions using an unbiased screening workflow. First, LNPs are complexed with plasma samples, from individual lean or obese male rats, and then functionally evaluated in vitro. Then, a fast, automated, and miniaturized method retrieves the LNPs with intact biocoronas, and multiomics analysis of the LNP-corona complexes reveals the particle corona content arising from each individual plasma sample. We find that the most efficacious LNP-corona complexes were enriched with high-density lipoprotein (HDL) and, compared to the commonly used corona-biomarker Apolipoprotein E, corona HDL content was a superior predictor of in-vivo activity. Using technically challenging and clinically relevant lipid nanoparticles, these methods reveal a previously unreported role for HDL as a source of ApoE and, form a framework for improving LNP therapeutic efficacy by controlling corona composition.

Therapeutic opportunities for the treatment of NASH with genetically validated targets.

The identification of genetic variants associated with fatty liver disease (FLD) from genome-wide association studies started in 2008 when single nucleotide polymorphisms in PNPLA3, the gene encoding patatin-like phospholipase domain-containing 3, were found to be associated with altered hepatic fat content. Since then, several genetic variants associated with protection from, or an increased risk of, FLD have been identified. The identification of these variants has provided insight into the metabolic pathways that cause FLD and enabled the identification of potential therapeutic targets. In this mini-review, we will examine the therapeutic opportunities derived from genetically validated targets in FLD, including oligonucleotide-based therapies targeting PNPLA3 and HSD17B13 that are currently being evaluated in clinical trials for the treatment of NASH (non-alcoholic steatohepatitis).

Outcomes and clinical features associated with surgically excised canine salivary gland carcinoma: A multi-institutional, retrospective, Veterinary Society of Surgical Oncology study.

OBJECTIVE: The objective of this study was to describe the clinical features, prognostic factors, and outcomes in dogs with surgically treated salivary gland carcinoma. STUDY DESIGN: Multi-institutional retrospective case series. ANIMALS: Seventy-two client-owned dogs from 16 institutions with surgically excised salivary gland carcinoma. METHODS: Medical records of dogs undergoing sialoadenectomy from January 1, 2000 to January 1, 2020 were reviewed for signalment, clinical signs, preoperative staging results, preoperative mass evaluation, complications, histopathologic diagnosis, local recurrence, metastatic disease, and survival times. Survival functions were estimated using the Kaplan-Meier estimator. Factors related to survival were individually tested using the log-rank test. RESULTS: The overall median survival time (MST) associated with salivary carcinoma was 1886 days. Local recurrence occurred in 29/69 (42%) dogs with an overall disease-free interval (DFI) of 191 days. Metastatic disease occurred in 22/69 (31.9%) dogs, with an overall DFI of 299 days. Lymph node metastasis was present at the time of surgery in 11/38 (28.9%) dogs in which lymphadenectomy was performed at the time of surgery; these dogs had a shorter DFI at 98 days (P = .03) and MST at 248 days (P < .001). CONCLUSION: The prognosis for dogs with salivary gland carcinoma treated surgically was more favorable than previously reported. Nodal metastasis was a negative prognostic factor for canine salivary gland carcinoma. CLINICAL SIGNIFICANCE: Surgical intervention should be considered for dogs with salivary carcinoma.

Including a spatial predictive process in band recovery models improves inference for Lincoln estimates of animal abundance.

Abundance estimation is a critical component of conservation planning, particularly for exploited species where managers set regulations to restrict harvest based on current population size. An increasingly common approach for abundance estimation is through integrated population modeling (IPM), which uses multiple data sources in a joint likelihood to estimate abundance and additional demographic parameters. Lincoln estimators are one commonly used IPM component for harvested species, which combine information on the rate and total number of individuals harvested within an integrated band-recovery framework to estimate abundance at large scales. A major assumption of the Lincoln estimator is that banding and recoveries are representative of the whole population, which may be violated if major sources of spatial heterogeneity in survival or harvest rates are not incorporated into the model. We developed an approach to account for spatial variation in harvest rates using a spatial predictive process, which we incorporated into a Lincoln estimator IPM. We simulated data under different configurations of sample sizes, harvest rates, and sources of spatial heterogeneity in harvest rate to assess potential model bias in parameter estimates. We then applied the model to data collected from a field study of wild turkeys (Meleagris gallapavo) to estimate local and statewide abundance in Maine, USA. We found that the band recovery model that incorporated a spatial predictive process consistently provided estimates of adult and juvenile abundance with low bias across a variety of spatial configurations of harvest rate and sampling intensities. When applied to data collected on wild turkeys, a model that did not incorporate spatial heterogeneity underestimated the harvest rate in some subregions. Consistent with simulation results, this led to overestimation of both local and statewide abundance. Our work demonstrates that a spatial predictive process is a viable mechanism to account for spatial variation in harvest rates and limit bias in abundance estimates. This approach could be extended to large-scale band recovery data sets and has applicability for the estimation of population parameters in other ecological models as well.

Hepatic patatin-like phospholipase domain-containing 3 levels are increased in I148M risk allele carriers and correlate with NAFLD in humans.

In nonalcoholic fatty liver disease (NAFLD) the patatin-like phospholipase domain-containing 3 (PNPLA3) rs738409 variant is a contributor. In mice, the Pnpla3 148M variant accumulates on lipid droplets and probably leads to sequestration of a lipase cofactor leading to impaired mobilization of triglycerides. To advance our understanding of the localization and abundance of PNPLA3 protein in humans, we used liver biopsies from patients with NAFLD to investigate the link to NAFLD and the PNPLA3 148M genotype. We experimentally qualified an antibody against human PNPLA3. Hepatic PNPLA3 protein fractional area and localization were determined by immunohistochemistry in biopsies from a well-characterized NAFLD cohort of 67 patients. Potential differences in hepatic PNPLA3 protein levels among patients related to degree of steatosis, lobular inflammation, ballooning, and fibrosis, and PNPLA3 I148M gene variants were assessed. Immunohistochemistry staining in biopsies from patients with NAFLD showed that hepatic PNPLA3 protein was predominantly localized to the membranes of small and large lipid droplets in hepatocytes. PNPLA3 protein levels correlated strongly with steatosis grade (p = 0.000027) and were also significantly higher in patients with lobular inflammation (p = 0.009), ballooning (p = 0.022), and significant fibrosis (stage 2-4, p = 0.014). In addition, PNPLA3 levels were higher in PNPLA3 rs738409 148M (CG, GG) risk allele carriers compared to 148I (CC) nonrisk allele carriers (p = 0.0029). Conclusion: PNPLA3 protein levels were associated with increased hepatic lipid content and disease severity in patients with NAFLD and were higher in PNPLA3 rs738409 (148M) risk allele carriers. Our hypothesis that increased hepatic levels of PNPLA3 may be part of the pathophysiological mechanism of NAFLD is supported.

Influence of stapler type on in vitro leakage pressures and location of functional end-to-end stapled anastomoses.

The objective of this study was to compare maximal leakage pressures and locations of 2 functional end-to-end stapled anastomosis (FEESA) constructs. Grossly normal jejunum was harvested from 4 large breed dogs. Thirty-two 8-cm segments of bowel were used to construct 16 FEESA. Construct type was divided into 2 groups: traditional FEESA (tFEESA) and modified FEESA (mFEESA). Leakage pressures and locations were recorded and compared for the 2 groups. There was no difference in the leakage pressures of the tFEESA and the mFEESA. However, 1 tFEESA did leak at subphysiologic intestinal peristaltic pressures. Although no difference in maximal leakage pressure was detected between the 2 constructs, mFEESA is an attractive alternative to tFEESA, as it requires less equipment and none of the mFEESA constructs leaked at subphysiologic pressures.

L'objectif de cette étude était de comparer les pressions de fuite maximales et les emplacements de deux assemblages fonctionnels d'anastomose agrafée bout à bout (FEESA). Du jéjunum macroscopiquement normal a été prélevé sur quatre chiens de grande race. Trente-deux segments de 8 cm d'intestin ont été utilisés pour produire 16 FEESA. Le type d'assemblage a été divisé en deux groupes : FEESA traditionnel (tFEESA) et FEESA modifié (mFEESA). Les pressions et emplacements des fuites ont été enregistrés et comparés pour les deux groupes. Il n'y avait aucune différence dans les pressions de fuite du tFEESA et du mFEESA. Cependant, un tFEESA a fui à des pressions péristaltiques intestinales sous-physiologiques. Bien qu'aucune différence de pression de fuite maximale n'ait été détectée entre les deux types d'assemblage, mFEESA est une alternative attrayante à tFEESA, car elle nécessite moins d'équipement et aucun des assemblages mFEESA n'a fui à des pressions sous-physiologiques.(Traduit par Docteur Serge Messier).

Inhibition of SGLT2 Preserves Function and Promotes Proliferation of Human Islets Cells In Vivo in Diabetic Mice.

Dapagliflozin is a sodium-glucose co-transporter 2 (SGLT2) inhibitor used for the treatment of diabetes. This study examines the effects of dapagliflozin on human islets, focusing on alpha and beta cell composition in relation to function in vivo, following treatment of xeno-transplanted diabetic mice. Mouse beta cells were ablated by alloxan, and dapagliflozin was provided in the drinking water while controls received tap water. Body weight, food and water intake, plasma glucose, and human C-peptide levels were monitored, and intravenous arginine/glucose tolerance tests (IVarg GTT) were performed to evaluate islet function. The grafted human islets were isolated at termination and stained for insulin, glucagon, Ki67, caspase 3, and PDX-1 immunoreactivity in dual and triple combinations. In addition, human islets were treated in vitro with dapagliflozin at different glucose concentrations, followed by insulin and glucagon secretion measurements. SGLT2 inhibition increased the animal survival rate and reduced plasma glucose, accompanied by sustained human C-peptide levels and improved islet response to glucose/arginine. SGLT2 inhibition increased both alpha and beta cell proliferation (Ki67+glucagon+ and Ki67+insulin+) while apoptosis was reduced (caspase3+glucagon+ and caspase3+insulin+). Alpha cells were fewer following inhibition of SGLT2 with increased glucagon/PDX-1 double-positive cells, a marker of alpha to beta cell transdifferentiation. In vitro treatment of human islets with dapagliflozin had no apparent impact on islet function. In summary, SGLT2 inhibition supported human islet function in vivo in the hyperglycemic milieu and potentially promoted alpha to beta cell transdifferentiation, most likely through an indirect mechanism.

The SGLT2 inhibitor dapagliflozin promotes systemic FFA mobilization, enhances hepatic ß-oxidation, and induces ketosis.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors have been shown to increase ketone bodies in patients with type 2 diabetes; however, the underlying mechanisms have not been fully elucidated. Here we examined the effect of the SGLT2 inhibitor dapagliflozin (1 mg/kg/day, formulated in a water, PEG400, ethanol, propylene glycol solution, 4 weeks) on lipid metabolism in obese Zucker rats. Fasting FFA metabolism was assessed in the anesthetized state using a [9,10-3H(N)]-palmitic acid tracer by estimating rates of plasma FFA appearance (Ra), whole-body FFA oxidation (Rox), and nonoxidative disposal (Rst). In the liver, clearance (Kß-ox) and flux (Rß-ox) of FFA into ß-oxidation were estimated using [9,10-3H]-(R)-bromopalmitate/[U-14C]palmitate tracers. As expected, dapagliflozin induced glycosuria and a robust antidiabetic effect; treatment reduced fasting plasma glucose and insulin, lowered glycated hemoglobin, and increased pancreatic insulin content compared with vehicle controls. Dapagliflozin also increased plasma FFA, Ra, Rox, and Rst with enhanced channeling toward oxidation versus storage. In the liver, there was also enhanced channeling of FFA to ß-oxidation, with increased Kß-ox, Rß-ox and tissue acetyl-CoA, compared with controls. Finally, dapagliflozin increased hepatic HMG-CoA and plasma ß-hydroxybutyrate, consistent with a specific enhancement of ketogenesis. Since ketogenesis has not been directly measured, we cannot exclude an additional contribution of impaired ketone body clearance to the ketosis. In conclusion, this study provides evidence that the dapagliflozin-induced increase in plasma ketone bodies is driven by the combined action of FFA mobilization from adipose tissue and diversion of hepatic FFA toward ß-oxidation.

PSD3 downregulation confers protection against fatty liver disease.

Fatty liver disease (FLD) is a growing health issue with burdening unmet clinical needs. FLD has a genetic component but, despite the common variants already identified, there is still a missing heritability component. Using a candidate gene approach, we identify a locus (rs71519934) at the Pleckstrin and Sec7 domain-containing 3 (PSD3) gene resulting in a leucine to threonine substitution at position 186 of the protein (L186T) that reduces susceptibility to the entire spectrum of FLD in individuals at risk. PSD3 downregulation by short interfering RNA reduces intracellular lipid content in primary human hepatocytes cultured in two and three dimensions, and in human and rodent hepatoma cells. Consistent with this, Psd3 downregulation by antisense oligonucleotides in vivo protects against FLD in mice fed a non-alcoholic steatohepatitis-inducing diet. Thus, translating these results to humans, PSD3 downregulation might be a future therapeutic option for treating FLD.

Ex vivo comparison of different thoracoabdominal stapler sizes for typhlectomy in canine cadavers.

OBJECTIVE: To determine the influence of staple size on leakage pressure of typhlectomy sites in canine cadavers. STUDY DESIGN: Randomized, experimental cadaveric study. ANIMALS: Twenty-four fresh canine cadavers. METHODS: Ileocecocolic segments were exteriorized following right paracostal laparotomy after euthanasia. Cecal base length and wall thickness were measured. Each cecum was randomly assigned to 1 of 3 groups (TA 30 V3 2.5 mm, TA 60 3.5 mm, and TA 60 4.8 mm). The cecal base was stapled and the cecum was removed. A 10 cm segment including the stapled cecal excision site was tested for initial leak pressure. RESULTS: The mean ± standard deviation body weights across the groups were 18.7 ± 6.1 kg, 16.2 ± 7.5 kg, and 14.2 ± 5.5 kg for the TA 30 V3 2.5 mm, TA 60 3.5 mm, and TA 60 4.8 mm groups, respectively (P = .48). There were no differences for mean cecal base length or wall thickness. Mean initial leak pressure (ILP) across groups was 182 ± 111 mmHg (TA 30 V3 2.5 mm), 112 ± 57 mmHg (TA 60 3.5 mm), and 77 ± 60 mmHg (TA 60 4.8 mm) (P = .78). CONCLUSION: Each stapler size that was evaluated resulted in a mean ILP in excess of typical intraluminal pressures under normal circumstances. There were no differences among groups. CLINICAL SIGNIFICANCE: The results of this cadaveric study support the use of any of the stapler sizes evaluated in similarly sized dogs. A prospective study is needed to be able to correlate stapler size and clinical outcome.

Improved inferences about landscape connectivity from spatial capture-recapture by integration of a movement model.

Understanding how broad-scale patterns in animal populations emerge from individual-level processes is an enduring challenge in ecology that requires investigation at multiple scales and perspectives. Complementary to this need for diverse approaches is the recent focus on integrated modeling in statistical ecology. Population-level processes represent the core of spatial capture-recapture (SCR), with many methodological extensions that have been motivated by standing ecological theory and data-integration opportunities. The extent to which these recent advances offer inferential improvements can be limited by the data requirements for quantifying individual-level processes. This is especially true for SCR models that use non-Euclidean distance to relax the restrictive assumption that individual space use is stationary and symmetrical to make inferences about landscape connectivity. To meet the challenges of scale and data quality, we propose integrating an explicit movement model with non-Euclidean SCR for joint estimation of a shared cost parameter between individual and population processes. Here, we define a movement kernel for step selection that uses "ecological distance" instead of Euclidean distance to quantify availability for each movement step in terms of landscape cost. We compare performance of our integrated model to that of existing SCR models using realistic animal movement simulations and data collected on black bears. We demonstrate that an integrated approach offers improvements both in terms of bias and precision in estimating the shared cost parameter over models fit to spatial encounters alone. Simulations suggest these gains were only realized when step lengths were small relative to home range size, and estimates of density were insensitive to whether or not an integrated approach was used. By combining the fine spatiotemporal scale of individual movement processes with the estimation of population density in SCR, integrated approaches such as the one we develop here have the potential to unify the fields of movement, population, and landscape ecology and improve our understanding of landscape connectivity.

Modelling human liver fibrosis in the context of non-alcoholic steatohepatitis using a microphysiological system.

Non-alcoholic steatohepatitis (NASH) is a common form of chronic liver disease characterised by lipid accumulation, infiltration of immune cells, hepatocellular ballooning, collagen deposition and liver fibrosis. There is a high unmet need to develop treatments for NASH. We have investigated how liver fibrosis and features of advanced clinical disease can be modelled using an in vitro microphysiological system (MPS). The NASH MPS model comprises a co-culture of primary human liver cells, which were cultured in a variety of conditions including+/- excess sugar, fat, exogenous TGFß or LPS. The transcriptomic, inflammatory and fibrotic phenotype of the model was characterised and compared using a system biology approach to identify conditions that mimic more advanced clinical disease. The transcriptomic profile of the model was shown to closely correlate with the profile of patient samples and the model displayed a quantifiable fibrotic phenotype. The effects of Obeticholic acid and Elafibranor, were evaluated in the model, as wells as the effects of dietary intervention, with all able to significantly reduce inflammatory and fibrosis markers. Overall, we demonstrate how the MPS NASH model can be used to model different aspects of clinical NASH but importantly demonstrate its ability to model advanced disease with a quantifiable fibrosis phenotype.

Allostatic hypermetabolic response in PGC1α/ß heterozygote mouse despite mitochondrial defects.

Aging, obesity, and insulin resistance are associated with low levels of PGC1α and PGC1ß coactivators and defective mitochondrial function. We studied mice deficient for PGC1α and PGC1ß [double heterozygous (DH)] to investigate their combined pathogenic contribution. Contrary to our hypothesis, DH mice were leaner, had increased energy dissipation, a pro-thermogenic profile in BAT and WAT, and improved carbohydrate metabolism compared to wild types. WAT showed upregulation of mitochondriogenesis/oxphos machinery upon allelic compensation of PGC1α4 from the remaining allele. However, DH mice had decreased mitochondrial OXPHOS and biogenesis transcriptomes in mitochondria-rich organs. Despite being metabolically healthy, mitochondrial defects in DH mice impaired muscle fiber remodeling and caused qualitative changes in the hepatic lipidome. Our data evidence first the existence of organ-specific compensatory allostatic mechanisms are robust enough to drive an unexpected phenotype. Second, optimization of adipose tissue bioenergetics is sufficient to maintain a healthy metabolic phenotype despite a broad severe mitochondrial dysfunction in other relevant metabolic organs. Third, the decrease in PGC1s in adipose tissue of obese and diabetic patients is in contrast with the robustness of the compensatory upregulation in the adipose of the DH mice.

An oral antisense oligonucleotide for PCSK9 inhibition.

Inhibitors of proprotein convertase subtilisin/kexin type 9 (PCSK9) reduce low-density lipoprotein (LDL) cholesterol and are used for treatment of dyslipidemia. Current PCSK9 inhibitors are administered via subcutaneous injection. We present a highly potent, chemically modified PCSK9 antisense oligonucleotide (ASO) with potential for oral delivery. Past attempts at oral delivery using earlier-generation ASO chemistries and transient permeation enhancers provided encouraging data, suggesting that improving potency of the ASO could make oral delivery a reality. The constrained ethyl chemistry and liver targeting enabled by N-acetylgalactosamine conjugation make this ASO highly potent. A single subcutaneous dose of 90 mg reduced PCSK9 by >90% in humans with elevated LDL cholesterol and a monthly subcutaneous dose of around 25 mg is predicted to reduce PCSK9 by 80% at steady state. To investigate the feasibility of oral administration, the ASO was coformulated in a tablet with sodium caprate as permeation enhancer. Repeated oral daily dosing in dogs resulted in a bioavailability of 7% in the liver (target organ), about fivefold greater than the plasma bioavailability. Target engagement after oral administration was confirmed by intrajejunal administration of a rat-specific surrogate ASO in solution with the enhancer to rats and by plasma PCSK9 and LDL cholesterol lowering in cynomolgus monkey after tablet administration. On the basis of an assumption of 5% liver bioavailability after oral administration in humans, a daily dose of 15 mg is predicted to reduce circulating PCSK9 by 80% at steady state, supporting the development of the compound for oral administration to treat dyslipidemia.