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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. Funding Agencies|ALF Grants, Region Ostergotland; Astra Zeneca; Forskningsradet i Sydostra Sverige

Increased saturated fatty acid levels in membrane phospholipids have been implicated in the development of metabolic disease. Here, we tested the hypothesis that increased saturated fatty acid (SFA) content in cell membranes negatively impacts adipocyte insulin signaling. Pre-adipocyte cell models with elevated SFA levels in phospholipids were generated by disrupting the ADIPOR2 locus, which resulted in a striking two-fold increase in SFA-containing phosphatidylcholines and phosphatidylethanolamines, which persisted in differentiated adipocytes. Similar changes in phospholipid composition were observed in white adipose tissues isolated from the ADIPOR2 knockout mice. The SFA levels in phospholipids could be further increased by treating ADIPOR2-deficient cells with palmitic acid and resulted in reduced membrane fluidity and endoplasmic reticulum stress in mouse and human pre-adipocytes. Strikingly, increased SFA levels in differentiated adipocyte phospholipids had no effect on adipocyte gene expression or insulin signaling in vitro. Similarly, increased adipocyte phospholipid saturation did not impair white adipose tissue function in vivo, even in mice fed a high saturated fat diet at thermoneutrality. We conclude that increasing SFA levels in adipocyte phospholipids is well tolerated and does not affect adipocyte insulin signaling in vitro and in vivo.

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., Employing a candidate gene approach, Mancina et al. identify a genetic variant of the Pleckstrin and Sec7 domain-containing 3 (PSD3) gene that reduces susceptibility to fatty liver disease. Functional studies in vitro and in vivo demonstrate that targeting PSD3 protects against fatty liver disease.

Background. There is a lack of predictive preclinical animal models combining atherosclerosis and type 2 diabetes. APOE∗3-Leiden (E3L) mice are a well-established model for diet-induced hyperlipidemia and atherosclerosis, and glucokinase+/− (GK+/−) mice are a translatable disease model for glucose control in type 2 diabetes. The respective mice respond similarly to lipid-lowering and antidiabetic drugs as humans. The objective of this study was to evaluate/characterize the APOE∗3-Leiden.glucokinase+/− (E3L.GK+/−) mouse as a novel disease model to study the metabolic syndrome and diabetic complications. Methods. Female E3L.GK+/−, E3L, and GK+/− mice were fed fat- and cholesterol-containing diets for 37 weeks, and plasma parameters were measured throughout. Development of diabetic macro- and microvascular complications was evaluated. Results. Cholesterol and triglyceride levels were significantly elevated in E3L and E3L.GK+/− mice compared to GK+/− mice, whereas fasting glucose was significantly increased in E3L.GK+/− and GK+/− mice compared to E3L. Atherosclerotic lesion size was increased 2.2-fold in E3L.GK+/− mice as compared to E3L (p=0.037), which was predicted by glucose exposure (R2=0.636, p=0.001). E3L and E3L.GK+/− mice developed NASH with severe inflammation and fibrosis which, however, was not altered by introduction of the defective GK phenotype, whereas mild kidney pathology with tubular vacuolization was present in all three phenotypes. Conclusions. We conclude that the E3L.GK+/− mouse is a promising novel diet-inducible disease model for investigation of the etiology and evaluation of drug treatment on diabetic atherosclerosis.

Background and Aims: The proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a key role in cholesterol homeostasis, and its inhibition represents an effective therapy to lower low-density lipoprotein cholesterol (LDL-C) levels. In this study, we examined the impact of the PCSK9 rs11591147 loss-of-function (LOF) variant on liver damage in a multicenter collection of patients at risk of nonalcoholic steatohepatitis (NASH), in clinical samples and experimental models. Methods: We considered 1874 consecutive individuals at risk of NASH as determined by histology. The SNP rs11591147, encoding for the p.R46L variant of PCSK9, was genotyped by TaqMan assays. We also evaluated 1) PCSK9 mRNA hepatic expression in human liver, and 2) the impact of a NASH-inducing diet in mice with hepatic overexpression of human PCSK9. Results: Carriers of PCSK9 rs11591147 had lower circulating LDL-C levels and were protected against nonalcoholic fatty liver disease (NAFLD) (OR: 0.42; 95% CI: 0.22-0.81; P=.01), NASH (OR: 0.48; 95% CI: 0.26-0.87; P=.01) and more severe fibrosis (OR: 0.55; 95% CI: 0.32-0.94; P=.03) independently of clinical, metabolic and genetic confounding factors. PCSK9 hepatic expression was directly correlated with liver steatosis (P=.03). Finally, liver-specific overexpression of human PCSK9 in male mice drives NAFLD and fibrosis upon a dietary challenge. Conclusions: In individuals at risk of NASH, PCSK9 was induced with hepatic fat accumulation and PCSK9 rs11591147 LOF variant was protective against liver steatosis, NASH and fibrosis, suggesting that PCSK9 inhibition may be a new therapeutic strategy to treat NASH.

Background and Aims: The proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a key role in cholesterol homeostasis, and its inhibition represents an effective therapy to lower LDL-C levels. In this study, we examined the impact of PCSK9 rs11591147 loss-of-function (LOF) variant on liver damage in a multicenter collection of patients at risk of nonalcoholic steatohepatitis (NASH), in clinical samples and experimental models. Methods: We considered 1,874 consecutive individuals at risk of NASH as determined by histology. The SNP rs11591147, encoding for the p.R46L variant of PCSK9, was genotyped by TaqMan assays. We also evaluated 1) PCSK9 mRNA hepatic expression in human liver, and 2) the impact of a NASH-inducing diet in mice with hepatic overexpression of human PCSK9. Results: Carriers of PCSK9 rs11591147 had lower circulating LDL-C levels and were protected against NAFLD (OR 0.42;95%C.I0.22-0.81;P=0.01), NASH (OR 0.39;95%C.I.0.20-0.75;P=0.005) and more severe fibrosis (OR 0.44;95%C.I.0.26-0.74;P=0.002) independently of clinical, metabolic and genetic confounding factors. PCSK9 hepatic expression was directly correlated with steatosis (P=0.03). Finally, liver-specific overexpression of human PCSK9 in mice drives NAFLD and fibrosis upon a dietary challenge. Conclusions: In individuals at risk, PCSK9 was induced with hepatic fat accumulation and PCSK9 rs11591147 LOF variant was protective against liver steatosis, NASH and fibrosis, suggesting PCSK9 inhibition may be a new therapeutic strategy to treat NASH. Funding Statement: This work was supported by project grant by the Swedish Research Council [Vetenskapsradet (VR), 2016-01527], the Swedish state under the Agreement between the Swedish government and the county councils (the ALF-agreement) [SU 2018-04276], the Novonordisk Foundation Grant for Excellence in Endocrinology [Excellence Project, 9321- 430], the Swedish Diabetes Foundation [DIA 2017-205], the Swedish Heart Lung Foundation [20120533], the Wallenberg Academy Fellows from the Knut and Alice Wallenberg Foundation [KAW 2017.0203]. LV was supported by MyFirst Grant AIRC n.16888, Ricerca Finalizzata Ministero della Salute [RF-2016-02364358], Ricerca corrente Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Fondazione Sviluppo Ca Granda [PR-0316], the European Union Programme Horizon 2020 [No. 777377]. Declaration of Interests: SR has been consulting for AMGEN, SANOFI, Astra Zeneca, Pfizer, AKCEA, Celgene, Medacorp, Foresite Labs, CAMP4 and received research grants from Astra Zeneca, AMGEN, SANOFI. SB, DK-P, A-CA, GP, MB-Y and DL are AstraZeneca employees. LV reports having received during the last 5 years speaking fees from MSD, Gilead, AlfaSigma, AbbVie, having served as a consultant of: Gilead, Pfizer, Astra Zeneca, Novo Nordisk, Diatech Pharmacogenetics and Intercept, and having received research grants from: Gilead. Ethics Approval Statement: The study was carried out in accordance with the principles of the Helsinki Declaration, and with local and national laws. Approval was obtained from Internal Review Boards and their Ethics Committees, and written informed consent for the study was obtained from all patients All animal experiments were performed with humane care and were approved by the Gothenburg Ethics Committee for Experimental Animals in Sweden. The holding facility has received full accreditation from the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).

Objective: Nonalcoholic fatty liver disease (NAFLD) is becoming a leading cause of advanced chronic liver disease. The progression of NAFLD, including nonalcoholic steatohepatitis (NASH), has a strong genetic component, and the most robust contributor is the patatin-like phospholipase domain-containing 3 (PNPLA3) rs738409 encoding the 148M protein sequence variant. We hypothesized that suppressing the expression of the PNPLA3 148M mutant protein would exert a beneficial effect on the entire spectrum of NAFLD. Methods: We examined the effects of liver-targeted GalNAc3-conjugated antisense oligonucleotide (ASO)-mediated silencing of Pnpla3 in a knock-in mouse model in which we introduced the human PNPLA3 I148M mutation. Results: ASO-mediated silencing of Pnpla3 reduced liver steatosis (p = 0.038) in homozygous Pnpla3 148M/M knock-in mutant mice but not in wild-type littermates fed a steatogenic high-sucrose diet. In mice fed a NASH-inducing diet, ASO-mediated silencing of Pnpla3 reduced liver steatosis score and NAFLD activity score independent of the Pnpla3 genotype, while reductions in liver inflammation score (p = 0.018) and fibrosis stage (p = 0.031) were observed only in the Pnpla3 knock-in 148M/M mutant mice. These responses were accompanied by reduced liver levels of Mcp1 (p = 0.026) and Timp2 (p = 0.007) specifically in the mutant knock-in mice. This may reduce levels of chemokine attracting inflammatory cells and increase the collagenolytic activity during tissue regeneration. Conclusion: This study provides the first evidence that a Pnpla3 ASO therapy can improve all features of NAFLD, including liver fibrosis, and suppress the expression of a strong innate genetic risk factor, Pnpla3 148M, which may open up a precision medicine approach in NASH. Keywords: PNPLA3, NAFLD, NASH, Fibrosis, Liver

Summary Hormone-secreting cells within pancreatic islets of Langerhans play important roles in metabolic homeostasis and disease. However, their transcriptional characterization is still incomplete. Here, we sequenced the transcriptomes of thousands of human islet cells from healthy and type 2 diabetic donors. We could define specific genetic programs for each individual endocrine and exocrine cell type, even for rare δ, γ, ε, and stellate cells, and revealed subpopulations of α, β, and acinar cells. Intriguingly, δ cells expressed several important receptors, indicating an unrecognized importance of these cells in integrating paracrine and systemic metabolic signals. Genes previously associated with obesity or diabetes were found to correlate with BMI. Finally, comparing healthy and T2D transcriptomes in a cell-type resolved manner uncovered candidates for future functional studies. Altogether, our analyses demonstrate the utility of the generated single-cell gene expression resource., Graphical Abstract Image 1, Highlights • Single-cell RNA-seq enabled molecular profiling of rare human pancreatic cells • Subpopulations were identified within endocrine and exocrine cell types • Genes associated with obesity or diabetes had correlating expression with BMI • Transcriptional alterations found in type 2 diabetic individuals, Segerstolpe et al. use single-cell transcriptomics to generate transcriptional profiles of individual pancreatic endocrine and exocrine cells of healthy and type 2 diabetic donors. They revealed cell-type-specific gene expression and novel subpopulations, as well as gene correlations to BMI and gene expression alterations in diabetes.

Background There is a lack of predictive preclinical animal models combining atherosclerosis and type 2 diabetes. APOE∗3-Leiden (E3L) mice are a well-established model for diet-induced hyperlipidemia and atherosclerosis, and glucokinase+/− (GK+/−) mice are a translatable disease model for glucose control in type 2 diabetes. The respective mice respond similarly to lipid-lowering and antidiabetic drugs as humans. The objective of this study was to evaluate/characterize the APOE∗3-Leiden.glucokinase+/− (E3L.GK+/−) mouse as a novel disease model to study the metabolic syndrome and diabetic complications. Methods Female E3L.GK+/−, E3L, and GK+/− mice were fed fat- and cholesterol-containing diets for 37 weeks, and plasma parameters were measured throughout. Development of diabetic macro- and microvascular complications was evaluated. Results Cholesterol and triglyceride levels were significantly elevated in E3L and E3L.GK+/− mice compared to GK+/− mice, whereas fasting glucose was significantly increased in E3L.GK+/− and GK+/− mice compared to E3L. Atherosclerotic lesion size was increased 2.2-fold in E3L.GK+/− mice as compared to E3L (p = 0.037), which was predicted by glucose exposure (R 2 = 0.636, p = 0.001). E3L and E3L.GK+/− mice developed NASH with severe inflammation and fibrosis which, however, was not altered by introduction of the defective GK phenotype, whereas mild kidney pathology with tubular vacuolization was present in all three phenotypes. Conclusions We conclude that the E3L.GK+/− mouse is a promising novel diet-inducible disease model for investigation of the etiology and evaluation of drug treatment on diabetic atherosclerosis.

Aim. Models combining diabetes and atherosclerosis are important in evaluating the cardiovascular (CV) effects and safety of antidiabetes drugs in the development of treatments targeting CV complications. Our aim was to evaluate if crossing the heterozygous glucokinase knockout mouse (GK+/−) and hyperlipidemic mouse deficient in apolipoprotein E (ApoE−/−) will generate a disease model exhibiting a diabetic and macrovascular phenotype.Methods. The effects of defective glucokinase on the glucose metabolism and on the progression and regression of atherosclerosis on high-fat diets were studied in both genders of GK+/−ApoE−/−and ApoE−/−mice. Coronary vascular function of the female GK+/−ApoE−/−and ApoE−/−mice was also investigated.Results. GK+/−ApoE−/−mice show a stable hyperglycemia which was increased on Western diet. In oral glucose tolerance test, GK+/−ApoE−/−mice showed significant glucose intolerance and impaired glucose-stimulated insulin secretion. Plasma lipids were comparable with ApoE−/−mice; nevertheless the GK+/−ApoE−/−mice showed slightly increased atherosclerosis development.Conclusions. The GK+/−ApoE−/−mice showed a stable and reproducible hyperglycemia, accelerated atherosclerotic lesion progression, and no lesion regression after lipid lowering. This novel model provides a promising tool for drug discovery, enabling the evaluation of compound effects against both diabetic and cardiovascular endpoints simultaneously in one animal model.

Atherosclerotic lesion size in mice is routinely evaluated by morphometrical measurements on serial sections in order to obtain volume measurements. The technique of confocal scanning laser microscopy (CSLM) makes it possible to optically scan and thereby evaluate a tissue sample. We here describe a method for measuring lesion volume in ApoE/LDLr deficient mice at 20 and 30 weeks of age using the non-destructive procedure of CSLM. Whole mount preparations of opened aorta with the lumen side facing the cover slip were analysed under 10x magnification in a CSLM (Leica). The autofluorescence of the elastic fibres of the lamina interna as opposed to the non-fluorescing lesion was used to define the bottom and top of the lesion during scanning. Ten to forty images were collected 2.4 microm apart, depending on the size of the lesion, and the stack of images was then analysed using Imaris (Bitplane). After the CSLM evaluation, the aortas were de-mounted, embedded in paraffin, sectioned, stained in hematoxylin and eosin and the volume re-evaluated with conventional morphometry. Statistical evaluation showed that the results obtained with CSLM and the results of morphometry were positively correlated. Area measurements of the plaques using en face preparations of aorta showed that the plaque area was generally larger at the left side and a significant increase of plaque area along the length of the thoracic aorta. Our results showed that atherosclerotic lesions in mice can be quantitatively evaluated by CSLM.

G protein coupled receptor 55 (GPR55) is expressed throughout the body, and although its exact physiological function is unknown, studies have suggested a role in the cardiovascular system. In particular, GPR55 has been proposed as mediating the haemodynamic effects of a number of atypical cannabinoid ligands; however this data is conflicting. Thus, given the incongruous nature of our understanding of the GPR55 receptor and the relative paucity of literature regarding its role in cardiovascular physiology, this study was carried out to examine the influence of GPR55 on cardiac function. Cardiac function was assessed via pressure volume loop analysis, and cardiac morphology/composition assessed via histological staining, in both wild-type (WT) and GPR55 knockout (GPR55(-/-)) mice. Pressure volume loop analysis revealed that basal cardiac function was similar in young WT and GPR55(-/-) mice. In contrast, mature GPR55(-/-) mice were characterised by both significant ventricular remodelling (reduced left ventricular wall thickness and increased collagen deposition) and systolic dysfunction when compared to age-matched WT mice. In particular, the load-dependent parameter, ejection fraction, and the load-independent indices, end-systolic pressure-volume relationship (ESPVR) and Emax, were all significantly (P