Molecular imaging of liver inflammation using an anti-VCAM-1 nanobody.

To date, a biopsy is mandatory to evaluate parenchymal inflammation in the liver. Here, we evaluated whether molecular imaging of vascular cell adhesion molecule-1 (VCAM-1) could be used as an alternative non-invasive tool to detect liver inflammation in the setting of chronic liver disease. To do so, we radiolabeled anti-VCAM-1 nanobody (99mTc-cAbVCAM1-5) and used single-photon emission computed tomography (SPECT) to quantify liver uptake in preclinical models of non-alcoholic fatty liver disease (NAFLD) with various degree of liver inflammation: wild-type mice fed a normal or high-fat diet (HFD), FOZ fed a HFD and C57BL6/J fed a choline-deficient or -supplemented HFD. 99mTc-cAbVCAM1-5 uptake strongly correlates with liver histological inflammatory score and with molecular inflammatory markers. The diagnostic power to detect any degree of liver inflammation is excellent (AUROC 0.85-0.99). These data build the rationale to investigate 99mTc-cAbVCAM1-5 imaging to detect liver inflammation in patients with NAFLD, a largely unmet medical need.

Ephrin-B1 regulates the adult diastolic function through a late postnatal maturation of cardiomyocyte surface crests.

The rod-shaped adult cardiomyocyte (CM) harbors a unique architecture of its lateral surface with periodic crests, relying on the presence of subsarcolemmal mitochondria (SSM) with unknown role. Here, we investigated the development and functional role of CM crests during the postnatal period. We found in rodents that CM crest maturation occurs late between postnatal day 20 (P20) and P60 through both SSM biogenesis, swelling and crest-crest lateral interactions between adjacent CM, promoting tissue compaction. At the functional level, we showed that the P20-P60 period is dedicated to the improvement of relaxation. Interestingly, crest maturation specifically contributes to an atypical CM hypertrophy of its short axis, without myofibril addition, but relying on CM lateral stretching. Mechanistically, using constitutive and conditional CM-specific knock-out mice, we identified ephrin-B1, a lateral membrane stabilizer, as a molecular determinant of P20-P60 crest maturation, governing both the CM lateral stretch and the diastolic function, thus highly suggesting a link between crest maturity and diastole. Remarkably, while young adult CM-specific Efnb1 KO mice essentially exhibit an impairment of the ventricular diastole with preserved ejection fraction and exercise intolerance, they progressively switch toward systolic heart failure with 100% KO mice dying after 13 months, indicative of a critical role of CM-ephrin-B1 in the adult heart function. This study highlights the molecular determinants and the biological implication of a new late P20-P60 postnatal developmental stage of the heart in rodents during which, in part, ephrin-B1 specifically regulates the maturation of the CM surface crests and of the diastolic function.

Diet-Induced Obesity and NASH Impair Disease Recovery in SARS-CoV-2-Infected Golden Hamsters.

Obese patients with non-alcoholic steatohepatitis (NASH) are prone to severe forms of COVID-19. There is an urgent need for new treatments that lower the severity of COVID-19 in this vulnerable population. To better replicate the human context, we set up a diet-induced model of obesity associated with dyslipidemia and NASH in the golden hamster (known to be a relevant preclinical model of COVID-19). A 20-week, free-choice diet induces obesity, dyslipidemia, and NASH (liver inflammation and fibrosis) in golden hamsters. Obese NASH hamsters have higher blood and pulmonary levels of inflammatory cytokines. In the early stages of a SARS-CoV-2 infection, the lung viral load and inflammation levels were similar in lean hamsters and obese NASH hamsters. However, obese NASH hamsters showed worse recovery (i.e., less resolution of lung inflammation 10 days post-infection (dpi) and lower body weight recovery on dpi 25). Obese NASH hamsters also exhibited higher levels of pulmonary fibrosis on dpi 25. Unlike lean animals, obese NASH hamsters infected with SARS-CoV-2 presented long-lasting dyslipidemia and systemic inflammation. Relative to lean controls, obese NASH hamsters had lower serum levels of angiotensin-converting enzyme 2 activity and higher serum levels of angiotensin II-a component known to favor inflammation and fibrosis. Even though the SARS-CoV-2 infection resulted in early weight loss and incomplete body weight recovery, obese NASH hamsters showed sustained liver steatosis, inflammation, hepatocyte ballooning, and marked liver fibrosis on dpi 25. We conclude that diet-induced obesity and NASH impair disease recovery in SARS-CoV-2-infected hamsters. This model might be of value for characterizing the pathophysiologic mechanisms of COVID-19 and evaluating the efficacy of treatments for the severe forms of COVID-19 observed in obese patients with NASH.

Alteration of the gut microbiota's composition and metabolic output correlates with COVID-19-like severity in obese NASH hamsters.

Obese patientss with nonalcoholic steatohepatitis (NASH) are particularly prone to developing severe forms of coronavirus disease 19 (COVID-19). The gut-to-lung axis is critical during viral infections of the respiratory tract, and a change in the gut microbiota's composition might have a critical role in disease severity. Here, we investigated the consequences of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on the gut microbiota in the context of obesity and NASH. To this end, we set up a nutritional model of obesity with dyslipidemia and NASH in the golden hamster, a relevant preclinical model of COVID-19. Relative to lean non-NASH controls, obese NASH hamsters develop severe inflammation of the lungs and liver. 16S rRNA gene profiling showed that depending on the diet, SARS-CoV-2 infection induced various changes in the gut microbiota's composition. Changes were more prominent and transient at day 4 post-infection in lean animals, alterations still persisted at day 10 in obese NASH animals. A targeted, quantitative metabolomic analysis revealed changes in the gut microbiota's metabolic output, some of which were diet-specific and regulated over time. Our results showed that specifically diet-associated taxa are correlated with disease parameters. Correlations between infection variables and diet-associated taxa highlighted a number of potentially protective or harmful bacteria in SARS-CoV-2-infected hamsters. In particular, some taxa in obese NASH hamsters (e.g. Blautia and Peptococcus) were associated with pro-inflammatory parameters in both the lungs and the liver. These taxon profiles and their association with specific disease markers suggest that microbial patterns might influence COVID-19 outcomes.

Highly Diluted Antibodies to eNOS Restore Endothelium Function in Aortic Rings From Hypertensive Rats.

Background: Nitric oxide (NO) as a vaso- and cardio-protective agent could reduce vasomotor dysfunction in different cardiovascular diseases. One of the current therapeutics targeted at NO availability in the vascular wall are highly diluted antibodies to endothelial NO-synthase (eNOS). This drug has previously shown its endothelium-protective effect and effectiveness in reducing hypertension. Current study was dedicated to evaluate the direct impact of highly diluted antibodies to eNOS on the vessel constriction and dilation ex vivo. Methods: For that purpose, we used thoracic aortas dissected from spontaneously hypertensive (SHR) rats. Endothelium-dependent relaxation in the presence of highly diluted antibodies to eNOS (1 mL) was examined after phenylephrine-induced pre-constriction of the aorta rings in response to gradually increased acetylcholine concentration (1 nM to 10 µM). Results: Highly diluted antibodies to eNOS enhanced acetylcholine-induced relaxation in a statistically significant manner. Moreover, it was demonstrated that observed effect was similar to perindopril, a well-known angiotensin-converting-enzyme inhibitor, which works through relaxing and widening blood vessels. Conclusions: Our findings indicate that highly diluted antibodies to eNOS restored impaired endothelium function, as demonstrated by increased relaxation of SHR rats aorta rings. The revealed results suggest beneficial effect of highly diluted antibodies to eNOS to ameliorate hypertension and related diseases.

Alteration of the gut microbiota following SARS-CoV-2 infection correlates with disease severity in hamsters.

Mounting evidence suggests that the gut-to-lung axis is critical during respiratory viral infections. We herein hypothesized that disruption of gut homeostasis during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may associate with early disease outcomes. To address this question, we took advantage of the Syrian hamster model. Our data confirmed that this model recapitulates some hallmark features of the human disease in the lungs. We further showed that SARS-CoV-2 infection associated with mild intestinal inflammation, relative alteration in intestinal barrier property and liver inflammation and altered lipid metabolism. These changes occurred concomitantly with an alteration of the gut microbiota composition over the course of infection, notably characterized by a higher relative abundance of deleterious bacterial taxa such as Enterobacteriaceae and Desulfovibrionaceae. Conversely, several members of the Ruminococcaceae and Lachnospiraceae families, including bacteria known to produce the fermentative products short-chain fatty acids (SCFAs), had a reduced relative proportion compared to non-infected controls. Accordingly, infection led to a transient decrease in systemic SCFA amounts. SCFA supplementation during infection had no effect on clinical and inflammatory parameters. Lastly, a strong correlation between some gut microbiota taxa and clinical and inflammation indices of SARS-CoV-2 infection severity was evidenced. Collectively, alteration of the gut microbiota correlates with disease severity in hamsters making this experimental model valuable for the design of interventional, gut microbiota-targeted, approaches for the control of COVID-19.Abbreviations: SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; COVID-19, coronavirus disease 2019; SCFAs, short-chain fatty acids; dpi, day post-infection; RT-PCR, reverse transcription polymerase chain reaction; IL, interleukin. ACE2, angiotensin converting enzyme 2; TMPRSS2, transmembrane serine protease 2.

The hepatic compensatory response to elevated systemic sulfide promotes diabetes.

Impaired hepatic glucose and lipid metabolism are hallmarks of type 2 diabetes. Increased sulfide production or sulfide donor compounds may beneficially regulate hepatic metabolism. Disposal of sulfide through the sulfide oxidation pathway (SOP) is critical for maintaining sulfide within a safe physiological range. We show that mice lacking the liver- enriched mitochondrial SOP enzyme thiosulfate sulfurtransferase (Tst-/- mice) exhibit high circulating sulfide, increased gluconeogenesis, hypertriglyceridemia, and fatty liver. Unexpectedly, hepatic sulfide levels are normal in Tst-/- mice because of exaggerated induction of sulfide disposal, with associated suppression of global protein persulfidation and nuclear respiratory factor 2 target protein levels. Hepatic proteomic and persulfidomic profiles converge on gluconeogenesis and lipid metabolism, revealing a selective deficit in medium-chain fatty acid oxidation in Tst-/- mice. We reveal a critical role of TST in hepatic metabolism that has implications for sulfide donor strategies in the context of metabolic disease.

CX3CR1 regulates gut microbiota and metabolism. A risk factor of type 2 diabetes.

OBJECTIVE: The intestinal microbiota to immune system crosstalk is a major regulator of metabolism and hence metabolic diseases. An impairment of the chemokine receptor CX3CR1, as a key regulator shaping intestinal microbiota under normal chow feeding, could be one of the early events of dysglycemia. METHODS: We studied the gut microbiota ecology by sequencing the gut and tissue microbiota. We studied its role in energy metabolism in CX3CR1-deficent and control mice using various bioassays notably the glycemic regulation during fasting and the respiratory quotient as two highly sensitive physiological features. We used antibiotics and prebiotics treatments, and germ free mouse colonization. RESULTS: We identify that CX3CR1 disruption impairs gut microbiota ecology and identified a specific signature associated to the genotype. The glycemic control during fasting and the respiratory quotient throughout the day are deeply impaired. A selected four-week prebiotic treatment modifies the dysbiotic microbiota and improves the fasting state glycemic control of the CX3CR1-deficent mice and following a glucose tolerance test. A 4 week antibiotic treatment also improves the glycemic control as well. Eventually, germ free mice colonized with the microbiota from CX3CR1-deficent mice developed glucose intolerance. CONCLUSIONS: CX3CR1 is a molecular mechanism in the control of the gut microbiota ecology ensuring the maintenance of a steady glycemia and energy metabolism. Its impairment could be an early mechanism leading to gut microbiota dysbiosis and the onset of metabolic disease.

Liraglutide targets the gut microbiota and the intestinal immune system to regulate insulin secretion.

AIMS: Liraglutide controls type 2 diabetes (T2D) and inflammation. Gut microbiota regulates the immune system and causes at least in part type 2 diabetes. We here evaluated whether liraglutide regulates T2D through both gut microbiota and immunity in dysmetabolic mice. METHODS: Diet-induced dysmetabolic mice were treated for 14 days with intraperitoneal injection of liraglutide (100 µg/kg) or with vehicle or Exendin 4 (10 µg/kg) as controls. Various metabolic parameters, the intestinal immune cells were characterized and the 16SrDNA gene sequenced from the gut. The causal role of gut microbiota was shown using large spectrum antibiotics and by colonization of germ-free mice with the gut microbiota from treated mice. RESULTS: Besides, the expected metabolic impacts liraglutide treatment induced a specific gut microbiota specific signature when compared to vehicle or Ex4-treated mice. However, liraglutide only increased glucose-induced insulin secretion, reduced the frequency of Th1 lymphocytes, and increased that of TReg in the intestine. These effects were abolished by a concomitant antibiotic treatment. Colonization of germ-free mice with gut microbiota from liraglutide-treated diabetic mice improved glucose-induced insulin secretion and regulated the intestinal immune system differently from what observed in germ-free mice colonized with microbiota from non-treated diabetic mice. CONCLUSIONS: Altogether, our result demonstrated first the influence of liraglutide on gut microbiota and the intestinal immune system which could at least in part control glucose-induced insulin secretion.

Elafibranor improves diet-induced nonalcoholic steatohepatitis associated with heart failure with preserved ejection fraction in Golden Syrian hamsters.

BACKGROUND: Cardiovascular disease is the leading cause of deaths in nonalcoholic steatohepatitis (NASH) patients. Mouse models, while widely used for drug development, do not fully replicate human NASH nor integrate the associated cardiac dysfunction, i.e. heart failure with preserved ejection fraction (HFpEF). To overcome these limitations, we established a nutritional hamster model developing both NASH and HFpEF. We then evaluated the effects of the dual peroxisome proliferator activated receptor alpha/delta agonist elafibranor developed for the treatment of NASH patients. METHODS: Male Golden Syrian hamsters were fed for 10 to 20 weeks with a free choice diet, which presents hamsters with a choice between control chow diet with normal drinking water or a high fat/high cholesterol diet with 10% fructose enriched drinking water. Biochemistry, histology and echocardiography analysis were performed to characterize NASH and HFpEF. Once the model was validated, elafibranor was evaluated at 15 mg/kg/day orally QD for 5 weeks. RESULTS: Hamsters fed a free choice diet for up to 20 weeks developed NASH, including hepatocyte ballooning (as confirmed with cytokeratin-18 immunostaining), bridging fibrosis, and a severe diastolic dysfunction with restrictive profile, but preserved ejection fraction. Elafibranor resolved NASH, with significant reduction in ballooning and fibrosis scores, and improved diastolic dysfunction with significant reduction in E/A and E/E' ratios. CONCLUSION: Our data demonstrate that the free choice diet induced NASH hamster model replicates the human phenotype and will be useful for validating novel drug candidates for the treatment of NASH and associated HFpEF.

Ezetimibe Enhances Macrophage-to-Feces Reverse Cholesterol Transport in Golden Syrian Hamsters Fed a High-Cholesterol Diet.

The aim of this work was to evaluate reverse cholesterol transport (RCT) in hamster, animal model expressing CETP under a high cholesterol diet (HF) supplemented with Ezetimibe using primary labelled macrophages. We studied three groups of hamsters (n=8/group) for 4 weeks: 1) chow diet group: Chow, 2) High cholesterol diet group: HF and 3) HF group supplemented with 0.01% of ezetimibe: HF+0.01%Ezet. Following intraperitoneal injection of 3H-cholesterol-labelled hamster primary macrophages, we measured the in vivo macrophage-to-feces RCT. .HF group exhibited an increase of triglycerides (TG), cholesterol, glucose in plasma and higher TG and cholesterol content in liver (p<0.01) compared to Chow group. Ezetimibe induced a significant decrease in plasma cholesterol with a lower LDL and VLDL cholesterol (p<0.001) and in liver cholesterol (p<0.001) and TG (p<0.01) content compared to HF. In vivo RCT essay showed an increase of tracer level in plasma and liver (p<0.05) but not in feces in HF compared to Chow group. The amount of labelled total sterol and cholesterol in liver and feces was significantly reduced (p<0.05) and increased (p=0.05) respectively with Ezetimibe treatment. No significant increase was obtained for labelled feces bile acids in HF+0.01%Ezet compared to HF. Ezetimibe decreased SCD1 gene expression and increased SR-B1 (p<0.05) in liver but did not affect NPC1L1 nor ABCG5 and ABCG8 expression in jejunum. In conclusion, ezetimibe exhibited an atheroprotective effect by enhancing RCT in hamster and decreasing LDL cholesterol. Ours findings showed also a hepatoprotective effect of ezetimibe by decreasing hepatic fat content. Significance Statement This work was assessed to determine the effect of ezetimibe treatment on high cholesterol diet induced disturbances and especially the effect on reverse cholesterol transport in animal model with CETP activity and using labelled primary hamster macrophages. We were able to demonstrate that ezetimibe exhibited an atheroprotective effect by enhancing RCT and by decreasing LDL cholesterol in hamster. We showed also a hepatoprotective effect of ezetimibe by decreasing hepatic fat content.

Liraglutide shows superior cardiometabolic benefits than lorcaserin in a novel free choice diet-induced obese rat model.

Lorcaserin (LORCA) and liraglutide (LIRA) were evaluated in a novel diet-induced obese (DIO) rat model fed a free choice (FC) diet, that presents rats with the options between control chow (CC) or high fat/cholesterol (HFC) diet, and normal water (NW) or 10% fructose water (FW). After 8 weeks of FC diet-induced obesity/insulin resistance, rats were maintained on FC diet and treated daily for 5 weeks with vehicle, LORCA 18 mg/kg orally or LIRA 0.4 mg/kg subcutaneously. Compared to CC diet, FC diet resulted in higher intake of HFC and FW, and significantly higher caloric intake and overweight. LIRA induced a lower HFC/FW and higher CC/NW intake, a 12% body weight loss (P < 0.01 vs. FC) and 40% lower visceral fat mass (P < 0.001). LORCA only reduced HFC intake and body weight gain (P < 0.001 vs. FC). FC diet raised HOMA-IR index and plasma leptinemia by 66% and 165% (both P < 0.05 vs. CC), which were 50% and 70% lower with LIRA (both P < 0.05 vs. FC), but unchanged by LORCA. LIRA and LORCA significantly improved FC diet-induced glucose intolerance. Only LIRA reduced liver fatty acids, triglycerides, and cholesterol by 68, 71 and 51% (all P < 0.001). FC diet also induced a diastolic dysfunction with reduced E/A ratio (P < 0.01 vs. CC), which was improved by LIRA and LORCA (both P < 0.01 vs. FC). LIRA also raised fractional shortening (P < 0.01 vs. FC). Overall, LIRA showed superior cardiometabolic benefits than LORCA in DIO rats under the FC diet, a model that will be useful to evaluate novel drugs targeting obesity and co-morbidities.

A 3-week nonalcoholic steatohepatitis mouse model shows elafibranor benefits on hepatic inflammation and cell death.

The long duration of animal models represents a clear limitation to quickly evaluate the efficacy of drugs targeting nonalcoholic steatohepatitis (NASH). We, therefore, developed a rapid mouse model of liver inflammation (i.e., the mouse fed a high-fat/high-cholesterol diet, where cyclodextrin is co-administered to favor hepatic cholesterol loading, liver inflammation, and NASH within 3 weeks), and evaluated the effects of the dual peroxisome proliferator-activated receptor alpha/delta agonist elafibranor (ELA). C57BL6/J mice were fed a 60% high-fat, 1.25% cholesterol, and 0.5% cholic acid diet with 2% cyclodextrin in drinking water (HFCC/CDX diet) for 3 weeks. After 1 week of the diet, mice were treated orally with vehicle or ELA 20 mg/kg q.d. for 2 weeks. Compared with vehicle, ELA markedly reduced liver lipids and nonalcoholic fatty liver disease activity scoring, through steatosis, inflammation, and fibrosis (all P < 0.01 vs. vehicle). Flow cytometry analysis showed that ELA significantly improved the HFCC/CDX diet-induced liver inflammation by preventing the increase in total number of immune cells (CD45+), Kupffer cells, dendritic cells, and monocytes population, as well as the reduction in natural killer and natural killer T cells, and by blocking conversion of T cells in regulatory T cells. ELA did not alter pyroptosis (Gasdermin D), but significantly reduced necroptosis (cleaved RIP3) and apoptosis (cleaved caspase 3) in the liver. In conclusion, ELA showed strong benefits on NASH, including improvement in hepatic inflammation, necroptosis, and apoptosis in the 3-week NASH mouse. This preclinical model will be useful to rapidly detect the effects of novel drugs targeting NASH.

Liraglutide improves hepatic steatosis and metabolic dysfunctions in a 3-week dietary mouse model of nonalcoholic steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is an emerging health problem worldwide. However, efficacious pharmacological treatment for NASH is lacking. A major issue for preclinical evaluation of potential therapeutics for NASH is the limited number of appropriate animal models, i.e., models that do not require long-term dietary intervention and adequately mimic disease progression in humans. The present study aimed to evaluate a 3-wk dietary mouse model of NASH and validate it by studying the effects of liraglutide, a compound in advanced clinical development for NASH. C57BL6/J mice were fed a diet high in fat (60%), cholesterol (1.25%), and cholic acid (0.5%), along with 2% hydroxypropyl-ß-cyclodextrin in drinking water (HFCC-CDX diet). Histological and biological parameters were measured at 1 and 3 wk. After 1-wk diet induction, liraglutide was administrated daily for 2 wk and then NASH-associated phenotypic aspects were evaluated in comparison with control mice. Prior to treatment with liraglutide, mice fed the HFCC-CDX diet for 1 wk developed liver steatosis and had increased levels of oxidative-stress markers and hepatic and systemic inflammation. For mice not treated with liraglutide, these aspects were even more pronounced after 3 wk of the dietary period, with additional liver insulin resistance and fibrosis. Liraglutide treatment corrected the diet-induced alterations in glucose metabolism and significantly reduced hepatic steatosis and inflammation. This study provides a novel 3-wk dietary model of mice that rapidly develop NASH features, and this model will be suitable for evaluating the therapeutic efficacy of compounds in preclinical drug development for NASH.NEW & NOTEWORTHY We propose a diet high in fat (60%), cholesterol (1.25%), and cholic acid (0.5%) along with 2% hydroxypropyl-ß-cyclodextrin in drinking water (HFCC-CDX diet) as a new dietary model of nonalcoholic steatohepatitis. We used the HFCC-CDX model to reproduce the main features of disease development in humans for the purpose of facilitating the rapid screening of drug candidates and prioritizing the more promising candidates for advanced preclinical assessment and subsequent clinical trials.

Nephropathy in diabetic db/db mice is accelerated by high protein diet and improved by the SGLT2 inhibitor dapagliflozin.

The widely used db/db mouse as a model of diabetic nephropathy (DN) only mimics the early changes in human DN with a slow disease progression. Since high protein diet (HPD) has been reported to affect progression of nephropathy in both humans and mice, we investigated whether HPD could accelerate nephropathy in db/db mice. Diabetic (C57BLKS-Leprdb/db) and non-diabetic (C57BLKS-Leprdb/+) mice were fed either HPD (60 kcal% protein) or control diet (22 kcal% protein), from 7 to 22 weeks of age. In db/db mice, HPD was found to significantly increase all measured readouts of renal injury including albuminuria, renal hypertrophy, mesangial expansion and expression of a panel of DN related markers, including KIM-1, Ki67 and Collagen III, which increased on both gene and protein levels. Furthermore, HPD activated the Renin-angiotensin system significantly and increased hyperfiltration, measured as reduced plasma Cystatin C. Usefulness of the HPD db/db mouse as a model for faster drug efficacy studies was investigated in a 5-week treatment study with the SGLT2 inhibitor, dapagliflozin. Expectedly, dapagliflozin normalised blood glucose levels and improved glucose intolerance in both HPD and control diet mice. Only HPD db/db mice, not the control diet db/db mice, showed clear hyperfiltration that was significantly reduced with dapagliflozin treatment at both 2 and 4 weeks of treatment. In conclusion, these studies confirm that HPD can significantly accelerate progression of nephropathy in db/db mice, and that this model could be useful for rapid evaluation of drug targets with potential to ameliorate features of DN, especially glomerular hyperfiltration.

Interaction between hormone-sensitive lipase and ChREBP in fat cells controls insulin sensitivity.

Impaired adipose tissue insulin signalling is a critical feature of insulin resistance. Here we identify a pathway linking the lipolytic enzyme hormone-sensitive lipase (HSL) to insulin action via the glucose-responsive transcription factor ChREBP and its target, the fatty acid elongase ELOVL6. Genetic inhibition of HSL in human adipocytes and mouse adipose tissue results in enhanced insulin sensitivity and induction of ELOVL6. ELOVL6 promotes an increase in phospholipid oleic acid, which modifies plasma membrane fluidity and enhances insulin signalling. HSL deficiency-mediated effects are suppressed by gene silencing of ChREBP and ELOVL6. Mechanistically, physical interaction between HSL, independent of lipase activity, and the isoform activated by glucose metabolism ChREBPα impairs ChREBPα translocation into the nucleus and induction of ChREBPß, the isoform with high transcriptional activity that is strongly associated with whole-body insulin sensitivity. Targeting the HSL-ChREBP interaction may allow therapeutic strategies for the restoration of insulin sensitivity.

Obeticholic acid raises LDL-cholesterol and reduces HDL-cholesterol in the Diet-Induced NASH (DIN) hamster model.

The use of rat and mouse models limits the translation to humans for developing novel drugs targeting nonalcoholic steatohepatitis (NASH). Obeticholic acid (OCA) illustrates this limitation since its dyslipidemic effect in humans cannot be observed in these rodents. Conversely, Golden Syrian hamsters have a lipoprotein metabolism mimicking human dyslipidemia since it does express the cholesteryl ester transfer protein (CETP). We therefore developed a Diet-Induced NASH (DIN) hamster model and evaluated the impact of OCA. Compared with chow fed controls, hamsters fed for 20 weeks with a free-choice (FC) diet, developed obesity, insulin resistance, dyslipidemia and NASH (microvesicular steatosis, inflammation, hepatocyte ballooning and perisinusoidal to bridging fibrosis). After 20 weeks of diet, FC fed hamsters were treated without or with obeticholic acid (15mg/kg/day) for 5 weeks. Although a non-significant trend towards higher dietary caloric intake was observed, OCA significantly lowered body weight after 5 weeks of treatment. OCA significantly increased CETP activity and LDL-C levels by 20% and 27%, and reduced HDL-C levels by 20%. OCA blunted hepatic gene expression of Cyp7a1 and Cyp8b1 and reduced fecal bile acids mass excretion by 64% (P < 0.05). Hamsters treated with OCA showed a trend towards higher scavenger receptor Class B type I (SR-BI) and lower LDL-receptor hepatic protein expression. OCA reduced NAS score for inflammation (P < 0.01) and total NAS score, although not significantly. Compared to mouse and rat models, the DIN hamster replicates benefits and side effects of OCA as observed in humans, and should be useful for evaluating novel drugs targeting NASH.

Evaluation of Antiatherogenic Properties of Ezetimibe Using 3H-Labeled Low-Density-Lipoprotein Cholesterol and 99mTc-cAbVCAM1-5 SPECT in ApoE-/- Mice Fed the Paigen Diet.

The addition of ezetimibe, an intestinal cholesterol absorption inhibitor, to statin therapy has recently shown clinical benefits in the Improved Reduction of Outcomes: Vytorin Efficacy International Trial by reducing low-density-lipoprotein (LDL) cholesterol levels more than statin therapy alone. Here, we investigated the mechanisms by which inhibition of intestinal cholesterol absorption might contribute to the clinically observed reduction in cardiovascular events by evaluating its effect on inflammatory plaque development in apolipoprotein E-/- mice. Methods: Apolipoprotein E-/- mice were fed the Paigen diet (1.25% cholesterol, 0.5% cholic acid, and 15% fat) without or with ezetimibe (7 mg/kg/d) for 6 wk. In a first set of mice (n = 15), we intravenously injected 3H-cholesteryl oleate-labeled human LDL to test whether ezetimibe promotes LDL-derived cholesterol fecal excretion. In a second set (n = 20), we used the imaging agent 99mTc-cAbVCAM1-5 to evaluate expression of an inflammatory marker, vascular cell adhesion molecule 1 (VCAM-1), in atherosclerotic plaques. In a third set (n = 21), we compared VCAM-1 expression with 99mTc-cAbVCAM1-5 uptake in various tissues. Results: Mice treated with ezetimibe showed a 173% higher LDL-cholesteryl ester plasma disappearance rate (P < 0.001 vs. control) after 3H-cholesteryl oleate-labeled LDL injection. At 96 h after injection, the hepatic fraction of 3H-tracer was 61% lower in mice treated with ezetimibe (P < 0.001). Meanwhile, LDL-derived 3H-cholesterol excretion in the feces was 107% higher (P < 0.001). The antiatherogenic effect of ezetimibe monitored by 99mTc-cAbVCAM1-5 SPECT showed a 49% reduction in aortic tracer uptake (percentage injected dose per cubic centimeter, 0.95 ± 0.04 vs. 1.87 ± 0.11; P < 0.01). In addition to hypercholesterolemia, the proinflammatory Paigen diet significantly increased VCAM-1 expression with respect to the control group in various tissues, including the aorta, and this expression correlated strongly with 99mTc-cAbVCAM1-5 uptake (r = 0.75; P < 0.05). Conclusion: Inhibition of intestinal cholesterol absorption with ezetimibe promotes antiatherosclerotic effects through increased LDL cholesterol catabolism and LDL-derived cholesterol fecal excretion and reduces inflamed atherosclerotic plaques. These mechanisms may contribute to the benefits of adding ezetimibe to a statin therapy.

Artemisinins Target GABAA Receptor Signaling and Impair α Cell Identity.

Type 1 diabetes is characterized by the destruction of pancreatic ß cells, and generating new insulin-producing cells from other cell types is a major aim of regenerative medicine. One promising approach is transdifferentiation of developmentally related pancreatic cell types, including glucagon-producing α cells. In a genetic model, loss of the master regulatory transcription factor Arx is sufficient to induce the conversion of α cells to functional ß-like cells. Here, we identify artemisinins as small molecules that functionally repress Arx by causing its translocation to the cytoplasm. We show that the protein gephyrin is the mammalian target of these antimalarial drugs and that the mechanism of action of these molecules depends on the enhancement of GABAA receptor signaling. Our results in zebrafish, rodents, and primary human pancreatic islets identify gephyrin as a druggable target for the regeneration of pancreatic ß cell mass from α cells.

Empagliflozin, via Switching Metabolism Toward Lipid Utilization, Moderately Increases LDL Cholesterol Levels Through Reduced LDL Catabolism.

In clinical trials, a small increase in LDL cholesterol has been reported with sodium-glucose cotransporter 2 (SGLT2) inhibitors. The mechanisms by which the SGLT2 inhibitor empagliflozin increases LDL cholesterol levels were investigated in hamsters with diet-induced dyslipidemia. Compared with vehicle, empagliflozin 30 mg/kg/day for 2 weeks significantly reduced fasting blood glucose by 18%, with significant increase in fasting plasma LDL cholesterol, free fatty acids, and total ketone bodies by 25, 49, and 116%, respectively. In fasting conditions, glycogen hepatic levels were further reduced by 84% with empagliflozin, while 3-hydroxy-3-methylglutaryl-CoA reductase activity and total cholesterol hepatic levels were 31 and 10% higher, respectively (both P < 0.05 vs. vehicle). A significant 20% reduction in hepatic LDL receptor protein expression was also observed with empagliflozin. Importantly, none of these parameters were changed by empagliflozin in fed conditions. Empagliflozin significantly reduced the catabolism of (3)H-cholesteryl oleate-labeled LDL injected intravenously by 20%, indicating that empagliflozin raises LDL levels through reduced catabolism. Unexpectedly, empagliflozin also reduced intestinal cholesterol absorption in vivo, which led to a significant increase in LDL- and macrophage-derived cholesterol fecal excretion (both P < 0.05 vs. vehicle). These data suggest that empagliflozin, by switching energy metabolism from carbohydrate to lipid utilization, moderately increases ketone production and LDL cholesterol levels. Interestingly, empagliflozin also reduces intestinal cholesterol absorption, which in turn promotes LDL- and macrophage-derived cholesterol fecal excretion.