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Introduction: Combination of therapies is a promising strategy for the treatment of NASH and liver fibrosis. Here we evaluated whether the Farnesoid X Receptor agonist obeticholic acid (OCA) combined with the glucagon-like peptide-1 (GLP-1) receptor agonist semaglutide (SEMA) would show superior effects than weight loss induction with time-restricted feeding (TRF) in mice. Methods: Mice were fed a 60% high fat/2% cholesterol diet with 10% fructose supplemented drinking water (HFCF diet) for 25 weeks to induce obesity and NASH/liver fibrosis. After the diet-induction period, mice were kept on HFCF diet and treated with vehicle (control) or OCA 30mg/kg p.o. QD + SEMA 0.06mg/kg s.c. QD, or placed on TRF from the last 3 hours of the dark cycle till the end of the light cycle, without access to food, but free access to normal drinking water (i.e. without fructose) every day, for 6 weeks. Results: OCA+SEMA induced a 20% lower caloric intake, which led to a 27% lower body weight (p Conclusion: OCA+SEMA combination reduced body weight and NAFLD activity score but did not improve hepatic fibrosis, while TRF improved both NASH and liver fibrosis. These TRF benefits should be further investigated in obese NASH patients. Disclosure F.Briand: None. E.Grasset: None. N.Breyner: None. T.Sulpice: None.

BACKGROUND AND AIMS Beyond the glucose control, anti-diabetic drugs need to demonstrate benefits on metabolic comorbidities. An animal model that recapitulates obesity and type 2 diabetes (T2D) comorbidities [i.e. non-alcoholic steato-hepatitis (NASH), nephropathy and heart failure with preserved ejection fraction (HFpEF)] is still needed for preclinical drug development. To overcome this limitation, we evaluated the effects of a high fat/cholesterol/fructose (HFCF) diet in the Lund MetS rat, a congenic BBDR.cg-lepr.cp model generated by introgression of the Koletsky leptin receptor mutation into the BioBreeding Diabetes Resistant (BBDR) rat. METHOD 17-week-old, male, lean control (ctrl) or Lund MetS obese T2D rats were fed a control chow (CC) diet or a HFCF diet, respectively for 8 weeks. Blood biochemistry was measured at 0, 4 and 8 weeks of diet. Kidney, heart and liver parameters were assessed at the end of the 8-week diet period. RESULTS Compared with ctrl, Lund MetS rats were obese (56% higher body weight) and diabetic with significantly higher %HbA1c (up to + 3%) and blood glucose levels (up to 3-fold higher) during the 8-week HFCF diet period. Significantly higher plasma insulin (up to 11-fold), total cholesterol (up to 5-fold), triglycerides (up to 8-fold), transaminases (up to 10-fold higher) levels were also observed in Lund MetS rats, as compared with ctrl. Hepatic total cholesterol, triglycerides and fatty acids levels were significantly higher in Lund Mets rats (14-, 7.3- and 6.7-fold higher as compared to ctrl, respectively). Liver histopathological scoring confirmed a NASH phenotype in Lund MetS rats fed the HFCF diet with strong liver steatosis, hepatic inflammation and portal to bridging fibrosis. Kidney function was substantially altered with a 60% decline in glomerular filtration rate and a 16-fold increase in urine albumin-to-creatinine ratio (both P CONCLUSION The present data demonstrate that the Lund MetS rat fed an HFCF diet that recapitulates the major metabolic comorbidities of obesity/T2D and may be a useful model for preclinical drug development.

BACKGROUND AND AIMS The Glucagon-like peptide 1 (GLP-1) receptor agonist liraglutide (LIRA) has cardioprotective effects and may reduce the development of kidney disease in type 2 diabetic patients. We here evaluated the effects of LIRA on both kidney and heart function in the Spontaneously Diabetic Torii (SDT) fatty rat, a type 2 diabetic cardiorenal model. METHOD SDT fatty male rats were treated subcutaneously with vehicle or LIRA 0.4 mg/kg QD for 10 weeks. To measure the effects of LIRA on glomerular hyperfiltration, rats were injected with FITC-sinistrin to measure glomerular filtration rate (GFR) at 4 weeks of treatment. At 5 weeks of treatment, rats underwent unilateral nephrectomy and were put on a 0.3% salt diet to induce a GFR decline. GFR was then measured at 10 weeks of treatment, before hemodynamics measurement and echocardiography. RESULTS Compared with vehicle, LIRA induced significant body weight loss, as well as blood glucose levels reduction by up to ∼20%. During the hyperfiltration phase, LIRA attenuated hyperfiltration, with a 19% lower GFR versus vehicle (P CONCLUSION In the SDT fatty rat, LIRA shows significant benefits by reducing renal hyperfiltration, preventing GFR decline, and improving cardiac hypertrophy, blood pressure and diastolic dysfunction. This preclinical model will be useful to evaluate drugs targeting the cardiorenal axis in type 2 diabetes.

Background: Obesity, non-alcoholic steatohepatitis (NASH) and dyslipidemia have been associated with severe forms of COVID-19. To better understand the impact of SARS-CoV-2 infection on dyslipidemia and NASH, we investigated the impact of infection with the beta variant of the SARS-CoV-2 in lean and high fat/cholesterol/fructose diet-induced obese Golden Syrian hamsters. Methods: Lean (chow diet fed) or diet-induced obese hamsters were infected intranasally with the beta variant of the SARS-CoV-2. At baseline (before infection) and at 4-, 7-, 10- and 25-days post-infection (dpi), blood and organs were collected for biochemistry and histology analyses. Results: Early after SARS-CoV-2 infection, lung viral load and pulmonary inflammation were not different between lean and obese hamsters. However, compared to lean hamsters, obese hamsters showed significantly higher MCP-1 serum levels at baseline and after infection, had impaired recovery (lower resolution of lung lesions at 10 dpi, lower body weight regain at 25 dpi), and exhibited higher pulmonary fibrosis at 25 dpi. In both lean and obese hamsters, SARS-CoV-2 infection led to reduction in serum triglycerides and HDL-cholesterol levels at 4 and 7 dpi. However, obese hamsters remained hypertriglyceridemic and hypercholesterolemic, while SARS-CoV-2 infection led to significant elevation of serum free fatty acids and LDL-cholesterol levels at 4 and 7 dpi in those obese individuals, as compared to baseline levels. Despite the substantial weight loss induced by SARS-CoV-2 infection, the NASH phenotype of obese hamsters was maintained, with significantly higher liver steatosis, inflammation, hepatocyte ballooning and fibrosis scores from day 0 to 25 dpi. Additionally, obese hamsters showed significantly higher liver fibrosis at 25 dpi, as compared to lean hamsters. Conclusion: Our data indicate that SARS-CoV-2 infection promotes dyslipidemia and non-alcoholic steatohepatitis in diet-induced obese Golden Syrian hamsters. This model will be useful to investigate the mechanisms leading to severe forms of COVID-19 seen in obese patients with dyslipidemia and NASH.

RATIONALEIn addition to its typical rod-shape, the mammalian adult cardiomyocyte (CM) harbors a unique lateral membrane surface architecture with periodic crests, relying on the presence of subsarcolemmal mitochondria (SSM) the role of which is still unknown.OBJECTIVETo investigate the development and functional role of CM crests during the postnatal period.METHODS AND RESULTSElectron/confocal microscopy and western-blot of left ventricular tissues from rat hearts indicated a late CM surface crest maturation, between postnatal day 20 (P20) and P60, as shown by substantial SSM swelling and increased claudin-5 cell surface expression. The P20-P60 postnatal stage also correlates with an ultimate maturation of the T-Tubules and the intercalated disk. At the cellular level, we identified an atypical CM hypertrophy characterized by an increase in long- and short-axes without myofibril addition and with sarcomere lateral stretching, indicative of lateral stretch-based CM hypertrophy. We confirmed the P20-P60 hypertrophy at the organ level by echocardiography but also demonstrated a transcriptomic program after P20 targeting all the cardiac cell populations. At the functional level, using Doppler echocardiography, we found that the P20-P60 period is specifically dedicated to the improvement of relaxation. Mechanistically, using CM-specific knock-out mice, we identified ephrin-B1 as a determinant of CM crest maturation after P20 controlling lateral CM stretch-hypertrophy and relaxation. Interestingly, while young adultEfnb1CMspe−/−mice essentially show a relaxation impairment with exercise intolerance, they progressively switch toward heart failure with 100% KO mice dying after 13 months.CONCLUSIONSThis study highlights a new late P20-P60 postnatal developmental stage of the heart in rodents during which the CM surface crests mature through an ephrin-B1-dependant mechanism and regulate the diastolic function. Moreover, we demonstrate for the first time that the CM crest architecture is cardioprotective.

Summary 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., Graphical abstract, Highlights • TST deficiency elevates sulfide, invoking exaggerated hepatic sulfide disposal • Exaggerated sulfide disposal triggers global hepatic protein underpersulfidation • Skewed persulfidation is associated with higher gluconeogenesis and impaired fat oxidation • Diabetogenic hepatic metabolism dominates over apparent peripheral insulin sensitization, Carter et al. show that mice lacking the mitochondrial sulfide oxidation pathway enzyme TST have high systemic sulfide levels that invoke an alternative hepatic sulfide disposal strategy. Consequently, hepatic metabolism is dominantly skewed toward a diabetogenic profile despite peripheral insulin sensitization. This has implications for sulfide donor therapeutic agents.

International audience; 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.

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. 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. 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. 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.

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

The Glucagon-Like Peptide 1 (GLP-1) receptor agonist liraglutide (LIRA) may reduce the rate of development and progression of diabetic kidney disease in type 2 diabetic patients. To evaluate its impact on kidney function, we evaluated the effects of LIRA on glomerular filtration rate (GFR), in both the hyperfiltration and GFR decline phases in the Spontaneously Diabetic Torii (SDT) fatty rat, a preclinical model of type 2 diabetes. SDT fatty male rats were treated subcutaneously, once daily with vehicle or LIRA 0.4mg/kg for 10 weeks. To measure the effects of LIRA on glomerular hyperfiltration, rats were injected with FITC-sinistrin at 4 weeks of treatment to measure GFR. After 5 weeks of treatment, rats underwent unilateral nephrectomy and were put on a 0.3% salt diet to induce a GFR decline for the last weeks of treatment, then GFR was measured at 10 weeks of treatment. Urine biochemistry and kidney histology analysis were also performed. Compared with vehicle, LIRA resulted in significant body weight loss and lower body weight. LIRA also reduced blood glucose levels significantly by up to ∼20%, as expected. During the hyperfiltration phase, LIRA attenuated hyperfiltration, with a 19% lower GFR vs. vehicle (vehicle: 21 ± 1.1 mL/min/kg; LIRA: 17.1 ± 1.0 mL/min/kg, p In conclusion, LIRA shows significant benefits on GFR, renal inflammation and fibrosis in the SDT fatty rat model. Disclosure F. Briand: Employee; Self; Physiogenex. Stock/Shareholder; Self; Physiogenex. M. Shinohara: None. E. Brousseau: Employee; Self; Physiogenex. Y. Kageyama: None. T. Sulpice: Employee; Self; Physiogenex. Stock/Shareholder; Self; Physiogenex.

International audience; 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.

Les activites antidiabetiques de Capparis spinosa (CS) et de Calamintha officinalis Moench (CO) ont ete evaluees sur un modele de souris de diabete de type 2 (HFD, high fat diet), presentant un etat prediabetique. L’administration des extraits aqueux bruts (coEA 100 mg/kg, csEA 100 mg/kg) et des extraits aqueux traites a la chaleur (coEAC 100 mg/kg, csEAC 100 mg/kg) pendant trois semaines a montre une activite antidiabetique, une baisse du poids ainsi qu’une baisse des concentrations plasmatiques d’acides gras circulants. Cette activite resiste au traitement a la chaleur. Les extraits bruts ont ete extraits par deux solvants differents, dichloromethane, butanol, produisant trois fractions de chaque extrait. Les activites antidiabetique et hypolipidemiante observees par traitement coEA (100 mg/kg) ont ete retrouvees chez les souris traitees avec la fraction eau residuelle (coER, 100 mg/kg) avec la meme efficacite, permettant d’affirmer que des molecules hydrophiles sont responsables des effets observes. L’activite antidiabetique observee chez les souris csEA (100 mg/kg) a ete retrouvee chez les souris traitees avec la fraction butanol (csBUT, 100 mg/kg) non correlee a l’activite hypolipidemiante retrouvee dans la fraction dichloromethane (csDC, 100 mg/kg), permettant d’affirmer que ces deux activites sont dues a des molecules lipophiles. Enfin, pour les deux plantes, l’activite sur le poids est retrouvee dans l’ensemble des fractions, ce qui montre que cette activite est due a des molecules de polarites differentes.

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-β-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-β-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.

To evaluate drugs targeting obesity/type 2 diabetes on weight loss and cardiometabolic benefits, and mimic human hyperenergetic nutritional behavior, we set-up a new diet-induced obese (DIO) rat model fed a free choice (FC) diet: control chow (CC) or high fat/cholesterol (HFC) diet, and normal water (NW) or 10% fructose water (FW). After an 8-week FC diet period, rats were treated with vehicle, liraglutide 0.4mg/kg s.c. QD (LIRA, a GLP-1 receptor agonist), lorcaserin 18mg/kg p.o. QD (LORCA, a 5-HT2C receptor agonist) and pioglitazone 10mg/kg p.o. QD (PIO, a PPARgamma agonist) for 5 weeks. FC diet induced high intake of HFC and FW, resulting in significantly higher caloric intake and overweight, as compared with rats fed a CC diet alone. LIRA induced a 12% body weight loss (p In conclusion, LIRA showed superior cardiometabolic benefits than LORCA and PIO in our DIO rat fed a FC diet. This novel model mimicking human overfeeding will be useful to evaluate drugs targeting obesity/type 2 diabetes. Disclosure F. Briand: Employee; Self; Physiogenex. E. Brousseau: Employee; Self; Physiogenex. T. Sulpice: Employee; Self; Physiogenex.

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.

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.

In addition to their benefits on glycemia, the specific actions of insulin analogs on glucose and lipid metabolism should be depicted. Here we analyzed the effects of Lispro (LIS) and Glulisine (GLU) insulin-analogs in diabetic rats. Diabetes was induced in Wistar male rats following an i.v. injection of streptozotocin (STZ) at 60mg/kg. Conscious rats underwent a 2-hour hyperinsulinemic (LIS or GLU at 0.8U/kg/h) euglycemic clamp with a constant infusion of 3H-glucose (4µCi/kg/min), with n=8 rats per each group. Glycemia was monitored continuously to adjust glucose infusion rate (GIR) to maintain euglycemia during the LIS or GLU perfusion. Blood, muscle and liver samples were collected for biochemical and radioactivity analysis. Compared to GLU, mean GIR was 51% higher in LIS treated rats (LIS: 18 +/- 1mg/kg/min, GLU: 12 +/- 1 mg/kg/min, p In conclusion, hyperinsulinemic euglycemic clamps in STZ rats enabled to detect a strong inhibition of hepatic glucose production by LIS, while GLU better suppresses lipolysis. Our experimental setting should help to differentiate other insulin analogs and dissect their mechanisms of action. Disclosure F. Briand: Employee; Self; Physiogenex S.A.S.. Stock/Shareholder; Self; Physiogenex S.A.S. E. Brousseau: Employee; Self; Physiogenex S.A.S. R. Burcelin: Stock/Shareholder; Self; Physiogenex S.A.S. T. Sulpice: Employee; Self; Physiogenex S.A.S.. Stock/Shareholder; Self; Physiogenex S.A.S..

Background: Beside its effect on LDL-cholesterol levels, proprotein convertase subtilisin/kexin9 (PCSK9) could also be linked to glucose metabolism and non-alcoholic fatty liver diseases. Here we evaluated whether Evolocumab (EVO), a monoclonal antibody targeting PCSK9, may alter glycemic profile and liver lipids in hamsters. Methods: Hamsters were maintained on either a control chow (CC) or high fat/cholesterol/fructose (HFCF) diet for 5 weeks. After 2 weeks of diet, hamsters were randomized into 2 groups, control or EVO at 30mg/kg s.c. once weekly for 3 weeks, to evaluate the effects on lipoprotein/glycemic profiles, and liver lipids. Results: When compared with CC diet, HFCF diet significantly increased total cholesterol and LDL-cholesterol levels by 87% and 102%. Hamsters fed the HFCF diet also showed higher HOMA-IR index of insulin resistance, higher blood glucose area under the curve (AUC) during an oral glucose tolerance test (OGTT), and higher liver lipids (triglycerides: +197%, total cholesterol: +421%, both p Conclusion: Our data indicate that diet-induced insulin resistance and liver steatosis in hamsters are not associated with elevated plasma PCSK9 levels, and are not altered by Evolocumab. Whether these results are specific to our hamster model and can translate to humans remains to be further investigated.

International audience; 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.

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.

Summary Type 1 diabetes is characterized by the destruction of pancreatic β 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 β-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 β cell mass from α cells., Graphical Abstract, Highlights • Artemisinins inhibit ARX function and impair α cell identity • Compounds act by stabilizing gephyrin, thus enhancing GABAA receptor signaling • Artemisinins increase β cell mass in zebrafish and rodent models • Functional and transcriptional data indicate a conserved phenotype in human islets, The anti-malarial drug Artemisinin can drive the in vivo conversion of pancreatic α cells into functional β-like cells through enhanced GABA signaling and may have potential as a therapeutic for diabetes.

Cathepsin S (CatS) belongs to a family of proteases that have been implicated in several disease processes. We previously identified CatS as a protein that is markedly overexpressed in adipose tissue of obese individuals and downregulated after weight loss and amelioration of glycaemic status induced by gastric bypass surgery. This prompted us to test whether the protease contributes to the pathogenesis of type 2 diabetes using mouse models with CatS inactivation. CatS knockout mice and wild-type mice treated with orally active small-molecule CatS inhibitors were fed chow or high-fat diets and explored for change in glycaemic status. CatS deletion induced a robust reduction in blood glucose, which was preserved in diet-induced obesity and with ageing and was recapitulated with CatS inhibition in obese mice. In vivo testing of glucose tolerance, insulin sensitivity and glycaemic response to gluconeogenic substrates revealed that CatS suppression reduced hepatic glucose production despite there being no improvement in insulin sensitivity. This phenotype relied on downregulation of gluconeogenic gene expression in liver and a lower rate of hepatocellular respiration. Mechanistically, we found that the protein ‘regulated in development and DNA damage response 1’ (REDD1), a factor potentially implicated in reduction of respiratory chain activity, was overexpressed in the liver of mice with CatS deficiency. Our results revealed an unexpected metabolic effect of CatS in promoting pro-diabetic alterations in the liver. CatS inhibitors currently proposed for treatment of autoimmune diseases could help to lower hepatic glucose output in obese individuals at risk for type 2 diabetes.

We investigated whether raising HDL-cholesterol levels with cholesteryl ester transfer protein (CETP) inhibition improves glucose homeostasis in dyslipidemic and insulin resistant hamsters. Compared with vehicle, torcetrapib 30 mg/kg/day (TOR) administered for 10 days significantly increased by ∼40% both HDL-cholesterol levels and 3 H-tracer appearance in HDL after 3 H-cholesterol labeled macrophages i.p. injection. TOR significantly reduced fasting plasma triglycerides, glycerol and free fatty acids levels by 65%, 31% and 23%, respectively. TOR also reduced blood glucose levels and plasma insulin by 20% and 49% respectively, which led to a 60% reduction in HOMA-IR index (all p 3 H-2-deoxyglucose and insulin injection, TOR significantly increased glucose uptake in oxidative soleus muscle, liver and heart by 26, 33 and 70%, respectively. Raising HDL levels with the CETP inhibitor torcetrapib improves glucose homeostasis in dyslipidemic and insulin resistant hamsters. Whether similar effect would be observed with other CETP inhibitors should be investigated.

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

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 3H-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.

Reverse cholesterol transport (RCT) promotes the egress of cholesterol from peripheral tissues to the liver for biliary and fecal excretion. Although not demonstrated in vivo, RCT is thought to be impaired in patients with metabolic syndrome, in which liver steatosis prevalence is relatively high. Golden Syrian hamsters were fed a nonpurified (CON) diet and normal drinking water or a high-fat (HF) diet containing 27% fat, 0.5% cholesterol, and 0.25% deoxycholate as well as 10% fructose in drinking water for 4 wk. Compared to CON, the HF diet induced insulin resistance and dyslipidemia, with significantly higher plasma non-HDL-cholesterol concentrations and cholesteryl ester transfer protein activity. The HF diet induced severe liver steatosis, with significantly higher cholesterol and TG levels compared to CON. In vivo RCT was assessed by i.p. injecting ³H-cholesterol labeled macrophages. Compared to CON, HF hamsters had significantly greater ³H-tracer recoveries in plasma, but not HDL. After 72 h, ³H-tracer recovery in HF hamsters was 318% higher in liver and 75% lower in bile (P < 0.01), indicating that the HF diet impaired hepatic cholesterol fluxes. However, macrophage-derived cholesterol fecal excretion was 45% higher in HF hamsters than in CON hamsters. This effect was not related to intestinal cholesterol absorption, which was 89% higher in HF hamsters (P < 0.05), suggesting a possible upregulation of transintestinal cholesterol excretion. Our data indicate a significant increase in macrophage-derived cholesterol fecal excretion in a hamster model of metabolic syndrome, which may not compensate for the diet-induced dyslipidemia and liver steatosis.

Cholesteryl ester transfer protein (CETP) transfers cholesteryl ester (CE) and triglyceride between HDL and apoB-containing lipoproteins. Anacetrapib (ANA), a reversible inhibitor of CETP, raises HDL cholesterol (HDL-C) and lowers LDL cholesterol in dyslipidemic patients; however, the effects of ANA on cholesterol/lipoprotein metabolism in a dyslipidemic hamster model have not been demonstrated. To test whether ANA (60 mg/kg/day, 2 weeks) promoted reverse cholesterol transport (RCT), ³H-cholesterol-loaded macrophages were injected and (3)H-tracer levels were measured in HDL, liver, and feces. Compared to controls, ANA inhibited CETP (94%) and increased HDL-C (47%). ³H-tracer in HDL increased by 69% in hamsters treated with ANA, suggesting increased cholesterol efflux from macrophages to HDL. ³H-tracer in fecal cholesterol and bile acids increased by 90% and 57%, respectively, indicating increased macrophage-to-feces RCT. Mass spectrometry analysis of HDL from ANA-treated hamsters revealed an increase in free unlabeled cholesterol and CE. Furthermore, bulk cholesterol and cholic acid were increased in feces from ANA-treated hamsters. Using two independent approaches to assess cholesterol metabolism, the current study demonstrates that CETP inhibition with ANA promotes macrophage-to-feces RCT and results in increased fecal cholesterol/bile acid excretion, further supporting its development as a novel lipid therapy for the treatment of dyslipidemia and atherosclerotic vascular disease.

International audience; Inhibition of dipeptidyl peptidase-4 (DPP-4) activity improves glucose homeostasis through a mode of action related to the stabilization of the active forms of DPP-4-sensitive hormones such as the incretins that enhance glucose-induced insulin secretion. However, the DPP-4 enzyme is highly expressed on the surface of intestinal epithelial cells; hence, the role of intestinal vs. systemic DPP-4 remains unclear. To analyze mechanisms through which the DPP-4 inhibitor sitagliptin regulates glycemia in mice, we administered low oral doses of the DPP-4 inhibitor sitagliptin that selectively reduced DPP-4 activity in the intestine. Glp1r(-/-) and Gipr(-/-) mice were studied and glucagon-like peptide (GLP)-1 receptor (GLP-1R) signaling was blocked by an i.v. infusion of the corresponding receptor antagonist exendin (9-39). The role of the dipeptides His-Ala and Tyr-Ala as DPP-4-generated GLP-1 and glucose-dependent insulinotropic peptide (GIP) degradation products was studied in vivo and in vitro on isolated islets. We demonstrate that very low doses of oral sitagliptin improve glucose tolerance and plasma insulin levels with selective reduction of intestinal but not systemic DPP-4 activity. The glucoregulatory action of sitagliptin was associated with increased vagus nerve activity and was diminished in wild-type mice treated with the GLP-1R antagonist exendin (9-39) and in Glp1r(-/-) and Gipr(-/-) mice. Furthermore, the dipeptides liberated from GLP-1 (His-Ala) and GIP (Tyr-Ala) deteriorated glucose tolerance, reduced insulin, and increased portal glucagon levels. The predominant mechanism through which DPP-4 inhibitors regulate glycemia involves local inhibition of intestinal DPP-4 activity, activation of incretin receptors, reduced liberation of bioactive dipeptides, and activation of the gut-to-pancreas neural axis.

Eur J Clin Invest 2011; 41 (9): 921–928 Abstract Background Reverse cholesterol transport (RCT) is an anti-atherogenic process by which cholesterol is effluxed from peripheral tissues by high-density lipoprotein (HDL) and returned to the liver for excretion into the bile and faeces. Dyslipidemia is thought to impair RCT through higher triglyceride-rich lipoprotein (TRL), low HDL-cholesterol and higher activity of cholesteryl ester transfer protein (CETP), which transfers cholesteryl esters from HDL to TRL for further hepatic uptake. As CETP pathway would represent a major route in human RCT, we therefore investigated whether diet-induced dyslipidemia impairs RCT in hamster, a CETP-expressing species. Materials and methods Golden Syrian hamsters were fed a chow or chow+0·3% cholesterol diet over 4 weeks. Biochemical parameters and in vivo VLDL-triglycerides secretion (Triton WR-1339 injection) were then measured. In vitro macrophage cholesterol efflux was measured, and in vivo macrophage-to-faeces RCT was also assessed after an intraperitoneal injection of 3H-cholesterol-labelled hamster primary macrophages. Results Cholesterol-enriched diet increased plasma total cholesterol (144%), triglycerides (101%), VLDL-triglycerides secretion (175%), CETP activity (44%) and reduced HDL-cholesterol/total cholesterol ratio by 20% (P

International audience; A fat-enriched diet modifies intestinal microbiota and initiates a low-grade inflammation, insulin resistance and type-2 diabetes. Here, we demonstrate that before the onset of diabetes, after only one week of a high-fat diet (HFD), live commensal intestinal bacteria are present in large numbers in the adipose tissue and the blood where they can induce inflammation. This translocation is prevented in mice lacking the microbial pattern recognition receptors Nod1 or CD14, but overtly increased in Myd88 knockout and ob/ob mouse. This 'metabolic bacteremia' is characterized by an increased co-localization with dendritic cells from the intestinal lamina propria and by an augmented intestinal mucosal adherence of non-pathogenic Escherichia coli. The bacterial translocation process from intestine towards tissue can be reversed by six weeks of treatment with the probiotic strain Bifidobacterium animalis subsp. lactis 420, which improves the animals' overall inflammatory and metabolic status. Altogether, these data demonstrate that the early onset of HFD-induced hyperglycemia is characterized by an increased bacterial translocation from intestine towards tissues, fuelling a continuous metabolic bacteremia, which could represent new therapeutic targets.