Germline manipulation using CRISPR/Cas9 genome editing has dramatically accelerated the generation of new mouse models. Nonetheless, many metabolic disease models still depend upon laborious germline targeting, and are further complicated by the need to avoid developmental phenotypes. We sought to address these experimental limitations by generating somatic mutations in the adult liver using CRISPR/Cas9, as a new strategy to model metabolic disorders. As proof-of-principle, we targeted the low-density lipoprotein receptor (Ldlr), which when deleted, leads to severe hypercholesterolemia and atherosclerosis. Here we show that hepatic disruption of Ldlr with AAV-CRISPR results in severe hypercholesterolemia and atherosclerosis. We further demonstrate that co-disruption of Apob, whose germline loss is embryonically lethal, completely prevented disease through compensatory inhibition of hepatic LDL production. This new concept of metabolic disease modeling by somatic genome editing could be applied to many other systemic as well as liver-restricted disorders which are difficult to study by germline manipulation.

BackgroundRecent studies suggest that the reported protective effects of statins (HMG-CoA reductase inhibitors) against community-acquired pneumonia (CAP) and sepsis in humans may be due to confounders and a healthy user-effect. To directly test whether statins are protective against Streptococcus pneumoniae, the leading cause of CAP, we examined the impact of prolonged oral simvastatin therapy at physiologically relevant doses in a mouse model of pneumococcal pneumonia. BALB/c mice were placed on rodent chow containing 0 mg/kg (control), 12 mg/kg (low simvastatin diet [LSD]; corresponds to 1.0 mg/kg/day), or 120 mg/kg (high simvastatin diet [HSD]; corresponds to 10 mg/kg/day) simvastatin for four weeks, infected intratracheally with S. pneumoniae serotype 4 strain TIGR4, and sacrificed at 24, 36, or 42 h post-infection for assessment of lung histology, cytokine production, vascular leakage and edema, bacterial burden and bloodstream dissemination. Some mice received ampicillin at 12-h intervals beginning at 48 h post-infection and were monitored for survival. Immunoblots of homogenized lung samples was used to assess ICAM-1 production.ResultsMice receiving HSD had reduced lung consolidation characterized by less macrophage and neutrophil infiltration and a significant reduction in the chemokines MCP-1 (P = 0.03) and KC (P = 0.02) and ICAM-1 in the lungs compared to control mice. HSD mice also had significantly lower bacterial titers in the blood at 36 (P = 0.007) and 42 (P = 0.03) hours post-infection versus controls. LSD had a more modest effect against S. pneumoniae but also resulted in reduced bacterial titers in the lungs and blood of mice after 42 h and a reduced number of infiltrated neutrophils. Neither LSD nor HSD mice had reduced mortality in a pneumonia model where mice received ampicillin 48 h after challenge.ConclusionsProlonged oral simvastatin therapy had a strong dose-dependent effect on protection against S. pneumoniae as evidenced by reduced neutrophil infiltration, maintenance of vascular integrity, and lowered chemokine production in the lungs of mice on HSD. Statin therapy also protected through reduced bacterial burden in the lungs. Despite these protective correlates, mortality in the simvastatin-receiving cohorts was equivalent to controls. Thus, oral simvastatin at physiologically relevant doses only modestly protects against pneumococcal pneumonia.

Risk of type 1 diabetes at 3 years is high for initially multiple and single Ab+ IT and multiple Ab+ NT. Genetic predisposition, age, and male sex are significant risk factors for development of Ab+ in twins.

Plasma high density lipoprotein-cholesterol (HDL-C) concentrations negatively correlate with atherosclerotic cardiovascular disease. HDL is thought to have several atheroprotective functions, which are likely distinct from the epidemiological inverse relationship between HDL-C levels and risk. Specifically, strategies that reduce HDL-C while promoting reverse cholesterol transport (RCT) may have therapeutic value. The major product of the serum opacity factor (SOF) reaction versus HDL is a cholesteryl ester (CE)-rich microemulsion (CERM), which contains apo E and the CE of ∼400,000 HDL particles. Huh7 hepatocytes take up CE faster when delivered as CERM than as HDL, in part via the LDL-receptor (LDLR). Here we compared the final RCT step, hepatic uptake and subsequent intracellular processing to cholesterol and bile salts for radiolabeled HDL-, CERM- and LDL-CE by Huh7 cells and in vivo in C57BL/6J mice. In Huh7 cells, uptake from LDL was greater than from CERM (2-4×) and HDL (5-10×). Halftimes for [14C]CE hydrolysis were 3.0 ± 0.2, 4.4 ± 0.6 and 5.4 ± 0.7 h respectively for HDL, CERM and LDL-CE. The fraction of sterols secreted as bile acids was ∼50% by 8 h for all three particles. HDL, CERM and LDL-CE metabolism in mice showed efficient plasma clearance of CERM-CE, liver uptake and metabolism, and secretion as bile acids into the gall bladder. This work supports the therapeutic potential of the SOF reaction, which diverts HDL-CE to the LDLR, thereby increasing hepatic CE uptake, and sterol disposal as bile acids.

Baboons (Papio hamadryas Sp.) exhibit significant sexual dimorphism in body size. Sexual dimorphism is also exhibited in a number of circulating factors associated with risk of cardiometabolic disease. We investigated whether sexual dimorphism in body size and composition underlie these differences. We examined data from 28 male and 24 female outdoor group-housed young adult baboons enrolled in a longitudinal observational study of cardiometabolic disease risk factors. Animals were sedated with ketamine HCl (10mg/Kg) before undergoing venous blood draws, basic body measurements, and dual-energy X-ray absorptiometry (DXA) body composition scans. Percentage glycated hemoglobin (%HbA1C) was measured in whole blood. Serum samples were analyzed for glucose, insulin, C-peptide, HDL-, and triglyceride concentrations. Males were heavier and had greater body length and lean tissue mass than females. Females had a greater body fat percentage relative to males (10.8 ±6.4 vs. 6.9 ±4.0, P=0.01). Although C-peptide, fasting glucose, and %HbA1C did not differ between the sexes, females had greater fasting insulin and triglyceride compared to their male counterparts. Insulin and percentage body fat were significantly correlated in males (r=0.61, P=0.001) and to a lesser extent in females (r=0.43, P=0.04). Overall, relations between adiposity and fasting insulin and fasting triglyceride were stronger in males. After accounting for differences in percentage body fat, fasting insulin and triglyceride were no longer statistically different between males and females. Despite stronger correlations between relative adiposity and insulin and triglyceride in males, the higher fasting insulin and triglyceride of female baboons may be underlain by their greater relative body fat masses.