A medium-chain fatty acid analogue prevents hepatosteatosis and decreases inflammatory lipid metabolites in a murine model of parenteral nutrition-induced hepatosteatosis.

BACKGROUND: Parenteral (intravenous) nutrition is lifesaving for patients with intestinal failure, but long-term use of parenteral nutrition often leads to liver disease. SEFA-6179 is a synthetic medium-chain fatty acid analogue designed to target multiple fatty acid receptors regulating metabolic and inflammatory pathways. We hypothesized that SEFA-6179 would prevent hepatosteatosis and lipotoxicity in a murine model of parenteral nutrition-induced hepatosteatosis. METHODS: Two in vivo experiments were conducted. In the first experiment, six-week-old male mice were provided an ad lib fat-free high carbohydrate diet (HCD) for 19 days with orogastric gavage of either fish oil, medium-chain triglycerides, or SEFA-6179 at a low (0.3mmol/kg) or high dose (0.6mmol/kg). In the second experiment, six-week-old mice were provided an ad lib fat-free high carbohydrate diet for 19 days with every other day tail vein injection of saline, soybean oil lipid emulsion, or fish oil lipid emulsion. Mice then received every other day orogastric gavage of medium-chain triglyceride vehicle or SEFA-6179 (0.6mmol/kg). Hepatosteatosis was assessed by a blinded pathologist using an established rodent steatosis score. Hepatic lipid metabolites were assessed using ultra-high-performance liquid chromatography-mass spectrometry. Effects of SEFA-6179 on fatty acid oxidation, lipogenesis, and fatty acid uptake in human liver cells were assessed in vitro. RESULTS: In the first experiment, mice receiving the HCD with either saline or medium-chain triglyceride treatment developed macrovesicular steatosis, while mice receiving fish oil or SEFA-6179 retained normal liver histology. In the second experiment, mice receiving a high carbohydrate diet with intravenous saline or soybean oil lipid emulsion, along with medium chain triglyceride vehicle treatment, developed macrovescular steatosis. Treatment with SEFA-6179 prevented steatosis. In each experiment, SEFA-6179 treatment decreased arachidonic acid metabolites as well as key molecules (diacylglycerol, ceramides) involved in lipotoxicity. SEFA-6179 increased both ß- and complete fatty oxidation in human liver cells, while having no impact on lipogenesis or fatty acid uptake. CONCLUSIONS: SEFA-6179 treatment prevented hepatosteatosis and decreased toxic lipid metabolites in a murine model of parenteral nutrition-induced hepatosteatosis. An increase in both ß- and complete hepatic fatty acid oxidation may underlie the reduction in steatosis.

Metabolic and Inflammatory Effects of an ω-3 Fatty Acid-Based Eucaloric Ketogenic Diet in Mice With Endotoxemia.

BACKGROUND: Dietary strategies can aid in the management of critically ill patients. Very-low-carbohydrate diets have been shown to improve glucose control and the inflammatory response. We aimed to determine the effects of a eucaloric ketogenic diet (EKD) enriched with ω-3 fatty acids (O3KD) on glucose levels and inflammation in mice with endotoxemia. METHODS: Adult mice were fed 1 of 3 diets (control diet [CD], EKD, or O3KD). After 4 weeks, each group received saline or Escherichia coli lipopolysaccharide (LPS) (5 mg/kg) intraperitoneally during the postprandial (PPP) or postabsorptive (PAP) periods. Blood glucose was measured at 0, 15, 30, 60, 90, 120, 180, and 240 minutes. Serum tumor necrosis factor (TNF)-α and interleukin (IL) 6 were measured by enzyme-linked immunosorbent assay. Distribution of serum fatty acids was determined by gas liquid chromatography. Hepatic expression of genes involved in inflammation, as well as glucose and lipid metabolism, were determined by quantitative polymerase chain reaction. RESULTS: During the PPP, glucose curves were comparable among the experimental groups. During the PAP, EKD showed a more pronounced increase in glucose levels at the first hour after LPS challenge compared with the CD-LPS group. During the PAP, IL6 was lower in O3KD-LPS compared with CD-LPS and EKD-LPS groups. These differences disappeared in the PPP. Similarly, TNF-α was lower in the O3KD-LPS group compared with the EKD-LPS group. The O3KD significantly increased the serum levels of the ω-3 eicosapentaenoic and docosahexaenoic acids and decreased the ω-6 arachidonic acid. CONCLUSION: An O3KD leads to reduced inflammation and maintains glucose homeostasis in mice with endotoxemia.

Alpha-tocopherol in intravenous lipid emulsions imparts hepatic protection in a murine model of hepatosteatosis induced by the enteral administration of a parenteral nutrition solution.

Intestinal failure-associated liver disease (IFALD) is a risk of parenteral nutrition (PN)-dependence. Intravenous soybean oil-based parenteral fat can exacerbate the risk of IFALD while intravenous fish oil can minimize its progression, yet the mechanisms by which soybean oil harms and fish oil protects the liver are uncertain. Properties that differentiate soybean and fish oils include α-tocopherol and phytosterol content. Soybean oil is rich in phytosterols and contains little α-tocopherol. Fish oil contains abundant α-tocopherol and little phytosterols. This study tested whether α-tocopherol confers hepatoprotective properties while phytosterols confer hepatotoxicity to intravenous fat emulsions. Utilizing emulsions formulated in the laboratory, a soybean oil emulsion (SO) failed to protect from hepatosteatosis in mice administered a PN solution enterally. An emulsion of soybean oil containing α-tocopherol (SO+AT) preserved normal hepatic architecture. A fish oil emulsion (FO) and an emulsion of fish oil containing phytosterols (FO+P) protected from steatosis in this model. Expression of hepatic acetyl CoA carboxylase (ACC) and peroxisome proliferator-activated receptor gamma (PPARγ), was increased in animals administered SO. ACC and PPARγ levels were comparable to chow-fed controls in animals receiving SO+AT, FO, and FO+P. This study suggests a hepatoprotective role for α-tocopherol in liver injury induced by the enteral administration of a parenteral nutrition solution. Phytosterols do not appear to compromise the hepatoprotective effects of fish oil.

Omega-3 fatty acids are protective in hepatic ischemia reperfusion injury in the absence of GPR120 signaling.

BACKGROUND: A single dose of IV fish oil (FO) before hepatic ischemia reperfusion injury (HIRI) increases hepatocyte proliferation and reduces necrosis in wild type (WT) mice. It has been suggested that the GPR120 receptor on Kupffer cells mediates FO's ability to reduce HIRI. The purpose of this study was to determine whether GPR120 is required for FO to reduce HIRI. METHODS: Sixty-four (n = 8/group) adult male WT (C57BL/6) and GPR120 knockout (KO) mice received IV FO (1 g/kg) or saline 1 h prior to HIRI or sham operation. Mice were euthanized 24 h postoperatively for analysis of hepatic histology, NFκB activity, and serum alanine transaminase (ALT) levels. RESULTS: FO pretreated livers had less necrosis after HIRI than saline pretreated livers in both WT (mean ±â€¯SEM 25.9 ±â€¯7.3% less, P = 0.007) and KO (36.6 ±â€¯7.3% less, P < 0.0001) mice. There was no significant difference in percent necrosis between WT-FO and KO-FO groups. Sham groups demonstrated minimal necrosis (0-1.9%). Mean [95% CI] ALT after HIRI was significantly higher (P = 0.04) in WT-Saline mice (1604 U/L [751-3427]) compared to WT-FO (321 U/L [150-686]) but was not significantly higher in KO-Saline mice compared to KO-FO. There were no differences in ALT between WT-FO and KO-FO mice who underwent HIRI or between groups who underwent sham surgery. There were no differences in NFκB or IKKß activation among groups as measured by Western blot analysis. CONCLUSIONS: IV FO pretreatment was able to reduce HIRI in GPR120 KO mice, suggesting the hepatoprotective effects of FO are not mediated by GPR120 alone.

Fish oil protects the liver from parenteral nutrition-induced injury via GPR120-mediated PPARγ signaling.

Intravenous fish oil lipid emulsions (FOLE) can prevent parenteral nutrition (PN)-induced liver injury in murine models and reverse PN-induced cholestasis in pediatric patients. However, the mechanisms by which fish oil protects the liver are incompletely characterized. Fish oil is rich in omega-3 fatty acids, which are ligands for the G-protein coupled receptor 120 (GPR120), expressed on hepatic Kupffer cells. This study tested the hypothesis that FOLE protects the liver from PN-induced injury through GPR120 signaling. Utilizing a previously described murine model of PN-induced liver injury in which mice develop steatosis in response to an oral parenteral nutrition diet, FOLE was able to preserve normal hepatic architecture in wild type mice, but not in congenic GPR120 knockout (gpr120-/-) mice. To further characterize the requirement of intact GPR120 for FOLE-mediated hepatic protection, gene expression profiles of key regulators of fat metabolism were measured. PPARγ was identified as a gene that is up-regulated by the PN diet and normalized with the addition of FOLE in wild type, but not in gpr120-/- mice. This was confirmed at the protein expression level. A PPARγ expression array further identified CD36 and SCD1, both down-stream effectors of PPARγ, to be up-regulated in PN-fed wild type mice yet normalized upon FOLE administration in wild type but not in gpr120-/- mice. Together, these results suggest that FOLE protects the liver, in part, through activation of GPR120 and the downstream effectors PPARγ and CD36. Identification of key genetic determinants of FOLE-mediated hepatic protection may provide targets for small molecule-based hepatic protection strategies.

Effects of dietary omega-3 fatty acids on bones of healthy mice.

BACKGROUND & AIMS: Altering the lipid component in diets may affect the incidence of metabolic bone disease in patients dependent on parenteral nutrition. Consumption of polyunsaturated fatty acids (PUFA) can impact bone health by modulating calcium metabolism, prostaglandin synthesis, lipid oxidation, osteoblast formation, and osteoclastogenesis. The aim of this study was to evaluate the dietary effects of PUFA on murine bone health. METHODS: Three-weeks-old male (n = 30) and female (n = 30) C57BL/6J mice were randomized into one of three dietary groups. The diets differed only in fat composition: soybean oil (SOY), rich in ω-6 PUFA; docosahexaenoic acid alone (DHA), an ω-3 PUFA; and DHA with arachidonic acid, an ω-6 PUFA, at a 20:1 ratio (DHA/ARA). After 9 weeks of dietary treatment, femurs were harvested for micro-computed tomographic analysis and mechanical testing via 3-point bending. Separate mice from each group were used solely for serial blood draws for measurement of biomarkers of bone formation and resorption. RESULTS: At the microstructural level, although some parameters in cortical bone reached differences that were statistically significant in female mice, these were too small to be considered biologically relevant. Similarly, trabecular bone parameters in male mice were statistically different in some dietary groups, although the biological interpretation of such subtle changes translate into a lack of effect in favor of any of the experimental diets. No differences were noted at the mechanical level and in blood-based biomarkers of bone metabolism across dietary groups within gender. CONCLUSIONS: Subtle differences were noted at the bones' microstructural level, however these are likely the result of random effects that do not translate into changes that are biologically relevant. Similarly, differences were not seen at the mechanical level, nor were they reflected in blood-based biomarkers of bone metabolism. Altogether, dietary consumption of PUFA do not seem to affect bone structure or metabolism in a healthy model of growing mice.

Vascular Endothelial Growth Factor Enhances Compensatory Lung Growth in Piglets.

BACKGROUND: Vascular endothelial growth factor has been found to accelerate compensatory lung growth after left pneumonectomy in mice. The aim of this study was to determine the natural history and the effects of vascular endothelial growth factor on compensatory lung growth in a large animal model. METHODS: To determine the natural history of compensatory lung growth, female Yorkshire piglets underwent a left pneumonectomy on days of life 10-11. Tissue harvest and volume measurement of the right lung were performed at baseline (n = 5) and on postoperative days 7 (n = 5), 14 (n = 4), and 21 (n = 5). For pharmacokinetic studies, vascular endothelial growth factor was infused via a central venous catheter, with plasma vascular endothelial growth factor levels measured at various time points. To test the effect of vascular endothelial growth factor on compensatory lung growth, 26 female Yorkshire piglets underwent a left pneumonectomy followed by daily infusion of vascular endothelial growth factor at 200 µg/kg or isovolumetric 0.9% NaCl (saline control). Lungs were harvested on postoperative day 7 for volume measurement and morphometric analyses. RESULTS: Compared with baseline, right lung volume after left pneumonectomy increased by factors of 2.1 ± 0.6, 3.3 ± 0.6, and 3.6 ± 0.4 on postoperative days 7, 14, and 21, respectively. The half-life of VEGF ranged from 89 to 144 minutes. Lesser doses of vascular endothelial growth factor resulted in better tolerance, volume of distribution, and clearance. Compared with the control group, piglets treated with vascular endothelial growth factor had greater lung volume (P < 0.0001), alveolar volume (P = 0.001), septal surface area (P = 0.007) and total alveolar count (P = 0.01). CONCLUSION: Vascular endothelial growth factor enhanced alveolar growth in neonatal piglets after unilateral pneumonectomy.

Pretreatment with intravenous fish oil reduces hepatic ischemia reperfusion injury in a murine model.

BACKGROUND: Ischemia reperfusion injury is a barrier to liver surgery and transplantation, particularly for steatotic livers. The purpose of this study was to determine if pretreatment with a single dose of intravenous fish oil decreases hepatic ischemia reperfusion injury and improves recovery of injured livers. METHODS: Sixty adult male C57BL/6 mice received 1 g/kg intravenous fish oil (Omegaven, Fresenius Kabi) or isovolumetric 0.9% NaCl (saline) via tail vein 1 hour before 30 minutes of 70% hepatic ischemia. Animals were killed 4, 8, or 24 hours postreperfusion, and livers were harvested for histologic analysis. RESULTS: Four hours postreperfusion, saline-treated livers demonstrated marked ischemia diffusely around the central veins, while intravenous fish oil-treated livers demonstrated only patchy necrosis with intervening normal parenchyma. Eight hours postreperfusion, all livers demonstrated pale areas of cell loss with surrounding regenerating hepatocytes. Ki67 staining confirmed 14.4/10 high-powered field (95% confidence interval, 3.2-25.6) more regenerating hepatocytes around areas of necrosis in intravenous fish oil-treated livers. Twenty-four hours postreperfusion, all livers demonstrated patchy areas of necrosis, with an 89% (95% confidence interval, 85-92) decrease in the area of necrosis in intravenous fish oil-treated livers. CONCLUSION: Intravenous fish oil treatment prior to hepatic ischemia reperfusion injury decreased the area of hepatic necrosis and increased hepatocyte regeneration compared to saline treatment in a mouse model.

Parenteral Soybean Oil Induces Hepatosteatosis Despite Addition of Fish Oil in a Mouse Model of Intestinal Failure-Associated Liver Disease.

BACKGROUND: Replacement of parenteral soybean oil (SO) with fish oil (FO) is an effective therapy for intestinal failure-associated liver disease (IFALD) in children. However, practitioners remain concerned about the risk of essential fatty acid deficiency (EFAD) and sometimes treat IFALD with a combination of 50% SO and 50% FO emulsions. The purpose of this study was to determine if mixing 50% SO and 50% FO emulsions would prevent hepatosteatosis in a murine model of parenteral nutrition (PN)-induced hepatosteatosis. METHODS: C57BL/6 mice were randomized to receive oral PN with parenteral saline, FO, SO, or a mixture of 50% FO and 50% SO for 19 days. Fatty acid analysis, histologic evaluation, Nonalcoholic Steatohepatitis Clinical Research Network (NSCRN) scores, and reverse-transcriptase polymerase chain reaction for key lipogenic genes were performed. RESULTS: The PN + saline group was the only group with EFAD, with a serum and hepatic triene/tetraene ratio of 0.53. NSCRN scores were highest in the PN + SO group (5.5; 95% confidence interval [CI], 4.9-6.1), followed by the PN + FO/SO (4.5; 95% CI, 3.5-5.5) group, with the lowest score in the PN + FO (2.0; 95% CI, 1.1-2.9) group. Acetyl CoA carboxylase α and acetyl CoA carboxylase ß expression was lower in the PN + FO group than in the PN + FO/SO or PN + SO groups. CONCLUSIONS: Our data demonstrate that a mixed fat emulsion of 50% SO and 50% FO is inferior to 100% FO in reducing hepatosteatosis in this model. These data suggest that use of parenteral SO with parenteral FO, in a 1:1 ratio, may still contribute to liver injury, although it is less hepatotoxic than pure SO.

A Comparison of Fish Oil Sources for Parenteral Lipid Emulsions in a Murine Model.

BACKGROUND: Fat emulsions are important components of parenteral nutrition (PN). Fish oil (FO) emulsions reverse cholestasis in PN-associated liver disease. There are 2 FO monographs. One is "FO; rich in omega-3 fatty acids" (NFO). The other, "omega-3 acids," (PFO), is enriched in omega-3 fatty acids, particularly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The purpose of this study is to compare the effects of 20% NFO and PFO emulsions produced in the laboratory in a murine model. METHODS: Emulsions were compounded containing different oils: soybean oil (SO), NFO, and two PFOs differing in percentage of fatty acids as triglycerides (PFO66 and PFO90). Chow-fed mice received saline, one of the above emulsions, or a commercial FO (OM) intravenously (2.4 g/kg/day) for 19 days. On day 19, animals were euthanized. Livers, spleens, and lungs were procured for histologic analysis. RESULTS: OM, SO, NFO, and PFO90 were well-tolerated clinically. PFO66 resulted in tachypnea and lethargy for ~1 minute following injections. At euthanasia, PFO66 and PFO90 groups had organomegaly. Histologically, these groups had splenic and hepatic fat-laden macrophages, and lungs had scattered fat deposits. Other groups had normal organs. CONCLUSIONS: PFO emulsions present an attractive possibility for improving inflammation in PN-dependent patients by concentrating anti-inflammatory EPA and DHA. However, 20% PFO emulsions were poorly tolerated and precipitated adverse end organ sequelae, suggesting that they may not be safe. Development of novel manufacturing methods may achieve safe 20% PFO parenteral emulsions, but by established formulation methods, these emulsions were clinically suboptimal despite meeting pharmacopeial standards.

The effect of docosahexaenoic acid on bone microstructure in young mice and bone fracture in neonates.

BACKGROUND: As low bone mineral density is a risk factor for fracture in childhood, optimizing age appropriate bone mass is recommended and might lower the impact of bone loss related to age. Consumption of omega-3 polyunsaturated fatty acids (PUFAs), including eicosapentaenoic and docosahexaenoic (DHA) acids have been shown to beneficially modulate bone metabolism. The objective of this study was to determine the incidence of fracture in neonates receiving a fish compared with soybean oil-based intravenous lipid emulsion and evaluate the effect of varying dietary omega-3 PUFA consumption on growing bone in young mice. MATERIALS AND METHODS: Eligibility criteria for the clinical study included gestational age ≤37 wk and parenteral nutrition-dependence for ≥4 wk. Radiographs were reviewed after lipid initiation to identify radiologic bone fracture. The animal study evaluated female C57/Bl6 mice randomized into one of five groups from age 3-12 wk, at which time femurs were harvested for micro-computed tomography and light microscopy analysis. RESULTS: A lower incidence of bone fracture was found in neonates maintained on fish compared with soybean oil. In the animal study, findings suggest the DHA diet provides the best protection against trabecular bone loss as evidenced by increased bone volume fraction, increased trabecular number, and decreased trabecular separation on micro-computed tomography. These protective effects appeared to affect the bone microstructure alone. CONCLUSIONS: The lower fracture risk observed in fish oil fed neonates in combination with the protective effects of DHA observed in the femurs of young C57/BL6 mice suggest an important role for omega-3 PUFAs on bone growth.

The effect of varying ratios of docosahexaenoic acid and arachidonic acid in the prevention and reversal of biochemical essential fatty acid deficiency in a murine model.

OBJECTIVE: Essential fatty acids (EFA) are necessary for growth, development, and biological function, and must be acquired through the diet. While linoleic acid (LA) and alpha-linolenic acid (ALA) have been considered the true EFAs, we previously demonstrated that docosahexaenoic acid (DHA) and arachidonic acid (AA) taken together as the sole source of dietary fatty acids can prevent biochemical essential fatty acid deficiency (EFAD). This study evaluates the effect of varying dietary ratios of DHA:AA in the prevention and reversal of biochemical EFAD in a murine model. METHODS: Using a murine model of EFAD, we provided mice with 2.1% of daily caloric intake in varying DHA:AA ratios (1:1, 5:1, 10:1, 20:1, 200:1, 100:0) for 19 days in association with a liquid high-carbohydrate fat-free diet to evaluate the effect on fatty acid profiles. In a second experiment, we evaluated the provision of varying DHA:AA ratios (20:1, 200:1, 100:0) on the reversal of biochemical EFAD. RESULTS: Mice provided with DHA and AA had no evidence of biochemical EFAD, regardless of the ratio (1:1, 5:1, 10:1, 20:1, 200:1, 100:0) administered. Biochemical EFAD was reversed with DHA:AA ratios of 20:1, 200:1, and 100:0 following 3 and 5 weeks of dietary provision, although the 20:1 ratio was most effective in the reversal and stabilization of the triene:tetraene ratio. CONCLUSION: Provision of DHA and AA, at 2.1% of daily caloric intake in varying ratios can prevent biochemical evidence of EFAD and hepatic steatosis over the short-term, with a ratio of 20:1 DHA:AA most effectively reversing EFAD.

Prolonging the female reproductive lifespan and improving egg quality with dietary omega-3 fatty acids.

Women approaching advanced maternal age have extremely poor outcomes with both natural and assisted fertility. Moreover, the incidence of chromosomal abnormalities and birth defects increases with age. As of yet, there is no effective and practical strategy for delaying ovarian aging or improving oocyte quality. We demonstrate that the lifelong consumption of a diet rich in omega-3 fatty acids prolongs murine reproductive function into advanced maternal age, while a diet rich in omega-6 fatty acids is associated with very poor reproductive success at advanced maternal age. Furthermore, even short-term dietary treatment with a diet rich in omega-3 fatty acids initiated at the time of the normal age-related rapid decline in murine reproductive function is associated with improved oocyte quality, while short-term dietary treatment with omega-6 fatty acids results in very poor oocyte quality. Thus, omega-3 fatty acids may provide an effective and practical avenue for delaying ovarian aging and improving oocyte quality at advanced maternal age.