Plasma Free Fatty Acids Research Articles

Title: Involvement of unsaturated fatty acid biosynthesis in CRC progression based on in vitro and in silico studies

Obesity is one of the risk factors concerns of colorectal cancer (CRC), the most common type of gastrointestinal cancer, due to the changing lifestyle and especially diet. There are various molecular pathways associated with obesity and the risk of CRC incidence, such as insulin resistance or elevated plasma free fatty acids, which alter the signaling pathways of intestinal epithelial cells. The aim of this study was to better understand the significance of unsaturated fatty acid biosynthesis on pathogenesis of colon cancer in obese. Based on GSE20931 dataset, obese individuals affected by CRC had higher increased gene expression than non-obese individuals. The analysis showed that in obese individuals, the 16 signaling pathway genes were activated and increased (FDR <0.05) significantly. The biosynthetic pathway of unsaturated fatty acids showed a cross-talk with the arachidonic acid metabolism pathway and the PPAR signaling pathway is influenced and regulated via these pathways. The biosynthetic pathway of unsaturated fatty acids consisting of 22 genes, were analyzed using GEO data and revealed that 4 genes (HSD17B12, TECR, FADS2, ELOVL5) from this pathway were significantly increased (FDR <0.05). These data were validated based on TCGA data (Adj.p.value <0.001). The expression level of candidate genes in HT-29 cells decreased significantly (P.value <0.01), and PPARγ expression increased under linoleic acid treatment (200 μM) compared to control cells. Moreover, in presence of linoleic acid treatment, migration, colony formation, and proliferation decreased (P.value <0.01) in presence of treatment. In summary, the Biosynthesis pathway of unsaturated fatty acids is an interesting and critical pathway in CRC.

Fecal carriage of vanB antibiotic resistance gene affects adipose tissue function under vancomycin use

ABSTRACT Detrimental consequences of antibiotic treatment may include long-lasting disruption of the gut microbiota. Previous studies found no negative effects of antibiotics on metabolic health, although individualized responses were observed. Here, we aimed to investigate the subject-specific response to vancomycin use in tissue-specific insulin sensitivity by stratifying individuals based on the presence of antibiotic resistance genes (ARGs) or opportunistic pathogens (OPs) in the baseline fecal microbiota. Quantitative Polymerase Chain Reaction (qPCR) was used to detect ARGs and OPs in DNA isolated from fecal samples of 56 males with overweight/obesity (Body Mass Index: 25–35 kg/m2) and impaired glucose metabolism (fasting plasma glucose ≥5.6 mmol/L and/or 2-hour glucose 7.8–11.1 mmol/L). A two-step hyperinsulinemic-euglycemic clamp was performed to determine tissue-specific insulin sensitivity. Abdominal subcutaneous adipose tissue (AT) gene expression was assessed using Affymetrix microarray. Gut microbial composition was determined using the Human Intestinal Tract Chip (HITChip) microarray. At baseline, the vancomycin resistance gene vanB was present in 60% of our population. In individuals that were vanB-negative at baseline, AT insulin sensitivity (insulin-mediated suppression of plasma free fatty acids) improved during vancomycin use, while it decreased among vanB-positive individuals (% change post versus baseline: 14.1 ± 5.6 vs. −6.7 ± 7.5% (p = .042)). The vancomycin-induced increase in AT insulin sensitivity was accompanied by downregulation of inflammatory pathways and enrichment of extracellular matrix remodeling pathways in AT. In the vanB-positive group, well-known vanB-carrying bacteria, Enterococcus and Streptococcus, expanded in the gut microbiome. In conclusion, microbiome composition and adipose tissue biology were differentially affected by vancomycin treatment based on fecal vanB carriage.

Palmitic acid promotes endothelial-to-mesenchymal transition via activation of the cytosolic DNA-sensing cGAS-STING pathway

Elevated levels of plasma free fatty acids (FFAs) lead to endothelial dysfunction, a process that is involved in the pathogenesis of atherosclerosis. Endothelial-to-mesenchymal transformation (EndMT) has been reported to accelerate endothelial dysfunction during the process of atherosclerosis. However, the underlying mechanisms of EndMT remain poorly understood. The present study aimed to investigate the role of the cytosolic DNA-sensing cyclic GMP-AMP synthase-stimulator interferon gene (cGAS-STING) pathway in palmitic acid (PA)-induced EndMT. Human aortic endothelial cells (HAECs) were exposed to different concentrations of PA, and subsequently its effects on EndMT and the cGAS-STING pathway were assessed. To investigate the role of cGAS-STING pathway on PA-induced EndMT, RNA interference was used to knockdown the expression of cGAS in HAECs prior to their exposure to PA. First, it was observed that PA reduced cell viability and intracellular nitric oxide production, and increased migratory capacity of the HAECs as well as the cellular oxidative stress response, leading to EndMT. Moreover, it was observed that the cGAS-STING pathway was activated in PA-exposed primary HAECs. Activating cGAS-STING pathway via mtDNA directing lead to EndMT in HAECs. Interestingly, cGAS knockdown by RNA interference attenuated PA-induced inflammation, oxidative stress and EndMT in HAECs. Taken together, the results of the present study suggested that the cytosolic DNA-sensing cGAS-STING pathway may have important roles in PA-induced EndMT in endothelial cells.

Statins effect on insulin resistance after a meal and exercise in hypercholesterolemic pre-diabetic individuals.

AimTo study if statins, a widely prescribed, inexpensive medication to prevent coronary artery diseases may cause insulin resistance (IR).MethodsFasted (HOMA‐IR) and post‐meal insulin resistance were assessed in 21 pre‐diabetic hypercholesterolemic individuals treated with statins (STA trial). Measurements were compared to another trial conducted 96 h after statin withdrawal using placebo pills (PLAC trial). Trials were duplicated 16–18 h after a bout of moderate‐intensity exercise (500 kcal of energy expenditure) to reduce IR and better appreciate statin effects (EXER+STA and EXER+PLAC trials).ResultsStatin withdrawal did not affect fasting (HOMA‐IR; 2.35 ± 1.05 vs. 2.18 ± 0.87 for STA vs. PLAC trials; p = 0.150) or post‐meal insulin resistance (i.e., Matsuda‐index, STA 6.23 ± 2.83 vs. PLAC 6.49 ± 3.74; p = 0.536). A bout of aerobic exercise lowered post‐meal IR (p = 0.043), but statin withdrawal did not add to the exercise actions (p = 0.564). Statin withdrawal increased post‐meal plasma free glycerol concentrations (0.136 ± 0.073 vs. 0.185 ± 0.090 mmol·L−1 for STA vs. PLAC trials; p < 0.001) but not plasma free fatty acids or fat oxidation (p = 0.981, and p = 0.621, respectively). Post‐meal fat oxidation was higher in the exercise trials (p = 0.002).ConclusionsWithdrawal of statin medication does not affect fasting or post‐meal insulin resistance in pre‐diabetic hypercholesterolemic individuals. Furthermore, statin use does not interfere with the beneficial effects of exercise on lowering IR.

1411-P: Increased 24-Hour Plasma Free Fatty Acids in People with Obesity and Prediabetes

The importance of increased plasma free fatty acids (FFAs) in the pathogenesis of insulin resistance in people with prediabetes is unclear. It has been proposed that the increase in plasma insulin associated with obesity should be adequate to suppress adipose tissue lipolytic rates and maintain normal plasma FFA levels. We evaluated serial plasma FFA concentrations for 24 h in 20 adults with obesity and normal fasting glucose and glucose tolerance (metabolically healthy obesity [MHO]) and 20 adults with prediabetes, matched on age, sex, BMI and percent body fat with the MHO group. Insulin sensitivity, assessed as the glucose infusion rate during an insulin clamp, was greater in the MHO than the prediabetes group (48.1 ± 3.0 vs. 23.5 ± 1.3 μmol/kg fat-free mass/min, respectively, P &amp;lt; 0.0001) . Plasma FFA were greater throughout 24 h in the prediabetes group than the MHO group (Figure) . The 24-h areas under the curve were greater for plasma FFA (5.59 ± 0.37 vs. 4.58 ± 0.30 mmol/L x hour, P = 0.04) and plasma insulin (13,5± 1720 vs. 6,541 ± 558 pmol/L x hour, P = 0.0004) in the prediabetes group than the MHO group. We conclude that plasma FFA concentrations are greater throughout the day in people with prediabetes than those with MHO, despite a marked increase in plasma insulin concentration in those with prediabetes. These data support the notion that increased 24-h plasma FFAs contribute to insulin resistance associated with obesity. Disclosure M.C.Petersen: None. S.S.Farabi: n/a. G.Smith: None. S.Klein: Advisory Panel; Altimmune, Consultant; Janssen Research &amp; Development, LLC, ProSciento, Research Support; Janssen Research &amp; Development, LLC. Funding NIH T32DK007120Endocrine Fellows Foundation

1322-P: Role of Macrophage NOD1 in Fat-Induced ß-Cell Dysfunction

In individuals with obesity, free fatty acids (FFA) are chronically raised resulting in ß-cell dysfunction, leading to type 2 diabetes (T2D) . Recent data implicate the role of inflammation in developing ß-cell dysfunction, which is in part mediated by nucleotide-oligomerization domain 1 (NOD1) , an intracellular pattern recognition receptor. NOD1 is activated by peptidoglycans of bacterial cell walls; however, in vitro studies have documented that NOD1 can also be activated by saturated FFA. Previous data from our lab have shown that palmitate-induced ß-cell dysfunction was prevented in whole body and ß-cell specific NOD1 null mice. We have seen earlier reports on increased macrophage infiltration in islets after elevation in circulating palmitate, and much higher expression of NOD1 in macrophages than ß-cells. Hence, we hypothesized that macrophage NOD1 receptors play a pivotal role in FFA-induced ß-cell dysfunction. We have generated myeloid specific NOD1 null mice to target macrophage NOD1 receptors by crossing NOD1 floxed mice with Lyz2 Cre+ mice. Both knockouts and their controls (floxed and Cre) were infused for 48 h with ethylpalmitate (hydrolyzed to palmitate and ethanol in plasma) , to achieve elevated circulating FFA in a non-toxic manner. Ethanol vehicle was infused as control treatment. To assess in vivo ß-cell function, hyperglycemic clamp was performed following the 48 h infusion, and the disposition index (DI) was calculated. Ethylpalmitate resulted in elevated plasma FFA compared to vehicle infusion. Ethylpalmitate caused marked reduction in DI in control mice, while knockout mice displayed no significant changes in DI between ethylpalmitate and vehicle infused groups. These results suggest that macrophage NOD1 plays a causal role in palmitate-induced ß-cell dysfunction in vivo. Overall, our data reveal NOD1 as a key factor and a potential therapeutic target for obesity-associated diabetes. Disclosure S.Rahman: None. J.Yung: None. Y.Zhang: None. A.Giacca: None.

1379-P: Combination of FGF21 and ß3-Adrenergic Treatment in Obese Mice

An induction of lipolysis by β3-adrenergic agonist CL316,243 (CL) increases plasma free fatty acid (FFA) level resulting later in ectopic lipid accumulation in liver. The peak of FFA stimulates insulin secretion and causes acute drop of blood glucose (BG) in chow-fed mice. This glucose-lowering effect is attenuated in insulin-resistant dietary obese (DIO) mice. Fibroblast growth factor 21 (FGF21) treatment increases insulin sensitivity while inhibiting lipolysis. Both FGF21 and CL induce uncoupling protein 1 (UCP1) in brown adipose tissue, increasing capacity for combustion of glucose. Thus, the FGF21 treatment has a potential to modulate the glucoregulatory action of CL. Here we aim to characterize the effect of the CL+FGF21 combination and to investigate the role of UCP1 in this effect. DIO C57Bl/6 mice were housed at 30 °C in order to minimize basal adrenergic tone. Animals were treated 2x daily with 65 μg FGF21 or its vehicle for 8 days. This treatment lowered insulin (increased insulin sensitivity) . Subsequently, the mice were injected with 35 μg CL or with saline. CL increased FFA and insulin in control mice, but did not affect BG (probably because of impaired insulin sensitivity) . The day after CL injection, 4-fold rise in ectopic lipid content in liver was observed in comparison to saline-treated group. In FGF21-treated mice, the CL-induced rise of NEFA and insulin was only transient, but sufficient to cause a drop of BG. The hepatic lipid accumulation after CL was completely preveted by FGF21. The same experimental set-up was repeated in DIO UCP1+/+ and -/- mice. Although the BG-lowering effect of the CL+FGF21combination was observed in both genotypes, the effect was significantly stronger in UCP1+/+. Thus, FGF21 sensitizes DIO mice to the BG-lowering effect of adrenergic stimulation while it prevents ectopic lipid accummulation. The drop of BG is observed even in the absence of UCP1, but UCP1 activity is required to reach the maximal BG-lowering effect of the combination. Disclosure P.Zouhar: None. Funding Czech Science Foundation (19-05356Y)

Maternal dietary methionine restriction alters the expression of energy metabolism genes in the duckling liver

BackgroundIn mammals, the nutritional status experienced during embryonic development shapes key metabolic pathways and influences the health and phenotype of the future individual, a phenomenon known as nutritional programming. In farmed birds as well, the quantity and quality of feed offered to the dam can impact the phenotype of the offspring. We have previously reported that a 38% reduction in the intake of the methyl donor methionine in the diet of 30 female ducks during the growing and laying periods - from 10 to 51 weeks of age - reduced the body weight of their 180 mule ducklings compared to that of 190 ducklings from 30 control females. The maternal dietary methionine restriction also altered the hepatic energy metabolism studied in 30 of their ducklings. Thus, their plasma glucose and triglyceride concentrations were higher while their plasma free fatty acid level was lower than those measured in the plasma of 30 ducklings from the control group. The objective of this new study was to better understand how maternal dietary methionine restriction affected the livers of their newly hatched male and female ducklings by investigating the hepatic expression levels of 100 genes primarily targeting energy metabolism, amino acid transport, oxidative stress, apoptotic activity and susceptibility to liver injury.ResultsSixteen of the genes studied were differentially expressed between the ducklings from the two groups. Maternal dietary methionine restriction affected the mRNA levels of genes involved in different pathways related to energy metabolism such as glycolysis, lipogenesis or electron transport. Moreover, the mRNA levels of the nuclear receptors PPARGC1B, PPARG and RXRA were also affected.ConclusionsOur results show that the 38% reduction in methionine intake in the diet of female ducks during the growing and egg-laying periods impacted the liver transcriptome of their offspring, which may explain the previously observed differences in their liver energy metabolism. These changes in mRNA levels, together with the observed phenotypic data, suggest an early modulation in the establishment of metabolic pathways.

Effects of Orlistat or Telmisartan on the Serum Free Fatty Acids in Non-alcoholic Fatty Liver Disease Patients: An Open-Labeled Randomized Controlled Study.

One of the important inducers of inflammatory responses and accumulation of fat in hepatocytes is free fatty acids which ultimately lead to the development of non-alcoholic fatty liver disease. Patients with non-alcoholic fatty liver disease have high levels of plasma free fatty acids which are usually associated with type 2 diabetes and components of metabolic syndrome including dyslipidemia. Objective of this research is to investigate the effects of orlistat (a lipase enzyme inhibitor) or telmisartan (an angiotensin receptor blocker) on the serum free fatty acids in non-alcoholic fatty liver disease patients taking into consideration the baseline lipid profile. This open-label clinical trial was carried out in the Department of Pharmacology, College of Medicine at the University of Sulaimani in cooperation with Shar Teaching Hospital in Sulaimani city-Kurdistan Region of Iraq. A total number of 74 non-alcoholic fatty liver disease patients were recruited and grouped randomly into group I (n = 25) treated with orlistat (120 mg/day orally) for 12 weeks, group II (n = 24) treated with telmisartan (20 mg/day orally) for 8 weeks, and group III (n = 25) treated with placebo (carboxy- methyl cellulose) once daily. Fasting serum level of free fatty acid and lipid profile including total cholesterol, triglyceride, high-density lipoprotein, and non-high-density lipoproteins were determined. Orlistat and telmisartan significantly reduced the triglyceride-glucose index and free fatty acid levels (P < .001) in patients with non-alcoholic fatty liver diseases. Short-term treatment with orlistat or telmisartan produce effective and significant reductions in FFAs in patients with non-alcoholic fatty liver disease compared to placebo. Orlistat effectively reduces the free fatty acid irrespective of the baseline lipid profile.

Lipidomics Analysis of Free Fatty Acids in Human Plasma of Healthy and Diabetic Subjects by Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS).

Targeted analytical methods for the determination of free fatty acids (FFAs) in human plasma are of high interest because they may help in identifying biomarkers for diseases and in monitoring the progress of a disease. The determination of FFAs is of particular importance in the case of metabolic disorders because FFAs have been associated with diabetes. We present a liquid chromatography-high resolution mass spectrometry (LC-HRMS) method, which allows the simultaneous determination of 74 FFAs in human plasma. The method is fast (10-min run) and straightforward, avoiding any derivatization step and tedious sample preparation. A total of 35 standard saturated and unsaturated FFAs, as well as 39 oxygenated (either hydroxy or oxo) saturated FFAs, were simultaneously detected and quantified in plasma samples from 29 subjects with type 2 diabetes mellitus (T2D), 14 with type 1 diabetes mellitus (T1D), and 28 healthy subjects. Alterations in the levels of medium-chain FFAs (C6:0 to C10:0) were observed between the control group and T2D and T1D patients.

Increased plasma fatty acid clearance, not fatty acid concentration, is associated with muscle insulin resistance in people with obesity

BackgroundAlthough it is well-accepted that increased plasma free fatty acid (FFA) concentration causes lipid overload and muscle insulin resistance in people with obesity, plasma FFA concentration poorly predicts insulin-resistant glucose metabolism. It has been proposed that hyperinsulinemia in people with obesity sufficiently inhibits adipose tissue triglyceride lipolysis to prevent FFA-induced insulin resistance. However, we hypothesized enhanced FFA clearance in people with obesity, compared with lean people, prevents a marked increase in plasma FFA even when FFA appearance is high. MethodsWe assessed FFA kinetics during basal conditions and during a hyperinsulinemic-euglycemic clamp procedure in 14 lean people and 46 people with obesity by using [13C]palmitate tracer infusion. Insulin-stimulated muscle glucose uptake rate was evaluated by dynamic PET-imaging of skeletal muscles after [18F]fluorodeoxyglucose injection. ResultsPlasma FFA clearance was accelerated in participants with obesity and correlated negatively with muscle insulin sensitivity without a difference between lean and obese participants. Furthermore, insulin infusion increased FFA clearance and the increase was greater in obese than lean participants. ConclusionsOur findings suggest plasma FFA extraction efficiency, not just plasma FFA concentration, is an important determinant of the cellular fatty acid load and the stimulatory effect of insulin on FFA clearance counteracts some of its antilipolytic effect.

Effects of Individual Circulating FFAs on Plasma Oxylipin Levels: Implications for FFA Epoxide‐diol Ratios as Indices of Soluble Epoxide Hydrolase Activity

Oxylipins, oxidation products of essential free fatty acids (FFAs), are involved in various cellular signaling. Among them, FFA epoxides are associated with beneficial effects in metabolic and cardiovascular health. FFA epoxides are metabolized to usually biologically less active diols by soluble epoxide hydrolase (sEH). Inhibition of sEH, which increases FFA epoxides, improves glucose homeostasis and cardiovascular health. Plasma epoxide‐diol ratios have been used as indices of sEH activity.Objective and HypothesisThe objective of this study was to examine the effects of acute elevation of plasma FFAs on oxylipins, particularly, epoxides, diols, and their ratios. We tested if FFA epoxide‐diol ratios are altered by circulating FFA levels (i.e., substrate availability) independent of sEH activity.MethodsMale Wistar rats received a constant intravenous infusion of olive (70% oleic acid [OA]), safflower seed (72% linoleic acid [LA]), and fish (rich in w‐3 FFAs) oils (20% wt/vol emulsion, 0.5 ml/h; n = 6 for each infusion) for 2 hours to selectively raise OA, LA, and w‐3 FFAs (eicosapentaenoic acid [EPA] and docosahexaenoic acid [DHA]), respectively. Blood samples were collected before and after the infusions and analyzed for oxylipins using LC‐MS and individual FFAs using GC‐MS. As LA, DHA, and EPA are metabolized to epoxides and diols, we evaluated their mass‐action effects to alter epoxides, diols, or their ratios. In contrast, OA is not metabolized to epoxides and diols detected in our assays, and the olive oil infusion served as control for any non‐mass‐action effects.ResultsAs expected, olive, safflower seed, and fish oil infusions selectively raised plasma OA (57%), LA (87%), and EPA (70%) and DHA (54%), respectively (P &lt; 0.05 for all). Interestingly, olive oil infusion had significant effects to decrease FFA epoxides and diols with more dramatic effects on epoxides than diols (P &lt; 0.01), resulting in large (up to 6‐fold) increases in diol‐to‐epoxide ratios. We found similar effects with safflower seed and fish oil infusions, although these infusions also showed mass action effects to increase epoxide and diol levels derived from LA (safflower seed oil) and DHA and EPA (fish oil). The decreases in FFA epoxides with acute elevation of circulating FFAs (i.e., increased substrate availability) were unexpected and appeared to be due to increased epoxide metabolism independent of sEH. We are currently examining the possibility that acute elevation of plasma FFAs stimulates glucuronidation of FFA epoxides to increase their removal via urinary excretion.ConclusionsRaising plasma FFAs has profound effects to decrease plasma FFA epoxides, significantly altering plasma epoxide‐diol ratios. These data suggest that caution should be taken in comparing epoxide‐diol ratios, as indices of sEH activity, between conditions with different circulating FFA levels. Our data also suggest that plasma FFAs regulate epoxide levels by stimulating a metabolic process independent of sEH, possibly glucuronidation followed by renal excretion.

Hepatic Lipidomic Analysis of Dietary Different Type of Fats and Fructose Interactions in the Pathogenesis of Nonalcoholic Fatty Liver Diseases

ObjectiveDietary nutrient interactions are key determinants to the development of nonalcoholic fatty liver disease (NAFLD). Fat and fructose are two major components of Western diet. This study aimed to determine the combined effects of different type of dietary fats and fructose on the development of NAFLD in a murine model.MethodsMale adult C57BL/6J mice were assigned to six groups and fed with diets (AIN‐93G based) enriched with different types of fat, such as saturated fat (SF‐beef tallow), n‐6 polyunsaturated fat (n6USF‐corn oil), or n‐3 polyunsaturated fat (n3USF‐menhaden fish oil), respectively, with or without fructose supplementation through 10% fructose (w/v) in the drinking water ad lib for 20 weeks. 42% of calories was derived from the fat. Metabolic phenotypes were characterized by liver histology, plasma ALT and AST, glucose tolerance test, hepatic mRNA expression of genes involved in lipid metabolism, and lipidomic analysis of hepatic fatty acid composition.ResultsWhile both SF and n6USF fed mice developed obesity and hepatic steatosis with or without fructose, SFA fed mice were characterized with hepatomegaly, liver injury and fibrosis, which were further exacerbated by fructose; conversely, plasma triglyceride levels were lower compared to controls. Interestingly, n6USF fed mice exhibited steatosis without hepatomegaly, and this was associated with markedly elevated plasma triglyceride and free fatty acid levels, which were blunted by fructose. Moreover, fructose and n6USF led to markedly elevated glucose intolerance tests compared to controls. Hepatic lipidomic analysis revealed that total monounsaturated fatty acids, but not total SFA, exhibited a stepwise decrease from SF to n6USF and n3USF fed mice across most of the lipid pools, which parallels the hepatic triglyceride content, irrespective the presence of fructose. In line with this, hepatic mRNA levels of lipogenesis genes, including Srebf1, Scd1, Fasn, Acacaand Acacb, displayed a similar pattern of alterations (SF&gt;n6USF&gt;n3USF), suggesting that increased lipogenesis contributes to hepatic steatosis in SF and n6USF fed mice. Of note, fructose play a mild, but significant role in the alterations of specific individual fatty acids in certain fatty acid pools, despite the predominant role played by the dietary fats.ConclusionOur data suggest that excess energy from SF intake results in fat storage in the liver, likely due to impaired triglyceride secretion; whereas excess energy from n6USF intake is stored in the periphery. Both effects were exacerbated by fructose supplementation. n3USF is beneficial even in the presence of fructose.

Lack of serum albumin improves insulin sensitivity in both male and female mice

Insulin resistance is a global health problem. It has been shown that free fatty acids (FFA) play a major role in insulin resistance. Albumin acts as main fatty acid binding protein in blood. To improve understanding of the role of FFA in insulin resistance, we aimed to determine whether lack of serum albumin and the associated suppression of plasma FFA concentration would improve insulin sensitivity in albumin knockout mice compared to wild type mice. Male and female homozygous albumin knockout mice and C57BL/6J wild‐type controls, each fed a chow diet, were studied at 6‐8 weeks of age. Glucose tolerance test, insulin tolerance test, tissue analyses, and plasma proteomics were performed. In both sexes of albumin knockout mice compared to wild‐type mice, we observed lower levels of hepatic diacylglycerol content, as well as lower blood glucose during glucose tolerance test and insulin tolerance test (P &lt; 0.05). The level of mRNA measurement showed higher expression of adiponectin, lipoprotein lipase, diacylglycerol acyltransferase‐1 and ‐2, and glucose transporter‐4 in adipose tissue, as well as lower hepatic perilipin‐2 expression (P&lt;0.05). In agreement with adipose mRNA level analysis, plasma proteomics indicated that circulating adiponectin concentration was higher in albumin knockout compared to wild‐type mice (P&lt;0.05). The results indicate an important role of albumin and plasma FFA concentration in lipid and glucose metabolism. We suggest that the lower plasma FFA concentration in albumin knockout mice compared to wild‐type mice leads to less hepatic diacylglycerol accumulation, and the lower level of this lipotoxic compound may contribute to the enhanced insulin sensitivity.

Ablation of pigment epithelium‐derived factor receptor, PEDF‐R, causes photoreceptor degeneration

Photoreceptor cells produce a receptor protein for pigment epithelium‐derived factor (PEDF‐R), which is encoded by the PNPLA2 gene of the patatin‐like phospholipase domain‐containing 2 family. PEDF‐R is a membrane‐linked lipase detected in the inner segments of photoreceptors. We aim to reveal the physiological role of PEDF‐R in photoreceptors by characterizing CRISPR Pnpla2 knock‐out mouse lines.CRISPR‐derived Pnpla2 knock‐out mouse lines were generated in a C57BL/6 and rd8 free background. RT‐PCR was performed from dissected retinas. Mice were evaluated for fundoscopy, electroretinography and angiography. Organs of the thorax were harvested and imaged using a LEICA microscope. Plasma was collected for lipid analyses. Eyes were enucleated and processed for histology, immunofluorescence, confocal microscopy, and transmission electron microscopy. Pnpla2‐/‐ mice had undetectable retinal Pnpla2 expression levels, enlarged whiter hearts, and lower plasma free fatty acids and triglycerides levels than their Pnpla2+/+ littermate controls. Their retinal pigment epithelial cells accumulated lipid droplets and their photoreceptors were deformed. Fundi of Pnpla2+/‐ eyes had white spots. The optic nerve area of Pnpla2‐/‐ and Pnpla2+/‐ mice had an excess of pigment. The a‐wave in Pnpla2‐/‐and Pnpla2+/‐ mice, and b‐wave in Pnpla2+/‐ mice were attenuated relative to controls. Retinal rhodopsin and opsin gene expression of Pnpla2‐/‐and Pnpla2+/‐ were lower than in controls. The immunofluorescent intensities of rhodopsin and opsin antibodies decreased in Pnpla2+/‐and Pnpla2‐/‐ photoreceptors relative to controls. The immunofluorescence of PKCα and synaptophysin antibodies (markers for bipolar cells and ribbon synapse) in the Pnpla2+/‐ retinas were lower than in controls. Ablation of Pnpla2 in mice causes malformation of photoreceptors and affects photoreceptor performance, identifying PEDF‐R as an important component for photoreceptor structure and function. These findings suggest that the absence of PEDF‐R in photoreceptor cells interrupts PEDF neurotrophic action, thereby heightening degeneration in the mice.

ATP10A has a protective and sex‐specific role in lipid metabolism

Genetic predisposition and environment play substantial roles in obesity, type 2 diabetes and cardiovascular disease (CVD). Genetic association studies have linked ATP10A, encoding a type IV P‐type ATPase (P4‐ATPase), to human metabolic disease. ATP10A is a lipid flippase that catalyzes the membrane translocation of phosphatidylcholine and glucosylceramide. These lipids and their respective metabolites have been independently implicated in metabolic dysfunction. To explore the role of this flippase in metabolism, we created a novel Atp10aknockout (KO) mouse model. Atp10a KO mice display a female‐specific weight gain during high‐fat diet feeding and this is attributable to increased adiposity. Female Atp10a KO mice also exhibit elevated plasma free fatty acids, cholesterol, and triglycerides, as well as a depletion in eicosanoid species compared to the wild type (WT) littermates. Additionally, female Atp10a KO mice exhibit elevated fasting blood glucose levels without compensatory elevation of insulin. We also found that the liver of female Atp10aKO mice displays larger lipid droplets, which was associated with increased diacylglycerol acyltransferase‐2 (DGAT2) expression and an attenuation of the insulin signaling pathway compared to the WT littermates. Thus far, our studies have shown that knocking out Atp10a in mice on a high fat diet results in sex‐specific perturbations to body composition, plasma lipid levels, glucose homeostasis, and liver metabolism. We have recently found that ATP10A is specifically expressed in the endothelial cells of multiple tissues. We are now exploring these metabolic phenotypes through the lens of endothelial cell dysfunction. These studies suggest mechanisms by which this flippase contributes to the development of CVD with obesity.

A Comparison of Substrate Utilization Profiles During Maximal and Submaximal Exercise Tests in Athletes.

BackgroundExercise is primarily sustained by energy derived from lipids (plasma free fatty acids and intramuscular triglycerides), and glucose (plasma glucose and muscle glycogen). Substrate utilization is the pattern by which these fuel sources are used during activity. There are many factors that influence substrate utilization. We aim to delineate the effect of exercise intensity and body composition on substrate utilization.ObjectiveThe objective of our study was to discern the differences in substrate utilization profiles during a maximal and submaximal graded exercise test, and to determine the extent to which body composition influences substrate utilization during the exercise tests.MethodsA total of 27 male athletes, 32.5 ± 11 years of age, were recruited for this study. Body composition was analyzed using a bioelectrical impedance analyzer. Maximal and submaximal exercise tests were performed on a treadmill. A novel graded submaximal treadmill protocol was used for the submaximal test.ResultsAverage percent body fat (PBF) was 15.8 ± 5%. Average maximal oxygen consumption (VO2max) was 47.6 ± 9 mL/kg/min, while the average exercise intensity (percent VO2max) at which participants were shifting to glucose predominance for energy during the maximal and submaximal tests were 76 ± 8.3% and 58.4 ± 21.1%, respectively. A paired-samples t-test was conducted to compare percent VO2max at crossover point in maximal and submaximal graded exercise tests. There was a significant difference in percent VO2max at the crossover point for maximal (76 ± 8.3%) and submaximal (58 ± 21.1%) tests (t = 4.752, p = 0.001). A linear regression was performed to elucidate the interaction between exercise intensity at the crossover point and body composition during a maximal and submaximal graded exercise test. There was a significant effect of PBF on percent VO2max at crossover point during the maximal graded exercise test [F(1,24) = 9.10, P = 0.006] with an R2 of 0.245. However, there was no significant effect of PBF on percent VO2max at crossover point during the submaximal graded exercise test (P > 0.05).ConclusionSubstrate utilization, represented by the crossover point, is dependent on the rate of increase in exercise intensity. At maximal efforts, the crossover to carbohydrates from fats as the predominant fuel source occurs at a significantly later stage of percent VO2max than at submaximal efforts. Furthermore, body composition represented by PBF is a significant predictor of substrate utilization during maximal efforts. Athletes with a relatively higher PBF are more likely to have increased lipid oxidation during high intensity exercises than those with a lower body fat percentage.

A method for assessing plasma free fatty acids from C2 to C18 and its application for the early detection of colorectal cancer

The targeted analysis of free fatty acids (FFAs) is attracting interest since several years with a plenty of studies. However, most of them are devoted to the solely determination of the short-chain fatty acids (SCFAs) arising from the symbiotic gut microbiota metabolism. Recently, the FFAs analysis highlighted changes in the plasma levels of octanoic and decanoic acids (medium-chain fatty acids or MCFAs) may be associated to gastrointestinal diseases, including colorectal cancer (CRC). Then, the simultaneous quantification of both SCFAs and MCFAs could be useful to put in evidence the interconnection between microbiota and metabolic alterations during hosts’ disease. To this aim, it was developed an isotopic dilution gas-chromatography coupled mass spectrometry (ID/GC-MS) method for the targeted analysis of both linear and branched FFAs (SCFAs, MCFAs, and LCFAs) in human plasma samples as specific markers for both microbiota and host metabolic alterations. In order to minimize sample manipulation procedures, an efficient, sensible and low time-consuming procedure is presented, which relies in a simple liquid-liquid extraction before the determination of underivatized free acids (FFAs) by Single Ion Monitoring (SIM) acquisition. The reached detection limits (LODs) were less than 100 μg L-1 for most of analytes, except for acetic, hexadecanoic and octadecanoic acids that showed a LOD > 1 mg L-1. Methods accuracy and precision, obtained by the analysis of the FFAs mixtures showed accuracy values between 84% and 100% and precision (RSD %) between 0.1% and 12.4% at the concentration levels tested. The proposed ID/GC-MS method was applied in a case study to evaluate the FFAs as specific markers for both microbiota and host alterations in CRC patients. Obtained results highlight the advantage of present method for its rapidity, simplicity, and robustness.

Subchronic Exposure to Benzene Induced Lipotoxic and Non-Lipotoxic Perturbations of Lipid Metabolic Dynamics in Male Wistar Rats

Benzene is a ubiquitous environmental pollutant. In this study, the effect of benzene exposure on lipid metabolism was investigated. Male albino rats were exposed to 100, 200, and 300 mg/kg body weight benzene through oral gavage for 12 weeks. At the end of the exposure, changes in lipid dynamics in the plasma, lipoprotein fractions, liver, and brain were investigated spectrophotometrically. One-way analysis of variance, followed by Tukey’s test was used to analyze the results with p &lt; 0.05 considered significant. Exposure to benzene for 12 weeks significantly (p &lt; 0.05) increased the concentration of plasma free fatty acids and reduced the plasma concentration of cholesterol and triacylglycerol. Benzene exposure also increased the triacylglycerol concentration in the plasma and lipoprotein fractions of the exposed animals. Furthermore, the exposure significantly (p &lt; 0.05) reduced the phospholipid concentrations in the plasma and lipoprotein fractions of the benzene-exposed animals. Benzene exposure significantly (p &lt; 0.05) reduced HDL cholesterol concentration. The exposure also resulted in a significant (p &lt; 0.05) elevation of cholesterol and phospholipids in the liver and brain of the exposed animals. The activity of HMG CoA reductase in the liver and brain of benzene-exposed animals was also enhanced significantly. Findings from the present study indicated that exposure to benzene perturbs lipid metabolic homeostasis through the induction of lipotoxic and non-lipotoxic dyslipidemia. Exposure to benzene may therefore represent an important risk factor in the pathogenesis of cardiovascular diseases.

Adipocyte Gq signaling is a regulator of glucose and lipid homeostasis in mice

Obesity is the major driver of the global epidemic in type 2 diabetes (T2D). In individuals with obesity, impaired insulin action leads to increased lipolysis in adipocytes, resulting in elevated plasma free fatty acid (FFA) levels that promote peripheral insulin resistance, a hallmark of T2D. Here we show, by using a combined genetic/biochemical/pharmacologic approach, that increased adipocyte lipolysis can be prevented by selective activation of adipocyte Gq signaling in vitro and in vivo (in mice). Activation of this pathway by a Gq-coupled designer receptor or by an agonist acting on an endogenous adipocyte Gq-coupled receptor (CysLT2 receptor) greatly improved glucose and lipid homeostasis in obese mice or in mice with adipocyte insulin receptor deficiency. Our findings identify adipocyte Gq signaling as an essential regulator of whole-body glucose and lipid homeostasis and should inform the development of novel classes of GPCR-based antidiabetic drugs.