GPAT1 Activity and Abundant Palmitic Acid Impair Insulin Suppression of Hepatic Glucose Production in Primary Mouse Hepatocytes.

BACKGROUND: Glycerol-3-phosphate acyltransferase (GPAT) activity is correlated with obesity and insulin resistance in mice and humans. However, insulin resistance exists in people with normal body weight, and individuals with obesity may be metabolically healthy, implying the presence of complex pathophysiologic mechanisms underpinning insulin resistance. OBJECTIVE: We asked what conditions related to GPAT1 must be met concurrently for hepatic insulin resistance to occur. METHODS: Mouse hepatocytes were overexpressed with GPATs via adenoviral infection or exposed to high or low concentrations of glucose. Glucose production by the cells and phosphatidic acid (PA) content in the cells were assayed, GPAT activity was measured, relative messenger RNA expressions of sterol-regulatory element-binding protein 1c (SREBP1c), carbohydrate response element-binding protein (ChREBP), and GPAT1 were analyzed, and insulin signaling transduction was examined. RESULTS: Overexpressing GPAT1 in mouse hepatocytes impaired insulin's suppression of glucose production, together with an increase in both N-ethylmaleimide-resistant GPAT activity and the content of di-16:0 PA. Akt-mediated insulin signaling was inhibited in hepatocytes that overexpressed GPAT1. When the cells were exposed to high-glucose concentrations, insulin suppression of glucose production was impaired, and adding palmitic acid exacerbated this impairment. High-glucose exposure increased the expression of SREBP1c, ChREBP, and GPAT1 by ∼2-, 5-, and 5.7-fold, respectively. The addition of 200 mM palmitic acid or linoleic acid to the culture media did not change the upregulation of expression of these genes by high glucose. High-glucose exposure increased di-16:0 PA content in the cells, and adding palmitic acid further increased di-16:0 PA content. The effect was specific to palmitic acid because linoleic acid did not show these effects. CONCLUSION: These data demonstrate that high-GPAT1 activity, whether induced by glucose exposure or acquired by transfection, and abundant palmitic acid can impair insulin's ability to suppress hepatic glucose production in primary mouse hepatocytes.

Pre-exercise protein intake is associated with reduced time in hypoglycaemia among adolescents with type 1 diabetes.

AIM: Secondary analyses were conducted from a randomized trial of an adaptive behavioural intervention to assess the relationship between protein intake (g and g/kg) consumed within 4 h before moderate-to-vigorous physical activity (MVPA) bouts and glycaemia during and following MVPA bouts among adolescents with type 1 diabetes (T1D). MATERIALS AND METHODS: Adolescents (n = 112) with T1D, 14.5 (13.8, 15.7) years of age and 36.6% overweight/obese, provided measures of glycaemia using continuous glucose monitoring [percentage of time above range (>180 mg/dl), time in range (70-180 mg/dl), time below range (TBR; <70 mg/dl)], self-reported physical activity (previous day physical activity recalls), and 24 h dietary recall data at baseline and 6 months post-intervention. Mixed effects regression models adjusted for design (randomization assignment, study site), demographic, clinical, anthropometric, dietary, physical activity and timing covariates estimated the association between pre-exercise protein intake on percentage of time above range, time in range and TBR during and following MVPA. RESULTS: Pre-exercise protein intakes of 10-19.9 g and >20 g were associated with an absolute reduction of -4.41% (p = .04) and -4.83% (p = .02) TBR during physical activity compared with those who did not consume protein before MVPA. Similarly, relative protein intakes of 0.125-0.249 g/kg and ≥0.25 g/kg were associated with -5.38% (p = .01) and -4.32% (p = .03) absolute reductions in TBR during physical activity. We did not observe a significant association between protein intake and measures of glycaemia following bouts of MVPA. CONCLUSIONS: Among adolescents with T1D, a dose of ≥10 g or ≥0.125 g/kg of protein within 4 h before MVPA may promote reduced time in hypoglycaemia during, but not following, physical activity.

Post-Exercise Protein Intake May Reduce Time in Hypoglycemia Following Moderate-Intensity Continuous Exercise among Adults with Type 1 Diabetes.

Little is known about the role of post-exercise protein intake on post-exercise glycemia. Secondary analyses were conducted to evaluate the role of post-exercise protein intake on post-exercise glycemia using data from an exercise pilot study. Adults with T1D (n = 11), with an average age of 33.0 ± 11.4 years and BMI of 25.1 ± 3.4, participated in isoenergetic sessions of high-intensity interval training (HIIT) or moderate-intensity continuous training (MICT). Participants completed food records on the days of exercise and provided continuous glucose monitoring data throughout the study, from which time in range (TIR, 70-180 mg/dL), time above range (TAR, >180 mg/dL), and time below range (TBR, <70 mg/dL) were calculated from exercise cessation until the following morning. Mixed effects regression models, adjusted for carbohydrate intake, diabetes duration, and lean mass, assessed the relationship between post-exercise protein intake on TIR, TAR, and TBR following exercise. No association was observed between protein intake and TIR, TAR, or TBR (p-values ≥ 0.07); however, a borderline significant reduction of -1.9% (95% CI: -3.9%, 0.0%; p = 0.05) TBR per 20 g protein was observed following MICT in analyses stratified by exercise mode. Increasing post-exercise protein intake may be a promising strategy to mitigate the risk of hypoglycemia following MICT.

Non-esterified erythrocyte linoleic acid, arachidonic acid, and subjective sleep outcomes.

OBJECTIVE: This study investigated whether non-esterified erythrocyte omega-6 PUFAs were associated with subjective assessment of sleep quality and duration, and risk for obstructive sleep apnea. METHODS: In this secondary analysis of the cross-sectional OPPERA-II study, 538 adults completed the Pittsburgh Sleep Quality Index (PSQI), reported their usual hours of sleep, and answered STOP screening questions for obstructive sleep apnea. Circulating non-esterified erythrocyte concentrations of omega-6 PUFA linoleic acid and arachidonic acid were quantified by liquid chromatography tandem mass spectroscopy. Sleep outcomes were dichotomized as poor (PSQI ≤5) vs good (PSQI ≥6) sleep quality, insufficient or excessive (≤6 or >9 h) vs good (7-9 h) sleep duration, and high (≥2 affirmative responses) vs low (<2 affirmative responses) risk for obstructive sleep apnea. Non-esterified omega-6 PUFAs and the continuous covariates of body mass index, Short Form (SF) 12 Health Survey Physical and Mental Component scores and resting measures of systolic and diastolic blood pressure were standardized for multivariable analysis. Categorical covariates were study site, age, sex, and race/ethnicity. Multivariable-adjusted logistic regression first estimated odds ratios (OR) and 95% confidence limits (CL) for sleep outcomes using linoleic acid as the main exposure. Analysis was then repeated using arachidonic acid as the main exposure. RESULTS: In the multivariable-adjusted model, each standard deviation increase in non-esterified erythrocyte linoleic acid was associated with higher odds of poor sleep quality (OR=1.2, 95% CL: 1.1, 1.5), insufficient or excessive sleep (OR= 1.3, 95% CL: 1.1, 1.6) and high-risk for obstructive sleep apnea (OR=1.3, 95% CL: 1.1, 1.6). Likewise, for each standard deviation increase in non-esterified erythrocyte arachidonic acid, odds increased of poor sleep quality (OR=1.2, 95% CL: 1.1, 1.5), and insufficient or excessive sleep (OR=1.2, 95% CL: 1.1, 1.5). Odds of being high risk for obstructive sleep apnea increased with greater circulating arachidonic acid, but the association did not reach statistical significance (OR=1.1, 95% CL: 0.9, 1.4). CONCLUSION: Non-esterified erythrocyte linoleic acid and arachidonic acid were associated with poor sleep quality and insufficient or excessive sleep duration. Linoleic acid, but not arachidonic acid, was also associated with high risk for obstructive sleep apnea.

Behavioral changes in FPR2/ALX and Chemr23 receptor knockout mice are exacerbated by prenatal alcohol exposure.

Introduction: Prenatal alcohol exposure (PAE) causes neuroinflammation that may contribute to the pathophysiology underlying Fetal Alcohol Spectrum Disorder. Supplementation with omega-3 polyunsaturated fatty acids (PUFAs) has shown success in mitigating effects of PAE in animal models, however, the underlying mechanisms are unknown. Some PUFA metabolites, specialized pro-resolving mediators (SPMs), play a role in the resolution phase of inflammation, and receptors for these are in the brain. Methods: To test the hypothesis that the SPM receptors FPR2 and ChemR23 play a role in PAE-induced behavioral deficits, we exposed pregnant wild-type (WT) and knockout (KO) mice to alcohol in late gestation and behaviorally tested male and female offspring as adolescents and young adults. Results: Maternal and fetal outcomes were not different among genotypes, however, growth and behavioral phenotypes in the offspring did differ and the effects of PAE were unique to each line. In the absence of PAE, ChemR23 KO animals showed decreased anxiety-like behavior on the elevated plus maze and FPR2 KO had poor grip strength and low activity compared to age-matched WT mice. WT mice showed improved performance on fear conditioning between adolescence and young adulthood, this was not seen in either KO. Discussion: This PAE model has subtle effects on WT behavior with lower activity levels in young adults, decreased grip strength in males between test ages, and decreased response to the fear cue indicating an effect of alcohol exposure on learning. The PAE-mediated decreased response to the fear cue was also seen in ChemR23 KO but not FPR2 KO mice, and PAE worsened performance of adolescent FPR2 KO mice on grip strength and activity. Collectively, these findings provide mechanistic insight into how PUFAs could act to attenuate cognitive impairments caused by PAE.

A High Protein Diet Is Associated with Improved Glycemic Control Following Exercise among Adolescents with Type 1 Diabetes.

Nutritional strategies are needed to aid people with type 1 diabetes (T1D) in managing glycemia following exercise. Secondary analyses were conducted from a randomized trial of an adaptive behavioral intervention to assess the relationship between post-exercise and daily protein (g/kg) intake on glycemia following moderate-to-vigorous physical activity (MVPA) among adolescents with T1D. Adolescents (n = 112) with T1D, 14.5 (13.8, 15.7) years of age, and 36.6% overweight or obese, provided measures of glycemia using continuous glucose monitoring (percent time above range [TAR, >180 mg/dL], time-in-range [TIR, 70-180 mg/dL], time-below-range [TBR, <70 mg/dL]), self-reported physical activity (previous day physical activity recalls), and 24 h dietary recall data at baseline and 6 months post-intervention. Mixed effects regression models adjusted for design (randomization assignment, study site), demographic, clinical, anthropometric, dietary, physical activity, and timing covariates estimated the association between post-exercise and daily protein intake on TAR, TIR, and TBR from the cessation of MVPA bouts until the following morning. Daily protein intakes of ≥1.2 g/kg/day were associated with 6.9% (p = 0.03) greater TIR and -8.0% (p = 0.02) less TAR following exercise, however, no association was observed between post-exercise protein intake and post-exercise glycemia. Following current sports nutrition guidelines for daily protein intake may promote improved glycemia following exercise among adolescents with T1D.

Circulating Polyunsaturated Fatty Acids and Pain Intensity in Five Chronic Pain Conditions.

Pain intensity is well-known to be influenced by a wide range of biobehavioral variables. Nutritional factors, however, have not been generally considered for their potential importance. This cross-sectional study examined associations between erythrocyte omega-6 (n-6) and omega-3 (n-3) polyunsaturated fatty acids (PUFAs) and pain intensity in 605 adults. Pain intensity was computed on a 0 to 100 numeric rating scale from questions about 5 chronic pain conditions: orofacial pain, headache, low back pain, irritable bowel syndrome, and bodily pain. For each pain condition, multiple linear regression tested the hypothesis that a higher ratio of n-6 arachidonic acid to the sum of n-3 eicosapentaenoic acid and docosahexaenoic acid (AA/(EPA+DHA) was associated with greater pain intensity. In covariate-adjusted analysis, orofacial pain intensity increased 5.7 points (95% CI: 1.4, 9.9) per unit increase in n-6/n-3 PUFA ratio. Likewise, a 1 unit increase in n-6/n-3 PUFA ratio was associated with significant increases in pain intensity (range 5-8 points) of headache pain, low back pain, and bodily pain, but not abdominal pain. Separate multiple linear regression models investigated the independent strength of association of individual PUFAs to the intensity of each pain condition. Overall, n-3 docosahexaenoic acid was most strongly, and inversely, associated with pain intensity. PERSPECTIVE: A higher ratio of n-6/n-3 long-chain polyunsaturated fatty acids was associated greater pain intensity for orofacial pain, headache, low back pain, and bodily pain, but not abdominal pain. The n-6/n-3 PUFA ratio was more consistently associated with pain intensity than any individual constituent of the long-chain PUFA ratio.

Circulating polyunsaturated fatty acids, pressure pain thresholds, and nociplastic pain conditions.

OBJECTIVE: Polyunsaturated fatty acids (PUFAs) play a role in pain regulation. This study sought to determine whether free PUFAs found in red blood cells also play a role in nociceptive processing. We examined associations between circulating PUFAs and nociceptive thresholds to noxious mechanical stimuli. We also determined whether nociceptive thresholds were associated with nociplastic pain conditions. METHODS: This cross-sectional study used stored red bloods cells and data from 605 adult participants in the OPPERA-2 study of chronic overlapping pain conditions. In OPPERA-2 adults completed quantitative sensory testing in which pressure algometry measured deep muscular tissue sensitivity at six anatomical sites. Standardized protocols classified adults for presence or absence of five nociplastic pain conditions: temporomandibular disorder, headache, low back pain, irritable bowel syndrome and fibromyalgia. Liquid chromatography tandem mass spectroscopy quantified erythrocyte PUFAs. We conducted three sets of analyses. First, a multivariable linear regression model assessed the association between n-6/n-3 PUFA ratio and the number of overlapping nociplastic pain conditions. Second, a series of 36 multivariable linear regression models assessed covariate-adjusted associations between PUFAs and nociceptive thresholds at each of six anatomical sites. Third, a series of 30 multivariable linear regression models assessed covariate-adjusted associations between nociceptive thresholds at six anatomical sites and each of five pain conditions. RESULTS: In multiple linear regression, each unit increase in n-6/n-3 PUFA ratio was associated with more pain conditions (ß = 0.30, 95% confidence limits: 0.07, 0.53, p = 0.012). Omega-6 linoleic acid and arachidonic acid were negatively associated with lower nociceptive thresholds at three and at five, respectively, anatomical sites. In contrast, omega-3 alpha-linolenic acid, eicosapentaenoic acid, docosahexaenoic acid and the n-6/n-3 PUFA ratio were not associated with nociceptive thresholds at any site. Pain cases had significantly lower nociceptive thresholds than non-case controls at all anatomical sites. CONCLUSION: A higher n-6/n-3 PUFA ratio was associated with more pain conditions. Omega-6 PUFAs may promote a generalized upregulation of nociceptive processing.

Circulating Omega-6 and Omega-3 Polyunsaturated Fatty Acids in Painful Temporomandibular Disorder and Low Back Pain.

Preclinical studies demonstrate opposing effects of long-chain polyunsaturated fatty acid (PUFA) metabolites on inflammation and nociception. Omega-6 (n-6) PUFAs amplify both processes while omega-3 (n-3) PUFAs inhibit them. This cross-sectional study examined relationships between PUFAs in circulating erythrocytes and 2 chronic idiopathic pain conditions: temporomandibular disorder (TMD) and low back pain in a community-based sample of 503 U.S. adults. Presence or absence of TMD and low back pain, respectively, were determined by clinical examination and by responses to established screening questions. Liquid chromatography-tandem mass spectrometry quantified PUFAs. In multivariable logistic regression models, a higher ratio of n-6/n-3 long-chain PUFAs was associated with greater odds of TMD (odds ratio ((OR) = 1.75, 95% confidence limits (CL): 1.16, 2.64) and low back pain (OR = 1.63, 95% CL: 1.07, 2.49). Higher levels of the pronociceptive n-6 long-chain arachidonic acid (AA) were associated with a greater probability of both pain conditions for women, but not men. Higher levels of the antinociceptive long-chain n-3 PUFAs eicosapentaenoic and docosahexaenoic acids were associated with a lower probability of both pain conditions for men, but not women. As systemic inflammation is not a hallmark of these conditions, PUFAs may influence idiopathic pain through other mechanisms. PERSPECTIVE: This cross-sectional clinical study found that a higher ratio of circulating n-6/n-3 long-chain PUFAs was associated with greater odds of 2 common chronic overlapping pain conditions. This suggests that the pro and antinociceptive properties of n-6 and n-3 PUFAs, respectively, influence pain independently of their well-established inflammatory pathways.

Ratio of Omega-6/Omega-3 Polyunsaturated Fatty Acids Associated With Somatic and Depressive Symptoms in People With Painful Temporomandibular Disorder and Irritable Bowel Syndrome.

Somatic symptom disturbance is among the strongest predictors of painful temporomandibular disorder (TMD). Related psychological constructs, such as anxiety and depression, respond therapeutically to omega-3 polyunsaturated fatty acids (PUFAs) in clinical trials. This cross-sectional study investigated associations between the omega-6/omega-3 PUFA ratio and somatic symptom disturbance and depressive symptoms in a community-based sample of 501 adults and determined whether these associations differed between adults with and without TMD or irritable bowel syndrome (IBS). Liquid chromatography tandem mass spectrometry quantified PUFAs in circulating erythrocytes. Somatic symptoms and depression were quantified using Symptom Checklist-90-Revised subscales. Presence or absence of TMD and IBS, respectively, were determined by clinical examination and Rome III screening questions. The standardized beta coefficient for the omega-6/omega-3 long-chain PUFA ratio was 0.26 (95% confidence limits (CL): 0.08, 0.43) in a multivariable linear regression model in which somatic symptom disturbance was the dependent variable. When modelling depressive symptoms as the dependent variable, the standardized beta coefficient was 0.17 (95% CL:0.01, 0.34). Both associations were stronger among TMD cases and IBS cases than among non-cases. Future randomized control trials that lower the omega-6/omega-3 PUFA ratio could consider somatic or depressive symptoms as a therapeutic target for TMD or IBS pain. PERSPECTIVE: In people with TMD or IBS, a high n-6/n-3 PUFA ratio was positively associated with somatic symptom disturbance and depressive symptoms. Both measures of psychological distress were elevated in people with painful TMD and IBS. Future randomized clinical trials will determine whether lowering the n-6/n-3 ratio is therapeutic for pain.

Live cell imaging of oxidative stress in human airway epithelial cells exposed to isoprene hydroxyhydroperoxide.

Exposure to respirable air particulate matter (PM2.5) in ambient air is associated with morbidity and premature deaths. A major source of PM2.5 is the photooxidation of volatile plant-produced organic compounds such as isoprene. Photochemical oxidation of isoprene leads to the formation of hydroperoxides, environmental oxidants that lead to inflammatory (IL-8) and adaptive (HMOX1) gene expression in human airway epithelial cells (HAEC). To examine the mechanism through which these oxidants alter intracellular redox balance, we used live-cell imaging to monitor the effects of isoprene hydroxyhydroperoxides (ISOPOOH) in HAEC expressing roGFP2, a sensor of the glutathione redox potential (EGSH). Non-cytotoxic exposure of HAEC to ISOPOOH resulted in a rapid and robust increase in EGSH that was independent of the generation of intracellular or extracellular hydrogen peroxide. Our results point to oxidation of GSH through the redox relay initiated by glutathione peroxidase 4, directly by ISOPOOH or indirectly by ISOPOOH-generated lipid hydroperoxides. We did not find evidence for involvement of peroxiredoxin 6. Supplementation of HAEC with polyunsaturated fatty acids enhanced ISOPOOH-induced glutathione oxidation, providing additional evidence that ISOPOOH initiates lipid peroxidation of cellular membranes. These findings demonstrate that ISOPOOH is a potent environmental airborne hydroperoxide with the potential to contribute to oxidative burden of human airway posed by inhalation of secondary organic aerosols.

Omega 3-fatty acids: health benefits and cellular mechanisms of action.

Epidemiological evidence has established that ingestion of long-chain polyunsaturated omega-3 fatty acids (omega-3 PUFAs), abundant in fish oils, have profound effects on many human disorders and diseases, including cardiovascular disease and cancer. Here we briefly review the dietary recommendations and the food sources that are naturally enriched by these fatty acids. There are also a number of products including eggs, bread, and cereals available to supplement omega-3 fatty acid dietary intake. Some of these supplements are proposed to aid different pathological conditions. While the beneficial effects of omega-3 fatty acids can no longer be doubted, their molecular mechanism of action remains elusive. Without question, the action of omega-3 fatty acids is complex and involves a number of integrated signaling pathways. This review focuses on one of the possible cellular mechanisms by which the omega-3 PUFAs, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), may function. Studies with cancer cells suggest that DHA induces cell cycle arrest and apoptosis by activating protein phosphatases, leading to dephosphorylation of retinoblastoma protein (pRB). Protein phosphatases are also involved with the protein Bcl2, which regulates the release of cytochrome c from mitochondria, and eventually, activation of the apoptotic enzyme caspase 3.