Protective action of arachidonic acid against alloxan-induced cytotoxicity and diabetes mellitus

Previous studies showed that essential fatty acid (EFA) deficiency, conjugated linoleic acid and troglitazone exert a protective effect in animal models of diabetes mellitus. Here we show that alloxan-induced in vitro cytotoxicity and apoptosis in an insulin secreting rat insulinoma, RIN, cells can be prevented by arachidonic acid (AA) and that both cyclo-oxygenase and lipoxygenase inhibitors do not block this protective action. Alloxan-induced diabetes in male Wistar rats was also prevented by oral supplementation of AA, gamma-linolenic acid (GLA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). This protective action is best when the animals were pre-treated with the fatty acid. These results suggest that polyunsaturated fatty acids can prevent alloxan-induced diabetes mellitus in experimental animals and may be useful to prevent diabetes mellitus in the high-risk population.     

Arachidonic acid and lipoxin A4 as possible endogenous anti-diabetic molecules

In both type 1 and type 2 diabetes mellitus, increased production of pro-inflammatory cytokines and reactive oxygen species (ROS) occurs that induce apoptosis of β cells and cause peripheral insulin resistance respectively though the degree of their increased production is higher in type 1 and less in type 2 diabetes mellitus. Despite this, the exact mechanism(s) that lead to increased production of pro-inflammatory cytokines: interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) and ROS is not known. Studies showed that plasma concentrations of arachidonic acid (AA) and lipoxin A₄ (LXA₄) are low in alloxan-induced type 1 diabetes mellitus in experimental animals and patients with type 2 diabetes mellitus. Prior administration of AA, eicosapentaenoic and docosahexaenoic acids (EPA and DHA, respectively) and transgenic animals that produce increased amounts of EPA and DHA acids were protected from chemical-induced diabetes mellitus that was associated with enhanced formation of LXA₄ and resolvins, while protectin D₁ ameliorated peripheral insulin resistance. AA, LXA₄, resolvins and protectins inhibit IL-6 and TNF-α production and suppress ROS generation. Thus, AA and lipoxins, resolvins and protectins may function as endogenous anti-diabetic molecules implying that their administration could be useful in the prevention and management of both types of diabetes mellitus.     

Effect of prostaglandins against alloxan-induced diabetes mellitus

Previously, we observed that alloxan-induced in vitro cytotoxicity and apoptosis in an insulin secreting rat insulinoma, RIN, cells was prevented by prior exposure to prostaglandin (PG) E(1), PGE(2), PGI(2), PGF(1)(alpha), and PGF(3)(alpha) (P<0.05 compared to alloxan), whereas thromboxane B(2) (TXB(2)) and 6-keto-PGF(1)(alpha) were ineffective. In an extension of these studies, we now report that prior intraperitoneal administration of PGE(1), PGE(2), PGF(1)(alpha), and PGF(3)(alpha) prevented alloxan-induced diabetes mellitus in male Wistar rats, whereas PGI(2), TXB(2), and 6-keto PGF(1)(alpha) were not that effective. PGE(1), PGE(2), PGF(1)(alpha), and PGF(3)(alpha) not only attenuated chemical-induced diabetes mellitus but also restored the antioxidant status to normal range in red blood cells and pancreas. These results suggest that PGE(1), PGE(2), PGF(1)(alpha), and PGF(3)(alpha) can abrogate chemically induced diabetes mellitus in experimental animals and attenuate the oxidant stress that occurs in diabetes mellitus.     

Suppressive effects of electrolyzed reduced water on alloxan-induced apoptosis and type 1 diabetes mellitus

Electrolyzed reduced water, which is capable of scavenging reactive oxygen species, is attracting recent attention because it has shown improved efficacy against several types of diseases including diabetes mellitus. Alloxan produces reactive oxygen species and causes type 1 diabetes mellitus in experimental animals by irreversible oxidative damage to insulin-producing β-cells. Here, we showed that electrolyzed reduced water prevented alloxan-induced DNA fragmentation and the production of cells in sub-G1 phase in HIT-T15 pancreatic β-cells. Blood glucose levels in alloxan-induced type 1 diabetes model mice were also significantly suppressed by feeding the mice with electrolyzed reduced water. These results suggest that electrolyzed reduced water can prevent apoptosis of pancreatic β-cells and the development of symptoms in type 1 diabetes model mice by alleviating the alloxan-derived generation of reactive oxygen species.     

Effect of L-arginine-nitric oxide system on the metabolism of essential fatty acids in chemical-induced diabetes mellitus

Several studies have shown that the activities of delta-6-desaturase and delta-5-desaturase are depressed in experimental diabetes and in humans with insulin- and non-insulin-dependent diabetes mellitus (type I and type II diabetes mellitus respectively). Furthermore, treatment with insulin is known to correct the defects in desaturases in rats and humans with diabetes, especially in type I. In a recent study, we demonstrated that L-arginine and nitric oxide can prevent alloxan-induced beta cell damage, and the severity of diabetes, and restore the antioxidant status to near normal levels. But, no information is available as to the relationship between L-arginine-nitric oxide system and the metabolism of essential fatty acids in diabetes mellitus. In the present study, it was noted that the plasma levels of saturated fatty acids: stearic and palmitic were increased where as unsaturated fatty acids such as oleic, linoleic, gamma-linolenic and eicosapentaenoic acids (OA, LA, GLA and EPA respectively) were decreased in alloxan-induced diabetic rats. In the liver phospholipid (PL) fraction, GLA, DGLA (dihomo-GLA) and alpha-linolenic acid (ALA) were decreased in the alloxan-treated group; in the muscle PL fraction, LA, GLA and DGLA were low, whereas an increase in the saturated fatty acid content was noted. L-arginine (the precursor of nitric oxide) and sodium nitroprusside (a nitric oxide donor) treatment of alloxan-induced diabetic rats enhanced the levels of LA, GLA and DGLA. Further, nitric oxide synthase inhibitor, L-NMMA, prevented this beneficial action of L-arginine-nitric oxide system on essential fatty acid metabolism. The abnormalities in the essential fatty acid metabolism observed also reverted to normalcy following control of diabetes with insulin. These results indicate that nitric oxide can enhance the activities of delta-6- and delta-5 desaturases, which are depressed in diabetes, and suggests that there is a close interaction between L-arginine-nitric oxide system and the metabolism of essential fatty acids.     

Eicosapentaenoic acid and arachidonic acid: collaboration and not antagonism is the key to biological understanding

Much of the literature on omega-3 and omega-6 fatty acids suggests that desirable effects of omega-3 fatty acids are in part related to depletion of arachidonic acid (AA). However, in rats and humans, we have found that low doses of EPA actually elevate membrane AA phospholipid concentrations. In patients with schizophrenia, treatment with eicosapentaenoic acid (EPA) produced clinical improvement, but that improvement was greater at a dose of 2 g/day than at 4 g/day. The improvement was not significantly correlated with changes in either EPA or docosahexaenoic acid (DHA) but was highly significantly positively correlated with rises in red cell membrane AA. We suggest that elevation of concentrations of both AA and EPA in cell membranes may be important for health.     

Effects of omega-3 and -6 fatty acids on Mycobacterium tuberculosis in macrophages and in mice

We recently showed that treatment of macrophages prior to Mycobacterium tuberculosis infection with the pro-inflammatory omega-6 lipid, arachidonic acid (AA) enhanced bacterial killing whereas the anti-inflammatory, omega-3 lipid eicosapentaenoic acid (EPA) stimulated bacterial growth. Here we tested if these effects were depending on when lipids were added to macrophages: before or during Mycobacterium smegmatis or M. tuberculosis infection. Collectively, our data suggested that a high omega-6 diet might be beneficial against mycobacteriosis, while a high omega-3 diet might be detrimental. AA also stimulated TNF-alpha secretion in M. tuberculosis-infected macrophages whereas EPA inhibited this process. AA strongly activated the MAP kinase p38 in uninfected cells but M. tuberculosis infected cells blocked the ability of AA to activate p38; AA-dependent killing is therefore independent of p38. We therefore tested diets enriched in omega-3 and omega-6 lipids on a mouse model of tuberculosis. In contrast to the in vitro results, the omega-6 tended to increase survival of M. tuberculosis in mice, while omega-3- tended to increase pathogen killing. Overall our results together with those previously reported in the literature suggest that it is almost impossible to predict, at the whole organism level, if a diet enriched in omega-3 or -6 will be beneficial or detrimental to intracellular pathogens.     

Effects of fatty acid unsaturation numbers on membrane fluidity and α-secretase-dependent amyloid precursor protein processing

Fatty acids may integrate into cell membranes to change physical properties of cell membranes, and subsequently alter cell functions in an unsaturation number-dependent manner. To address the roles of fatty acid unsaturation numbers in cellular pathways of Alzheimer's disease (AD), we systematically investigated the effects of fatty acids on cell membrane fluidity and α-secretase-cleaved soluble amyloid precursor protein (sAPP(α)) secretion in relation to unsaturation numbers using stearic acid (SA, 18:0), oleic acid (OA, 18:1), linoleic acid (LA, 18:2), α-linolenic acid (ALA, 18:3), arachidonic acid (AA, 20:4), eicosapentaenoic acid (EPA, 20:5), and docosahexaenoic acid (DHA, 22:6). Treatments of differentiated human neuroblastoma (SH-SY5Y cells) with AA, EPA and DHA for 24h increased sAPP(α) secretion and membrane fluidity, whereas those treatments with SA, OA, LA and ALA did not. Treatments with AA and DHA did not alter the total expressions of amyloid precursor protein (APP) and α-secretases in SH-SY5Y cells. These results suggested that not all unsaturated fatty acids but only those with 4 or more double bonds, such as AA, EPA and DHA, are able to increase membrane fluidity and lead to increase in sAPP(α) secretion. This study provides insights into dietary strategies for the prevention of AD.     

Role of omega-3 polyunsaturated fatty acids on cyclooxygenase-2 metabolism in brain-metastatic melanoma

Cyclooxygenase-2 (COX-2) is important in the progression of epithelial tumors. Evidence indicates that omega-6 PUFAs such as arachidonic acid (AA) promote the growth of tumor cells; however, omega-3 fatty acids [eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)] inhibit tumor cell proliferation. We investigated the effects of omega-3 PUFA on the expression and function of COX-2 in 70W, a human melanoma cell line that metastasizes to the brain in nude mice. We show that 1) tumor necrosis factor-alpha upregulates the expression of both COX-2 mRNA and prostaglandin E2 (PGE2) production, and 2) omega-3 and omega-6 PUFA regulate COX-2 mRNA expression and PGE2 production. AA increased COX-2 mRNA expression and prostaglandin production in omega-6-stimulated 70W cells. Conversely, COX-2 mRNA expression decreased in cells incubated with EPA or DHA. AA increased Matrigel invasion 2.4-fold, whereas EPA or DHA did not. Additionally, PGE2 increased in vitro invasion 2.5-fold, whereas exposure to PGE3 significantly decreased invasion. Our results demonstrate that incubation of 70W cells with either AA or PGE2 increased invasiveness, whereas incubation with EPA or DHA downregulated both COX-2 mRNA and protein expression, with a subsequent decrease in Matrigel invasion. Taken together, these results indicate that omega-3 PUFA regulate COX-2-mediated invasion in brain-metastatic melanoma.     

Protective effects of arachidonic acid against palmitic acid-mediated lipotoxicity in HIT-T15 cells

Saturated fatty acids have been considered major contributing factors in type 2 diabetes, whereas unsaturated fatty acids have beneficial effects for preventing the development of diabetes. However, the effects of polyunsaturated fatty acids in pancreatic β cells have not been reported. Here, we examined the effects of arachidonic acid (AA) on palmitic acid (PA)-mediated lipotoxicity in clonal HIT-T15 pancreatic β cells. AA prevented the PA-induced lipotoxicity as indicated by cell viability, DNA fragmentation and mitochondrial membrane potential, whereas eicosatetraynoic acid (ETYA), a non-metabolizable AA, had little effect on PA-induced lipotoxicity. In parallel with its protective effects against PA-induced lipotoxicity, AA restored impaired insulin expression and secretion induced by PA. AA but not ETYA increased intracellular triglyceride (TG) in the presence of PA compared with PA alone, and xanthohumol, a diacylglycerol acyltransferase (DGAT) inhibitor, reversed AA-induced protection from PA. Taken together, our results suggest that AA protects against PA-induced lipotoxicity in clonal HIT-T15 pancreatic β cells, and the protective effects may be associated with TG accumulation, possibly through sequestration of lipotoxic PA into TG.     

Quantitative association between altered plasma esterified omega-6 fatty acid proportions and psychological stress.

Medical students (MS) tested during the first year of medical school showed both greater stress on the Brief Symptom Inventory and lower plasma proportions of total esterified arachidonic acid (AA, C20:4n-6), and its omega-6 fatty acid (FA) precursor, linoleic acid (C18:2n-6) than control laboratory workers. This association suggests that omega-6 FA metabolism may be affected during stress. Low AA values might result from depletion of plasma stores for immunoregulatory prostenoids formation or from modification of metabolic pathways by cortisol or other cytokine compounds implicated in stress. Values for other major FA and the omega-3 neuronal metabolic substrate, docosahexaenoic acid (DHA, C22:6n-3) were similar between students and controls. The clear preservation of the omega-3 FA pathway suggests their programmed availability for neuronal function during stress. Since plasma FA proportions may affect immune cell membrane function(s), we suggest that altered values of plasma FAs may be an important component of the physiological effects of psychological stress.     

Saturated free fatty acids,palmitic acid and stearic acid,induce apoptosis by stimulation of ceramide generation in rat testicular Leydig cell

In men, obesity has generally been associated with reduced plasma testosterone levels and with elevation of the plasma free fatty acids (FFAs). In this study, we investigated the effects of saturated FFAs including palmitic acid (PA) and stearic acid (SA), and polyunsaturated FFA arachidonic acid (AA) on the survival of rat testicular Leydig cell cultured in vitro. PA and SA markedly suppressed Leydig cell survival in a time- and dose-dependent manner. In contrast, AA stimulated the cell proliferation at 5-10 times of physiological concentration. The suppressive effect of PA and SA on cell survival was caused by apoptosis evidenced by DNA ladder formation and Annexin V-EGFP/propidium iodide staining of the cells. The apoptotic effect of PA was possibly mediated by ceramide generation because it could be completely blocked by ceramide synthase inhibitor fumonisin B1 and exogenous ceramide itself could directly induce apoptosis in vitro. Surprisingly, the apoptosis induced by PA could be partly prevented by AA. These results indicate that PA and SA induce apoptosis in testicular Leydig cells by ceramide production and these apoptotic effects may be a possible mechanism for reproductive abnormalities in obese men, and AA can partly prevent the apoptotic effect induced by saturated FFA.     

Long-chain acyl-CoA synthetase 6 preferentially promotes DHA metabolism

Previously we demonstrated that supplementation with the polyunsaturated fatty acids (PUFA) arachidonic acid (AA) or docosahexaenoic acid (DHA) increased neurite outgrowth of PC12 cells during differentiation, and that overexpression of rat acyl-CoA synthetase long-chain family member 6 (Acsl6, formerly ACS2) further increased PUFA-enhanced neurite outgrowth. However, whether Acsl6 overexpression enhanced the amount of PUFA accumulated in the cells or altered the partitioning of any fatty acids into phospholipids (PLs) or triacylglycerides (TAGs) was unknown. Here we show that Acsl6 overexpression specifically promotes DHA internalization, activation to DHA-CoA, and accumulation in differentiating PC12 cells. In contrast, oleic acid (OA) and AA internalization and activation to OA-CoA and AA-CoA were increased only marginally by Acsl6 overexpression. Additionally, the level of total cellular PLs was increased in Acsl6 overexpressing cells when the medium was supplemented with AA and DHA, but not with OA. Acsl6 overexpression increased the incorporation of [(14)C]-labeled OA, AA, or DHA into PLs and TAGs. These results do not support a role for Acsl6 in the specific targeting of fatty acids into PLs or TAGs. Rather, our data support the hypothesis that Acsl6 functions primarily in DHA metabolism, and that its overexpression increases DHA and AA internalization primarily during the first 24 h of neuronal differentiation to stimulate PL synthesis and enhance neurite outgrowth.     

In vitro mimicry of essential fatty acid deficiency in human endothelial cells by TNFalpha impact of omega-3 versus omega-6 fatty acids

Severe endothelial abnormalities are a prominent feature in sepsis with cytokines such as tumor necrosis factor (TNF)alpha being implicated in the pathogenesis. As mimic to inflammation, human umbilical vascular endothelial cells (HUVEC) were incubated with TNFalpha for 22 h, in the absence or presence of the omega-6 fatty acid (FA), arachidonic acid (AA), or the alternative omega-3 FA eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). TNFalpha caused marked alterations in the PUFA profile and long chain PUFA content of total phospholipids (PL) decreased. In contrast, there was a compensatory increase in mead acid [MA, 20:3(omega-9)], the hallmark acid of the essential fatty acid deficiency (EFAD) syndrome. Corresponding changes were noted in phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol, but not in the sphingomyelin fraction. Supplementation with AA, EPA, or DHA markedly increased the respective FA contents in the PL pools, suppressed the increase in MA, and resulted in a shift either toward further predominance of omega-6 or predominance of omega-3 FA. We conclude that short-term TNFalpha incubation of HUVEC causes an EFAD state hitherto only described for long-term malnutrition, and that endothelial cells are susceptible to differential influence by omega-3 versus omega-6 FA supplementation under these conditions.     

Reducing the Dietary Omega-6:Omega-3 Utilizing α-Linolenic Acid; Not a Sufficient Therapy for Attenuating High-Fat-Diet-Induced Obesity Development Nor Related Detrimental Metabolic and Adipose Tissue Inflammatory Outcomes

Aims

To examine the effect of manipulating the omega-6:omega-3 (1∶1, 5∶1, 10∶1, and 20∶1) utilizing only α-linolenic and linoleic acid within a clinically-relevant high-fat diet (HFD) composed of up to seven sources of fat and designed to be similar to the standard American diet (MUFA∶PUFA of 2∶1, 12% and 40% of calories from saturated and total fat, respectively) on body composition, macrophage polarization, inflammation, and metabolic dysfunction in mice.

Methods

Diets were administered for 20 weeks. Body composition and metabolism (HOMA index and lipid profile) were examined monthly. GC-MS was utilized to determine the eicosapentaenoic acid (EPA):arachidonic acid (AA) and the docosahexaenoic acid (DHA):AA in AT phospholipids. Adipose tissue (AT) mRNA expression of chemokines (MCP-1, Fetuin-A, CXCL14), marker genes for M1 and M2 macrophages (CD11c and CD206, respectively) and inflammatory markers (TNF-α, IL-6, IL-1β, TLR-2, TLR-4, IL-10, GPR120) were measured along with activation of NFκB, JNK, and STAT-3. Macrophage infiltration into AT was examined using F4/80 immunohistochemistry.

Results

Any therapeutic benefit produced by reducing the omega-6:omega-3 was evident only when comparing the 1∶1 to 20∶1 HFD; the 1∶1 HFD resulted in a lower TC:HDL-C and decreased AT CXCL14 gene expression and AT macrophage infiltration, which was linked to a higher EPA:AA and DHA:AA in AT phospholipids. However, despite these effects, and independent of the omega-6:omega-3, all HFDs, in general, led to similar levels of adiposity, insulin resistance, and AT inflammation.

Conclusion

Reducing the omega-6:omega-3 using α-linolenic acid is not an effective therapy for attenuating obesity and type II diabetes mellitus development.     

Fatty acid composition of the postmortem prefrontal cortex of adolescent male and female suicide victims

Prior epidemiological, prospective intervention, and peripheral and central fatty acid composition studies suggest that omega-3 fatty acid deficiency may be associated with the pathoaetiology of depression and suicide. In the present study, we determined the fatty acid composition of the postmortem prefrontal cortex (PFC) of adolescent male and female suicide victims and age-matched controls. Fatty acid composition (wt% total fatty acids) and concentrations (μmol/g) were determined in the postmortem PFC (Brodmann area 10) of male and female adolescent (aged 13–20 years) suicide victims (n=20) and age-matched controls (n=20) by gas chromatography. None of the major polyunsaturated fatty acids including the principle brain omega-3 fatty acid, docosahexaenoic acid (DHA), monounsaturated fatty acids, or saturated fatty acids differed significantly between adolescent suicide victims and controls before or after segregation by gender. The arachidonic acid (AA, 20:4n-6): DHA ratio and adrenic acid (22:4n-6) composition were negatively correlated with age at death in controls but not in suicides, and males exhibited a greater AA:DHA ratio irrespective of cause-of-death. These results demonstrate that adolescent male and female suicide victims do not exhibit DHA deficits in the postmortem PFC relative to age-matched controls, and suggest that suicide victims do not exhibit the normal age-related decrease in adrenic acid composition and the AA:DHA ratio.     

Maternal and fetal brain contents of docosahexaenoic acid (DHA) and arachidonic acid (AA) at various essential fatty acid (EFA), DHA and AA dietary intakes during pregnancy in mice

We investigated essential fatty acids (EFA) and long-chain polyunsaturated fatty acids (LCP) in maternal and fetal brain as a function of EFA/LCP availability to the feto-maternal unit in mice. Diets varying in parent EFA, arachidonic acid (AA), and docosahexaenoic acid (DHA) were administered from day 3 prior to conception till day 15 of pregnancy. We concentrated on DHA, AA, Mead acid, and EFA-index [(omega-3+omega-6)/(omega-7+omega-9)] in maternal erythrocytes, maternal brain, and fetal brain. It was found that erythrocyte EFA/LCP sensitively reflects declining EFA/LCP status in pregnancy, although this decline was not apparent in maternal brain. Differences in erythrocyte EFA/LCP coincided with larger differences in fetal brain EFA/LCP as compared to EFA/LCP in maternal brain. Both maternal and fetal brains were affected by short-term EFA/LCP intake, but the developing fetal brain proved most sensitive. The inverse relationship between fetal brain AA and DHA suggests the need of a maternal dietary DHA/AA balance, at least in mice.     

Fatty acid uptake by breast cancer cells (MDA-MB-231): Effects of insulin,leptin, adiponectin,and TNFα

In order to exert metabolic effects, fatty acids must be taken up by cells and metabolize effectively to different classes of cellular lipids (triacylglycerols, phospholipids, etc.) for incorporation into different cellular and intracellular compartments. Therefore, the main aim of the present study is to investigate the uptake and metabolism of fatty acids representing three different series of fatty acids such as oleic acid, 18:1n-9 (OA), arachidonic acid, 20:4n-6 (AA), and eicosapentaneoic acid, 20:5n-3 (EPA) by breast cancer cells, MDA-MB-231. Moreover, we investigated the effects of insulin and several adipokines on the fatty acid uptake by these cells as obesity and insulin resistance syndrome have been suggested to affect breast cancer risk. We report for the first time that AA was predominantly taken up by these cells compared with EPA and OA. Pre-incubation of these cells with TNFα stimulated most of the uptake of EPA (30%), whereas uptake of OA and AA was stimulated only 10–15% compared with the controls. Insulin, leptin, and adiponectin had no effect on fatty acid uptake by these cells. Together these results demonstrate that preferential uptake of AA in MDA-MB-231 cells, and the fatty acid uptake activity of these cells is influenced by TNFα.