Although there are studies demonstrating positive effects of PEs treatment in several diseases related with menopause, it is important to take into account that its excessive consumption can trigger harmful effects on health. Not to mention that not all PEs providedprotection or even increased women’s quality life during menopause. For this purpose, is critical to perform the screening of PEs in our experimental model in order to test the toxicity of the compounds in study (coumestrol and enterodiol) and also to roughly estimate safe concentrations. Thus, assessment of PEs toxicity on myoblast proliferation was also performed with different concentrations and two different incubation times. The results presented in Figure 14 showed that no toxicity occurs with any of the concentrations tested. As well as, no differences was observed between both time points (27 and 30 hours). These results are in agreement with several studies showing that some PEs, including genistein may have beneficial effect in the heart (Fan et al., 2013) and resveratrol (Zhao et al., 2008). Despite the reduced toxicity in this cell model, or in the whole tissue it is crucial to take into account that PEs, including genistein, coumestrol and resveratrol can exert genotoxic effects in several in vitro models (Stopper et al., 2005).
Type 2 diabetes mellitus (T2DM) is one of the most prevalent metabolic diseases worldwide caused by in- sulin resistance of the peripheral tissues and impaired insulin secretion of pancreatic β-cells . Physical in- activity and hypercaloric diets, rich in carbohydrates and saturated fats, are thought to be responsible for an increase in body weight and can cause the metabolic syndrome [2–4], which is characterized by obesity, hypertension and dyslipidemia, and often precedes the manifestation of T2DM. Chronically elevated plasma levels of non-esterified fatty acids (NEFAs), which are associated with the metabolic syndrome , can sup- press insulin secretion and cause β-cell dysfunction and loss by apoptosis. This so-called lipotoxicity is sub- ject to intensive research and scientific debate. Only long-chain saturated NEFAs induce lipotoxicity in ro- dent insulin-producing cells in in-vitro experiments, whereas unsaturated NEFAs are not toxic . Short- and medium-chain fatty acids are metabolized in the mitochondrial β-oxidation, whereas long-chain fatty acids can be metabolized in the mitochondria but also in the peroxisomes when cells were exposed to high NEFA concentrations [7, 8]. Very long-chain fatty acids are exclusive substrates for the peroxisomal β-oxidation [8–10]. The electron transfer in the first step of the per- oxisomal β-oxidation catalyzed by the enzyme acyl- CoA-oxidase results in high concentrations of hydrogen peroxide, which can cause cellular damage . In con- trast to other cell types pancreatic β-cells do not ex- press the H 2 O 2 inactivating enzyme catalase [11, 12].
We hypothesized that the cytoprotection provided by a -linolenic acid was a common function of rat primary hepatocytes and would be effective with clinically-rele- vant palmiticacidlipotoxicity. We have previously proved that a -linolenic acid protects against endoplas- mic reticulum stress-mediated apoptosis of stearic acidlipotoxicity . In this paper, we report that: (1) The characteristics of palmiticacid-mediated ER stress and apoptosis in primary hepatocytes; (2) a-linolenic acid could provide protectionagainst the cell death induced by palmiticacid; (3) Take the role of GRP78, GRP94 expression and induction of CHOP into consideration, the beneficial effects were mediated via modification of the ER stress process with specific attention.
Besides protective effects of unsaturated fatty acids against saturated FA load via TG accumulation, it cannot be ruled out that there may be other mechanisms re- sponsible for the beneficial effects of AA against PA- inducedlipotoxicity. The potent unsaturated fatty acid AA itself is readily metabolized into lipid second mes- sengers such as leukotrienes and prostaglandins. To as- sess whether AA metabolites could confer the protective action against PA-inducedlipotoxicity or what sort of AA metabolites could contribute to their protection, specific inhibitors interfering with 3 different pathways for AA metabolism could be employed. No inhibitors af- fected the protective effects of AA against PA load (data not shown), thereby suggesting that AA-CoA but not AA metabolites is essential metabolites for protection of AA against PA-mediated lipotoxicity. Secondly, cAMP accumulation is suggested to reverse PA-induced apop- tosis via both protein kinase A- and cAMP-guanine nu- cleotide exchange factor-dependent pathways in β-cells . However, either cAMP-generating agents or non- metabolizable cAMP analogue bromo-cAMP did not exert significantly protective effects on the PA-inflicted cell damage (data not shown). Thirdly, the cytotoxic effects of PA might be derived from its physicochemical property. A relatively large surge in partitioning of PA into the phospholipid membrane lowers the membrane fluidity due to high melting temperature (T M ) . In
C2C12 myoblasts and J774 macrophages were cultured in DMEM containing 4.5 mM glucose, 10% foetal bovine serum (FBS) and 1% antibiotic anti-fungal (ABAF) mixture. Before study, differ- entiation of myoblasts into myotubes was achieved by switching to DMEM containing 2% horse serum for 5 days. Macrophages were treated with 200 ng/ml phorbol myristate acetate (PMA) for 3 days before use (Karten et al., 1999). Macrophage treatment medium was generated by coupling DMEM containing 10% FBS, 1% ABAF and 2% bovine serum albumin (BSA) with 0.75 mM palmiticacid (SFA), 0.75 mM palmitoleic acid (UFA, chosen because of its identi- cal acyl chain length), a combination of both, or 10 ng/ml of lipo- polysaccharide (LPS) as positive control. This was added to J774 cells for 8 h, before being aspirated and the cells washed in PBS x3. Absence of carry-over of FAs into the CM was conﬁrmed by measurement using a kit (Wako Chemicals, Neuss, Germany). Fresh DMEM was then added for 16 h and the CM generated trans- ferred to C2C12 myotubes for a further 16 h. Myotubes were then serum-starved for 2 h and selected wells stimulated with 100nM insulin (Novo Nordisk, Crawley, UK) prior to measurement of glycogen synthesis or lysis and western blotting.
Activation the UPR caused ER stress and cell death, which is bring out by an excess of saturated fatty acids in many cell types [4, 27–29]. Palmiticacid activates ER stress and has been suggested to play a crucial role in the NAFLD. Therefore, dam- ages in ER stabilization is the cause of many dis- eases and contributes to lipotoxicity. The purpose of our study is to find out the relationship between the saturated fatty acids and ER stress. Our study indicates that (1) chlorogenic acid can reduce cellular dysfunction and apoptosis caused by thapsi- gargin. (2) chlorogenic acid can reduce cellular dysfunction and apoptosis caused by palmiticacid. (3) by reducing ER stress and apoptosis, chlorogenic acid protects hepatocytes from palmiticacid ’ s lipotoxicity.
Materials and methods: Fifty male Wistar rats assigned to five groups; control, ISO-treated group (100 mg/kg), ISO + vitamin E-treated group (100 IU/kg), ISO + L-carnitine (100 mg/kg) and ISO + vitamin E + L-carnitine treated group. At the end of the experiment, serum cardiac enzyme as well as the cardiac level malondi- aldehyde (MDA), antioxidant enzymes and inflammatory cytokines interleukin-6, tumour necrosis factor alpha (TNF-a) were assessed. Histopathological changes in the left ventricle wall were assessed using the light and electron microscopy. Results: Treating rats with vitamin E and L-carnitine could alleviate ISO-induced changes as it significantly reduced the serum level cardiac enzymes, MDA and IL-6, TNF-a and improved the antioxidants enzymes (SOD, GSPxase and GSRase). Histopathologically, they improved cardiac fibres atrophy, haemorrhages between cardiac fibres, lost striations, and disturbed sarcomere structure. The combined ef- fect of vitamin E and L-carnitine was more superior compared to the other groups. Conclusions: Combined administration of vitamin E, L-carnitine ameliorated the biochemical and histopathological cardiac injury induced by ISO. The effect seemed to be mediated through the antioxidant and anti-inflammatory effect of vitamin E, L-carnitine. Administration of these two elements is recommended for patient at risk for myocardial infarction. (Folia Morphol 2019; 78, 2: 274–282)
After treatment, cells (n = 150 mice, about 50 mice in each group) were collected and washed with an ice-cold phos- phate buffer solution (PBS) and lysed with radioimmunopre- cipitation assay (RIPA) lysis buffer containing 1% phenylmethylsulfonyl fluoride (PMSF), and the total protein concentration was measured by a bicinchoninic acid (BCA) assay according to the manufacturer’ s instructions. For each sample, 30 μg total protein was separated on a 12% poly- acrylamide gel before being transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). After blocking in Tris-buffered saline-Tween-20 (TBST) supplemented with 5% skim milk at 25 °C for 1 h, the membranes were incubated overnight at 4 °C with a pri- mary antibody (Additional file 1: Table S1). After washing, the membranes were incubated with a secondary antibody conjugated with horseradish peroxidase at 37 °C for 30 min. Finally, the immunoreactive bands were visualized using a Super Signal West Pico kit (Proteintech, Wuhan, China) with a Bio-Rad imaging system (Bio-Rad, CA, USA) according to the manufacturer’s instructions. The protein band densities were semiquantified by densitometric analysis using ImageJ software 1.48 (Bethesda, MD, USA).
Oxidative stress is well known to play the major role in the pathophysiology of IBD [49,50]. Induction of UC in experimental animals causes oxidative injury due to im- balance between the levels of pro-oxidant and antioxi- dant systems . UC is characterized by overproduction of reactive oxygen and nitrogen species leading to signifi- cant cellular adverse effects such as LPO and damage to tissue proteins and nucleic acids [10,52]. Furthermore, increased levels of free radicals were found in colonic tis- sue specimens of patients with UC [53,54]. Antioxidant enzymes such as SOD and CAT, and the non-enzymatic sulfhydryl groups play the major role in the organism defense against excess free radicals generated under dis- ease conditions . In the present investigation, concen- trations of protein and non-protein sulfhydryl groups as well as activities of the antioxidant enzymes such as SOD and CAT were severely reduced in the colon following AA administration, which clearly indicates increased level of oxidative stress which may damage cells by lipid peroxidation of membranes and oxidation of cellular pro- teins. Indeed, increased levels of TBARS and free radicals found in the study may damage cells as observed by histopathological investigations.
In addition to generating energy, there are several additional potential consequences to upregulation of the ETS in T cells. Increased reactive oxygen species production as a by-product of increased electron trans- port has been reported to have a negative effect on Th17 development (14) and, as such, may play an indi- rect positive role in Treg development. We find Tregs and Tact release ATP (data not shown), which can rapidly be converted to antiinflammatory adenosine via CD39 and CD73 (40–45). It remains to be demon- strated whether secreted ATP, rather than ATP released from dying cells, represents a significant substrate for membrane-bound ectonucleotidases. A further consequence that we have demonstrated in this study may be intracellular removal of potentially harmful oxidative substrates, such as long chain free fatty acids. We show that Foxp3-expressing cells are relatively resistant to fatty acid–induced death due to removal by β-oxidation. The combination of increased lipid uptake and increased ETS protein production with con- comitant elevated fatty acid β-oxidation is cytoprotective to Tregs. This mechanism may be physiologically important in multiple ways. In human and mouse plasma, the free fatty acid concentration ranges from 200-–600 μM but can be 3-fold higher in diabetes (46). Palmitate is the most abundant plasma-free fatty acid at around 25% (47, 48); however, elevated plasma free fatty acid levels are associated with obesity and the metabolic syndrome and cause inflammation, activation of macrophages, and death of multiple cell types including pancreatic β cells, neurons, and T lymphocytes (24, 49, 50). It will be important to ascertain whether resistance to lipotoxicity by Tregs plays a role in survival in vivo. Treg numbers in visceral fat may increase or decline depending on multiple factors, which might include inflammatory mediators, adipose cell types, and different polarized macrophages in the adipose tissue. Human Tregs have been shown to be either increased in visceral adipose tissue (51–53), diminished (51), or increased in subcutaneous fat (53, 54). They have also been shown to be decreased in visceral fat in humans and mice (54, 55). Dysregulation of lipid metabolism can lead to tissue-specific inflammatory pathology. Nonalcoholic fatty liver disease has been shown to result in release of linoleic acid (C18:2) from hepatocytes, leading to preferential CD4 + T
Under the stimulation of PA, the mitochondrial membrane potential of hepatocytes decreased, and the production of mitochondrial reactive oxygen species increased. No matter what the initial cause is, mitochondrial dysfunction plays an important role in nonalcoholic steatohepatitis, . Autophagy maintains the number and health of mitochondria in cells by clearing too many or damaged mitochondria. Mitochondria are the main organs of active oxygen generation in cells. When mitochondria damage, the membrane potential decreases, membrane permeability increases, and the release of reactive oxygen is increased. At the same time, the release of cytochrome c and other factors into the cytoplasm can induce mitochondrial apoptosis, and autophagy can reduce the occurrence of . In HepG2 hepatocytes, mitochondrial damage induced by peanut four enoic acid, sulfoxide or chloroform can be alleviated by autophagy agonist . Therefore, CBD can reduce the mitochondrial damage induced by PA and reduce mitochondrial related oxidative stress and apoptosis by promoting autophagy flow.
In this study, we treated MPC5 cells with PA to create an in vitro hyperlipidemia model, and found that PA could increase lipid accumulation, and induce cell death and apoptosis in podocytes.
The mechanism of podocyte apoptosis is complex. It has been reported that palmitate can induce podocyte apoptosis by activating the endoplasmic reticulum (ER) stress-mediated apoptotic pathway [37–41]; however, it remains unknown whether the two primary apoptosis signaling pathways (death receptor-mediated pathway and mitochondria-mediated pathway) are also involved in the execution of PA-induced podocyte apoptosis. The death receptor-mediated pathway is an extrinsic signaling pathway that is activated upon ligation of cell surface death receptors, including Fas, the TRAIL receptor, and tumor necrosis factor receptor , and recruits the adapter protein FADD and procaspase-8 to form the death- inducing signaling complex (DISC). This protein complex serves as a platform for caspase activation, and the auto-catalytic activation of caspase-8 at the DISC leads to activation of caspase-3, triggering the apoptotic process [43, 44]. In this study, we found that the levels of FADD, caspase-8, and Bid did not significantly change during the process of PA-induced podocyte apoptosis. Mitochondria-mediated pathway of apoptosis is the intrinsic pathway, which is triggered upon mitochondrial injury including loss of integrity of mitochondrial outer membrane. Then, cytochrome c is released from the mitochondria into the cytosol. The Bcl-2 protein family controls this process. Once in the cytosol, cytochrome c recruits procaspase-9 to the apoptosome, which induces cleavage of downstream effector caspase-3, resulting in apoptosis [45–47]. Here, we found that PA treatment resulted in the increased expression of Bax/Bcl-2, depolarization of ΔΨm, mitochondrial swelling and destruction, and release of cytochrome c from the mitochondria into the cytosol of podocytes. These perturbations, in turn, activate caspase cascades (caspase-9 and caspase-3) in a dose-dependent manner. PARP, a marker of caspase-3 activation during apoptosis , was also clearly cleaved after PA treatment in podocytes. This finding indicates that the mitochondria-mediated apoptotic pathway, rather than the death receptor pathway, is involved in the process of PA-induced apoptosis in podocytes.
Table 1 shows the amounts of oleic acid which were added to 1.25 g of palmiticacid, freezing temperatures (the temperatures which two-phase is appeared and the mixture be milky) and the mole fractions of palmiticacid. Figs. 2 and 4 indicate the temperature versus mole fraction of palmiticacid for oleic acid- palmiticacid and palmiticacid- pentadecanoic acid, respectively. Figs. 3 shows the natural log of the mole fraction of palmiticacid versus reverse absolute temperature for oleic acid- palmiticacid system.
In order to explore the molecular mechanisms, we detected the mTORC1 signaling and Akt activity by Western blots. As shown in Figure 6A-I, LPS treatment significantly reduced mTORC1 signaling indicated by phosphorylated ribosome protein 6 (pS6) and elevated phos- phorylated 4E-BP1, and Akt activity indicated by phosphorylated Akt (pAkt) both in mice and H9c2 cells. Expectedly, rapamycin treatment after LPS further reduced mTORC1 signaling and enhanced Akt activity significantly, which may prevent cardiomyocytes from LPS-induced injures.
species-rich phenotype. VD has been suggested to modulate energy pathways, including upregulating genes involved with fatty acid oxidation and anti-oxidation . HFD has been shown to cause a reduced PPARγ expres- sion and classical PPARγ targets including CD36 in neu- rons , as well as in the small intestine . In neurons PPARγ was suggested to act as neuronal lipid sensor, sensing and signaling to the central nervous sys- tem clues about the peripheral metabolic status . In the intestinal epithelial cells, PPARγ is suggested to regulate barrier function and microbiome related inflam- mation . We show that inhibition of PPARγ prevents the VD inducedprotectionagainst the PA-induced neur- onal loss in vitro, suggesting that high VD concentra- tions induce PPARγ activity.
A method is described for producing soybean varieties and lines exhibiting palmiticacid contents of at least about 18.0% up to 30.0% or more. The novel soybean lines are obtained from a soybean seed designated A1937NMU-85 and its descendants, particular desirable progeny resulting from the cross of A1937NMU-85 with ElginEMS-421, and further with the cross of selected progeny with A89-259098.
simultaneously elevated. Neither elevated saturated fatty acid concentration is negated by the expression of the other saturated fatty acid in the elevated concentration. The endogenously formed palmiticacid content is from about 14% to about 24% and the endogenously formed stearic acid content is from about 20% to about 30% by weight of the total fatty acid composition. In a preferred embodiment, the endogenously formed linolenic acid (C18:3) component of the soybean oil is no more than about 3% by weight of the total fatty acid composition.
Shh is a critical mediator of the angiogenic effects of EPO. We fur- ther investigated how EPO increases angiogenic cytokine levels in infarcted hearts. Since Akt and ERK, which are activated by EPO, have been reported to regulate VEGF expression (19, 20), we first determined whether EPO increased expression levels of VEGF by activating these kinases in cardiomyocytes. Although both Akt and ERK were activated by EPO in cultured cardiomyo- cytes, activation levels were not so high as compared with other growth factors such as insulin (Supplemental Figure 2B and data not shown). Since EPO-induced upregulation of VEGF was so robust, we hypothesized that other mitogens mediate the EPO- induced upregulation of VEGF. It has recently been reported that carbamylated EPO (CEPO) promotes neural progenitor cell proliferation and their differentiation into neurons through an upregulation of Shh expression (21). Shh, a critical regulator of patterning and growth in various tissues during embryogenesis, has been reported to show angiogenic effects in infarcted hearts (22, 23). We thus examined the involvement of Shh signaling in EPO-induced cardioprotection.