Toxicity of fatty acid profiles of popular edible oils in human EndoC-ßH1 beta-cells.

An inappropriate diet, particularly excessive consumption of dietary fats and oils, may have a major negative impact on beta-cell function and cause type 2 diabetes mellitus. To investigate this issue, we examined the toxicity of free fatty acid (FFA) compositions mirroring the FFA profiles of various popular edible oils in human EndoC-ßH1 beta-cells and in rat islets. For this purpose, we made compositions consisting exclusively of various FFAs in different volumetric percentages mimicking these oils and additionally mixtures of these compositions. Human EndoC-ßH1 beta-cells were incubated with different oil compositions and the toxicity, lipid droplet formation, ER-stress, and H2O2 production were analyzed. Compositions with prominent content of saturated as well as unsaturated long-chain FFAs showed moderate but significant toxicity both in human EndoC-ßH1 beta-cells and rat islets, however, without further measurable metabolic impairments. On the other hand compositions with high content of medium-chain FFAs revealed no toxicity. A composition with 50% of the very long-chain unsaturated FFA erucic acid caused high toxicity with concomitant peroxisomal H2O2 production. The toxicity of FFAs to human EndoC-ßH1 beta-cells was dampened in mixtures of FFA compositions with a significant content of medium-chain FFAs, but not with a significant proportion of unsaturated FFAs.

Attenuation of Free Fatty Acid-Induced Muscle Insulin Resistance by Rosemary Extract.

Elevated blood free fatty acids (FFAs), as seen in obesity, impair muscle insulin action leading to insulin resistance and Type 2 diabetes mellitus. Serine phosphorylation of the insulin receptor substrate (IRS) is linked to insulin resistance and a number of serine/threonine kinases including JNK, mTOR and p70 S6K have been implicated in this process. Activation of the energy sensor AMP-activated protein kinase (AMPK) increases muscle glucose uptake, and in recent years AMPK has been viewed as an important target to counteract insulin resistance. We reported recently that rosemary extract (RE) increased muscle cell glucose uptake and activated AMPK. However, the effect of RE on FFA-induced muscle insulin resistance has never been examined. In the current study, we investigated the effect of RE in palmitate-induced insulin resistant L6 myotubes. Exposure of myotubes to palmitate reduced the insulin-stimulated glucose uptake, increased serine phosphorylation of IRS-1, and decreased the insulin-stimulated phosphorylation of Akt. Importantly, exposure to RE abolished these effects and the insulin-stimulated glucose uptake was restored. Treatment with palmitate increased the phosphorylation/activation of JNK, mTOR and p70 S6K whereas RE completely abolished these effects. RE increased the phosphorylation of AMPK even in the presence of palmitate. Our data indicate that rosemary extract has the potential to counteract the palmitate-induced muscle cell insulin resistance and further studies are required to explore its antidiabetic properties.

Leonurus japonicus Houtt Attenuates Nonalcoholic Fatty Liver Disease in Free Fatty Acid-Induced HepG2 Cells and Mice Fed a High-Fat Diet.

We investigated the effects of a Leonurus japonicus ethanol extract (LJE) on nonalcoholic fatty liver disease (NAFLD). An in vitro model of hepatic steatosis was treated with 1 mM free fatty acid (FFA) in HepG2 cells. An in vivo NAFLD model was established using C57BL/6 mice fed a high-fat diet (HFD) and administered LJE (100 or 200 mg/kg) orally for 14 weeks. LJE treatment suppressed lipid accumulation and intracellular triglyceride levels significantly in a concentration-dependent manner in HepG2 cells. Moreover, LJE significantly reduced the expression of sterol regulatory element binding protein 1-c, and its downstream genes, which are associated with lipogenesis, in HepG2 cells. In HFD-fed mice, LJE treatment decreased body weight significantly and decreased serum alanine transaminase levels to normal values, concurrent with a decrease in hepatic lipid accumulation. Furthermore, LJE supplementation ameliorated insulin sensitivity by decreasing serum glucose and insulin levels. LJE improved hepatic steatosis by increasing the expression of phosphorylated AMP-activated protein kinase and peroxisome proliferator-activated receptor-α in HFD-fed mice and FFA-treated HepG2 cells. The results suggested that LJE might be a potential therapeutic agent to treat NAFLD.

1-Methylnicotinamide ameliorates lipotoxicity-induced oxidative stress and cell death in kidney proximal tubular cells.

Free fatty acid-bound albumin (FFA-albumin)-related oxidative stress is involved in the pathogenesis of proximal tubular cell (PTC) damage and subsequent renal dysfunction in patients with refractory proteinuria. Nicotinamide adenine dinucleotide (NAD) metabolism has recently been focused on as a novel therapeutic target for several modern diseases, including diabetes. This study was designed to identify a novel molecule in NAD metabolism to protect PTCs from lipotoxicity-related oxidative stress. Among 19 candidate enzymes involved in mammalian NAD metabolism, the mRNA expression level of nicotinamide n-methyltransferase (NNMT) was significantly increased in both the kidneys of FFA-albumin-overloaded mice and cultured PTCs stimulated with palmitate-albumin. Knockdown of NNMT exacerbated palmitate-albumin-induced cell death in cultured PTCs, whereas overexpression of NNMT inhibited it. Intracellular concentration of 1-Methylnicotinamide (1-MNA), a metabolite of NNMT, increased and decreased in cultured NNMT-overexpressing and -knockdown PTCs, respectively. Treatment with 1-MNA inhibited palmitate-albumin-induced mitochondrial reactive oxygen species generation and cell death in cultured PTCs. Furthermore, oral administration of 1-MNA ameliorated oxidative stress, apoptosis, necrosis, inflammation, and fibrosis in the kidneys of FFA-albumin-overloaded mice. In conclusion, NNMT-derived 1-MNA can reduce lipotoxicity-mediated oxidative stress and cell damage in PTCs. Supplementation of 1-MNA may have potential as a new therapy in patients with refractory proteinuria.

[Research of Herba Artemisiae Scoporiae inhibits the hepatic lipotoxicity].

OBJECTIVE: To study the efficiency and effect mechanism of Herba Artemisiae Scoporiae inhibits the hepatic lipotoxicity model in vitro. METHOD: Preparation rat regular serum and medicine serum. Under the safty of medicine thickness by toxicity testing, normal and model groups were added 10% normal rat serum, Herba Artemisiae Scoporiae group was added 10% medicine serum incubation for 24 h, FFA was added to all the groups but the normal incubation for 24 h. The indices were tested below: the content of serum tumor necrosis factor alpha (TNF-alpha) by ELISA, cellular triglyceride content (TG), Oil Red Staining; protein expression of cellular Bcl-2 Assaciated X protein (Bax), phospho-IKB (P-IkappaB) and Cathepsin B (ctsb) by Western Blotting; gene expression of cellular TNF-alpha, Bax and ctsb by real-time PCR; the expression and distribution of ctsb observed by immunofluorescence. RESULT: After being incubated with FFA for 24 hours, TG deposition of HepG2 in the model group increased markedly. Compared with normal group, not only the content of serum TNF-alpha, but also the protein expression of cellular ctsb, P-IkappaB and mRNA expression of ctsb, TNF-a increased significantly. Contrast to model group, TG deposition decreased markedly in the Herba Artemisiae Scoporiae group. The Herba Artemisiae Scoporiae inhibited TNF-alpha content, the protein expression of cellular ctsb, P-IkappaB and mRNA expression of TNF-alpha significantly. CONCLUSION: Herba Artemisiae Scoporiae has a direct inhibition on HepG2 steatosis and TNF-alpha secretion induced by long-chain FFA. The effect mechanism of Herba Artemisiae Scoporiae inhibits the hepatic lipotoxicity has close relationship with inhibition on the protein expression and mRNA expression of ctsb.

Cardiac metallothionein induction plays the major role in the prevention of diabetic cardiomyopathy by zinc supplementation.

BACKGROUND: Our previous studies showed that transgenic mice that overexpress cardiac-specific metallothionein (MT) are highly resistant to diabetes-induced cardiomyopathy. Zinc is the major metal that binds to MT under physiological conditions and is a potent inducer of MT. The present study therefore explored whether zinc supplementation can protect against diabetic cardiomyopathy through cardiac MT induction. METHODS AND RESULTS: Diabetes was induced in mice (C57BL/6J strain) by a single injection of streptozotocin. Half were supplemented intraperitoneally with zinc sulfate (5 mg/kg) every other day for 3 months. After zinc supplementation, mice were maintained for 3 more months and then examined for cardiomyopathy by functional and morphological analysis. Significant increases in cardiac morphological impairment, fibrosis, and dysfunction were observed in diabetic mice but not in diabetic mice supplemented with zinc. Zinc supplementation also induced a significant increase in cardiac MT expression. The role of MT in cardiac protection by zinc supplementation was examined in cultured cardiac cells that were directly exposed to high levels of glucose (HG) and free fatty acid (FFA) (palmitate), treatment that mimics diabetic conditions. Cell survival rate was significantly decreased for cells exposed to HG/FFA but did not change for cells exposed to HG/FFA and pretreated with zinc or low-dose cadmium, each of which induces significant MT synthesis. When MT expression was silenced with the use of MT small-interfering RNA, the preventive effect of pretreatment with zinc or low-dose cadmium was abolished. CONCLUSIONS: These results suggest that the prevention of diabetic cardiomyopathy by zinc supplementation is predominantly mediated by an increase in cardiac MT.

Effect of the fat composition of a single meal on the composition and cytotoxic potencies of lipolytically-releasable free fatty acids in postprandial plasma.

Ingestion of a meal increases plasma levels of triglyceride (TG)-rich lipoproteins through the secretion of intestine-derived chylomcirons and liver-derived very low density lipoproteins (VLDL). We have determined the effects of the fat composition of a single meal on the composition of TG in TG-rich lipoproteins (VLDL + chylomicrons) and circulating and lipolytically-releasable free fatty acids (FFA) in postprandial (PP) plasma and on the cytotoxic potencies of the lipolytically-released FFA to cultured arterial wall cells. PP lipemia was induced by feeding fasted normolipidemic human subjects with a meal rich in saturated fat (SF) and another meal rich in polyunsaturated fat (PUF), or vice versa; each meal provided 65% of energy as fat, and polyunsaturated to saturated fatty acid ratios (P/S) of the SF and PUF in the meals were 0.40 and 2.49, respectively. The mean P/S of TG in TG-rich lipoproteins (1.43) and circulating FFA (1.46) in 4 h PP plasma of PUF were significantly higher than those in PP plasma of SF (0.44 and 0.59, respectively) in fasting plasma (0.52 and 0.53, respectively). In vitro lipolysis of fasting and PP serum by purified bovine milk lipoprotein lipase (LpL) resulted in a marked (8.8-12.3-fold) increase in the serum FFA level. The P/S of serum FFA in postlipolysis fasting and PP serum were consistently higher than that of FFA or that of TG associated with TG-rich lipoproteins in prelipolysis fasting and PP serum, indicating that polyunsaturated TG in VLDL and/or chylomicrons is more susceptible than saturated TG to lipolysis. When postlipolysis serum was interacted with cultured endothelial cells and mouse peritoneal macrophages (MPM), the lipolytically-released FFA in PP serum of SF and PUF disrupted the barrier function of endothelial cells and were cytotoxic to cultured MPM; FFA in postlipolysis fasting serum was not cytotoxic. FFA in postlipolysis PP serum of PUF were consistently more potent than that in postlipolysis PP serum of SF. Further study showed that all long-chain monounsaturated FFA and polyunsaturated FFA, but not saturated FFA, incorporated into lipoproteins (LDL) were cytotoxic to cultured MPM. In conclusion, despite the generally well-accepted belief that SF is more atherogenic than PUF, the present study provides in vitro evidence that the lipolytic remnant products of TG-rich lipoproteins produced after a meal rich in PUF are more injurious to arterial wall cells than those produced after a meal rich in SF.

Effects of L-carnitine on ventricular arrhythmias in dogs with acute myocardial ischemia and a supplement of excess free fatty acids.

The effects of L-carnitine on ventricular arrhythmias were evaluated in dogs with acute myocardial ischemia and a supplement of excess free fatty acids (FFA). Acute myocardial ischemia was induced by ligation of left anterior descending coronary artery. After 80 minutes of coronary occlusion, high plasma FFA was induced by intravenous injection of heparin 200 mu/kg and Intralipid 5 ml/kg as a bolus. After additional 60 minutes, beating hearts were removed from animals and tissue levels of free carnitine, short and long chain acyl carnitine, FFA and adenosine triphosphate (ATP) were determined. L-carnitine 100 mg/kg was administered intravenously 5 minutes before coronary artery ligation. Electrocardiograms were recorded continuously by a Holter electrocardiographic recorder during the experiment and ventricular arrhythmias were quantified by an arbitrary scoring system. In ischemic and excess FFA supplemented myocardium, free carnitine and ATP decreased, whereas long chain acyl carnitine and FFA increased. And these metabolic changes tended to be reduced by L-carnitine. Pretreatment of L-carnitine also reduced the grade of ventricular arrhythmias induced both by acute myocardial ischemia and by supplemented of excess FFA. These results suggest that the administration of L-carnitine may be beneficial to prevent serious arrhythmias in ischemic heart disease, presumably by restoring the imparied FFA oxidation.