The Key Role of Akt Protein Kinase in Metabolic-Inflammatory Pathways Cross-Talk: TNF-α Down-Regulation and Improving of Insulin Resistance in HepG2 Cell Line.

BACKGROUND: Elevation of plasma free fatty acids as a principal aspect of type 2 diabetes maintains etiologically insulin insensitivity in target cells. TNF-α inhibitory effects on key insulin signaling pathway elements remain to be verified in insulinresistant hepatic cells. Thus, TNF-α knockdown effects on the key elements of insulin signaling were investigated in the palmitate-induced insulin-resistant hepatocytes. The Akt serine kinase, a key protein of the insulin signaling pathway, phosphorylation was monitored to understand the TNF-α effect on probable enhancing of insulin resistance. METHODS: Insulin-resistant HepG2 cells were produced using 0.5 mM palmitate treatment and shRNA-mediated TNF-α gene knockdown and its down-regulation confirmed using ELISA technique. Western blotting analysis was used to assess the Akt protein phosphorylation status. RESULTS: Palmitate-induced insulin resistance caused TNF-α protein overexpression 1.2-, 2.78, and 2.25- fold as compared to the control cells at post-treatment times of 8 h, 16 h, and 24 h, respectively. In the presence of palmitate, TNF-α expression showed around 30% reduction in TNF-α knockdown cells as compared to normal cells. In the TNF-α down-regulated cell, Akt phosphorylation was approximately 62% more than control cells after treatment with 100 nM insulin in conjugation with 0.5 mM palmitate. CONCLUSIONS: The obtained data demonstrated that TNF-α protein expression reduction improved insulin-stimulated Akt phosphorylation in the HepG2 cells and decreased lipidinduced insulin resistance of the diabetic hepatocytes.

MiR-155 inhibition alleviates suppression of osteoblastic differentiation by high glucose and free fatty acids in human bone marrow stromal cells by upregulating SIRT1.

Diabetic osteoporosis is a severe and chronic complication of diabetes in the bone and joint system, and its pathogenesis is needed to be explored. In the present study, we examined the effect and underlying mechanism of miR-155 on osteogenic differentiation in human bone marrow-derived mesenchymal stem cells (hBMSCs) under high glucose and free fatty acids (HG-FFA) conditions. It was shown that miR-155 levels in hBMSCs increased corresponding to the time of exposure to HG-FFA treatment. MiR-155 expression was altered by transfecting miR-155 mimic or miR-155 inhibitor. HG-FFA exposure resulted in an obviously decrease in cell viability and alkaline phosphatase (ALP) activity, and downregulated the expressionof runt-related transcription factor 2 (Runx2) and osteocalcin (OCN) in hBMSCs. Transfection of miR-155 mimic further exacerbated HG-FFA-induced inhibitory effect on osteogenic differentiation, and miR-155 inhibitor neutralized this inhibitory effect. Luciferase assays confirmed that SIRT1 was a direct target of miR-155 and can be negatively modulated by miR-155. Furthermore, SIRT1 siRNA partially counteracted miR-155 inhibitor-induced upregulation of SIRT1in HG-FFA-treated hBMSCs. SIRT1 siRNA also reversed the promotional effect of the miR-155 inhibitor on ALP activity and expression of the Runx2 and OCN proteins under HG-FFA conditions. In conclusion, the results suggest that miR-155 suppression promoted osteogenic differentiation of hBMSCs under HG-FFA conditions by targeting SIRT1. Inhibition of MiR-155 may provide a new therapeutic method for the prevention and treatment of diabetic osteoporosis.

Fibrillation of human islet amyloid polypeptide and its toxicity to pancreatic ß-cells under lipid environment.

BACKGROUND: Previous studies suggested that fibrillar human IAPP (hIAPP) is more likely to deposit in ß-cells, resulting in ß-cell injury. However, the changes in the conformation of hIAPP in lipid environment and the mechanism involved in ß-cell damage are unclear. METHODS: Synthetic hIAPP was incubated with five types of free fatty acids and phospholipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS), which constitute the cell membrane. Thioflavin-T fluorescence assay was conducted to analyze the degree of hIAPP fibrosis, and circular dichroism spectroscopy was performed to detect the ß-fold formation of hIAPP. Furthermore, INS-1 cells were infected with human IAPP delivered by a GV230-EGFP plasmid. The effects of endogenous hIAPP overexpression induced by sodium palmitate on the survival, endoplasmic reticulum (ER) stress, and apoptosis of INS-1 cells were evaluated. RESULTS: The five types of free fatty acids can accelerate the fibrosis of hIAPP. Sodium palmitate also maintained the stability of fibrillar hIAPP. POPS, not POPC, accelerated hIAPP fibrosis. Treatment of INS-1 cells with sodium palmitate increased the expression of hIAPP, activated ER stress and ER stress-dependent apoptosis signaling pathways, and increased the apoptotic rate. CONCLUSION: Free fatty acids and anionic phospholipid can promote ß-fold formation and fibrosis in hIAPP. High lipid induced the overexpression of hIAPP and aggravated ER stress and apoptosis in INS-1 cells, which caused ß-cell death in high lipid environment. GENERAL SIGNIFICANCE: Our study reveals free fatty acids and hIAPP synergistically implicated in endoplasmic reticulum stress and apoptosis of islet ß-cells.

Primary Metabolism and Medium-Chain Fatty Acid Alterations Precede Long-Chain Fatty Acid Changes Impacting Neutral Lipid Metabolism in Response to an Anticancer Lysophosphatidylcholine Analogue in Yeast.

The nonmetabolizable lysophosphatidylcholine (LysoPC) analogue edelfosine is the prototype of a class of compounds being investigated for their potential as selective chemotherapeutic agents. Edelfosine targets membranes, disturbing cellular homeostasis. Is not clear at this point how membrane alterations are communicated between intracellular compartments leading to growth inhibition and eventual cell death. In the present study, a combined metabolomics/lipidomics approach for the unbiased identification of metabolic pathways altered in yeast treated with sublethal concentrations of the LysoPC analogue was employed. Mass spectrometry of polar metabolites, fatty acids, and lipidomic profiling was used to study the effects of edelfosine on yeast metabolism. Amino acid and sugar metabolism, the Krebs cycle, and fatty acid profiles were most disrupted, with polar metabolites and short-medium chain fatty acid changes preceding long and very long-chain fatty acid variations. Initial increases in metabolites such as trehalose, proline, and γ-amino butyric acid with a concomitant decrease in metabolites of the Krebs cycle, citrate and fumarate, are interpreted as a cellular attempt to offset oxidative stress in response to mitochondrial dysfunction induced by the treatment. Notably, alanine, inositol, and myristoleic acid showed a steady increase during the period analyzed (2, 4, and 6 h after treatment). Of importance was the finding that edelfosine induced significant alterations in neutral glycerolipid metabolism resulting in a significant increase in the signaling lipid diacylglycerol.

Uncoupling lipid metabolism from inflammation through fatty acid binding protein-dependent expression of UCP2.

Chronic inflammation in obese adipose tissue is linked to endoplasmic reticulum (ER) stress and systemic insulin resistance. Targeted deletion of the murine fatty acid binding protein (FABP4/aP2) uncouples obesity from inflammation although the mechanism underlying this finding has remained enigmatic. Here, we show that inhibition or deletion of FABP4/aP2 in macrophages results in increased intracellular free fatty acids (FFAs) and elevated expression of uncoupling protein 2 (UCP2) without concomitant increases in UCP1 or UCP3. Silencing of UCP2 mRNA in FABP4/aP2-deficient macrophages negated the protective effect of FABP loss and increased ER stress in response to palmitate or lipopolysaccharide (LPS). Pharmacologic inhibition of FABP4/aP2 with the FABP inhibitor HTS01037 also upregulated UCP2 and reduced expression of BiP, CHOP, and XBP-1s. Expression of native FABP4/aP2 (but not the non-fatty acid binding mutant R126Q) into FABP4/aP2 null cells reduced UCP2 expression, suggesting that the FABP-FFA equilibrium controls UCP2 expression. FABP4/aP2-deficient macrophages are resistant to LPS-induced mitochondrial dysfunction and exhibit decreased mitochondrial protein carbonylation and UCP2-dependent reduction in intracellular reactive oxygen species. These data demonstrate that FABP4/aP2 directly regulates intracellular FFA levels and indirectly controls macrophage inflammation and ER stress by regulating the expression of UCP2.

Composition of fatty acids in virgin olive oils from cross breeding segregating populations by gas chromatography separation with flame ionization detection.

BACKGROUND: Recent technological advances to improve the quality of virgin olive oil (VOO) have been focused on olive breeding programs by selecting outstanding cultivars and target progenies. Fatty acid (FA) composition, with special emphasis on oleic acid (C18:1) and palmitic acid (C16:0), is one of the most critical quality factors to be evaluated in VOO. For this reason, the profile of FAs is frequently used as a decision tool in olive breeding programs. RESULTS: A method based on gas chromatography with flame ionization detection (GC-FID) was used to study the influence of genotype on the concentration of ten of the most important FAs in VOOs from target crosses Arbequina × Arbosana, Picual × Koroneiki and Sikitita × Arbosana and their corresponding genitors Arbequina, Arbosana, Koroneiki, Picual and Sikitita. For this purpose, a targeted approach was selected for determination of esterified FAs (EFAs) and non-esterified FAs (NEFAs) in a dual analysis by the same chromatographic method. A Pearson analysis revealed correlations between pairs of FAs, which allowed detecting metabolic connections through desaturation and elongation enzymes. An ANOVA test (with P < 0.01) led to identification of C16:0 EFA, C16:1 EFA and C18:1 EFA and also C16:1 NEFA and C18:0 NEFA as the FAs more influenced by cross breeding. Statistical analysis was carried out by unsupervised analysis using principal component analysis (PCA) and cluster analysis (CA) to look for variability sources. CONCLUSION: Crosses with a common genitor (Arbequina × Arbosana and Sikitita × Arbosana) were partially overlapped in the PCAs using the profile of FAs. The CA results revealed clear differences between Sikitita × Arbosana and Picual × Koroneiki crosses in the composition of the most significant FAs, while Arbequina × Arbosana was not properly discriminated from the other crosses.

PPARα modulates the TSH ß-subunit mRNA expression in thyrotrope TαT1 cells and in a mouse model.

SCOPE: Fasting leads to a significant downregulation of the hypothalamus-pituitary-thyroid axis, and peroxisome proliferator-activated receptor (PPAR) α is a key transcription factor in mediating a magnitude of adaptive responses to fasting. In this study, we examined the role of PPARα in regulation of the hypothalamus-pituitary-thyroid axis. METHODS AND RESULTS: Thyroid-stimulating hormone ß-subunit (TSHß) mRNA abundance was being reduced in response to treatment of TαT1 cells with PPARα agonists (p < 0.05), indicating an inhibitory transcriptional regulation of TSHß by PPARα. As expected, fasting significantly downregulated TSHß mRNA expression in a two-factorial study with fed or fasted wild-type (WT) and PPARα knockout mice (p < 0.05). In contrast to the in vitro data, fasted PPARα knockout mice revealed lower mRNA concentrations of pituitary TSHß (-64%) and TSH-regulated thyroid genes, and lower plasma concentrations of thyroxine (T4, -25%), triiodothyronine (T3, -25%), free T4 (-60%), and free T3 (-35%) than fasted WT mice (p < 0.05). Those differences were not observed in fed mice. CONCLUSIONS: Data from thyrotrope cells revealed that PPARα could contribute to the fasting-associated downregulation of the TSHß mRNA expression. In a mouse model, fasting led to a significant reduction in TSHß mRNA level, but unexpectedly this effect was stronger in mice lacking PPARα than in WT mice.