Differential Incorporation of Alpha-Linolenic Acid Into Phospholipid Classes in H4IIE Cells

Objectives Alpha-linolenic acid (ALA) is an essential fatty acid found in plant-based oils, with health benefits attributed to its metabolic conversion to very-long chain polyunsaturated fatty acids. Other routes of cellular ALA metabolism exist, but much of our knowledge of ALA metabolism stems from dated analytical techniques. Essential fatty acids are present in cells primarily as fatty acyls, glycerolipids, and glycerophospholipids, but finer details of ALA metabolism remain unexplored. The current study used unbiased metabolomics to profile the ALA metabolites produced in liver cells after treatment with ALA. Methods H4IIE hepatoma cells were incubated with or without 60 μM ALA for 0, 15, 30, 60, 120, 180, 260, 480, and 720 minutes. Samples were extracted and prepared for HPLC/LC-QTOF-MS analysis. MS data acquisitions were completed in both positive and negative modes. Data were analyzed with 2-way ANOVA (ALA and time as factors) followed by Bonferroni FWER corrections for multiple comparisons. Differences with p < 0.05 were considered significant. Results Among the metabolites detected, differences in patterns of ALA-containing phospholipids (PLs) were observed with ALA treatment. Consistent with a high proportion of phosphatidylcholine (PC) in mammalian cell membranes, ALA-containing PCs such as PC(18:3/18:3), PC(18:3/18:4), lysoPC(18:3/0:0), and PC(18:3/18:1) were significantly higher in ALA-treated cells at most time points. In addition, several phosphatidylethanolamine (PE) species with ALA were significantly higher in ALA-treated cells, including PE(18:3/18:3), PE(18:3/18:2), PE(18:3/16:0), PE(18:3/20:5), and PE(18:3/22:6). To a lesser degree, ALA was also found in phosphatidylserine (PS)(18:3/20:5), lysoPS(18:3/0:0), lysophosphatidylglycerol (18:3/0:0) and phosphatidic acid (PA)(18:3/18:2) and PA(18:3/22:6), suggesting that ALA incorporation into PLs is not limited to PC, contrary to the current known pathways of ALA metabolism. Conclusions Based on these results, there appears to be a preference for incorporation of ALA into PC and PE; however, ALA is also incorporated into PS, PG, and PA. Given the role of different PLs, and functionality based on their fatty acyl composition, future studies will explore the functional outcomes of ALA incorporation into different PL classes. Funding Sources NSERC Discovery Grant

Retinol binding protein 4 (Rbp4) mRNA translation in hepatocytes is enhanced by activation of mTORC1

Increased expression of the peptide hormone retinol-binding protein 4 (RBP4) has been implicated in the development of insulin resistance, type 2 diabetes, and visual dysfunction. Prior investigations of the mechanisms that influence RBP4 synthesis have focused solely on changes in mRNA abundance. Yet, the production of many secreted proteins is controlled at the level of mRNA translation, as it allows for a rapid and reversible change in expression. Herein, we evaluated Rbp4 mRNA translation using sucrose density gradient centrifugation. In the liver of fasted rodents, Rbp4 mRNA translation was low. In response to re-feeding, Rbp4 mRNA translation was enhanced and RBP4 levels in serum were increased. In H4IIE cells, refreshing culture medium promoted Rbp4 mRNA translation and expression of the protein. Rbp4 mRNA abundance was not increased by either experimental manipulation. Enhanced Rbp4 mRNA translation was associated with activation of the kinase mTORC1 and enhanced phosphorylation of the translational repressor 4E-BP1. In H4IIE cells, expression of a 4E-BP1 variant that is unable to be phosphorylated by mTORC1 or suppression of mTORC1 with rapamycin attenuated activity of a luciferase reporter encoding the Rbp4 mRNA 5′-untranslated region (UTR). Purine substitutions to disrupt a terminal oligopyrimidine (TOP)-like sequence in the Rbp4 5′-UTRprevented the suppressive effect of rapamycin on reporter activity. Rapamycin also prevented upregulation of Rbp4 mRNA translation in the liver, and reduced serum levels of RBP4 in response to feeding. Overall, the findings support a model in which nutrient-induced activation of mTORC1 up regulates Rbp4 mRNA translation to promote RBP4 synthesis.

Glucagon-dependent suppression of mTORC1 is associated with upregulation of hepatic FGF21 mRNA translation

Fibroblast growth factor 21 (FGF21) is a peptide hormone that acts to enhance insulin sensitivity and reverse many of the metabolic defects associated with consumption of a high-fat diet. Recent studies show that the liver is the primary source of FGF21 in the blood and that hepatic FGF21 expression is upregulated by glucagon. Interestingly, glucagon acts to upregulate FGF21 production by primary cultures of rat hepatocytes and H4IIE and HepG2 hepatocarcinoma cells independent of changes in FGF21 mRNA abundance, suggesting that FGF21 protein expression is regulated posttranscriptionally. Based on these observations, the goal of the present study was to assess whether or not FGF21 mRNA is translationally regulated. The results show that FGF21 mRNA translation and secretion of the hormone are significantly upregulated in H4IIE cells exposed to 25 nM glucagon, independent of changes in FGF21 mRNA abundance. Furthermore, the glucagon-induced upregulation of FGF21 mRNA translation is associated with suppressed activity of the mechanistic target of rapamycin in complex 1 (mTORC1). Similarly, the results show that rapamycin-induced suppression of mTORC1 leads to upregulation of FGF21 mRNA translation with no change in FGF21 mRNA abundance. In contrast, activation of mTORC1 by refreshing the culture medium leads to downregulation of FGF21 mRNA translation. Notably, refeeding fasted rats also leads to downregulation of FGF21 mRNA translation concomitantly with activation of mTORC1 in the liver. Overall, the findings support a model in which glucagon acts to upregulate FGF21 production by hepatocytes through suppression of mTORC1 and subsequent upregulation of FGF21 mRNA translation.

Study of the Hypoglycemic Activity of Derivatives of Isoflavones fromCicer arietinumL.

The chickpea, a food and medicine used by the people of Xinjiang, has a beneficial hypoglycemic effect. To better utilize this national resource and develop hypoglycemic agents from components of the chickpea, a series of new derivatives of isoflavone compounds from the chickpea were synthesized. An insulin-resistant (IR) HepG2 cell model was used to screen the hypoglycemic activities of these compounds. And the structure-activity relationships of these compounds were explored. Additionally, several combinations of these compound displayed higher hypoglycemic activity than any single compound, and they had similar hypoglycemic activity to that of the positive control group (p>0.05). In addition, combination 3 and combination 6 exerted different effects on the insulin sensitivity of H4IIE cells stimulated with resistin. And the results indicated that combination 3 would have higher hypoglycemic activity. These findings demonstrate the characteristics of multiple components and targets of Chinese herbal medicine. This evidence may provide new ideas for the development of hypoglycemic drugs.