BACKGROUND: Obesity is well-established as a significant contributor to the development of insulin resistance (IR) and diabetes, partially due to elevated plasma saturated free fatty acids like palmitic acid (PA). Grb10-interacting GYF Protein 2 (GIGYF2), an RNA-binding protein, is widely expressed in various tissues including the liver, and has been implicated in diabetes-induced cognitive impairment. Whereas, its role in obesity-related IR remains uninvestigated.
METHODS: In this study, we employed palmitic acid (PA) exposure to establish an in vitro IR model in the human liver cancer cell line HepG2 with high-dose chronic PA treatment. The cells were stained with fluorescent dye 2-NBDG to evaluate cell glucose uptake. The mRNA expression levels of genes were determined by real-time qRT-PCR (RT-qPCR). Western blotting was employed to examine the protein expression levels. The RNA immunoprecipitation (RIP) was used to investigate the binding between protein and mRNA. Lentivirus-mediated gene knockdown and overexpression were employed for gene manipulation. In mice, an IR model induced by a high-fat diet (HFD) was established to validate the role and action mechanisms of GIGYF2 in the modulation of HFD-induced IR in vivo.
RESULTS: In hepatocytes, high levels of PA exposure strongly trigger the occurrence of hepatic IR evidenced by reduced glucose uptake and elevated extracellular glucose content, which is remarkably accompanied by up-regulation of GIGYF2. Silencing GIGYF2 ameliorated PA-induced IR and enhanced glucose uptake. Conversely, GIGYF2 overexpression promoted IR, PTEN upregulation, and AKT inactivation. Additionally, PA-induced hepatic IR caused a notable increase in STAU1, which was prevented by depleting GIGYF2. Notably, silencing STAU1 prevented GIGYF2-induced PTEN upregulation, PI3K/AKT pathway inactivation, and IR. STAU1 was found to stabilize PTEN mRNA by binding to its 3\'UTR. In liver cells, tocopherol treatment inhibits GIGYF2 expression and mitigates PA-induced IR. In the in vivo mice model, GIGYF2 knockdown and tocopherol administration alleviate high-fat diet (HFD)-induced glucose intolerance and IR, along with the suppression of STAU1/PTEN and restoration of PI3K/AKT signaling.
CONCLUSIONS: Our study discloses that GIGYF2 mediates obesity-related IR by disrupting the PI3K/AKT signaling axis through the up-regulation of STAU1/PTEN. Targeting GIGYF2 may offer a potential strategy for treating obesity-related metabolic diseases, including type 2 diabetes.

Here, we investigated the complexation of short chain amylose (SCAs) and palmitic acid (PA), serving as polymeric building blocks that alter the selectivity and directionality of particle growth. This alteration affects the shape anisotropy of the particles, broadening their applications due to the increased surface area. By modifying the concentration of PA, we were able to make spherical, macaron, and disc-shaped particles, demonstrating that PA acts as a structure-directing agent. We further illustrated the lateral and longitudinal stacking kinetics between PA-SCA inclusion complexes during self-assembly, leading to anisotropy. Transmission electron microscope (TEM) and scanning electron microscope (SEM) revealed the structural difference between the initial and final morphologies of palmitic acid-short chain amylose particles (PA-SCAPs) compared to those of short-chain amylose particle (SCAPs). The presence of PA-SCA inclusion complex in the anisotropic particles was confirmed using nuclear magnetic resonance (NMR) and powder x-ray diffraction (XRD) analysis.

OBJECTIVE:  Metabolic-associated fatty liver disease (MAFLD) is a common cause of chronic liver disease worldwide. However, there is currently no recognized effective drugs for treating it.
METHODS:  In this study, we investigated the efficacy of Honokiol (HNK) in vitro for mitigating MAFLD. Then, 0.4 mM palmitic acid (PA) and LO2 cells were used to establish the MAFLD model. The protective effect of HNK on MAFLD was confirmed by Oil Red O staining and cell counting kit (CCK-8) assay in LO2 cell line. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were carried out to analyze the regulatory role of HNK on Nrf2 and RIPK3 signaling pathways. The effect of HNK and its downstream signaling pathways on oxidative stress were verified by the detection of reactive oxygen species (ROS), malondialdehyde (MDA), catalase (CAT), and superoxide dismutase (SOD). The concentration of IL-1β, IL-6L, and TNF-α was assessed by enzyme-linked immunosorbent assay (ELISA).
RESULTS:  The middle concentration of HNK (50 μmol/L) was selected as the best option for inhibiting lipidosis and oxidative stress in MAFLD models. Honokiol mitigates MAFLD via activation of nuclear factor E2-related factor 2 (Nrf2) signaling pathways in vitro. Honokiol suppressed MAFLD via activating the Nrf2 signaling pathway to play an antioxidant and anti-inflammatory role. Also, HNK regulates Nrf2 and RIPK3 signaling pathways to mitigate MAFLD.
CONCLUSIONS:  Our results showed that HNK may suppress the oxidative stress and inflammation in MAFLD via activation of Nrf2 signaling pathway.

The objective of our study was to evaluate the effect of altering the dietary supply of acetate, palmitic acid (PA), and cottonseed on the yields of milk components and milk fatty acids (FA) in lactating dairy cows. Thirty-two multiparous Holstein cows (133 ± 57 d in milk, 50.5 ± 7.2 kg/d milk) were used in a 4 × 4 Latin square split plot design with a 2 × 2 factorial arrangement of subplot treatments. Cows were blocked by energy-corrected milk (ECM) yield and allocated to a main plot receiving a basal diet (n = 16) with no supplemental PA (Low PA) or a basal diet (n = 16) with 1.5% inclusion of a FA supplement containing ~85% PA (High PA). In each main plot, the following subplots of treatment diets were fed in a Latin square arrangement consisting of 14-d periods: 1) a control diet (CON), 2) the control diet supplemented with 3% sodium acetate (AC), 3) the control diet supplemented with 12% whole cottonseed (CS), and 4) the control diet supplemented with 3% sodium acetate and 12% whole cottonseed (CS+AC). The PA supplement and sodium acetate replaced soyhulls, and whole cottonseed replaced cottonseed hulls and meal. All diets were balanced for 30% neutral detergent fiber (NDF), 23% forage NDF, 28% starch, and 17% crude protein (CP). Sources of FA were classified as de novo (<16 carbons), mixed (16-carbon), and preformed (>16 carbons). The statistical model included the random effect of cow nested within basal diet and fixed effect of period, basal diet, acetate, cottonseed, and their interactions. Three-way interactions among basal diet, acetate, and cottonseed were observed for the yields of milk fat, 3.5% fat-corrected milk (FCM), and the molar yields of de novo FA, mixed FA, and preformed FA. In the Low PA diets, AC and CS+AC increased the yields of milk fat and FCM compared with CON and CS, whereas, in the High PA diets, CS+AC increased the yields of milk fat and FCM compared with the other treatments and AC increased milk fat yield compared with CON and CS. Compared with Low PA, High PA increased milk fat content, mixed FA yield, and tended to increase C4:0 yield. Diets containing acetate increased DMI and the yields of milk fat, ECM, FCM, de novo FA, mixed FA, and preformed FA compared with diets without acetate. Diets containing cottonseed increased the yields of milk and preformed FA, tended to increase the yields of FCM and protein, and decreased DMI and the yields of de novo FA and mixed FA compared with diets without cottonseed. In summary, in high PA diets, the inclusion of acetate plus cottonseed increased milk fat yield compared with the other treatments. The CON diet in High PA increased milk fat yield to the same extent as AC and CS+AC in Low PA suggesting PA is important for initiating milk TG synthesis. Balancing the supply of de novo FA substrates and preformed FA is important for increasing the synthesis of milk fat triglycerides and milk fat production.

BACKGROUND: Negative energy balance (NEB) typically occurs in dairy cows after delivery. Cows with a high yield are more likely to experience significant NEB. This type of metabolic imbalance could cause ketosis, which is often accompanied by a decline in reproductive performance. However, the molecular mechanisms underlying NEB have yet to be fully elucidated. During excessive NEB, the body fat is extensively broken down, resulting in the abnormal accumulation of non-esterified fatty acids (NEFAs), represented by palmitic acid (PA), within the uterus. Such an abnormal accumulation has the potential to damage bovine endometrial epithelial cells (BEECs), while the molecular mechanisms underlying its involvement in the PA-induced injury of BEECs remains poorly understood. Melatonin (MT) is recognized for its regulatory role in maintaining the homeostasis of mitochondrial reactive oxygen species (mitoROS). However, little is known as to whether MT could ameliorate the damage incurred by BEECs in response to PA and the molecular mechanism involved.
RESULTS: Analysis showed that 0.2 mmol/L PA stress increased the level of cellular and mitochondrial oxidative stress, as indicated by increased reactive oxygen species (ROS) level. In addition, we observed mitochondrial dysfunction, including abnormal mitochondrial structure and respiratory function, along with a reduction in mitochondrial membrane potential and mitochondrial copy number, and the induction of apoptosis. Notably, we also observed the upregulation of autophagy proteins (PINK, Parkin, LC3B and Ubiquitin), however, the P62 protein was also increased. As we expected, 100 μmol/L of MT pre-treatment attenuated PA-induced mitochondrial ROS and restored mitochondrial respiratory function. Meanwhile, MT pretreatment reversed the upregulation of P62 induced by PA and activated the AMPK-mTOR-Beclin-1 pathway, contributing to an increase of autophagy and decline apoptosis.
CONCLUSIONS: Our findings indicate that PA can induce mitochondrial dysfunction and enhance autophagy in BEECs. In addition, MT is proved to not only reduce mitochondrial oxidative stress but also facilitate the clearance of damaged mitochondria by upregulating autophagy pathways, thereby safeguarding the mitochondrial pool and promoting cellular viability. Our study provides a better understanding of the molecular mechanisms underlying the effect of an excess of NEB on the fertility outcomes of high yielding dairy cows.

BACKGROUND: The fruit of Phyllanthus emblica L., a traditional medicine in China and India, is used to treat diabetes mellitus. Its water extract (WEPE) has demonstrated hypoglycemic effects in diabetic rats, but its mechanisms on glucose utilization and insulin resistance in skeletal muscle remain unclear. Therefore, this study aims to investigate the effects and underlying mechanisms of WEPE on glucose utilization and insulin resistance using C2C12 myotubes.
METHODS: Effects of WEPE on glucose uptake, GLUT4 translocation, and AMPK and AKT phosphorylation were investigated in C2C12 myotubes and palmitate-treated myotubes. An AMPK inhibitor and siRNA were used to explore the mechanisms of WEPE. Glucose uptake was determined using a 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-2-deoxyglucose (2-NBDG) uptake assay, and protein expression and GLUT4 translocation were assessed via western blotting.
RESULTS: In normal myotubes, WEPE significantly stimulated glucose uptake and GLUT4 translocation to the plasma membrane at concentrations of 125 and 250 µg/mL. This was accompanied by an increase in the phosphorylation of AMPK and its downstream targets. However, both compound C and AMPK siRNA blocked the WEPE-induced GLUT4 translocation and glucose uptake. Moreover, pretreatment with STO-609, a calcium/calmodulin-dependent protein kinase kinase β (CaMKKβ) inhibitor, inhibited WEPE-induced AMPK phosphorylation and attenuated the WEPE-stimulated glucose uptake and GLUT4 translocation. In myotubes treated with palmitate, WEPE prevented palmitate-induced insulin resistance by enhancing insulin-mediated glucose uptake and AKT phosphorylation. It also restored the insulin-mediated translocation of GLUT4 from cytoplasm to membrane. However, these effects of WEPE on glucose uptake and GLUT4 translocation were blocked by pretreatment with compound C.
CONCLUSIONS: WEPE significantly stimulated basal glucose uptake though CaMKKβ/AMPK pathway and markedly ameliorated palmitate-induced insulin resistance by activating the AMPK pathway in C2C12 myotubes.

Dietary intake of omega-3 polyunsaturated fatty acids (eicosapentaenoic acid, EPA) exerts antiarrhythmic effects, although the mechanisms are poorly understood. Here, we investigated the possible beneficial actions of EPA on saturated fatty acid-induced changes in the L-type Ca2+ channel in cardiomyocytes. Cardiomyocytes were cultured with an oleic acid/palmitic acid mixture (OAPA) in the presence or absence of EPA. Beating rate reduction in cardiomyocytes caused by OAPA were reversed by EPA. EPA also retrieved a reduction in Cav1.2 L-type Ca2+ current, mRNA, and protein caused by OAPA. Immunocytochemical analysis revealed a distinct downregulation of the Cav1.2 channel caused by OAPA with a concomitant decrease in the phosphorylated component of a transcription factor adenosine-3\',5\'-cyclic monophosphate (cAMP) response element binding protein (CREB) in the nucleus, which were rescued by EPA. A free fatty acid receptor 4 (FFAR4) agonist TUG-891 reversed expression of Cav1.2 and CREB mRNA caused by OAPA, whereas an FFAR4 antagonist AH-7614 abolished the effects of EPA. Excessive reactive oxygen species (ROS) accumulation caused by OAPA decreased Cav1.2 and CREB mRNA expressions, which was reversed by an ROS scavenger. Our data suggest that EPA rescues cellular Cav1.2-Ca2+ channel decline caused by OAPA lipotoxicity and oxidative stresses via both free fatty acid receptor 4-dependent and -independent pathways.

The present study was carried out to investigate the proximate composition, fatty acid (FA) profile and volatile compounds (VC) of cooked green licuri (Syagrus coronata) - an unripe stage that is then cooked - and naturally ripe licuri almonds. The FA profiles were determined by gas chromatography (GC) and the VC composition was evaluated using headspace-solid-phase microextraction coupled with GC-MS. The cooked green licuri presented higher moisture, and lower contents of ashes, proteins and lipids than naturally ripe licuri almonds. The FA profiles of cooked green licuri and naturally ripe licuri almonds showed that saturated FAs were predominant (80%) in both samples, and the concentrations of lauric, palmitic, and oleic acids in naturally ripe licuri almonds were higher than those in cooked green licuri. Limonene was the predominant compound in naturally ripe licuri almonds. The main class of VC in the cooked green licuri were aldehydes, with 3-methyl-butanal and furfural being the main species. Alcohols, such as 3-methyl-butanol and 2-heptanol, were the main class of VC in naturally ripe licuri almonds. Among the volatile compounds, 1-hexanol and 2-nonanone contributed to the aroma of cooked green licuri almonds, whereas 2-heptanone, ethanol, and limonene contributed to the aroma of naturally ripe licuri almonds (almonds not subjected to any cooking process). In a word, cooked green licuri and naturally riped licuri almonds, despite having different proximate compositions, present similar fatty acid profile and distinct aromatic characteristics. Therefore, cooked green licuri and naturally riped licuri almonds are an alternative source of nutrient and could be investigated for the use in the food industry to enhance flavor and aroma to new products.

The complexation of physically modified starch with fatty acids is favorable for the production of resistant starch. However, there is a lack of information on the effect of ultrasonication (UC) on the structure and properties of starch complexes and the molecular mechanism of the stabilization. Here, the multi-scale structure and in vitro digestive properties of starch-fatty acid complexes before and after UC were investigated, and the stabilization mechanisms of starch and fatty acids were explored. The results showed that the physicochemical properties and multi-scale structure of the starch-fatty acid complexes significantly changed with the type of fatty acids. The solubility and swelling power of the starch-fatty acid complexes were significantly decreased after UC (P < 0.05), which facilitated the binding of starch with fatty acids. The XRD results revealed that after the addition of fatty acids, the starch-fatty acid complexes showed typical V-shaped complexes. In addition, the starch-fatty acid complexes showed a significant increase in complexing index, improved short-range ordering and enhanced thermal stability. However, the differences in the structure and properties of the fatty acids themselves resulted in no significant improvement in the multi-scale structure of maize starch-palmitic acid by UC. In terms of digestibility, especially the complexes after UC were more compact in structure, which increased the difficulty of enzymatic digestion and thus slowed down the digestion process. DFT calculations and combined with FT-IR analysis showed that non-covalent interactions such as hydrogen bonding and hydrophobic interactions were the main driving force for the formation of the complexes, with binding energies (lauric acid, myristic acid and palmitic acid) of -30.50, -22.14 and -14.10 kcal/mol, respectively. Molecular dynamics simulations further confirmed the molecular mechanism of inclusion complex formation and stabilization. This study is important for the regulation of starchy foods by controlling processing conditions, and provides important information on the role of fatty acids in the regulation of starch complexes and the binding mechanism.

BACKGROUND: Hepatic stellate cells (HSC) have numerous critical roles in liver function and homeostasis, while they are also known for their importance during liver injury and fibrosis. There is therefore a need for relevant in vitro human HSC models to fill current knowledge gaps. In particular, the roles of vitamin A (VA), lipid droplets (LDs), and energy metabolism in human HSC activation are poorly understood.
METHODS: In this study, human pluripotent stem cell-derived HSCs (scHSCs), benchmarked to human primary HSC, were exposed to 48-hour starvation of retinol (ROL) and palmitic acid (PA) in the presence or absence of the potent HSC activator TGF-β. The interventions were studied by an extensive set of phenotypic and functional analyses, including transcriptomic analysis, measurement of activation-related proteins and cytokines, VA- and LD storage, and cell energy metabolism.
RESULTS: The results show that though the starvation of ROL and PA alone did not induce scHSC activation, the starvation amplified the TGF-β-induced activation-related transcriptome. However, TGF-β-induced activation alone did not lead to a reduction in VA or LD stores. Additionally, reduced glycolysis and increased mitochondrial fission were observed in response to TGF-β.
CONCLUSIONS: scHSCs are robust models for activation studies. The loss of VA and LDs is not sufficient for scHSC activation in vitro, but may amplify the TGF-β-induced activation response. Collectively, our work provides an extensive framework for studying human HSCs in healthy and diseased conditions.