CPT1B maintains redox homeostasis and inhibits ferroptosis to induce gemcitabine resistance via the KEAP1/NRF2 axis in pancreatic cancer.

BACKGROUND: Although we have made progress in treatment and have increased the 5-year survival by ≤30% in pancreatic cancer, chemotherapy resistance remains a major obstacle. However, whether reprogrammed lipid metabolism contributes to chemoresistance still needs to be further studied. METHODS: Gene expression was determined using Western blotting and quantitative reverse transcription polymerase chain reaction. Cell cloning formation assay, Cell Counting Kit-8, EdU assay, wound healing assay, transwell assay, and flow cytometry were used to detect apoptosis, cell proliferation capacity, migration capacity, and cytotoxicity of gemcitabine. Confocal fluorescence microscopy, transmission electron microscopy, etc., were used to detect the changes in intracellular reactive oxygen species, glutathione, lipid peroxidation level, and cell morphology. An animal study was performed to evaluate the effect of CPT1B knockdown on tumor growth and gemcitabine efficacy. RESULTS: In our study, we observed that the CPT1B expression level was higher in pancreatic ductal adenocarcinoma tissues than in normal tissues and correlated with a low rate of survival. Moreover, silencing of CPT1B significantly suppressed the proliferative ability and metastasis of pancreatic cancer cells. Furthermore, we discovered that CPT1B interacts with Kelch-like ECH-associated protein 1, and CPT1B knockdown led to decreased NRF2 expression and ferroptosis induction. In addition, CPT1B expression increased after gemcitabine treatment, and it was highly expressed in gemcitabine-resistant pancreatic ductal adenocarcinoma cells. Finally, we discovered that ferroptosis induced by CPT1B knockdown enhanced the gemcitabine toxicity in pancreatic ductal adenocarcinoma. CONCLUSION: CPT1B may act as a promising target in treating patients with gemcitabine-resistant pancreatic ductal adenocarcinoma .

Ketohexokinase-C regulates global protein acetylation to decrease carnitine palmitoyltransferase 1a-mediated fatty acid oxidation.

BACKGROUND & AIMS: The consumption of sugar and a high-fat diet (HFD) promotes the development of obesity and metabolic dysfunction. Despite their well-known synergy, the mechanisms by which sugar worsens the outcomes associated with a HFD are largely elusive. METHODS: Six-week-old, male, C57Bl/6 J mice were fed either chow or a HFD and were provided with regular, fructose- or glucose-sweetened water. Moreover, cultured AML12 hepatocytes were engineered to overexpress ketohexokinase-C (KHK-C) using a lentivirus vector, while CRISPR-Cas9 was used to knockdown CPT1α. The cell culture experiments were complemented with in vivo studies using mice with hepatic overexpression of KHK-C and in mice with liver-specific CPT1α knockout. We used comprehensive metabolomics, electron microscopy, mitochondrial substrate phenotyping, proteomics and acetylome analysis to investigate underlying mechanisms. RESULTS: Fructose supplementation in mice fed normal chow and fructose or glucose supplementation in mice fed a HFD increase KHK-C, an enzyme that catalyzes the first step of fructolysis. Elevated KHK-C is associated with an increase in lipogenic proteins, such as ACLY, without affecting their mRNA expression. An increase in KHK-C also correlates with acetylation of CPT1α at K508, and lower CPT1α protein in vivo. In vitro, KHK-C overexpression lowers CPT1α and increases triglyceride accumulation. The effects of KHK-C are, in part, replicated by a knockdown of CPT1α. An increase in KHK-C correlates negatively with CPT1α protein levels in mice fed sugar and a HFD, but also in genetically obese db/db and lipodystrophic FIRKO mice. Mechanistically, overexpression of KHK-C in vitro increases global protein acetylation and decreases levels of the major cytoplasmic deacetylase, SIRT2. CONCLUSIONS: KHK-C-induced acetylation is a novel mechanism by which dietary fructose augments lipogenesis and decreases fatty acid oxidation to promote the development of metabolic complications. IMPACT AND IMPLICATIONS: Fructose is a highly lipogenic nutrient whose negative consequences have been largely attributed to increased de novo lipogenesis. Herein, we show that fructose upregulates ketohexokinase, which in turn modifies global protein acetylation, including acetylation of CPT1a, to decrease fatty acid oxidation. Our findings broaden the impact of dietary sugar beyond its lipogenic role and have implications on drug development aimed at reducing the harmful effects attributed to sugar metabolism.

Capparis spinosa improves non-alcoholic steatohepatitis through down-regulating SREBP-1c and a PPARα-independent pathway in high-fat diet-fed rats.

OBJECTIVE: Non-alcoholic steatohepatitis (NASH) has become a global medical problem. Currently, there is no approved pharmacologic treatment for this condition. Previous studies have suggested that in the pathogenesis of this disease, regulatory pathways associated with de novo lipogenesis and ß-oxidation pathways genes are misregulated. Capparis spinosa (CS) belongs to the family of Capparidaceae and is a traditional plant used to treat various diseases, particularly dyslipidemia. The compounds and extracts of this plant in In vivo and in vitro studies resulted in a reduction in lipid profiles and glucose. However, the mechanism of these effects remains unknown. This study aimed to evaluate the effects of (CS) fruit extract on NASH compared to fenofibrate and explored the related molecular mechanism. RESULTS: In the rats (n = 40) model of NASH, biochemical and histopathological examinations showed that liver steatosis, inflammation, and hepatic fibrosis were markedly attenuated in response to CS and fenofibrate interventions. At the molecular level, CS treatment down-regulated sterol regulatory element-binding protein-1c (SREBP-1c) (p < 0.001), acetyl-CoA carboxylase (ACC) (p < 0.001), and up-regulated Carnitine palmitoyltransferase I (CPT1) expression (p < 0.001). In conclusion, CS has favorable therapeutic effects for NASH, which was associated with ameliorating steatosis and fibrosis via regulation of the DNL and ß-oxidation pathway genes.

Dietary L-arginine supplementation increases the hepatic expression of AMP-activated protein kinase in rats.

The goal of this study was to elucidate the molecular mechanisms responsible for the anti-obesity effect of L-arginine supplementation in diet-induced obese rats. Male Sprague-Dawley rats were fed either a low-fat or high-fat diet for 15 weeks. Thereafter, lean or obese rats were pair-fed their same respective diets and received drinking water containing either 1.51% L-arginine-HCl or 2.55% L-alanine (isonitrogenous control) for 12 weeks. Gene and protein expression of key enzymes in the metabolism of energy substrates were determined using real-time polymerase-chain reaction and western blotting techniques. The mRNA levels of hepatic fatty acid synthase and stearoyl-CoA desaturase were reduced (P < 0.05) but those of hepatic AMP-activated protein kinase-α (AMPKα), peroxisome proliferator activator receptor γ coactivator-1α, and carnitine palmitoyltransferase I (CPT-I), as well as skeletal muscle CPT-I were increased (P < 0.05) by L-arginine treatment. The protein expression and activity of hepatic AMPKα markedly increased (P < 0.05) but the activity of hepatic acetyl-CoA carboxylase (ACC) decreased (P < 0.05) in response to dietary L-arginine supplementation. Collectively, our results indicate that liver is the major target for the action of dietary L-arginine supplementation on reducing white-fat mass in diet-induced obese rats by inhibiting fatty acid synthesis and increasing fatty acid oxidation via the AMPK-ACC signaling pathway. Additionally, increased CPT-I expression in skeletal muscle may also contribute to the enhanced oxidation of long-chain fatty acids in L-arginine-supplemented rats.

Role of the transcriptional factor C/EBPbeta in free fatty acid-elicited beta-cell failure.

Fatty acids can favour the development of Type 2 diabetes by reducing insulin secretion and inducing apoptosis of pancreatic beta-cells. Here, we show that sustained exposure of the beta-cell line MIN6 or of isolated pancreatic islets to the most abundant circulating fatty acid palmitate increases the level of C/EBPbeta, an insulin transcriptional repressor. In contrast, two unsaturated fatty acids, oleate and linoleate were without effect. The induction of C/EBPbeta elicited by palmitate was prevented by inhibiting the ERK1/2 MAP kinase pathway or by reducing mitochondrial fatty acid oxidation with an inhibitor of Carnitine Palmitoyl Transferase-1. Overexpression of C/EBPbeta mimicked the detrimental effects of palmitate and resulted in a drastic reduction in insulin promoter activity, impairment in the capacity to respond to secretory stimuli and an increase in apoptosis. Our data suggest a potential involvement of C/EBPbeta as mediator of the deleterious effects of unsaturated free fatty acids on beta-cell function.

Carnitine palmitoyltransferase modulation of hepatic fatty acid metabolism and radio-HPLC evidence for low ketogenesis in neonatal pigs.

A neonatal piglet model was used to study hepatic fatty acid metabolism during the early postnatal period. Hepatocytes were isolated from pigs at birth or after 24 h, in fed or unfed states (n = 4 pigs/group). Cells were incubated with 1 mmol/L [1-(14)C]-octanoate (C8) or -palmitate (C16) in the presence or absence of 1 mmol/L L-carnitine, carnitine plus tetradecylglycidic acid (TDGA; 10 mumol/L) or carnitine plus glucagon (0.5 microgram/L). Accumulation of radiolabel [nmol/(h. 10(6) cells)] in CO2 and acid-soluble products (ASP) was higher (3.5- and 4.5-fold, respectively) from C8 than from C16 (P < 0.0001). Glucagon, carnitine and TDGA had no effect on the oxidation of C8 (P > 0.1). Carnitine addition tended to increase C16 flux to ASP [from 5.3 to 7.6 nmol/(h. 10(6) cells); P < 0.1], whereas carnitine plus TDGA decreased flux (from 7.6 to 2.1; P < 0.001). Esterified products accounted for 70% of metabolized label in control C16 incubations; this was reduced to 62% by carnitine (P < 0.05) and increased to 80% by the addition of carnitine plus TDGA (P < 0.0001). The 1-(14)C flux to CO2 in cells from 24-h-old unfed piglets was 47% lower than from fed pigs (P < 0.01) but 28% higher than in pigs at birth. Radiolabel contained in ASP and total metabolized label were 48% lower from unfed pigs compared with the piglets at birth and 24-h-old fed pigs (P < 0.01) and were paralleled by changes in oxygen consumption.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphatidic acid biosynthesis in rat liver mitochondria and microsomal fractions. Regulation of fatty acid positional specificity.

The positional and fatty acid specificity of phosphatidic acid biosynthesis in rat liver mitochondria and microsomal fractions was studied by using acylcarnitines, CoA and an excess of carnitine palmitoyltransferase (EC 2.3.1.21) as the source of acyl-CoA. In the mitochondria, the preference for palmitic acid at the 1-position is increased at high acyl-CoA concentrations, whereas it is decreased in the microsomal fraction. There was no change in the fatty acid specificity at the 2-position with different acyl-CoA concentrations in any of the factions. The preference in mitochondria for linoleic acid at the 2-position is strongly increased at high concentrations of lysophosphatidic acid.