ABSTRACT
MEK is a canonical effector of mutant KRAS; however, MEK inhibitors fail to yield satisfactory clinical outcomes in KRAS-mutant cancers. Here, we identified mitochondrial oxidative phosphorylation (OXPHOS) induction as a profound metabolic alteration to confer KRAS-mutant non-small cell lung cancer (NSCLC) resistance to the clinical MEK inhibitor trametinib. Metabolic flux analysis demonstrated that pyruvate metabolism and fatty acid oxidation were markedly enhanced and coordinately powered the OXPHOS system in resistant cells after trametinib treatment, satisfying their energy demand and protecting them from apoptosis. As molecular events in this process, the pyruvate dehydrogenase complex (PDHc) and carnitine palmitoyl transferase IA (CPTIA), two rate-limiting enzymes that control the metabolic flux of pyruvate and palmitic acid to mitochondrial respiration were activated through phosphorylation and transcriptional regulation. Importantly, the co-administration of trametinib and IACS-010759, a clinical mitochondrial complex I inhibitor that blocks OXPHOS, significantly impeded tumor growth and prolonged mouse survival. Overall, our findings reveal that MEK inhibitor therapy creates a metabolic vulnerability in the mitochondria and further develop an effective combinatorial strategy to circumvent MEK inhibitors resistance in KRAS-driven NSCLC.
ABSTRACT
ObjectiveTo investigate the effect and mechanism of Mori Folium extract on the glucose and lipid metabolism disorders in the liver of rats with type 2 diabetes mellitus （T2DM） through the phosphatidylinositol 3-kinase/protein kinase B/peroxisome proliferation-activated receptor α/carnitine palmitoyl transferase-1 （PI3K/Akt/PPARα/CPT-1） signaling pathway. MethodThe T2DM model was induced by the high-fat diet combined with the intraperitoneal injection of streptozotocin （STZ）. The model rats were randomly divided into a model group， a metformin （0.2 g·kg-1） group， and a Mori Folium water extract （4.0 g·kg-1） group according to blood glucose and body weight. In the 8-week administration， fasting blood glucose was measured at the same time every week. The histomorphological and fat changes in the rat liver were observed by hematoxylin-eosin （HE） staining and oil red O staining. The levels of total cholesterol （TC）， triglyceride （TG）， low-density lipoprotein cholesterol （LDL-C）， high-density lipoprotein cholesterol （HDL-C）， alanine aminotransferase （ALT）， and aspartate aminotransferase （AST） in the serum were measured by biochemical methods. Western blot （WB） was used to quantitatively detect the protein expression of p-PI3K，PI3K，p-Akt，Akt，PPARα，and CPT-1 in the rat liver. ResultAfter 8-week administration， the blood glucose of rats was higher in the model group than that in the control group （P<0.01）， and lower in the Mori Folium water extract group than that in the model group （P<0.01）. The results of HE staining showed that the liver tissue structure of the control group was complete， and the hepatocytes were arranged radially around the central vein， while the hepatocyte injury in the model group was obvious. Compared with the model group， the Mori Folium water extract group showed improved vacuolar degeneration and no lesions such as small bile duct hyperplasia. Oil red O staining showed that there was no obvious steatosis and necrosis in the hepatocytes of rats in the control group， and no lipid droplets in the hepatocytes were observed， while the model group showed increased lipid droplets. Mori Folium significantly reduced the lipid droplets in the liver. Biochemical analysis showed that the levels of TC， TG， LDL-C， AST， and ALT in the model group were significantly higher than those in control group （P<0.01）. The levels of TC， TG， LDL-C， AST， and ALT in the Mori Folium water extract group were significantly lower than those in the model group （P<0.05，P<0.01）. WB showed that the protein expression of p-PI3K/PI3K， p-Akt/Akt， PPARα， and CPT-1 in the model group were lower than those in the control group （P<0.01）. Mori Folium water extract could increase the protein expression of p-PI3K/PI3K， p-Akt/Akt， PPARα， and CPT-1 （P<0.05 or P<0.01）. ConclusionThe hypoglycemic mechanism of Mori Folium water extract may be related to the regulation of the PI3K/Akt/PPARα/CPT-1 signaling pathway.
ABSTRACT
Objective To investigate the protective effect of ligustrazine on the transporting function of hepatocellular mitochondria membrane in the rats with sepsis-induced acute liver injury (SALI). Methods The Sprague-Dawley (SD) rats were randomly divided into sham operation group, SALI group [established by cecal ligation and puncture (CLP)], ligustrazine treatment group (injection of ligustrazine 60 mg/kg through tail vein after CLP) and ligustrazine preventive group (7 days before CLP, ligustrazine was injected daily through tail vein for 60 mg/kg), and there were 12 rats in each group. Abdominal aorta blood and liver were harvested at 10 hours after operation. The content of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and mitochondrial aspartate aminotransferase (m-AST) were determined by enzyme coupling rate method. The content of ATP was detected by colorimetric and chemical fluorescein method. The activity of mitochondrial ATPase was detected by phosphorus quantification. The expressions of mitochondrial membrane aquaporin 8 (AQP8) and carnitine palmitoyl transferase (CPT) were detected by Western Blot. Results Compared with sham operation group, the levels of serum ALT, AST and m-AST were significantly increased in SALI group, ligustrazine treatment group and ligustrazine preventive group, and the content of ATP was reduced, the activity of mitochondrial membrane ATPase, the expressions of AQP8 and CPT-1A were significantly decreased. Compared with SALI group, the levels of serum ALT, AST and m-AST were significantly decreased in ligustrazine treatment and ligustrazine preventive groups [ALT (U/L): 123.8±32.8, 105.0±44.5 vs. 233.0±110.1; AST (U/L):427.0±117.9, 303.9±110.3 vs. 742.6±441.4; m-AST (U/L): 239.6±64.9, 168.2±60.0 vs. 412.8±252.6; all P <0.01], the content of ATP were significantly increased (nmol/mg: 29.5±10.3, 34.6±11.2 vs. 19.3±8.8, both P < 0.01), the activity of ATPase in hepatocellular mitochondrial membrane were significantly increased [Na+-K+-ATPase (U/mg):3.91±0.30, 3.97±0.35 vs. 2.87±0.82; Mg2+-ATPase (U/mg): 3.75±0.38, 3.88±0.35 vs. 2.64±1.06; Ca2+-ATPase (U/mg): 3.15±0.58, 2.98±0.31 vs. 1.75±1.25; Ca2+-Mg2+-ATPase (U/mg): 3.82±0.31, 3.91±0.42 vs. 2.57±1.01, all P < 0.01], the expressions of AQP8 and CPT-1A were significantly increased [percentage increase from sham operation group (100%), AQP8/COX-Ⅳ: (79.12±7.79)%, (88.40±9.22)% vs. (62.08±11.91)%; CPT-1A/COX-Ⅳ:(87.92±10.06)%, (84.91±17.48)% vs. (72.11±7.82)%, all P < 0.01]. The levels of serum AST and m-AST in ligustrazine preventive group were significant lower than those in ligustrazine treatment group [AST (U/L): 303.9± 110.3 vs. 427.0±117.9; m-AST (U/L): 168.2±60.0 vs. 239.6±64.9, both P < 0.05]. There was no significant difference in the expression of CPT-2 in mitochondrial membrane between the four groups. Conclusions Ligustrazine could play a protective role on the mitochondrial membrane function of transporting water, ion and fat in the rats with SALI. The preventive function of ligustrazine is better than the treatment effect of the rats with sepsis.
ABSTRACT
Objective@#To investigate the dynamic expression of hepatic carnitine palmitoyltransferase-II (CPT-II) in the mitochondrial inner membrane during hepatocyte malignant transformation induced by lipid accumulation.@*Methods@#Male Sprague-Dawley rats were divided randomly into control, fatty liver, and induced cancer groups, which were fed with normal, high-fat (HF), and HF containing 2-fluorenylacetamide (0.05%, 2-FAA) diets, respectively, for 14 weeks. One rat from each group was sacrificed every two weeks and the blood and liver samples were collected. Liver morphological changes were evaluated with hematoxylin and eosin staining, and the liver tissue samples were divided into control, fatty liver, degeneration, precancerous, and cancerous groups accordingly. Hepatic lipids were dyed by the oil red O method. The CPT-II expression was measured by immunohistochemistry and compared with the specific CPT-II concentration (ng/mg liver protein, ng/mg P) among different groups. Serum levels of circulating total cholesterol (Tch), triglyceride (TG), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were quantitatively analyzed.@*Results@#Massive lipid accumulation hepatocytes was seen in rats on HF and HF containing 2-FAA diets. The lipid levels in the control group were significantly lower than those in the fatty liver (t = -11.556, P < 0.001), degeneration (t = -4.847, P = 0.04), precancerous (t = -13.652, P = 0.005), and cancerous groups (t = -10.896, P = 0.008). The serum TG and Tch levels in the degeneration, precancerous, and cancerous groups were 2-3 times higher than those in the control group (P < 0.05). After 2-FAA treatment, the morphological changes of rat hepatocytes showed the progression from degeneration and precancerosis to cancerosis, with hepatocyte injury. The serum AST and ALT levels in the degeneration, precancerous, and cancerous groups were significantly higher (4-8 times) than those in the control group (P < 0.05). The specific concentration of liver CPT-II expression was significantly reduced during hepatocyte malignant transformation, as confirmed by immunohistochemistry, with the CPT-II levels significantly lower in the cancerous group than in any of other groups (P < 0.05).@*Conclusion@#Low hepatic CPT-II expression might lead to abnormal lipid accumulation in hepatocytes, which should promote the malignant transformation of hepatocytes.
ABSTRACT
We examined the capacity of high-intensity intermittent training (HI-IT) to facilitate the delivery of lipids to enzymes responsible for oxidation, a task performed by the carnitine palmitoyl transferase (CPT) system in the rat gastrocnemius muscle. Male adult Wistar rats (160-250 g) were randomly distributed into 3 groups: sedentary (Sed, N = 5), HI-IT (N = 10), and moderate-intensity continuous training (MI-CT, N = 10). The trained groups were exercised for 8 weeks with a 10% (HI-IT) and a 5% (MI-CT) overload. The HI-IT group presented 11.8% decreased weight gain compared to the Sed group. The maximal activities of CPT-I, CPT-II, and citrate synthase were all increased in the HI-IT group compared to the Sed group (P < 0.01), as also was gene expression, measured by RT-PCR, of fatty acid binding protein (FABP; P < 0.01) and lipoprotein lipase (LPL; P < 0.05). Lactate dehydrogenase also presented a higher maximal activity (nmol·min-1·mg protein-1) in HI-IT (around 83%). We suggest that 8 weeks of HI-IT enhance mitochondrial lipid transport capacity thus facilitating the oxidation process in the gastrocnemius muscle. This adaptation may also be associated with the decrease in weight gain observed in the animals and was concomitant to a higher gene expression of both FABP and LPL in HI-IT, suggesting that intermittent exercise is a "time-efficient" strategy inducing metabolic adaptation.