Ketogenic diet ameliorates lipid dysregulation in type 2 diabetic mice by downregulating hepatic pescadillo 1

Background Previous reports implied a possible link between PES1 and lipid metabolism. However, the role of PES1 in regulating T2DM related lipid metabolism and the effect of ketogenic diet (KD) on PES1 have not been reported. The aim of present study is to explore the role of PES1 in effects of KD on diabetic mice and its mediated mechanism. Methods Male C57BL/6J and KKAy mice were fed with standard diet (SD) and KD, respectively. Simultaneously, McArdle 7777 cells were treated by β-hydroxybutyric acid (β-HB), Pes1 siRNA or Pes1 overexpression plasmid, respectively. Additionally, liver-conditional knockout (CKO) of Pes1 in vivo was applied. Results Hepatic PES1 expression in diabetic mice was markedly increased, which was suppressed by KD feeding with an accompanying reduction of hepatic and plasma triglycerides (TG). In mice with CKO of Pes1, the protein levels of p300, SREBP1c, FASN, SCD1, Caspase1, NLRP3 and GSDMD were dramatically downregulated in livers, and the plasma and hepatic TG, IL-1β and IL-18 were decreased as well. The similar outcomes were also observed in β-HB and Pes1 knockdown treated hepatocytes. By contrast, Pes1 overexpression in cultured hepatocytes showed that these levels were significantly enhanced, which were, however reduced under β-HB treatment. Mechanistically, we discovered that β-HB decreased CHOP binding to the Pes1 promoters, resulting in the downregulation of PES1, thereby reducing PES1 binding to p300 and Caspase1 promoters. The inhibition of p300 and Caspase1 expression elicited the dramatic suppression of acetylation of SREBP1c via its interaction with p300, and the decreased GSDMD levels. Besides, knockdown of Caspase1 also alleviated the TG levels in cultured hepatocytes. Conclusion KD may improve lipid dysregulation in type 2 diabetic mice by downregulating hepatic PES1 expression.

Ketogenic Diet Ameliorates Lipid Dysregulation in Type 2 Diabetic Mice by Downregulating Hepatic Pescadillo 1

Background: Previous reports implied a possible link between PES1 and lipid metabolism. However, the role of PES1 in regulation of T2DM related lipid metabolism and the effect of KD on PES1 have not been reported. The aim of present study is to explore the role of PES1 in effects of ketogenic diet (KD) on diabetic mice and its mediated mechanism.Methods: Male C57BL/6J and KKAy mice were fed with standard diet (SD) and KD, respectively. Simultaneously, McArdle 7777 cells were treated by β-hydroxybutyric acid (β-HB), Pes1 siRNA or Pes1 overexpression plasmid, respectively. Additionally, liver-conditional knockout (CKO) of Pes1 in vivo were used.Results: We unexpectedly found that hepatic PES1 expression in T2DM patients was markedly elevated, but the elevated PES1 was suppressed by KD feeding in T2DM mice with the reduction of hepatic and plasma triglycerides (TG). In mice with CKO of Pes1, the protein levels of p300, SREBP1c, FASN, SCD1, caspase1, NLRP3 and GSDMD were dramatically downregulated in livers, and the plasma and hepatic TG, IL-1β and IL-18 were decreased as well. The similar phenomena were also observed in β-HB and Pes1 knockdown treated hepatocytes. By contrast, Pes1 overexpression in cultured hepatocytes showed that these levels were significantly enhanced, which however were reduced under β-HB treatment. Mechanistically, we discovered that β-HB decreased CHOP binding to the Pes1 promoters, resulting in the downregulation of PES1, thereby reducing PES1 binding to p300 and caspase1 promoters. The inhibition of p300 and caspase1 expressions elicited the dramatic suppression of acetylation of SREBP1c via its interaction with p300, and the decreased GSDMD levels. Besides, knockdown of caspase1 also alleviated the TG levels in cultured hepatocytes.Conclusion: KD may improve lipid dysregulation in type 2 diabetic mice by downregulating hepatic PES1 expression.

Analysis and Optimization of Conditions for the Use of 2′,7′-Dichlorofluorescein Diacetate in Cultured Hepatocytes

Numerous liver pathologies encompass oxidative stress as molecular basis of disease. The use of 2′,7′-dichlorodihydrofluorescein-diacetate (DCFH2-DA) as fluorogenic redox probe is problematic in liver cell lines because of membrane transport proteins that interfere with probe kinetics, among other reasons. The properties of DCFH2-DA were analyzed in hepatocytes (HepG2, HepaRG) to characterize methodological issues that could hamper data interpretation and falsely skew conclusions. Experiments were focused on probe stability in relevant media, cellular probe uptake/retention/excretion, and basal oxidant formation and metabolism. DCFH2-DA was used under optimized experimental conditions to intravitally visualize and quantify oxidative stress in real-time in HepG2 cells subjected to anoxia/reoxygenation. The most important findings were that: (1) the non-fluorescent DCFH2-DA and the fluorescent DCF are rapidly taken up by hepatocytes, (2) DCF is poorly retained in hepatocytes, and (3) DCFH2 oxidation kinetics are cell type-specific. Furthermore, (4) DCF fluorescence intensity was pH-dependent at pH < 7 and (5) the stability of DCFH2-DA in cell culture medium relied on medium composition. The use of DCFH2-DA to measure oxidative stress in cultured hepatocytes comes with methodological and technical challenges, which were characterized and solved. Optimized in vitro and intravital imaging protocols were formulated to help researchers conduct proper experiments and draw robust conclusions.

Growth Hormone Signaling Pathway Leading to the Induction of DNA Synthesis and Proliferation in Primary Cultured Hepatocytes of Adult Rats

Background: We investigated the signal transduction pathway associated with growth hormone (GH)-stimulated DNA synthesis and proliferation in primary cultured hepatocytes. Methods: Adult rat hepatocytes were isolated from normal livers by two-step in situ collagenase perfusion to facilitate disaggregation of the adult rat liver. Then hepatocytes were cultured in serum-free Williams’ medium E supplemented with GH (1-100 ng/ml) in the presence or absence of test reagents. GH-induced hepatocyte DNA synthesis and proliferation were determined, and the phosphorylation activities of Janus kinase (JAK) 2 (JAK2) (p125 kDa), p95-kDa RTK, and ERK1/2 were measured by western blotting. Results: Hepatocytes grown in serum-free defined medium proliferated within 5 h of culture in the presence of GH (100 ng/ml) in a concentration- and time-dependent manner (EC50 75 ng/ml). These proliferative effects of GH were almost completely blocked by an anti-GH receptor monoclonal antibody (85 ng/ml) and an anti-insulin-like growth factor (IGF)-I receptor monoclonal antibody. In addition, the proliferative effects of GH were significantly blocked by a JAK2 inhibitor (TG101209, 10−6 M), as well as specific signal-transducing inhibitors of phospholipase C (PLC; U-73122, 10−6 M), RTK (AG538, 10−6 M), phosphoinositide 3-kinase (PI3K; LY294002, 10−6 M), mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK; PD98059, 10−6 M), and mammalian target of rapamycin (mTOR; rapamycin, 10 ng/ml). GH significantly induced the phosphorylations of JAK2 (p125 kDa), p95-kDa IGF-I receptor tyrosine kinase (RTK), and ERK2 in this order according to western blotting analysis. Conclusions: The proliferative action of GH is mediated by two main signaling pathways. One includes activation of the GH receptor/JAK2/PLC/Ca2+ pathway, and the other involves activation of the p95-kDa IGF-I RTK/PI3K/ERK2/mTOR pathway in primary cultures of adult rat hepatocytes.