AMPK Phosphorylates and Inhibits SREBP Activity to Attenuate Hepatic Steatosis and Atherosclerosis in Diet-Induced Insulin-Resistant Mice

SummaryAMPK has emerged as a critical mechanism for salutary effects of polyphenols on lipid metabolic disorders in type 1 and type 2 diabetes. Here we demonstrate that AMPK interacts with and directly phosphorylates sterol regulatory element binding proteins (SREBP-1c and -2). Ser372 phosphorylation of SREBP-1c by AMPK is necessary for inhibition of proteolytic processing and transcriptional activity of SREBP-1c in response to polyphenols and metformin. AMPK stimulates Ser372 phosphorylation, suppresses SREBP-1c cleavage and nuclear translocation, and represses SREBP-1c target gene expression in hepatocytes exposed to high glucose, leading to reduced lipogenesis and lipid accumulation. Hepatic activation of AMPK by the synthetic polyphenol S17834 protects against hepatic steatosis, hyperlipidemia, and accelerated atherosclerosis in diet-induced insulin-resistant LDL receptor-deficient mice in part through phosphorylation of SREBP-1c Ser372 and suppression of SREBP-1c- and -2-dependent lipogenesis. AMPK-dependent phosphorylation of SREBP may offer therapeutic strategies to combat insulin resistance, dyslipidemia, and atherosclerosis

Exploration of the hypoglycemic mechanism of Fuzhuan brick tea based on integrating global metabolomics and network pharmacology analysis

Introduction: Fuzhuan brick tea (FBT) is a worldwide popular beverage which has the appreciable potential in regulating glycometabolism. However, the reports on the hypoglycemic mechanism of FBT remain limited.Methods: In this study, the hypoglycemic effect of FBT was evaluated in a pharmacological experiment based on Kunming mice. Global metabolomics and network pharmacology were combined to discover the potential target metabolites and genes. In addition, the real-time quantitative polymerase chain reaction (RT-qPCR) analysis was performed for verification.Results: Seven potential target metabolites and six potential target genes were screened using the integrated approach. After RT-qPCR analysis, it was found that the mRNA expression of VEGFA, KDR, MAPK14, and PPARA showed significant differences between normal and diabetes mellitus mice, with a retracement after FBT treatment.Conclusion: These results indicated that the hypoglycemic effect of FBT was associated with its anti-inflammatory activities and regulation of lipid metabolism disorders. The exploration of the hypoglycemic mechanism of FBT would be meaningful for its further application and development

The Antimicrobial Peptide Mastoparan X Protects Against Enterohemorrhagic Escherichia coli O157:H7 Infection, Inhibits Inflammation, and Enhances the Intestinal Epithelial Barrier

Escherichia coli can cause intestinal diseases in humans and livestock, destroy the intestinal barrier, exacerbate systemic inflammation, and seriously threaten human health and animal husbandry development. The aim of this study was to investigate whether the antimicrobial peptide mastoparan X (MPX) was effective against E. coli infection. BALB/c mice infected with E. coli by intraperitoneal injection, which represents a sepsis model. In this study, MPX exhibited no toxicity in IPEC-J2 cells and notably suppressed the levels of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), myeloperoxidase (MPO), and lactate dehydrogenase (LDH) released by E. coli. In addition, MPX improved the expression of ZO-1, occludin, and claudin and enhanced the wound healing of IPEC-J2 cells. The therapeutic effect of MPX was evaluated in a murine model, revealing that it protected mice from lethal E. coli infection. Furthermore, MPX increased the length of villi and reduced the infiltration of inflammatory cells into the jejunum. SEM and TEM analyses showed that MPX effectively ameliorated the jejunum damage caused by E. coli and increased the number and length of microvilli. In addition, MPX decreased the expression of IL-2, IL-6, TNF-α, p-p38, and p-p65 in the jejunum and colon. Moreover, MPX increased the expression of ZO-1, occludin, and MUC2 in the jejunum and colon, improved the function of the intestinal barrier, and promoted the absorption of nutrients. This study suggests that MPX is an effective therapeutic agent for E. coli infection and other intestinal diseases, laying the foundation for the development of new drugs for bacterial infections

Activation of sterol regulatory element binding protein and NLRP3 inflammasome in atherosclerotic lesion development in diabetic pigs.

Aberrantly elevated sterol regulatory element binding protein (SREBP), the lipogenic transcription factor, contributes to the development of fatty liver and insulin resistance in animals. Our recent studies have discovered that AMP-activated protein kinase (AMPK) phosphorylates SREBP at Ser-327 and inhibits its activity, represses SREBP-dependent lipogenesis, and thereby ameliorates hepatic steatosis and atherosclerosis in insulin-resistant LDLR(-/-) mice. Chronic inflammation and activation of NLRP3 inflammasome have been implicated in atherosclerosis and fatty liver disease. However, whether SREBP is involved in vascular lipid accumulation and inflammation in atherosclerosis remains largely unknown.The preclinical study with aortic pouch biopsy specimens from humans with atherosclerosis and diabetes shows intense immunostaining for SREBP-1 and the inflammatory marker VCAM-1 in atherosclerotic plaques. The cleavage processing of SREBP-1 and -2 and expression of their target genes are increased in the well-established porcine model of diabetes and atherosclerosis, which develops human-like, complex atherosclerotic plaques. Immunostaining analysis indicates an elevation in SREBP-1 that is primarily localized in endothelial cells and in infiltrated macrophages within fatty streaks, fibrous caps with necrotic cores, and cholesterol crystals in advanced lesions. Moreover, concomitant suppression of NAD-dependent deacetylase SIRT1 and AMPK is observed in atherosclerotic pigs, which leads to the proteolytic activation of SREBP-1 by diminishing the deacetylation and Ser-372 phosphorylation of SREBP-1. Aberrantly elevated NLRP3 inflammasome markers are evidenced by increased expression of inflammasome components including NLPR3, ASC, and IL-1β. The increase in SREBP-1 activity and IL-1β production in lesions is associated with vascular inflammation and endothelial dysfunction in atherosclerotic pig aorta, as demonstrated by the induction of NF-κB, VCAM-1, iNOS, and COX-2, as well as by the repression of eNOS.These translational studies provide in vivo evidence that the dysregulation of SIRT1-AMPK-SREBP and stimulation of NLRP3 inflammasome may contribute to vascular lipid deposition and inflammation in atherosclerosis

Yishen-tongbi decoction inhibits excessive activation of B cells by activating the FcγRIIb/Lyn/SHP-1 pathway and attenuates the inflammatory response in CIA rats

Rheumatoid arthritis (RA) is a chronic autoimmune disease. Strong evidence supports that excessive activation of B cells plays a critical role in the pathogenesis of RA. Fc gamma receptor b (FcγRIIb) is the B cell inhibitory receptor and inhibits BCR (B cell receptor) signalling in part by selectively dephosphorylating CD19 which is considered a co-receptor for BCR and is essential for B cell activation. Our previous study demonstrated that a FcγRIIb I232T polymorphism presented a strong genetic link to RA and may lead to the excessive activation of B cells. Therefore, novel therapeutic strategies and drugs that can effectively inhibit the excessive activation of B cells by regulating the FcγRIIb are necessary for the treatment of RA. Therefore, we used Burkitt’s lymphoma ST486 human B cells (lacking endogenous FcγRIIb) transfected with the 232Thr loss-of-function mutant to construct a FcγRIIb mutant cell line (ST486), and we demonstrated that YSTB treatment not only reduced proliferation and promoted apoptosis in ST486 cells but also did so in a dose-dependent manner. Furthermore, the intracellular Ca2+ flux of ST486 cells was decreased after treatment with YSTB, inhibiting the excessive activation of ST486 cells, and these effects correlated with the CD19/FcγRIIb-Lyn-SHP-1 pathways. Our data showed that YSTB treatment inhibited the expression of phosphorylated CD19 and upregulated the protein expression of FcγRIIb, Lyn, and SHP-1. Additionally, the CIA model was established to explore the anti-inflammatory and inhibitory effects of YSTB on bone destruction, and we found that YSTB decreased the paw oedema and arthritis index (AI) in CIA rats. It is worth mentioning that YSTB clearly decreased the AI earlier than methotrexate (MTX) (day 10 vs 16). Moreover, synovial hyperplasia, inflammatory cell infiltration and cartilage surface erosion in CIA rats were noticeably reduced after treatment with YSTB as evidenced by histopathological examination. Finally, we found that YSTB treatment suppressed bone erosion and joint space score (JNS) in CIA rats as evidenced by radiographic assessment. In summary, these data suggest that YSTB has great therapeutic potential for RA treatment

The cleavage processing of SREBP-1 and -2 as well as expression of their target genes are enhanced in the aorta of diabetic, atherosclerotic pigs.

<p><b>A</b>. The transcriptional regulation of <i>de novo</i> lipogenic enzymes (blue color) by SREBP-1. <b>B</b>. The levels of mature, active form of SREBP-1 and expression of its target lipogenic enzyme, fatty acid synthase (FAS), are increased in the aorta of diabetic and atherosclerotic pigs. Total aortic lysates (100 µg proteins) were resolved by 8% SDS-PAGE and immunoblotted with the specific antibody to recognize both precursor (<b>P</b>, ∼125 kDa) and active mature (<b>M</b>, ∼68 kDa) forms of SREBP-1. Representative immunoblots of aortic tissues from two pigs in each group are shown. <b>C</b>. Densitometric quantification of the mature form of SREBP-1 is normalized to that of β-actin and expressed as relative levels to control pigs. <b>D</b> and <b>E</b>. The transcription of SREBP-1 target genes is elevated in the aorta of diabetic, atherosclerotic pigs. Total RNA was isolated from pig aorta, and mRNA levels of genes encoding stearoyl-CoA desaturase1 (SCD1) (<b>D</b>) and glycerol-3-phosphate acyltransferase (GPAT) (<b>E</b>) were determined by real-time PCR. <b>F</b> and <b>G</b>. The levels of the cleaved form of SREBP-2 protein were quantified by densitometry, normalized to those of β-actin, and expressed as relative levels to normal pigs. <b>H</b> and <b>I</b>. The mRNA amounts of SREBP-2 and its target gene, HMGCoA synthase (HMGCS), are elevated in atherosclerotic pigs. The mRNA levels of SREBP-2 and cholestrogenic genes were determined by real-time PCR, normalized to those of β-actin, and presented as relative levels to control pigs. Data were presented as the mean ± S.E.M., n = 3, *P<0.05, vs control pigs.</p

NLRP3 inflammasome and inflammatory response are increased in the aorta of diabetic, atherosclerotic pigs.

<p><b>A–C</b>. expression of NLRP3 and apoptosis-associated speck-like protein (ASC) is increased in the aorta of diabetic, atherosclerotic pigs. <b>D</b>. The mRNA levels of proinflammatory cytokine interlucin-1 β (IL-1β) are increased in the aorta of diabetic, atherosclerotic pigs. <b>E</b> and <b>F</b>. The mRNA amounts of SREBP-1a and NF-κB are elevated in diabetic, atherosclerotic pigs. <b>G</b>. The expression of eNOS, indicative of endothelial function, is reduced in diabetic, atherosclerotic pigs. Data were presented as the mean ± S.E.M., n = 3, *P<0.05, vs control pigs.</p

Characterization of human-like, complicated atherosclerotic phenotypes in the well-established porcine model of diabetes-accelerated atherosclerosis.

<p>Histopathologic characteristics of early/intermediate lesions and advanced plaques in diabetic and atherosclerotic pigs. Representative H&E staining (<b>A</b>) and Oil Red O staining (<b>B</b>) of aortic sections of control pigs (<b>a,b</b>, <b>i</b> and <b>j</b>) and diabetic, atherosclerotic pigs (<b>c–h</b> and <b>k–o</b>) are shown (scale bars, 100 µm). H&E-staining (<b>d–h</b>) showed a human-like fibroatheroma with a fibrous cap at the shoulder and a necrotic lipid core at the base of lesion (L = lumen, FC = fibrous cap, NC = necrotic lipid core, and M = media). Note the clear space (yellow arrow) which represents an area of cholesterol crystals removed during tissue processing (<b>e</b>) and smooth muscle cell migration from the media to the intima (green arrow) (<b>g</b>). The advanced plaques have necrotic lipid cores containing large numbers of cholesterol crystals (<b>m</b>, <b>n</b> and <b>o</b>).</p