Kuai Li

Carnitine palmitoyltransferase 1A prevents fatty acid-induced adipocyte dysfunction through suppression of c-Jun N-terminal kinase.

The adipocyte is the principal cell type for fat storage. CPT1 (carnitine palmitoyltransferase-1) is the rate-limiting enzyme for fatty acid β-oxidation, but the physiological role of CPT1 in adipocytes remains unclear. In the present study, we focused on the specific role of CPT1A in the normal functioning of adipocytes. Three 3T3-L1 adipocyte cell lines stably expressing hCPT1A (human CPT1A) cDNA, mouse CPT1A shRNA (short-hairpin RNA) or GFP (green fluorescent protein) were generated and the biological functions of these cell lines were characterized. Alteration in CPT1 activity, either by ectopic overexpression or pharmacological inhibition using etomoxir, did not affect adipocyte differentiation. However, overexpression of hCPT1A significantly reduced the content of intracellular NEFAs (non-esterified fatty acids) compared with the control cells when adipocytes were challenged with fatty acids. The changes were accompanied by an increase in fatty acid uptake and a decrease in fatty acid release. Interestingly, CPT1A protected against fatty acid-induced insulin resistance and expression of pro-inflammatory adipokines such as TNF-α (tumour necrosis factor-α) and IL-6 (interleukin-6) in adipocytes. Further studies demonstrated that JNK (c-Jun N terminal kinase) activity was substantially suppressed upon CPT1A overexpression, whereas knockdown or pharmacological inhibition of CPT1 caused a significant enhancement of JNK activity. The specific inhibitor of JNK SP600125 largely abolished the changes caused by the shRNA- and etomoxir-mediated decrease in CPT1 activity. Moreover, C2C12 myocytes co-cultured with adipocytes pre-treated with fatty acids displayed altered insulin sensitivity. Taken together, our findings have identified a favourable role for CPT1A in adipocytes to attenuate fatty acid-evoked insulin resistance and inflammation via suppression of JNK.

Adipose tissue deletion of Gpr116 impairs insulin sensitivity through modulation of adipose function

G protein‐coupled receptor 116 (GPR116) is a novel member of the G protein‐coupled receptors and its function is largely unknown. To investigate the physiological function of GPR116 in vivo, we generated adipose tissue specific conditional Gpr116 knockout mice (CKO) and fed them on standard chow or high fat diets. Selective deletion of Gpr116 in adipose tissue caused a pronounced glucose intolerance and insulin resistance in mice, especially when challenged with a high fat diet. Biochemical analysis revealed a more severe hepatosteatosis in CKO mice. Additionally, we found that CKO mice showed a lowered concentration of circulating adiponectin and an increased level of serum resistin. Our study suggests that GPR116 may play a critical role in controlling adipocyte biology and systemic energy homeostasis.

Adipocyte SIRT1 controls systemic insulin sensitivity by modulating macrophages in adipose tissue

Adipose tissue inflammation, characterized by augmented infiltration and altered polarization of macrophages, contributes to insulin resistance and its associated metabolic diseases. The NAD+‐dependent deacetylase SIRT1 serves as a guardian against metabolic disorders in multiple tissues. To dissect the roles of SIRT1 in adipose tissues, metabolic phenotypes of mice with selective ablation of SIRT1 in adipocytes and myeloid cells were monitored. Compared to myeloid‐specific SIRT1 depletion, mice with adipocyte‐selective deletion of SIRT1 are more susceptible to diet‐induced insulin resistance. The phenotypic changes in adipocyte‐selective SIRT1 knockout mice are associated with an increased number of adipose‐resident macrophages and their polarization toward the pro‐inflammatory M1 subtype. Mechanistically, SIRT1 in adipocytes modulates expression and secretion of several adipokines, including adiponectin, MCP‐1, and interleukin 4, which in turn alters recruitment and polarization of the macrophages in adipose tissues. In adipocytes, SIRT1 deacetylates the transcription factor NFATc1 and thereby enhances the binding of NFATc1 to the Il4 gene promoter. These findings suggest that adipocyte SIRT1 controls systemic glucose homeostasis and insulin sensitivity via the cross talk with adipose‐resident macrophages.

Visualization and quantification of browning using a Ucp1-2A-luciferase knock-in mouse model

Both mammals and adult humans possess classic brown adipocytes and beige adipocytes, and the amount and activity of these adipocytes are considered key factors in combating obesity and its associated metabolic diseases. Uncoupling protein 1 (Ucp1) is the functional marker of both brown and beige adipocytes. To facilitate a reliable, easy, and sensitive measurement of Ucp1 expression both in vivo and in vitro, we generated a Ucp1-2A-luciferase knock-in mouse by deleting the stop codon for the mouse Ucp1 gene and replacing it with a 2A peptide. This peptide was followed by the luciferase coding sequence to recapitulate the expression of the Ucp1 gene at the transcriptional and translational levels. With this mouse, we discovered a cold-sensitive brown/beige adipose depot underneath the skin of the ears, which we named uBAT. Because of the sensitivity and high dynamic range of luciferase activity, the Ucp1-2A-luciferase mouse is useful for both in vitro quantitative determination and in vivo visualization of nonshivering thermogenesis. With the use of this model, we identified and characterized axitinib, an oral small-molecule tyrosine kinase inhibitor, as an effective browning agent.

Crystal structure of isoamyl acetate‐hydrolyzing esterase from Saccharomyces cerevisiae reveals a novel active site architecture and the basis of substrate specificity

Crystal structureof isoamyl acetate-hydrolyzing esterase from Saccharomyces cerevisiae reveals a novel active site architecture and thebasisof substratespecificity Jinming Ma, Qianda Lu, Ye Yuan, Honghua Ge, Kuai Li, Wei Zhao, Yongxiang Gao, Liwen Niu,* and Maikun Teng* 1Hefei National Laboratory for Physical Sciences at Microscale, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, China 2 Key Laboratory of Structural Biology, Chinese Academy of Sciences, Hefei, Anhui 230026, China

BMS309403 Stimulates Glucose Uptake in Myotubes through Activation of AMP-Activated Protein Kinase

BMS309403 is a biphenyl azole inhibitor against fatty acid binding protein 4 (FABP4) and regarded as a lead compound for effective treatment of obesity related cardio-metabolic diseases. Here we discovered an off-target activity of BMS309403 in that it stimulates glucose uptake in C2C12 myotubes in a temporal and dose dependent manner via activation of AMP-activated protein kinase (AMPK) signaling pathway but independent of FABPs. Further analysis indicated that BMS309403 activates AMPK through increasing the ratio of intracellular AMP:ATP while decreasing mitochondrial membrane potential. These findings provide mechanistic insights on the action of BMS309403.

The discovery of novel and selective fatty acid binding protein 4 inhibitors by virtual screening and biological evaluation.

Adipocyte fatty acid binding protein (AFABP, FABP4) has been proven to be a potential therapeutic target for diabetes, atherosclerosis and inflammation-related diseases. In this study, a series of new scaffolds of small molecule inhibitors of FABP4 were identified by virtual screening and were validated by a bioassay. Fifty selected compounds were tested, which led to the discovery of seven hits. Structural similarity-based searches were then performed based on the hits and led to the identification of one high affinity compound 33b (Ki=0.29±0.07μM, ΔTm=8.5°C). This compounds effective blockade of inflammatory response was further validated by its ability to suppress pro-inflammatory cytokines induced by lipopolysaccharide (LPS) stimulation. Molecular dynamics simulation (MD) and mutagenesis studies validated key residues for its inhibitory potency and thus provide an important clue for the further development of drugs.

Absence of Appl2 sensitizes endotoxin shock through activation of PI3K/Akt pathway

BackgroundThe adapter proteins Appl1 (adaptor protein containing pleckstrin homology domain, phosphotyrosine domain, and leucine zipper motif 1) and Appl2 are highly homologous and involved in several signaling pathways. While previous studies have shown that Appl1 plays a pivotal role in adiponectin signaling and insulin secretion, the physiological functions of Appl2 are largely unknown.ResultsIn the present study, the role of Appl2 in sepsis shock was investigated by using Appl2 knockout (KO) mice. When challenged with lipopolysaccharides (LPS), Appl2 KO mice exhibited more severe symptoms of endotoxin shock, accompanied by increased production of proinflammatory cytokines. In comparison with the wild-type control, deletion of Appl2 led to higher levels of TNF-α and IL-1β in primary macrophages. In addition, phosphorylation of Akt and its downstream effector NF-κB was significantly enhanced. By co-immunoprecipitation, we found that Appl2 and Appl1 interacted with each other and formed a complex with PI3K regulatory subunit p85α, which is an upstream regulator of Akt. Consistent with these results, deletion of Appl1 in macrophages exhibited characteristics of reduced Akt activation and decreased the production of TNFα and IL-1β when challenged by LPS.ConclusionsResults of the present study demonstrated that Appl2 is a critical negative regulator of innate immune response via inhibition of PI3K/Akt/NF-κB signaling pathway by forming a complex with Appl1 and PI3K.

High level expression and purification of active recombinant human interleukin-15 in Pichia pastoris

Interleukin-15 (IL-15) is a pleiotropic cytokine and a member of the four α-helix bundle family of cytokines which include IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21. IL-15 exhibits a broad biological activity and induces the differentiation and proliferation of T, B and natural killer (NK) cells. In this study, a DNA fragment containing the mature human IL-15 sequence was cloned into pPICZaA vector, generating a fusion protein with the alpha factor signal sequence in the N-terminus and 6×His as well as c-Myc tags in the C-terminus. The resulting plasmid was integrated into the genome of Pichia pastoris strain X-33. Recombinant yeast transformants with high-level recombinant human IL-15 (rhIL-15) production were identified, which secrete as much as 75 mg/L rhIL-15 after 3 days of induction by methanol. The rhIL-15 was purified by Ni(+)-NTA affinity chromatography, followed by DEAE anion exchange, yielding over 95% highly purified rhIL-15. Mass spectrometry and MALDI-TOF-TOF analysis showed the purified rhIL-15 had larger molecular weights than expected, due to different degrees of N-linked glycosylation. The biological activity of the rhIL-15 proteins was measured by its ability to enhance cellular proliferation of CTLL-2 and NK cells. The results demonstrate that the experimental procedure we have reported here can produce a large amount of active recombinant human IL-15 from P. pastoris.

Cloning, expression, purification, crystallization and preliminary crystallographic analysis of 5-aminolaevulinic acid dehydratase from Bacillus subtilis

5-aminolaevulinic acid dehydratase (ALAD), a crucial enzyme in the biosynthesis of tetrapyrrole, catalyses the condensation of two 5-aminolaevulinic acid (ALA) molecules to form porphobilinogen (PBG). The gene encoding ALAD was amplified from genomic DNA of Bacillus subtilis and the protein was overexpressed in Escherichia coli strain BL21 (DE3). The protein was purified and crystallized with an additional MGSSHHHHHHSSGLVPRGSH- tag at the N-terminus of the target protein. Diffraction-quality single crystals were obtained by the hanging-drop vapour-diffusion method. An X-ray diffraction data set was collected at a resolution of 2.7 A.