DsbA-L prevents obesity-induced inflammation and insulin resistance by suppressing the mtDNA release-activated cGAS-cGAMP-STING pathway.

Chronic inflammation in adipose tissue plays a key role in obesity-induced insulin resistance. However, the mechanisms underlying obesity-induced inflammation remain elusive. Here we show that obesity promotes mtDNA release into the cytosol, where it triggers inflammatory responses by activating the DNA-sensing cGAS-cGAMP-STING pathway. Fat-specific knockout of disulfide-bond A oxidoreductase-like protein (DsbA-L), a chaperone-like protein originally identified in the mitochondrial matrix, impaired mitochondrial function and promoted mtDNA release, leading to activation of the cGAS-cGAMP-STING pathway and inflammatory responses. Conversely, fat-specific overexpression of DsbA-L protected mice against high-fat diet-induced activation of the cGAS-cGAMP-STING pathway and inflammation. Taken together, we identify DsbA-L as a key molecule that maintains mitochondrial integrity. DsbA-L deficiency promotes inflammation and insulin resistance by activating the cGAS-cGAMP-STING pathway. Our study also reveals that, in addition to its well-characterized roles in innate immune surveillance, the cGAS-cGAMP-STING pathway plays an important role in mediating obesity-induced metabolic dysfunction.

Endoplasmic reticulum (ER) localization is critical for DsbA-L protein to suppress ER stress and adiponectin down-regulation in adipocytes.

Adiponectin is an adipokine with insulin-sensitizing and anti-inflammatory functions. We previously reported that adiponectin multimerization and stability are promoted by the disulfide bond A oxidoreductase-like protein (DsbA-L) in cells and in vivo. However, the precise mechanism by which DsbA-L regulates adiponectin biosynthesis remains elusive. Here we show that DsbA-L is co-localized with the endoplasmic reticulum (ER) marker protein disulfide isomerase and the mitochondrial marker MitoTracker. In addition, DsbA-L interacts with the ER chaperone protein Ero1-Lα in 3T3-L1 adipocytes. In silico analysis and truncation mapping studies revealed that DsbA-L contains an ER targeting signal at its N terminus. Deletion of the first 6 residues at the N terminus greatly impaired DsbA-L localization in the ER. Overexpression of the wild type but not the ER localization-defective mutant of DsbA-L protects against thapsigargin-induced ER stress and adiponectin down-regulation in 3T3-L1 adipocytes. In addition, overexpression of the wild type but not the ER localization-defective mutant of DsbA-L promotes adiponectin multimerization. Together, our results reveal that DsbA-L is localized in both the mitochondria and the ER in adipocytes and that its ER localization plays a critical role in suppressing ER stress and promoting adiponectin biosynthesis and secretion.

Phosphorylation of adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain, and leucine zipper motif 1 (APPL1) at Ser430 mediates endoplasmic reticulum (ER) stress-induced insulin resistance in hepatocytes.

APPL1 is an adaptor protein that plays a critical role in regulating adiponectin and insulin signaling. However, how APPL1 is regulated under normal and pathological conditions remains largely unknown. In this study, we show that APPL1 undergoes phosphorylation at Ser(430) and that this phosphorylation is enhanced in the liver of obese mice displaying insulin resistance. In cultured mouse hepatocytes, APPL1 phosphorylation at Ser(430) is stimulated by phorbol 12-myristate 13-acetate, an activator of classic PKC isoforms, and by the endoplasmic reticulum (ER) stress inducer, thapsigargin. Overexpression of wild-type but not dominant negative PKCα increases APPL1 phosphorylation at Ser(430) in mouse hepatocytes. In addition, suppressing PKCα expression by shRNA in hepatocytes reduces ER stress-induced APPL1 phosphorylation at Ser(430) as well as the inhibitory effect of ER stress on insulin-stimulated Akt phosphorylation. Consistent with a negative regulatory role of APPL1 phosphorylation at Ser(430) in insulin signaling, overexpression of APPL1(S430D) but not APPL1(S430A) impairs the potentiating effect of APPL1 on insulin-stimulated Akt phosphorylation at Thr(308). Taken together, our results identify APPL1 as a novel target in ER stress-induced insulin resistance and PKCα as the kinase mediating ER stress-induced phosphorylation of APPL1 at Ser(430).

A novel vessel segmentation algorithm for pathological en-face images based on matched filter.

The vascular information in fundus images can provide important basis for detection and prediction of retina-related diseases. However, the presence of lesions such as Coroidal Neovascularization can seriously interfere with normal vascular areas in optical coherence tomography (OCT) fundus images. In this paper, a novel method is proposed for detecting blood vessels in pathological OCT fundus images. First of all, an automatic localization and filling method is used in preprocessing step to reduce pathological interference. Afterwards, in terms of vessel extraction, a pore ablation method based on capillary bundle model is applied. The ablation method processes the image after matched filter feature extraction, which can eliminate the interference caused by diseased blood vessels to a great extent. At the end of the proposed method, morphological operations are used to obtain the main vascular features. Experimental results on the dataset show that the proposed method achieves 0.88 ± 0.03, 0.79 ± 0.05, 0.66 ± 0.04, results in DICE, PRECISION and TPR, respectively. Effective extraction of vascular information from OCT fundus images is of great significance for the diagnosis and treatment of retinal related diseases.

Negative regulation of insulin-stimulated mitogen-activated protein kinase signaling by Grb10.

Grb10 is a Pleckstrin homology and Src homology 2 (SH2) domain-containing protein that binds to the tyrosine-phosphorylated insulin receptor in response to insulin stimulation. Loss of Grb10 function in mice results in fetal and placental overgrowth; however, the molecular mechanism remains unknown. In the present study, we show that overexpression of Grb10 in Chinese hamster ovary cells expressing the insulin receptor or in 3T3-L1 adipocytes reduced insulin-stimulated phosphorylation of MAPK. Overexpression of Grb10 in rat primary adipocytes also inhibited insulin-stimulated phosphorylation of the MAPK downstream substrate Elk1. To determine the mechanism by which Grb10 inhibited insulin-stimulated MAPK signaling, we examined whether Grb10 affects the phosphorylation of MAPK upstream signaling components. We found that overexpression of Grb10 inhibited the insulin-stimulated phosphorylation of Shc, a positive regulator of the MAPK signaling pathway. The inhibitory effect was diminished when the SH2 domain of Grb10 was deleted. The negative role of Grb10 in insulin signaling was established by suppression of endogenous Grb10 by RNA interference in HeLa cells overexpressing the insulin receptor, which enhanced insulin-stimulated phosphorylation of MAPK, Shc, and Akt. Taken together, our findings suggest that Grb10 functions as a negative regulator in the insulin-stimulated MAPK signaling pathway. In addition, the inhibitory effect of Grb10 on the MAPK pathway is most likely due to a direct block of insulin-stimulated Shc tyrosine phosphorylation.

Grb10 promotes lipolysis and thermogenesis by phosphorylation-dependent feedback inhibition of mTORC1.

Identification of key regulators of lipid metabolism and thermogenic functions has important therapeutic implications for the current obesity and diabetes epidemic. Here, we show that Grb10, a direct substrate of mechanistic/mammalian target of rapamycin (mTOR), is expressed highly in brown adipose tissue, and its expression in white adipose tissue is markedly induced by cold exposure. In adipocytes, mTOR-mediated phosphorylation at Ser501/503 switches the binding preference of Grb10 from the insulin receptor to raptor, leading to the dissociation of raptor from mTOR and downregulation of mTOR complex 1 (mTORC1) signaling. Fat-specific disruption of Grb10 increased mTORC1 signaling in adipose tissues, suppressed lipolysis, and reduced thermogenic function. The effects of Grb10 deficiency on lipolysis and thermogenesis were diminished by rapamycin administration in vivo. Our study has uncovered a unique feedback mechanism regulating mTORC1 signaling in adipose tissues and identified Grb10 as a key regulator of adiposity, thermogenesis, and energy expenditure.

Grb10 mediates insulin-stimulated degradation of the insulin receptor: a mechanism of negative regulation.

Growth factor receptor-bound protein 10 (Grb10) is an adapter protein that interacts with a number of tyrosine-phosphorylated growth factor receptors, including the insulin receptor (IR). To investigate the role of Grb10 in insulin signaling, we generated cell lines in which the expression levels of Grb10 are either overexpressed by stable transfection or suppressed by RNA interference. We found that suppressing endogenous Grb10 expression led to increased IR protein levels, whereas overexpression of Grb10 led to reduced IR protein levels. Altering Grb10 expression levels had no effect on the mRNA levels of IR, suggesting that the modulation occurs at the protein level. Reduced IR levels were also observed in cells with prolonged insulin treatment, and this reduction was inhibited in Grb10-deficient cells. The insulin-induced IR reduction was greatly reversed by MG-132, a proteasomal inhibitor, but not by chloroquine, a lysosomal inhibitor. IR underwent insulin-stimulated ubiquitination in cells, and this ubiquitination was inhibited in the Grb10-suppressed cell line. Together, our results suggest that, in addition to inhibiting IR kinase activity by directly binding to the IR, Grb10 also negatively regulates insulin signaling by mediating insulin-stimulated degradation of the receptor.

Fine tuning PDK1 activity by phosphorylation at Ser163.

3-Phosphoinositide-dependent protein kinase-1 (PDK1) mediates phosphorylation and activation of members of the AGC protein kinase family and plays an essential role in insulin signaling and action. However, whether and how PDK1 activity is regulated in cells remains largely uncharacterized. In the present study, we show that PDK1 undergoes insulin-stimulated and phosphatidylinositol 3-kinase-dependent phosphorylation at Ser244 in the activation loop and at a novel site: Ser163 in the hinge region between the two lobes of the kinase domain. Sequence alignment studies revealed that the residue corresponding to Ser163 of PDK1 in all other AGC kinases is glutamate, suggesting that a negative charge at this site may be important for PDK1 function. Replacing Ser163 with a negatively charged residue, glutamate, led to a 2-fold increase in PDK1 activity. Molecular modeling studies suggested that phosphorylated Ser163 may form additional hydrogen bonds with Tyr149 and Gln223. In support of this, mutation of Tyr149 to Ala is sufficient to reduce PDK1 activity. Taken together, our results suggest that PDK1 phosphorylation of Ser163 may provide a mechanism to fine-tune PDK1 activity and function in cells.

[Antisense oligonucleotide targeting endostatin enhances hematopoiesis reconstitution in BMT mice].

OBJECTIVE: To explore the effect of antisense oligonucleotide targeting endostatin (endostatin-ASON) transfecting bone marrow stromal cells ( BMSC) on hematopoiesis reconstitution in BMT mice. METHODS: Inhibition of endostatin / VCAM-1 protein and mRNA expression was investigated by transfection of antisense oligonucleotide targeting endostatin with confocal microscopy, Western blot and RT-PCR. Bone marrow stromal cells were cultured and divided into 4 groups: group (1) without any treatment; group (2) BMT only; group (3) BMT + endostatin-ASON transfection; group (4) BMT + endostatin scrambled sequence transfection. RESULTS: (1) Endostatin-ASON was successfully introduced into BMSC in vitro, and the transfecting rate was 86% ;(2) After Endostatin-ASON transfected into BMSC, the expression of Endostatin mRNA and its protein on the BMSC was signficantly inhibited at different time point after BMT [the grey value of Endostatin was (0.09 +/- 0.03) - (1.44 +/- 1.19) and (0.02 + 0.02) - (0.14 +/- 0.05), respectively] (P < 0.01 and P < 0.05); (3) Transfecting with Endostatin-ASON effectively promoted the expression of VCAM-1 mRNA and its protein on the BMSC [the gray value of VCAM-1 was (1.60 +/- 0. 92) - (8.05 +/- 0.87) and (0.07 +/- 0.02) - (0.67 +/- 0.09) , respectively] (P <0.01 and P <0.05) ; (4) There was no effects of transfecting Endostatin scrambled sequence on the expression of Endostatin and VCAM-1 on the BMSC (P > 0.05). CONCLUSION: Endostatin-ASON could inhibit Endostatin expression and enhance VCAM-1 expression in BMSC after syngeneic-BMT in mice, which might be one of the mechanisms underlying the endostatin-ASON accelerating hematopoiesis reconstitution after allogeneic-BMT.