Preparation and characterization of genetic construct for the specific MCPIP1 protein expression in porcine adipose tissue

Otyłość charakteryzująca się nadmiernym gromadzeniem zbędnej tkanki tłuszczowej w organizmie, obecnie uznawana jest także za przewlekłą ogólnoustrojową chorobę zapalną w której tkanka tłuszczowa pełni funkcję narządu endokrynnego produkując liczne cząsteczki bioaktywne (cytokiny, chemokiny, hormony) nazywane ogólnie adipokinami. Prowadzi to do rozwoju zespołu metabolicznego, będącego głównym czynnikiem ryzyka dla cukrzycy typu II, nadciśnienia tętniczego czy chorób sercowonaczyniowych. Badania prowadzone w naszym Zakładzie wskazują na regulację procesu adipogenezy przez białko MCPIP1. Obserwujemy hamowanie różnicowania preadipocytów przy stabilnej ekspresji białka MCPIP1 w mysich embrionalnych fibroblastach 3T3-L1. Ponadto widoczna jest zmniejszona ekspresja C/EBPβ, biorącego udział w regulacji początkowego etapu adipogenezy, a także obniżony poziom PPARγ – głównego czynnika indukującego ten proces. Prowadzi to do zmniejszonej akumulacji lipidów, a w konsekwencji także do zmniejszonej liczby w pełni dojrzałych adipocytów. W związku z tym na potrzeby tej pracy powstał konstrukt genetyczny pozwalający na wprowadzenie specyficznej ekspresji białka MCPIP1 w tkance tłuszczowej u zwierząt. Wykonano klonowanie, w wyniku którego otrzymano transgen zawierający sekwencję kodującą białko MCPIP1 z wprowadzonym między 4 i 5 egzonem pojedynczym intronem. Sekwencja ta została umieszczona pod kontrolą mysiego promotora genu Fabp4, który ulega aktywacji głównie w adipocytach białej tkanki tłuszczowej. Przed wykorzystaniem otrzymanego konstruktu do generacji transgenicznych zwierząt, potwierdzono nadekspresję białka MCPIP1 w testach komórkowych. Następnie wprowadzono go do zarodków świń w procesie mikroiniekcji i tak przygotowane zygoty implantowano do jajowodów samic. Wyniki uzyskane w przyszłości w badaniach przeprowadzonych na wyprowadzonej transgenicznej linii z ekspresją białka MCPIP1 w tkance tłuszczowej posłużą do zrozumienia podstaw rozwoju otyłości i odkrycia nowej terapii leczenia chorób zespołu metabolicznego.Obesity, a condition characterized by the deposition of inert fat, now is also recognized as a chronic and systemic inflammatory disease, where adipose tissue acts as endocrine organ producing numerous bioactive molecules (cytokines, chemokines, hormones) known as adipokines. This leads to the development of metabolic syndrome, which is the main risk factor for type II diabetes, hypertension, or cardiovascular disease. Studies in our laboratory demonstrate the regulation of adipogenesis by MCPIP1 protein. We report that forced expression of MCPIP1 in mouse embryonic fibroblast 3T3-L1 inhibits C/EBPβ (involved in the regulation of the initial stage of adipogenesis) and PPARγ (universally accepted as the master regulator that is necessary and sufficient to induce adipogenesis) expression at both the protein and transcript level. Inhibition of adipogenesis is manifested by a weaker lipid accumulation and reduced amount of mature adipocytes. Therefore, the purpose of this work was to generate genetic construct for the specific MCPIP1 protein expression in animal adipose tissue. Transgene was prepared by introducing the coding sequence for the protein MCPIP1 (with single intron between 4 and 5 exon) under the murine Fabp4 promoter. FABP4 promoter is activated predominantly in adipocytes of white adipose tissue. Before the use of obtained construct for the generation of transgenic animals, MCPIP1 protein expression was confirmed in cell-based assays. Then, transgene was introduced to the embryos in the microinjection process and so prepared zygotes were implanted into the oviducts of females. The results obtained in the future studies on transgenic lines expressing MCPIP1 in adipose tissue let to understand the basis for the development of obesity and may help to generate new therapies against the metabolic syndrome diseases

Overexpression of sphingosine-1-phosphate lyase protects insulin-secreting cells against cytokine toxicity.

Increasing evidence suggests a crucial role of inflammation in cytokine-mediated β-cell dysfunction and death in type 1 diabetes mellitus, although the mechanisms are incompletely understood. Sphingosine 1-phosphate (S1P) is a multifunctional bioactive sphingolipid involved in the development of many autoimmune and inflammatory diseases. Here, we investigated the role of intracellular S1P in insulin-secreting INS1E cells by genetically manipulating the S1P-metabolizing enzyme S1P lyase (SPL). The expression of spl was down-regulated by cytokines in INS1E cells and rat islets. Overexpression of SPL protected against cytokine toxicity. Interestingly, the SPL overexpression did not suppress the cytokine-induced NFκB-iNOS-NO pathway but attenuated calcium leakage from endoplasmic reticulum (ER) stores as manifested by lower cytosolic calcium levels, higher expression of the ER protein Sec61a, decreased dephosphorylation of Bcl-2-associated death promoter (Bad) protein, and weaker caspase-3 activation in cytokine-treated (IL-1β, TNFα, and IFNγ) cells. This coincided with reduced cytokine-mediated ER stress, indicated by measurements of CCAAT/enhancer-binding protein homologous protein (chop) and immunoglobulin heavy chain binding protein (bip) levels. Moreover, cytokine-treated SPL-overexpressing cells exhibited increased expression of prohibitin 2 (Phb2), involved in the regulation of mitochondrial assembly and respiration. SPL-overexpressing cells were partially protected against cytokine-mediated ATP reduction and inhibition of glucose-induced insulin secretion. siRNA-mediated spl suppression resulted in effects opposite to those observed for SPL overexpression. Knockdown of phb2 partially reversed beneficial effects of SPL overexpression. In conclusion, the relatively low endogenous Spl expression level in insulin-secreting cells contributes to their extraordinary vulnerability to proinflammatory cytokine toxicity and may therefore represent a promising target for β-cell protection in type 1 diabetes mellitus

MCPIP1 regulates the sensitivity of pancreatic beta-cells to cytokine toxicity

The autoimmune-mediated beta-cell death in type 1 diabetes (T1DM) is associated with local inflammation (insulitis). We examined the role of MCPIP1 (monocyte chemotactic protein–induced protein 1), a novel cytokine-induced antiinflammatory protein, in this process. Basal MCPIP1 expression was lower in rat vs. human islets and beta-cells. Proinflammatory cytokines stimulated MCPIP1 expression in rat and human islets and in insulin-secreting cells. Moderate overexpression of MCPIP1 protected insulin-secreting INS1E cells against cytokine toxicity by a mechanism dependent on the presence of the PIN/DUB domain in MCPIP1. It also reduced cytokine-induced Chop and C/ebpβ expression and maintained MCL-1 expression. The shRNA-mediated suppression of MCPIP1 led to the potentiation of cytokine-mediated NFκB activation and cytokine toxicity in human EndoC-βH1 beta-cells. MCPIP1 expression was very high in infiltrated beta-cells before and after diabetes manifestation in the LEW.1AR1-iddm rat model of human T1DM. The extremely high expression of MCPIP1 in clonal beta-cells was associated with a failure of the regulatory feedback-loop mechanism, ER stress induction and high cytokine toxicity. In conclusion, our data indicate that the expression level of MCPIP1 affects the susceptibility of insulin-secreting cells to cytokines and regulates the mechanism of beta-cell death in T1DM.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

MCPIP1 is a novel link between diabetogenic conditions and impaired insulin secretory capacity

SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Drug screening identifies tazarotene and bexarotene as therapeutic agents in multiple sulfatase deficiency

Schlotawa L, Tyka K, Kettwig M, et al. Drug screening identifies tazarotene and bexarotene as therapeutic agents in multiple sulfatase deficiency. EMBO Molecular Medicine. 2023: e14837.Multiple sulfatase deficiency (MSD, MIM #272200) results from pathogenic variants in the SUMF1 gene that impair proper function of the formylglycine-generating enzyme (FGE). FGE is essential for the posttranslational activation of cellular sulfatases. MSD patients display reduced or absent sulfatase activities and, as a result, clinical signs of single sulfatase disorders in a unique combination. Up to date therapeutic options for MSD are limited and mostly palliative. We performed a screen of FDA-approved drugs using immortalized MSD patient fibroblasts. Recovery of arylsulfatase A activity served as the primary readout. Subsequent analysis confirmed that treatment of primary MSD fibroblasts with tazarotene and bexarotene, two retinoids, led to a correction of MSD pathophysiology. Upon treatment, sulfatase activities increased in a dose- and time-dependent manner, reduced glycosaminoglycan content decreased and lysosomal position and size normalized. Treatment of MSD patient derived induced pluripotent stem cells (iPSC) differentiated into neuronal progenitor cells (NPC) resulted in a positive treatment response. Tazarotene and bexarotene act to ultimately increase the stability of FGE variants. The results lay the basis for future research on the development of a first therapeutic option for MSD patients. © 2023 The Authors. Published under the terms of the CC BY 4.0 license