Maternal hyperglycemia induces alterations in hepatic amino acid, glucose and lipid metabolism of neonatal offspring: Multi-omics insights from a diabetic pig model.

OBJECTIVE: To gain mechanistic insights into adverse effects of maternal hyperglycemia on the liver of neonates, we performed a multi-omics analysis of liver tissue from piglets developed in genetically diabetic (mutant INS gene induced diabetes of youth; MIDY) or wild-type (WT) pigs. METHODS: Proteome, metabolome and lipidome profiles of liver and clinical parameters of serum samples from 3-day-old WT piglets (n = 9) born to MIDY mothers (PHG) were compared with those of WT piglets (n = 10) born to normoglycemic mothers (PNG). Furthermore, protein-protein interaction network analysis was used to reveal highly interacting proteins that participate in the same molecular mechanisms and to relate these mechanisms with human pathology. RESULTS: Hepatocytes of PHG displayed pronounced lipid droplet accumulation, although the abundances of central lipogenic enzymes such as fatty acid-synthase (FASN) were decreased. Additionally, circulating triglyceride (TG) levels were reduced as a trend. Serum levels of non-esterified free fatty acids (NEFA) were elevated in PHG, potentially stimulating hepatic gluconeogenesis. This is supported by elevated hepatic phosphoenolpyruvate carboxykinase (PCK1) and circulating alanine transaminase (ALT) levels. Even though targeted metabolomics showed strongly elevated phosphatidylcholine (PC) levels, the abundances of multiple key enzymes involved in major PC synthesis pathways - most prominently those from the Kennedy pathway - were paradoxically reduced in PHG liver. Conversely, enzymes involved in PC excretion and breakdown such as PC-specific translocase ATP-binding cassette 4 (ABCB4) and phospholipase A2 were increased in abundance. CONCLUSIONS: Our study indicates that maternal hyperglycemia without confounding obesity induces profound molecular changes in the liver of neonatal offspring. In particular, we found evidence for stimulated gluconeogenesis and hepatic lipid accumulation independent of de novo lipogenesis. Reduced levels of PC biosynthesis enzymes and increased levels of proteins involved in PC translocation or breakdown may represent counter-regulatory mechanisms to maternally elevated PC levels. Our comprehensive multi-omics dataset provides a valuable resource for future meta-analysis studies focusing on liver metabolism in newborns from diabetic mothers.

Combating Drug Resistance by Exploiting miRNA-200c-Controlled Phase II Detoxification.

Acquired drug resistance constitutes a serious obstacle to the successful therapy of cancer. In the process of therapy resistance, microRNAs can play important roles. In order to combat resistance formation and to improve the efficacy of chemotherapeutics, the mechanisms of the multifaceted hsa-miR-200c on drug resistance were elucidated. Upon knockout of hsa-miR-200c in breast carcinoma cells, a proteomic approach identified altered expression of glutathione S-transferases (GSTs) when cells were treated with the chemotherapeutic drug doxorubicin. In different hsa-miR-200c expression systems, such as knockout, inducible sponge and inducible overexpression, the differential expression of all members of the GST family was evaluated. Expression of hsa-miR-200c in cancer cells led to the repression of a multitude of these GSTs and as consequence, enhanced drug-induced tumor cell death which was evaluated for two chemotherapeutic drugs. Additionally, the influence of hsa-miR-200c on the glutathione pathway, which is part of the phase II detoxification mechanism, was investigated. Finally, the long-term effects of hsa-miR-200c on drug efficacy were studied in vitro and in vivo. Upon doxycycline induction of hsa-miR-200c, MDA-MB 231 xenograft mouse models revealed a strongly reduced tumor growth and an enhanced treatment response to doxorubicin. A combined treatment of these tumors with hsa-miR-200c and doxorubicin resulted in complete regression of the tumor in 60% of the animals. These results identify hsa-miR-200c as an important player regulating the cellular phase II detoxification, thus sensitizing cancer cells not expressing this microRNA to chemotherapeutics and reversing drug resistance through suppression of GSTs.

Profound Effects of Dexamethasone on the Immunological State, Synthesis and Secretion Capacity of Human Testicular Peritubular Cells.

The functions of human testicular peritubular cells (HTPCs), forming a small compartment located between the seminiferous epithelium and the interstitial areas of the testis, are not fully known but go beyond intratesticular sperm transport and include immunological roles. The expression of the glucocorticoid receptor (GR) indicates that they may be regulated by glucocorticoids (GCs). Herein, we studied the consequences of the GC dexamethasone (Dex) in cultured HTPCs, which serves as a unique window into the human testis. We examined changes in cytokines, mainly by qPCR and ELISA. A holistic mass-spectrometry-based proteome analysis of cellular and secreted proteins was also performed. Dex, used in a therapeutic concentration, decreased the transcript level of proinflammatory cytokines, e.g., IL6, IL8 and MCP1. An siRNA-mediated knockdown of GR reduced the actions on IL6. Changes in IL6 were confirmed by ELISA measurements. Of note, Dex also lowered GR levels. The proteomic results revealed strong responses after 24 h (31 significantly altered cellular proteins) and more pronounced ones after 72 h of Dex exposure (30 less abundant and 42 more abundant cellular proteins). Dex also altered the composition of the secretome (33 proteins decreased, 13 increased) after 72 h. Among the regulated proteins were extracellular matrix (ECM) and basement membrane components (e.g., FBLN2, COL1A2 and COL3A1), as well as PTX3 and StAR. These results pinpoint novel, profound effects of Dex in HTPCs. If transferrable to the human testis, changes specifically in ECM and the immunological state of the testis may occur in men upon treatment with Dex for medical reasons.

Necroptosis in primate luteolysis: a role for ceramide.

The corpus luteum (CL) is a transient endocrine organ, yet molecular mechanisms resulting in its demise are not well known. The presence of phosphorylated mixed lineage kinase domain-like pseudokinase pMLKL(T357/S358) in human and nonhuman primate CL samples (Macaca mulatta and Callithrix jacchus) implied that necroptosis of luteal cells may be involved. In M. mulatta CL, pMLKL positive staining became detectable only from the mid-late luteal phase onwards, pointing to necroptosis during regression of the CL. Cell death, including necroptosis, was previously observed in cultures of human luteal granulosa cells (GCs), an apt model for the study of the human CL. To explore mechanisms of necroptotic cell death in GCs during culture, we performed a proteomic analysis. The levels of 50 proteins were significantly altered after 5 days of culture. Interconnectivity analysis and immunocytochemistry implicated specifically the ceramide salvage pathway to be enhanced. M. mulatta CL transcriptome analysis indicated in vivo relevance. Perturbing endogenous ceramide generation by fumonisin B1 (FB1) and addition of soluble ceramide (C2-CER) yielded opposite actions on viability of GCs and therefore supported the significance of the ceramide pathway. Morphological changes indicated necrotic cell death in the C2-CER treated group. Studies with the pan caspase blocker zVAD-fmk or the necroptosis blocker necrosulfonamid (NSA) further supported that C2-CER induced necroptosis. Our data pinpoint necroptosis in a physiological process, namely CL regression. This raises the possibility that the primate CL could be rescued by pharmacological inhibition of necroptosis or by interaction with ceramide metabolism.