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A phosphoproteomic approach reveals that PKD3 controls PKA-mediated glucose and tyrosine metabolism
(2021)
Members of the protein kinase D (PKD) family (PKD1, 2, and 3) integrate hormonal and nutritional inputs to regulate complex cellular metabolism. Despite the fact that a number of functions have been annotated to particular PKDs, their molecular targets are relatively poorly explored. PKD3 promotes insulin sensitivity and suppresses lipogenesis in the liver of animals fed a high-fat diet. However, its substrates are largely unknown. Here we applied proteomic approaches to determine PKD3 targets. We identified more than 300 putative targets of PKD3. Furthermore, biochemical analysis revealed that PKD3 regulates cAMP-dependent PKA activity, a master regulator of the hepatic response to glucagon and fasting. PKA regulates glucose, lipid, and amino acid metabolism in the liver, by targeting key enzymes in the respective processes. Among them the PKA targets phenylalanine hydroxylase (PAH) catalyzes the conversion of phenylalanine to tyrosine. Consistently, we showed that PKD3 is activated by glucagon and promotes glucose and tyrosine levels in hepatocytes. Therefore, our data indicate that PKD3 might play a role in the hepatic response to glucagon.
That the human brain contains magnetite is well established; however, its spatial distribution in the brain has remained unknown. We present room temperature, remanent magnetization measurements on 822 specimens from seven dissected whole human brains in order to systematically map concentrations of magnetic remanence carriers. Median saturation remanent magnetizations from the cerebellum were approximately twice as high as those from the cerebral cortex in all seven cases (statistically significantly distinct, p = 0.016). Brain stems were over two times higher in magnetization on average than the cerebral cortex. The ventral (lowermost) horizontal layer of the cerebral cortex was consistently more magnetic than the average cerebral cortex in each of the seven studied cases. Although exceptions existed, the reproducible magnetization patterns lead us to conclude that magnetite is preferentially partitioned in the human brain, specifically in the cerebellum and brain stem.
Deregulated expression of MYC is a driver of colorectal carcinogenesis, necessitating novel strategies to inhibit MYC function. The ubiquitin ligase HUWE1 (HECTH9, ARF-BP1, MULE) associates with both MYC and the MYC-associated protein MIZ1. We show here that HUWE1 is required for growth of colorectal cancer cells in culture and in orthotopic xenograft models. Using high-throughput screening, we identify small molecule inhibitors of HUWE1, which inhibit MYC-dependent transactivation in colorectal cancer cells, but not in stem and normal colon epithelial cells. Inhibition of HUWE1 stabilizes MIZ1. MIZ1 globally accumulates on MYC target genes and contributes to repression of MYC-activated target genes upon HUWE1 inhibition. Our data show that transcriptional activation by MYC in colon cancer cells requires the continuous degradation of MIZ1 and identify a novel principle that allows for inhibition of MYC function in tumor cells.
To facilitate true regeneration, a vascular graft should direct the evolution of a neovessel to obtain the function of a native vessel. For this, scaffolds have to permit the formation of an intraluminal endothelial cell monolayer, mimicking the tunica intima. In addition, when attempting to mimic a tunica media‐like outer layer, the stacking and orientation of vascular smooth muscle cells (vSMCs) should be recapitulated. An integral scaffold design that facilitates this has so far remained a challenge. A hybrid fabrication approach is introduced by combining solution electrospinning and melt electrowriting. This allows a tissue‐structure mimetic, hierarchically bilayered tubular scaffold, comprising an inner layer of randomly oriented dense fiber mesh and an outer layer of microfibers with controlled orientation. The scaffold supports the organization of a continuous luminal endothelial monolayer and oriented layers of vSM‐like cells in the media, thus facilitating control over specific and tissue‐mimetic cellular differentiation and support of the phenotypic morphology in the respective layers. Neither soluble factors nor a surface bioactivation of the scaffold is needed with this approach, demonstrating that heterotypic scaffold design can direct physiological tissue‐like cell organization and differentiation.
West African torrent-frogs of the genus Odontobatrachus currently belong to a single species: Odontobatrachus natator (Boulenger, 1905). Recently, molecular results and biogeographic separation led to the recognition of five Operational Taxonomic Units (OTUs) thus identifying a species-complex. Based on these insights, morphological analyses on more than 150 adult specimens, covering the entire distribution of the family and all OTUs, were carried out. Despite strong morphological congruence, combinations of morphological characters made the differentiation of OTUs successful and allowed the recognition of five distinct species: Odontobatrachus natator, and four species new to science: Odontobatrachus arndti sp. n., O. fouta sp. n., O. smithi sp. n. and O. ziama sp. n. All species occur in parapatry: Odontobatrachus natator is known from western Guinea to eastern Liberia, O. ziama sp. n. from eastern Guinea, O. smithi sp. n. and O. fouta sp. n. from western Guinea, O. arndti sp. n. from the border triangle Guinea-Liberia-Cote d'Ivoire. In addition, for the first time the advertisement call of a West African torrent-frog (O. arndti sp. n.) is described.
The measurement of transepithelial electrical resistance (TEER) is a common technique to determine the barrier integrity of epithelial cell monolayers. However, it is remarkable that absolute TEER values of similar cell types cultured under comparable conditions show an immense heterogeneity. Based on previous observations, we hypothesized that the heterogeneity of absolute TEER measurements can not only be explained by maturation of junctional proteins but rather by dynamics in the absolute length of cell junctions within monolayers. Therefore, we analyzed TEER in epithelial cell monolayers of Caco2 cells during their differentiation, with special emphasis on both changes in the junctional complex and overall cell morphology within monolayers. We found that in epithelial Caco2 monolayers TEER increased until confluency, then decreased for some time, which was then followed by an additional increase during junctional differentiation. In contrast, permeability of macromolecules measured at different time points as 4 kDA fluorescein isothiocyanate (FITC)-dextran flux across monolayers steadily decreased during this time. Detailed analysis suggested that this observation could be explained by alterations of junctional length along the cell borders within monolayers during differentiation. In conclusion, these observations confirmed that changes in cell numbers and consecutive increase of junctional length have a critical impact on TEER values, especially at stages of early confluency when junctions are immature.
In the last few years, quantitative analysis of metabolites in body fluids using LC/MS has become an established method in laboratory medicine and toxicology. By preparing metabolite profiles in biological specimens, we are able to understand pathophysiological mechanisms at the biochemical and thus the functional level. An innovative investigative method, which has not yet been used widely in the forensic context, is to use the clinical application of metabolomics. In a metabolomic analysis of 41 samples of postmortem cerebrospinal fluid (CSF) samples divided into cohorts of four different causes of death, namely, cardiovascular fatalities, isoIated torso trauma, traumatic brain injury, and multi-organ failure, we were able to identify relevant differences in the metabolite profile between these individual groups. According to this preliminary assessment, we assume that information on biochemical processes is not gained by differences in the concentration of individual metabolites in CSF, but by a combination of differently distributed metabolites forming the perspective of a new generation of biomarkers for diagnosing (fatal) TBI and associated neuropathological changes in the CNS using CSF samples.
The aim of this study was to investigate if the biomarkers myelin basic protein (MBP) and neurofilament-H (NF-H) yielded informative value in forensic diagnostics when examining cadaveric cerebrospinal fluid (CSF) biochemically via an enzyme-linked immunosorbent assay (ELISA) and comparing the corresponding brain tissue in fatal traumatic brain injury (TBI) autopsy cases by immunocytochemistry versus immunohistochemistry. In 21 trauma and 19 control cases, CSF was collected semi-sterile after suboccipital puncture and brain specimens after preparation. The CSF MBP (p = 0.006) and NF-H (p = 0.0002) levels after TBI were significantly higher than those in cardiovascular controls. Immunohistochemical staining against MBP and against NF-H was performed on cortical and subcortical samples from also biochemically investigated cases (5 TBI cases/5 controls). Compared to the controls, the TBI cases showed a visually reduced staining reaction against MBP or repeatedly ruptured neurofilaments against NF-H. Immunocytochemical tests showed MBP-positive phagocytizing macrophages in CSF with a survival time of > 24 h. In addition, numerous TMEM119-positive microglia could be detected with different degrees of staining intensity in the CSF of trauma cases. As a result, we were able to document that elevated levels of MBP and NF-H in the CSF should be considered as useful neuroinjury biomarkers of traumatic brain injury.
Hepatic activation of protein kinase C (PKC) isoforms by diacylglycerol (DAG) promotes insulin resistance and contributes to the development of type 2 diabetes (T2D). The closely related protein kinase D (PKD) isoforms act as effectors for DAG and PKC. Here, we showed that PKD3 was the predominant PKD isoform expressed in hepatocytes and was activated by lipid overload. PKD3 suppressed the activity of downstream insulin effectors including the kinase AKT and mechanistic target of rapamycin complex 1 and 2 (mTORC1 and mTORC2). Hepatic deletion of PKD3 in mice improved insulin-induced glucose tolerance. However, increased insulin signaling in the absence of PKD3 promoted lipogenesis mediated by SREBP (sterol regulatory element-binding protein) and consequently increased triglyceride and cholesterol content in the livers of PKD3-deficient mice fed a high-fat diet. Conversely, hepatic-specific overexpression of a constitutively active PKD3 mutant suppressed insulin-induced signaling and caused insulin resistance. Our results indicate that PKD3 provides feedback on hepatic lipid production and suppresses insulin signaling. Therefore, manipulation of PKD3 activity could be used to decrease hepatic lipid content or improve hepatic insulin sensitivity.