Substrate Delivery Research Articles

Neuroprotective effects of lactate and ketone bodies in acute brain injury.

The goal of neurocritical care is to prevent and reverse the pathologic cascades of secondary brain injury by optimizing cerebral blood flow, oxygen supply and substrate delivery. While glucose is an essential energetic substrate for the brain, we frequently observe a strong decrease in glucose delivery and/or a glucose metabolic dysregulation following acute brain injury. In parallel, during the last decades, lactate and ketone bodies have been identified as potential alternative fuels to provide energy to the brain, both under physiological conditions and in case of glucose shortage. They are now viewed as integral parts of brain metabolism. In addition to their energetic role, experimental evidence also supports their neuroprotective properties after acute brain injury, regulating in particular intracranial pressure control, decreasing ischemic volume, and leading to an improvement in cognitive functions as well as survival. In this review, we present preclinical and clinical evidence exploring the mechanisms underlying their neuroprotective effects and identify research priorities for promoting lactate and ketone bodies use in brain injury.

SGLT2-Inhibitors and Kidney Protection: Mechanisms Beyond Tubulo-Glomerular Feedback.

Sodium-glucose cotransporter 2 (SGLT2)-inhibitors reduce the risk for kidney failure and are a key component of guideline-directed therapy for chronic kidney disease. While SGLT2-inhibitors' ability to activate tubulo-glomerular feedback and reduce hyperfiltration-mediated kidney injury is considered to be the central mechanism for kidney protection, recent data from experimental studies raises questions on the primacy of this mechanism. This review examines SGLT2-inhibitors' role in tubulo-glomerular feedback and summarizes emerging evidence on following of SGLT2-inhibitors' other putative mechanisms for kidney protection: optimization of kidney's energy substrate utilization and delivery, regulation of autophagy and maintenance of cellular homeostasis, attenuation of sympathetic hyperactivity, and improvement in vascular health and microvascular function. It is imperative to examine the impact of SGLT2 inhibition on these different physiologic processes to help our understanding of mechanisms underpinning kidney protection with this important class of drugs.

Economic supply chain modelling of industrial insect production in the Netherlands

Abstract Defatted larvae meal (dLM), specifically from black soldier flies, could help overcome the animal protein gap. As insect production is an emerging sector, current economic research is scarce and very diverse. Thus, the aim of this research was to develop a simulation model that enables the analysis of full industrial scale costs of producing dLM and to provide insight in the distribution of these costs in the insect supply chain. The deterministic supply chain model is built on three modules, technical, transition and economic module, which all follow a previously defined supply chain structure and allow to extract quantity and price information for intermediate or final products. The model was parameterized and checked for plausibility in multiple consultation rounds with the INSECTFEED consortium and business partners. Additionally, model behaviour was checked with scenario, sensitivity, and break-even price analyses. In the default situation 5.57 tDM raw substrate and 26.7 million neonates are required to produce 1 tDM dLM for a price of €5,116/tDM. Most costs are added in the raw substrate delivery (€1,952/tDM) and production and collection (€821/tDM) step. Important cost factors are the raw substrate (€1,939/tDM) and building and inventory (€1,459/tDM). Parameters with high relative response rate towards the price of dLM are the feed conversion rate, dry matter percentage of larvae, raw substrate price, larvae density, labour wage and growth rate. To reach break-even prices for substituting fish meal with alive grown larvae (AGL) (€1,318/tDM AGL), improving production parameters is not sufficient. Just changing prices of raw substrate to −€78/tDM or frass to €1,175/tDM would enable a profitable operation. However, these are not deemed as realistic in mass production. Although there is some insecurity in data, the model results are the most realistic representation of industrial scale production amounts and costs.

Abstract A040: Suppressing the cytomatrix elastic solid phase of cytoplasm will eliminate the root cause of cancer

Abstract The Warburg effect, the increase of glucose uptake rate and lactate production, is a tremendous metabolic switch, a prominent cancer hallmark, and a century-old mystery. We recently solved an age-old problem of biochemistry, how myriad diverse chemical reactions coincide in the cell, by chemically separating elastic solid (cytomatrix) and viscous fluid (cytosol) phases of cytoplasm (iScience, 2023, PMID:36824274) that can elucidate a role of the Warburg effect in tumors. Biochemical reactions within the cytoplasm are catalytic and performed by enzyme complexes. Catalytic complexes are immobilized on the solid platform, and the cytomatrix physically separates them from each other, but the substrate delivery mechanisms have remained unknown. Using the multiomic approach, we found that cytomatrix proteins actomyosins are responsible for cellular micromechanics and cytosolic motion. Cytosolic motion delivers substrates to catalytic complexes. Cytomatrix micromechanics requires energy maintained by mitochondrial respiration in physiological conditions. However, due to mutational and epigenetic alterations, malignant cells need additional energy to stir biochemical processes. Actomyosin and actin-binding and regulating proteome, a structural base of the cytomatrix and primary motor proteins, exploits aerobic glycolysis to meet the energy demand of cytomatrix, just as muscle fiber utilizes the aerobic glycolysis during intense exercise. Therefore, synchronizing energy output for cytomatrix micromechanics is a limiting step for the malignant transformation involving the Warburg effect. Unless mutations coalesce with the increased energy production for cytomatrix mechanics, potential cancer may only survive in the quiescent state (or benign), or it will be eliminated. Cancer development appears to be a two-sided coin relying on both the mutation and the energy demand, suggesting that not everyone gets cancer with the same mutation without the involvement of aerobic glycolysis. By uniting the Warburg effect with the cytomatrix micromechanics and genetic and epigenetic alterations, our concept of cancer development better explains the transformation of healthy cells to malignant cells and malignant cell resistance to therapy and recurrence. Revealing the cytomatrix allowed us to find its practical application. We developed technology that works on the cytomatrix level and can improve cancer treatment and prevent tumor recurrence. Suppression of the cytomatrix activity will inhibit cancer resistance and recurrence and increase the efficiency of the current anticancer therapy. It is impossible to know if cancer will return after treatment. Cancers come back when small numbers of malignant cells can remain in the body after treatment. These cells are too rare to find with current tests. We have developed a method to help detect this small number of malignant cells. Our fast and sensitive method can isolate malignant cells from a frozen biopsy about 10 – 20 mg weight. Thus, revealing the cytomatrix opens a new chapter in cancer research and therapy. Citation Format: Tattym E. Shaiken. Suppressing the cytomatrix elastic solid phase of cytoplasm will eliminate the root cause of cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr A040.

Research Progress on hCNT3 Structure/Function and Nucleoside Anticancer Drugs.

Membrane protein human concentrative nucleoside transporter 3 (hCNT3) can not only transport extracellular nucleosides into the cell but also transport various nucleoside-derived anticancer drugs to the focus of infection for therapeutic effects. Typical nucleoside anticancer drugs, including fludarabine, cladabine, decitabine, and clofarabine, are recognized by hCNT3 and then delivered to the lesion site for their therapeutic effects. hCNT3 is highly conserved during the evolution from lower to higher vertebrates, which contains scaffold and transport domains in structure and delivers substrates by coupling with Na+ and H+ ions in function. In the process of substrate delivery, the transport domain rises from the lower side of transmembrane 9 (TM9) in the inward conformation to the upper side of the outward conformation, accompanied by the collaborative motion of TM7b/ TM4b and hairpin 1b (HP1b)/ HP2b. With the report of a series of three-dimensional structures of homologous CNTs, the structural characteristics and biological functions of hCNT3 have attracted increasing attention from pharmacists and biologists. Our research group has also recently designed an anticancer lead compound with high hCNT3 transport potential based on the structure of 5-fluorouracil. In this work, the sequence evolution, conservation, molecular structure, cationic chelation, substrate recognition, elevator motion pattern and nucleoside derivative drugs of hCNT3 were reviewed, and the differences in hCNT3 transport mode and nucleoside anticancer drug modification were summarized, aiming to provide theoretical guidance for the subsequent molecular design of novel anticancer drugs targeting hCNT3.

Helicobacter pylori FlgN binds its substrate FlgK and the flagellum ATPase FliI in a similar manner observed for the FliT chaperone.

In bacterial flagellum biogenesis, secretion of the hook-filament junction proteins FlgK and FlgL and completion of the flagellum requires the FlgN chaperone. Similarly, the related FliT chaperone is necessary for the secretion of the filament cap protein FliD and binds the flagellar export gate protein FlhA and the flagellum ATPase FliI. FlgN and FliT require FliJ for effective substrate secretion. In Helicobacter pylori, neither FlgN, FliT, nor FliJ have been annotated. We demonstrate that the genome location of HP1120 is identical to that of flgN in other flagellated bacteria and that HP1120 is the homolog of Campylobacter jejuni FlgN. A modeled HP1120 structure contains three α-helices and resembles the FliT chaperone, sharing a similar substrate-binding pocket. Using pulldowns and thermophoresis, we show that both HP1120 and a HP1120Δ126-144 deletion mutant bind to FlgK with nanomolar affinity, but not to the filament cap protein FliD, confirming that HP1120 is FlgN. Based on size-exclusion chromatography and multi-angle light scattering, H. pylori FlgN binds to FlgK with 1:1 stoichiometry. Overall structural similarities between FlgN and FliT suggest that substrate recognition on FlgN primarily involves an antiparallel coiled-coil interface between the third helix of FlgN and the C-terminal helix of the substrate. A FlgNΔ126-144 N100A, Y103A, S111I triple mutant targeting this interface significantly impairs the binding of FlgK. Finally, we demonstrate that FlgNΔ126-144 , like FliT, binds with sub-micromolar affinity to the flagellum ATPase FliI or its N-terminal domain. Hence FlgN and FliT likely couple delivery of low-abundance export substrates to the flagellum ATPase FliI.

Electro-metabolic signaling.

Precise matching of energy substrate delivery to local metabolic needs is essential for the health and function of all tissues. Here, we outline a mechanistic framework for understanding this critical process, which we refer to as electro-metabolic signaling (EMS). All tissues exhibit changes in metabolism over varying spatiotemporal scales and have widely varying energetic needs and reserves. We propose that across tissues, common signatures of elevated metabolism or increases in energy substrate usage that exceed key local thresholds rapidly engage mechanisms that generate hyperpolarizing electrical signals in capillaries that then relax contractile elements throughout the vasculature to quickly adjust blood flow to meet changing needs. The attendant increase in energy substrate delivery serves to meet local metabolic requirements and thus avoids a mismatch in supply and demand and prevents metabolic stress. We discuss in detail key examples of EMS that our laboratories have discovered in the brain and the heart, and we outline potential further EMS mechanisms operating in tissues such as skeletal muscle, pancreas, and kidney. We suggest that the energy imbalance evoked by EMS uncoupling may be central to cellular dysfunction from which the hallmarks of aging and metabolic diseases emerge and may lead to generalized organ failure states-such as diverse flavors of heart failure and dementia. Understanding and manipulating EMS may be key to preventing or reversing these dysfunctions.

Mastering the brain in critical conditions: an update

Acute brain injuries, such as traumatic brain injury and ischemic and hemorragic stroke, are a leading cause of death and disability worldwide. While characterized by clearly distict primary events—vascular damage in strokes and biomechanical damage in traumatic brain injuries—they share common secondary injury mechanisms influencing long-term outcomes. Growing evidence suggests that a more personalized approach to optimize energy substrate delivery to the injured brain and prognosticate towards families could be beneficial. In this context, continuous invasive and/or non-invasive neuromonitoring, together with clinical evaluation and neuroimaging to support strategies that optimize cerebral blood flow and metabolic delivery, as well as approaches to neuroprognostication are gaining interest. Recently, the European Society of Intensive Care Medicine organized a 2-day course focused on a practical case-based clinical approach of acute brain-injured patients in different scenarios and on future perspectives to advance the management of this population. The aim of this manuscript is to update clinicians dealing with acute brain injured patients in the intensive care unit, describing current knowledge and clinical practice based on the insights presented during this course.

Characterization and Validation of Radiotracer Kinetics Using the Langendorff Isolated Perfused Heart.

The Langendorff isolated perfused heart is a physiologically relevant and controllable ex vivo model well suited to characterizing and validating novel radiotracers for a wide range of molecular imaging applications. It allows the monitoring of first pass tracer uptake kinetics either as a bolus injection or as a continuous infusion in beating myocardial tissue with a high degree of experimental control in terms of cardiac workload, perfusion, energy substrate delivery and composition, and drug co-administration. The radiotracer pharmacokinetic data that it provides is not contaminated by confounding factors such as off-target tracer metabolism, and as a non-imaging technique, time activity curves can be acquired with very high temporal resolution. In this chapter, we describe the basic principles and practice for setting up and using Langendorff isolated perfused hearts for the assessment of novel radiotracers and outline their potential for modeling pathophysiological conditions relevant to cardiovascular disease.

Structural and bioinformatics analysis of single-domain substrate-binding protein from Rhodothermus marinus

Substrate-binding proteins (SBPs) are key elements in determining the substrate specificity and high affinity of the ATP-binding cassette uptake system. A typical SBP has two domains that recognize substrates and are responsible for the specific substrate delivery. Conversely, in GenBank, genes for SBPs constituting a single domain SBP are often found in vicinity of a methyl-accepting chemotaxis protein gene. However, the molecular function and mechanism of single domain SBPs are not fully elucidated. To understand their molecular functions, we performed a crystallographic study of single domain SBP from Rhodothermus marinus (RmSBP). RmSBP crystals were soaked in solution containing NaBr or HgCl2 and their structures determined at 1.75 and 2.3 Å resolution, respectively. RmSBP soaked in NaBr exhibited disorder of the α2-helix, β5-to β6-strand loop, and C-terminus region, showing the structural dynamic region of RmSBP. RmSBP soaked in HgCl2 showed that Hg2+ bound to Cys145 located between the α5-and α6-helices. The structural properties of RmSBP were compared with those of single domain SBP homologs. These results will contribute to continued identification of the molecular function and mechanism of single domain SBPs.

Suppression of tumour growth from transplanted astrocytoma cells transfected with luciferase in mice by bioluminescence mediated, systemic, photodynamic therapy

BackgroundGrade 4 astrocytomas are usually incurable due to their diffusely infiltrative nature. Photodynamic therapy (PDT) is a promising therapeutic option, but external light delivery is impractical when cancer cells infiltrate unknown areas of normal brain. Hence the search for endogenous sources to generate light at cancer cells. In vitro, astrocytoma cells, transfected with firefly luciferase, can be killed by bioluminescence-mediated PDT (bPDT). This study asks if bPDT can suppress tumour growth In vivo, when all components of treatment are administered systemically. MethodsTransfected astrocytoma cells were injected subcutaneously or intra-cranially in athymic CD1 nu/nu mice. bPDT required ip bolus of mTHPC (photosensitiser) and delivery of the d-luciferin substrate over 7 days via an implanted osmotic pump. Control animals had no treatment, photosensitiser only or d-luciferin only. For subcutaneous tumours, size and BLI (light emitted after d-luciferin bolus) were measured before and every 2 days after PDT. For intracranial tumours, monitoring was weekly BLI. ResultsFor subcutaneous tumours, there was significant suppression of the tumour growth rate (P<0.05), and absolute tumour size (P<0.01) after bPDT. Proliferation of subcutaneous and intracranial tumours (monitored by BrdU uptake) was significantly reduced in treated mice. (P<0.001) ConclusionsThis study reports bPDT suppression of tumour growth from luciferase transfected astrocytoma cells with all components of treatment given systemically, as required for effective management of recurrent astrocytomas in unknown sites. However, research on systemic bPDT is needed to establish whether effects on non-transfected tumours can be achieved without any unacceptable effects on normal tissues.

DNAzyme Nanomachine with Fluorogenic Substrate Delivery Function: Advancing Sensitivity in Nucleic Acid Detection.

We have developed a hook-equipped DNA nanomachine (HDNM) for the rapid detection of specific nucleic acid sequences without a preamplification step. HDNM efficiently unwinds RNA structures and improves the detection sensitivity. Compared to the hookless system, HDNM offers an 80-fold and 13-fold enhancement in DNA and RNA detection, respectively, reducing incubation time from 3 to 1 h.

Pparβ/δ Has an Indispensable Role in T Cell Function and Anti-Tumor Responses

The peroxisome proliferator-activated receptors (PPARs) are a group of transcriptional factor of the nuclear hormone receptor superfamily, which promote ligand-dependent transcription of target genes that regulate substrate delivery, oxidative phosphorylation (OXPHOS), and energy balance. The PPAR superfamily consists of three subtypes, PPARalpha (PPARα), PPARgamma (PPARγ), and PPARbeta/delta (PPARβ/δ), with distinct tissue allocations. In addition to their roles in energy homeostasis and carbohydrate and lipid metabolism in metabolic organs, an emerging role of PPARs involves the differentiation and fate of immune cells. PPARβ/δ is ubiquitously expressed and activated by fatty acid ligands. While the effects of PPARα and PPARγ on M2 macrophage polarization and Th1 inhibition are well established, only few studies have investigated the effects of PPARβ/δ on the immune responses. It has been proposed that PPARβ/δ might inhibit T cell differentiation towards T helper 1 cells (Th1) and T helper 17 cells (Th17), and synthetic ligands of PPARβ/δ might alleviate inflammation in autoimmune diseases by downregulating pro-inflammatory mediators. Conversely, other studies have observed that PPARβ/δ agonists might enhance anti-tumor T cell responses by reprogramming immunometabolism toward fatty acid oxidation and increasing T cell longevity. In the present study, we sought to determine the role of PPARβ/δ in T cell function by combining genetic and pharmacologic approaches. We generated mice with conditional targeting of Pparβ/δ gene and crossed them with CD4Cre ( Pparβ/δ f/fCD4Cre) to delete Pparβ/δ in T cells. In vitro stimulation of T cells from Pparβ/δ f/fCD4Cre mice with anti-CD3/TCR-and-CD28 mediated signaling resulted in diminished expansion of CD4 + and CD8 + T cells. Consistent with these findings, treatment of antigen-specific CD4 + and CD8 + T cells from OTII and OTI TCR-transgenic mice with the Pparβ/δ-specific antagonist GSK3787 diminished responses to cognate antigen as determined by DNA synthesis, expression of activation markers and differentiation to T effector cells. Furthermore, under these conditions, GSK 3787 induced a dose-dependent decrease in the production of IL-2, TNF-α, and IFN-γ by OTII antigen-specific CD4 + T cells. To investigate the T cell-specific role of Pparβ/δ in anti-tumor responses, we used Pparβ/δ f/fCD4Cre and control Pparβ/δ f/f mice and implanted them with MC38 colon adenocarcinoma tumor cells. We found that Pparβ/δ f/fCD4Cre mice had enhanced tumor growth compared to control. No quantitative differences were detected in tumor infiltrating CD4 +, CD8 + and Treg cells. However, Treg cells from tumor-bearing Pparβ/δ f/fCD4Cre mice had a diminished activation state as determined by lower expression of CD44 and PD-1. CD4 + T cells in tumors and tumor draining lymph nodes also had decreased expression of CD44 and ICOS but no differences in other activation or checkpoint markers, including GITR, TIGIT, CD69 and CD44 compared to their counterparts from tumor-bearing control mice. In contrast, CD8 + T cells from tumor-bearing Pparβ/δ f/fCD4Cre mice had increased expression of Tox and multiple checkpoint inhibitors including CTLA4, PD-1, and Lag3 and decreased expression of IFN-g compared to CD8 + cells from control tumor-bearing mice. These results reveal a previously unappreciated indispensable role of PPARβ/δ in regulating anti-tumor immune responses and protecting CD8 + tumor infiltrating T cells from developing an exhaustion immunological and functional phenotype. Our findings suggest that pharmacologic PPARβ/δ-specific agonists might prevent tumor-mediated exhaustion and support anti-tumor function of CD8 + T effector cells.

NMR characterization of novel interactions between p97 AAA+ ATPase and the p47 adaptor revealing insights into substrate delivery mechanism

p97/VCP is an essential AAA+ ATPase involved in diverse cellular activities by interacting with an array of protein adaptors that recruit p97 for specific tasks. p47 is one of the adaptors that targets p97 for membrane remodeling by forming a stable complex with p97 through multivalent interactions. Here, we report a pair of previously unidentified interactions between the N-terminal part of p47 (residues 1–94) and the N-terminal domain (NTD) of p97. Using nuclear magnetic resonance (NMR) spectroscopy, we identify two binding sites on p47, one located on the ubiquitin-associated (UBA) domain and the other on the intrinsically disordered linker, that interact with the same basic patch on p97 NTD, driven by electrostatic forces. Reciprocal NMR titration experiments between p47 (residues 1–94) and p97 NTD reveal that these interactions are relatively weak in nature with dissociation constants on the order of hundreds of micromolar to millimolar in trans. Structural models of the two interactions are developed based on NMR chemical shift perturbations, which reveal details of the tentative binding interfaces. Our findings provide new insights into the mechanism by which ubiquitinated substrates are delivered from p47 to p97 for unfolding.

Abstract 16878: Exercise Intolerance in Heart Failure With Preserved Ejection Fraction is More Closely Related to Energetic Decline During Exercise Than to Mitochondrial Oxidative Capacity in Recovery

Introduction: Heart failure (HF) with preserved ejection fraction (HFpEF) is associated with severe exercise intolerance (EI) and exertional fatigue. Impaired skeletal muscle high-energy phosphate metabolism has been reported in HFpEF patients and postulated as a potential contributor to EI. However, whether EI is more closely related to rapid energetic decline during exercise, or reduced mitochondrial oxidative capacity remains unknown. Hypothesis: We hypothesized that six-minute walk distance (6MWD) is more closely related to the rate of skeletal muscle energetic decline during exercise than mitochondrial oxidative capacity in HFpEF. Methods: Calf muscle energetics were measured by phosphorus magnetic resonance spectroscopy ( 31 PMRS). The rate of phosphocreatine (PCr) decline normalized to work performed during graded plantar flexion exercise to volitional fatigue and the time for PCr post-exercise recovery, an established metric inversely related to mitochondrial oxidative capacity, were measured in 64 HFpEF patients (age: 65 ± 11 years; 53% women). Results: The mean rate of PCr decline, PCr recovery time, and 6MWD were 52 ± 108 μmol/g wet weight/KJ, 80 ± 45 seconds, and 296 ± 109 meters, respectively. The 6MWD was inversely related to the rate of PCr decline (r = -0.46; P = 0.0002; Figure, A ) but not significantly related to the PCr recovery time (r = -0.06; P = 0.66; Figure, B ), as determined by Pearson’s correlation coefficients. Conclusions: EI, as measured by 6MWD, in HFpEF patients is closely associated with impaired skeletal muscle metabolism, specifically rapid skeletal muscle PCr decline during exercise rather than mitochondrial oxidative capacity. This suggests that impaired glycolytic metabolism and substrate delivery may contribute metabolically to EI in HFpEF patients.

Insulin-induced gene 2 protects against hepatic ischemia–reperfusion injury via metabolic remodeling

BackgroundHepatic ischemia–reperfusion (IR) injury is the primary reason for complications following hepatectomy and liver transplantation (LT). Insulin-induced gene 2 (Insig2) is one of several proteins that anchor the reticulum in the cytoplasm and is essential for metabolism and inflammatory responses. However, its function in IR injury remains ambiguous.MethodsInsig2 global knock-out (KO) mice and mice with adeno-associated-virus8 (AAV8)-delivered Insig2 hepatocyte-specific overexpression were subjected to a 70% hepatic IR model. Liver injury was assessed by monitoring hepatic histology, inflammatory responses, and apoptosis. Hypoxia/reoxygenation stimulation (H/R) of primary hepatocytes and hypoxia model induced by cobalt chloride (CoCl2) were used for in vitro experiments. Multi-omics analysis of transcriptomics, proteomics, and metabolomics was used to investigate the molecular mechanisms underlying Insig2.ResultsHepatic Insig2 expression was significantly reduced in clinical samples undergoing LT and the mouse IR model. Our findings showed that Insig2 depletion significantly aggravated IR-induced hepatic inflammation, cell death and injury, whereas Insig2 overexpression caused the opposite phenotypes. The results of in vitro H/R experiments were consistent with those in vivo. Mechanistically, multi-omics analysis revealed that Insig2 is associated with increased antioxidant pentose phosphate pathway (PPP) activity. The inhibition of glucose-6-phosphate-dehydrogenase (G6PD), a rate-limiting enzyme of PPP, rescued the protective effect of Insig2 overexpression, exacerbating liver injury. Finally, our findings indicated that mouse IR injury could be attenuated by developing a nanoparticle delivery system that enables liver-targeted delivery of substrate of PPP (glucose 6-phosphate).ConclusionsInsig2 has a protective function in liver IR by upregulating the PPP activity and remodeling glucose metabolism. The supplementary glucose 6-phosphate (G6P) salt may serve as a viable therapeutic target for alleviating hepatic IR.

Total and Regional Brain Volumes in Fetuses With Congenital Heart Disease.

Congenital heart disease (CHD) is common and is associated with impaired early brain development and neurodevelopmental outcomes, yet the exact mechanisms underlying these associations are unclear. To utilize MRI data from a cohort of fetuses with CHD as well as typically developing fetuses to test the hypothesis that expected cerebral substrate delivery is associated with total and regional fetal brain volumes. Retrospective case-control study. Three hundred eighty fetuses (188 male), comprising 45 healthy controls and 335 with isolated CHD, scanned between 29 and 37 weeks gestation. Fetuses with CHD were assigned into one of four groups based on expected cerebral substrate delivery. T2-weighted single-shot fast-spin-echo sequences and a balanced steady-state free precession gradient echo sequence were obtained on a 1.5 T scanner. Images were motion-corrected and reconstructed using an automated slice-to-volume registration reconstruction technique, before undergoing segmentation using an automated pipeline and convolutional neural network that had undergone semi-supervised training. Differences in total, regional brain (cortical gray matter, white matter, deep gray matter, cerebellum, and brainstem) and brain:body volumes were compared between groups. ANOVA was used to test for differences in brain volumes between groups, after accounting for sex and gestational age at scan. PFDR -values <0.05 were considered statistically significant. Total and regional brain volumes were smaller in fetuses where cerebral substrate delivery is reduced. No significant differences were observed in total or regional brain volumes between control fetuses and fetuses with CHD but normal cerebral substrate delivery (all PFDR  > 0.12). Severely reduced cerebral substrate delivery is associated with lower brain:body volume ratios. Total and regional brain volumes are smaller in fetuses with CHD where there is a reduction in cerebral substrate delivery, but not in those where cerebral substrate delivery is expected to be normal. 3 TECHNICAL EFFICACY: Stage 3.

Structural and functional diversity of type IV secretion systems.

Considerable progress has been made in recent years in the structural and molecular biology of type IV secretion systems in Gram-negative bacteria. The latest advances have substantially improved our understanding of the mechanisms underlying the recruitment and delivery of DNA and protein substrates to the extracellular environment or target cells. In this Review, we aim to summarize these exciting structural and molecular biology findings and to discuss their functional implications for substrate recognition, recruitment and translocation, as well as the biogenesis of extracellular pili. We also describe adaptations necessary for deploying a breadth of processes, such as bacterial survival, host-pathogen interactions and biotic and abiotic adhesion. We highlight the functional and structural diversity that allows this extremely versatile secretion superfamily to function under different environmental conditions and in different bacterial species. Additionally, we emphasize the importance of further understanding the mechanism of type IV secretion, which will support us in combating antimicrobial resistance and treating type IV secretion system-related infections.

THU047 Growth Hormone (GH) Signaling In Hepatocytes Controls Hepatic Insulin Sensitivity And Nutrient Disposition

Abstract Disclosure: M. del Rio-Moreno: None. M. Vazquez-Borrego: None. J. Cordoba-Chacon: None. O.P. McGuinness: None. R.D. Kineman: None. It is commonly accepted that GH reduces the actions of insulin. However, we reported plasma GH levels within a physiological range are negatively associated with insulin-mediated glucose uptake, but positively associated with insulin-mediated suppression of hepatic glucose production (HGP) [Cordoba-Chacon J. et al. 2014. AJP Endocrinol Metab]. These findings suggest that GH may promote insulin’s actions on the liver. To determine the direct contribution of GH signaling on hepatic insulin sensitivity, hyperinsulinemic-euglycemic clamps were performed with stable isotope tracers in chow-fed adult-onset, hepatocyte-specific GHR knockdown (aHepGHRkd) mice. aHepGHRkd mice show reduced IGF1 and increased GH levels [Cordoba-Chacon J. et al 2015, Diabetes] which indirectly impacts systemic insulin sensitivity and substrate flux to the liver. To normalize plasma IGF1 and GH levels, aHepGHRkd mice were co-treated with adeno-associated virus (AAV) that express IGF1 or a constitutively active form of STAT5b (STAT5bCA). These AAVs have a thyroxin binding globulin promoter, leading to hepatocyte-specific expression. Under both basal and clamped conditions, aHepGHRkd mice exhibited hyperinsulinemia, that was normalized by AAV-mediated IGF1 and STAT5bCA expression. Interestingly, glucose disappearance (Rd), tissue-specific glucose uptake, or HGP (gluconeogenesis and glycogenolysis) did not differ across all groups. However, higher insulin tone in aHepGHRkd mice was required to achieve the same level of Rd and suppression of HGP in the steady-state, which was indicative of systemic and hepatic insulin resistance. These observations are consistent with our previous findings that GH reduces tissue-specific glucose uptake but promotes insulin-mediated control of HGP. In contrast, aHepGHRkd mice exhibited an increased rate of hepatic de novo lipogenesis, and an increase in plasma levels of newly formed triglyceride and triglyceride-bound palmitate, that was normalized by IGF1 and STAT5bCA expression. Taken together, these results indicate loss of hepatocyte GH signaling leads to pathway selective hepatic insulin resistance. This pathway selective hepatic insulin resistance has also been reported in mouse models due to altered substrate delivery to the liver as consequence of excess dietary intake and/or increased adipose tissue lipolysis. However, chow-fed aHepGHRkd mice show hyperinsulinemia that was sufficient to normalize systemic glucose uptake and suppress WAT lipolysis, as noted by normal plasma NEFA levels under both basal and clamped conditions. Therefore, changes in substrate delivery to the liver cannot readily explain the changes observed, suggesting hepatocyte GHR-signaling plays a direct role in partitioning of hepatic nutrients in response to insulin. Presentation: Thursday, June 15, 2023

Substrate recruitment mechanism by gram-negative type III, IV, and VI bacterial injectisomes.

Bacteria use a wide arsenal of macromolecular substrates (DNA and proteins) to interact with or infect prokaryotic and eukaryotic cells. To do so, they utilize substrate-injecting secretion systems or injectisomes. However, prior to secretion, substrates must be recruited to specialized recruitment platforms and then handed over to the secretion apparatus for secretion. In this review, we provide an update on recent advances in substrate recruitment and delivery by gram-negative bacterial recruitment platforms associated with Type III, IV, and VI secretion systems.