Biology subjects

Sweet, I. R.

Publications and source records attributed to Sweet, I. R..

10 recordsLinked to original sources

bioRxiv2023

A Compound that Inhibits Glycolysis in Prostate Cancer Controls Growth of Advanced Prostate Cancer

PurposeMetastatic castration-resistant prostate cancer remains incurable regardless of recent therapeutic advances. Prostate cancer tumors display highly glycolytic phenotypes as the cancer progresses. Non-specific inhibitors of glycolysis have not been utilized successfully for chemotherapy, because of their penchant to cause systemic toxicity. This study reports the preclinical activity, safety, and pharmacokinetics of a novel small molecule preclinical candidate, BKIDC-1553, with antiglycolytic activity. Experimental designWe tested a large battery of prostate cancer cell lines for inhibition of cell proliferation, in vitro. Cell cycle, metabolic and enzymatic assays were used to demonstrate their mechanism of action. A human PDX model implanted in mice and a human organoid were studied for sensitivity to our BKIDC preclinical candidate. A battery of pharmacokinetic experiments, absorption, distribution, metabolism, and excretion experiments, and in vitro and in vivo toxicology experiments were carried out to assess readiness for clinical trials. ResultsWe demonstrate a new class of small molecule inhibitors where antiglycolytic activity in prostate cancer cell lines is mediated through inhibition of hexokinase 2. These compounds display selective growth inhibition across multiple prostate cancer models. We describe a lead BKIDC-1553 that demonstrates promising activity in a preclinical xenograft model of advanced prostate cancer, equivalent to that of enzalutamide. BKIDC-1553 demonstrates safety and pharmacologic properties consistent with a compound that can be taken into human studies with expectations of a good safety margin and predicted dosing for efficacy. ConclusionThis work supports testing BKIDC-1553 and its derivatives in clinical trials for patients with advanced prostate cancer.

cancer biology↗

bioRxiv2023

Elamipretide Improves ADP Sensitivity in Aged Mitochondria by Increasing Uptake through the Adenine Nucleotide Translocator (ANT)

Aging muscle experiences functional decline in part mediated by impaired mitochondrial ADP sensitivity. Elamipretide (ELAM) rapidly improves physiological and mitochondrial function in aging and binds directly to the mitochondrial ADP transporter ANT. We hypothesized that ELAM improves ADP sensitivity in aging leading to rescued physiological function. We measured the response to ADP stimulation in young and old muscle mitochondria with ELAM treatment, in vivo heart and muscle function, and compared protein abundance, phosphorylation, and S-glutathionylation of ADP/ATP pathway proteins. ELAM treatment increased ADP sensitivity in old muscle mitochondria by increasing uptake of ADP through the ANT and rescued muscle force and heart systolic function. Protein abundance in the ADP/ATP transport and synthesis pathway was unchanged, but ELAM treatment decreased protein s-glutathionylation incuding of ANT. Mitochondrial ADP sensitivity is rapidly modifiable. This research supports the hypothesis that ELAM improves ANT function in aging and links mitochondrial ADP sensitivity to physiological function. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/525989v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1980dc7org.highwire.dtl.DTLVardef@1b0c22eorg.highwire.dtl.DTLVardef@19c9244org.highwire.dtl.DTLVardef@46e79b_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract.C_FLOATNO ELAM Binds Directly to ANT and ATP Synthase and ELAM Treatment Improves ADP Sensitivity, Increases ATP Production, and Improves Physiological Function in Old Muscles. ADP (adenosine diphosphate), ATP (adenosine triphosphate), VDAC (voltage-dependent anion channel), ANT (Adenine nucleotide translocator), H+ (proton), ROS (reactive oxygen species), NADH (nicotinamide adenine dinucleotide), FADH2 (flavin adenine dinucleotide), O2 (oxygen), ELAM (elamipretide), -SH (free thiol), -SSG (glutathionylated protein). C_FIG

physiology↗

bioRxiv2023

Mitochondrial diabetes in mice expressing a dominant-negative allele of nuclear respiratory factor-1 (NRF-1) in pancreatic and β-cells

Genetic polymorphisms in nuclear respiratory factor-1 (NRF1), a key transcriptional regulator of nuclear-encoded mitochondrial proteins, have been linked to diabetes. Homozygous deletion of Nrf1 is embryonic lethal in mice. Our goal was to generate mice with {beta}-cell-specific reduction in NRF1 function to investigate the relationship between NRF1 and diabetes. We report the generation of mice expressing a dominant-negative allele of Nrf1 (DNNRF1) in pancreatic {beta}-cells. Heterozygous transgenic mice had high fed blood glucose levels detected at 3 wks of age, which persisted through adulthood. Plasma insulin levels in DNNRF1 transgenic mice were reduced, while insulin sensitivity remained intact in young animals. Islet size was reduced with increased numbers of apoptotic cells, and insulin content in islets by immunohistochemistry was low. Glucose-stimulated insulin secretion in isolated islets was reduced in DNNRF1-mice, but partially rescued by KCl, suggesting that decreased mitochondrial function contributed to the insulin secretory defect. Electron micrographs demonstrated abnormal mitochondrial morphology in {beta}- cells. Expression of NRF1 target genes Tfam, T@1m and T@2m, and islet cytochrome c oxidase and succinate dehydrogenase activities were reduced in DNNRF1-mice. Rescue of mitochondrial function with low level activation of transgenic c-Myc in {beta}-cells was sufficient to restore {beta}-cell mass and prevent diabetes. This study demonstrates that reduced NRF1 function can lead to loss of {beta}-cell function and establishes a model to study the interplay between regulators of bi- genomic gene transcription in diabetes.

molecular biology↗

bioRxiv2022

Contemporary human H3N2 influenza A viruses require a low threshold of suitable glycan receptors for efficient infection

Recent human H3N2 influenza A viruses (IAV) have evolved to employ elongated glycans terminating in 2,6-linked sialic acid as their receptors. These glycans are displayed in low abundancies by cells commonly employed to propagate these viruses (MDCK and hCK), resulting in low or no viral propagation. Here, we examined whether the overexpression of the glycosyltransferases B3GNT2 and B4GALT1, which are responsible for the elongation of poly-N-acetyllactosamines (LacNAc), would result in improved A/H3N2 propagation. Stable overexpression of B3GNT2 and B4GALT1 in MDCK and hCK cells was achieved by lentiviral integration and subsequent antibiotic selection and confirmed by qPCR and protein mass spectrometry experiments. Flow cytometry and glycan mass spectrometry experiments using the B3GNT2 and/or B4GALT1 knock-in cells demonstrated increased binding of viral hemagglutinins and the presence of a larger number of LacNAc repeating units, especially on hCK-B3GNT2 cells. An increase in the number of glycan receptors did, however, not result in a greater infection efficiency of recent human H3N2 viruses. Based on these results, we propose that H3N2 IAVs require a low number of suitable glycan receptors to infect cells and that an increase in the glycan receptor display above this threshold does not result in improved infection efficiency.

microbiology↗

bioRxiv2022

A Peptide Triple Agonist of GLP-1, Neuropeptide Y1, and Neuropeptide Y2 Receptors Promotes Glycemic Control and Weight Loss

Mechanisms underlying long-term sustained weight loss and glycemic normalization after obesity surgery include changes in gut hormone levels, including glucagon-like peptide 1 (GLP-1) and peptide YY (PYY). We demonstrate that two peptide biased agonists (GEP44 and GEP12) of the GLP-1, neuropeptide Y1, and neuropeptide Y2 receptors (GLP-1R, Y1-R, and Y2-R, respectively) elicit Y1-R antagonist-controlled, GLP-1R-dependent stimulation of insulin secretion in both rat and human pancreatic islets, thus revealing the counteracting effects of Y1-R and GLP-1R agonism. These agonists also promote insulin-independent Y1-R-mediated glucose uptake in muscle tissue ex vivo and more profound reductions in food intake and body weight than liraglutide when administered to diet-induced obese rats. Our findings support a role for Y1-R signaling in glucoregulation and highlight the therapeutic potential of simultaneous receptor targeting to achieve long-term benefits for millions of patients.

pharmacology and toxicology↗

bioRxiv2021

Metabolic function in aging retina and retinal pigment epithelium remains robust despite vision loss

PurposeCharacterize how metabolic function in the murine retina and retinal pigment epithelium-choroid-sclera (eyecup) complex is impacted by natural aging. MethodsWe examined scotopic and photopic visual function of young (3-6 months) and aged (23-26 months) C57Bl/6J mice using electroretinograms (ERGs). Metabolic changes in retina and eyecup explants were characterized by measuring uptake and usage of U-13C-glucose or U-13C-glutamine at different timepoints by gas chromatography-mass spectrometry (GC-MS), measuring oxygen consumption rate (OCR) using a perifusion apparatus, and determining ATP levels with a bioluminescence assay. ResultsScotopic and photopic ERG responses declined in aged mice. Glucose metabolism, glutamine metabolism, OCR, and ATP pools in retinal explants were mostly unaffected by the age of the mouse. In eyecups, glutamine usage in the Krebs Cycle decreased while glucose metabolism, OCR, and ATP pools remained stable. ConclusionsThe ex vivo approach in our study to examine aging glucose and glutamine metabolism in retina and RPE showed negligible impact of age on retina and an impairment of glutamine anaplerosis in eyecups. The surprising metabolic stability of these tissues ex vivo suggests age-related metabolic alterations in these tissues may not be intrinsic. Future experiments should focus on determining whether external factors including nutrient supply, oxygen availability, or other structural changes influence ocular metabolism in vivo.

biochemistry↗

bioRxiv2021

Fluidics System for Resolving Concentration-Dependent Effects of Dissolved Gases on Tissue Metabolism

Oxygen (O2) and other dissolved gases such as the gasotransmitters H2S, CO and NO affect cell metabolism and function. To evaluate effects of dissolved gases on processes in tissue, we developed a fluidics system that controls dissolved gases while simultaneously measuring parameters of electron transport, metabolism and secretory function. We use pancreatic islets, retina and liver to highlight its ability to assess effects of O2 and H2S. Protocols aimed at emulating hypoxia-reperfusion conditions resolved a previously unrecognized transient spike in O2 consumption rate (OCR) following replenishment of O2, and tissue-specific recovery of OCR following hypoxia. The system revealed both inhibitory and stimulatory effects of H2S on insulin secretion rate from isolated islets. The unique ability of this new system to quantify metabolic state and cell function in response to precise changes in dissolved gases provides a powerful platform for cell physiologists to study a wide range of disease states.

cell biology↗

bioRxiv2021

Succinate is Broadly Tissue-Permeant and Uncouples Mitochondrial Respiration from ATP Synthesis

Succinate is central in energy metabolism. It is thought to be made and consumed within mitochondria, and not to cross the plasma membrane. Contrary to the canonical role of succinate, it is produced in the retina and released into the extracellular milleau. It is unknown how this affects adjacent retinal pigment epithelium, within eyecup tissue. By measuring consumption of 13C4-succinate and O2 we probe metabolism of the eyecup, we find robust succinate consumption by eyecup but not retina tissue. Succinate-dependant stimulation of eyecup respiration is oligomycin resistant; indicating that in eyecup tissue succinate uncouples mitochondrial ATP synthesis from respiration.

biochemistry↗

bioRxiv2020

HIF1α stability in hypoxia is not oxidant-initiated but can be tuned by redox regulation of the proteasome

Hypoxic adaptation mediated by HIF transcription factors has been shown to require mitochondria. Current models suggest that mitochondria regulate oxygen sensor (HIF prolyl hydroxylase) activity and HIF1 stability during hypoxia by either increasing mitochondrial peroxide as a second messenger or by serving as oxygen consumers that enhance the kinetics of cytoplasmic oxygen reduction. Here, we address the role of mitochondrial peroxide specifically in regulating HIF1 stability. We use state-of-the-art tools to evaluate the role of peroxide and other reactive oxygen species (ROS) in regulating HIF1 stability. We show that antioxidant enzymes are not homeostatically induced nor are peroxide levels increased in hypoxia. Forced expression of diverse antioxidant enzymes, all of which diminish peroxide, had disparate effects on HIF1 protein stability. Reduction of lipid peroxides by glutathione peroxidase-4 or superoxide by mitochondrial SOD failed to influence HIF1 protein stability. These data showed that mitochondrial, cytosolic and lipid ROS are dispensable for HIF1 stability and should affirm therapeutic efforts to activate the HIF pathway in disease states by HIF prolyl hydroxylase inhibition.

cell biology↗

bioRxiv2020

Succinate can shuttle reducing power from the hypoxic retina to the O2-rich pigment epithelium.

When O2 is plentiful, the mitochondrial electron transport chain uses it as a terminal electron acceptor. However, the mammalian retina thrives within a hypoxic niche in the eye. We find that mitochondria in retinas adapt to their hypoxic environment by relying on the "reverse" succinate dehydrogenase reaction, where fumarate accepts electrons instead of O2. Reverse succinate dehydrogenase activity produces succinate and is enhanced by down-regulation of cytochrome oxidase subunits. Retinas can export the succinate to the neighboring retinal pigment epithelium/choroid complex. There, succinate stimulates O2 consumption several fold and enhances synthesis and release of malate. Malate released from the pigment epithelium can be imported into the retina, where it is converted to fumarate and again used to accept electrons in the reverse succinate dehydrogenase reaction. Our findings show how a malate/succinate shuttle can sustain these two tissues by transferring reducing power from an O2-poor tissue (retina) to an O2-rich one (retinal pigment epithelium/choroid).

biochemistry↗