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Subarachnoid hemorrhage (SAH) can be associated with neurological deficits and has profound consequences for mortality and morbidity. Cerebral vasospasm (CVS) and delayed cerebral ischemia affect neurological outcomes in SAH patients, but their mechanisms are not fully understood, and effective treatments are limited. Here, we report that urotensin II receptor UT plays a pivotal role in both early events and delayed mechanisms following SAH in male mice. Few days post-SAH, UT expression is triggered by blood or hemoglobin in the leptomeningeal compartment. UT contributes to perimeningeal glia limitans astrocyte reactivity, microvascular alterations and neuroinflammation independent of CNS-associated macrophages (CAMs). Later, CAM-dependent vascular inflammation and subsequent CVS develop, leading to cognitive dysfunction. In an SAH model using humanized UTh+/h+ male mice, we show that post-SAH CVS and behavioral deficits, mediated by UT through Gq/PLC/Ca2+ signaling, are prevented by UT antagonists. These results highlight the potential of targeting UT pathways to reduce early meningeal response and delayed cerebral ischemia in SAH patients.
© 2024. The Author(s).

Examination of INPP5A in uveal melanoma uncovers novel calcium transients

Preprint on BioRxiv : the Preprint Server for Biology on 22 September 2024 by Onken, M. D., Kaltenbronn, K. M., et al.

Uveal melanoma is a highly aggressive intraocular cancer that metastasizes in about half of patients whereupon it is inexorably fatal. Uveal melanomas (UM) are distinct from other melanomas because they are driven by constitutively activating mutation in the heterotrimeric G protein alpha subunits Gq (GNAQ) and G11 (GNA11). This results in constitutive production of inositol trisphosphate (IP3) by phospholipase C-beta downstream of Gq/11. In normal cells, increased IP3 causes calcium release from the endoplasmic reticulum, which would be cytotoxic if maintained chronically, but UM cells are able to survive constitutive IP3 production. INPP5A, which dephosphorylates and thus inactivates IP3, is highly upregulated in UM cells compared to other melanomas and another study has shown that INPP5A is necessary for UM cell survival. To understand the mechanism of calcium regulation in response to IP3, we collected single-cell calcium measurements and found that UM cells driven by constitutively active Gq/11 produce spontaneous calcium transients. These calcium oscillations are not seen in any other melanoma cell lines unless induced by an agonist, but they are present in patient UM tumor samples. Moreover, these calcium oscillations are lost in UM cells treated with the Gq/11 inhibitor FR900359, demonstrating their dependence on constitutive Gq/11 activity. We found that the INPP5A inhibitor YU144369 causes significant changes in calcium oscillations in UM cells, demonstrating a role for INPP5A in this system. INPP5A is tethered to membranes by C-terminal prenylation and palmitoylation, suggesting that localization may play a role in INPP5A regulation of IP3 levels. GFP-tagged INPP5A was localized to plasma membrane, nuclear envelop, endoplasmic reticulum, and lysosomes. Mutation of the palmitoylation site significantly reduced localization to the plasma membrane, while mutation of the prenylation site resulted in purely nucleoplasmic localization of INPP5A. These results demonstrate a role of palmitoylation in the regulation of INPP5A localization and mobilization in UM cells.

Pyruvate kinase modulates the link between β-cell fructose metabolism and insulin secretion

Preprint on BioRxiv : the Preprint Server for Biology on 15 August 2024 by Murao, N., Morikawa, R., et al.

2 ABSTRACT Glucose triggers insulin secretion from pancreatic β-cells through intracellular glucose metabolism, ATP production, and closure of ATP-sensitive K + channels (K ATP channels). Fructose also stimulates insulin secretion, but the underlying mechanisms remain unclear. This study investigated the contribution of phospholipase C (PLC) signaling and fructose metabolism to fructose-stimulated insulin secretion (FSIS) using MIN6-K8 clonal β-cells and mouse islets. Fructose-induced PLC activation, assessed by inositol 1-phosphate accumulation, was reduced in fructose-unresponsive β-cell models, such as diabetic mouse islets and K ATP channel-deficient β-cells, suggesting that β-cell fructose responsiveness is primarily determined by PLC signaling. Although FSIS was dependent on K ATP channels and Ca 2+ influx, the ATP/ADP ratio was unexpectedly lowered by fructose, and suppression of intracellular fructose metabolism hardly affected FSIS. Metabolic flux analysis revealed that the accumulation of fructose 1-phosphate (F1P) suppressed pyruvate kinase (PK) activity, contributing to ATP depletion. Strikingly, a small-molecule PK activator, TEPP-46, antagonized F1P-mediated PK suppression, prevented the drop in the ATP/ADP ratio, and restored FSIS in MIN6-K8 cells, normal mouse islets, and fructose-unresponsive diabetic mouse islets. These findings revealed the metabolic effects of fructose in β-cells and identified PK as a key regulator linking β-cell fructose metabolism and FSIS, thereby providing new insights into the mechanisms of insulin secretion and potential therapeutic targets for fructose-associated metabolic diseases. 1 GRAPHICAL ABSTRACT Left: Fructose-stimulated insulin secretion (FSIS) is driven by sweet taste receptor (STR)-mediated PLC signaling in pancreatic β-cells. Meanwhile, fructose metabolism does not promote FSIS because fructose causes accumulation of fructose 1-phosphate (F1P), which suppresses pyruvate kinase M2 (PKM2), lowering the ATP/ADP ratio. Right: A small-molecule PK activator counteracted F1P-mediated PKM2 inhibition, prevented ATP decrease, and substantially enhanced FSIS in normal and diabetic mouse β-cells. Thus, PK has been identified as a key regulator linking β-cell fructose metabolism and FSIS.

Diversity, a hallmark of G protein-coupled receptor (GPCR) signaling, partly stems from alternative splicing of a single gene generating more than one isoform for a receptor. Additionally, receptor responses to ligands can be attenuated by desensitization upon prolonged or repeated ligand exposure. Both phenomena have been demonstrated and exemplified by the deuterostome tachykinin signaling system, although the role of phosphorylation in desensitization remains a subject of debate. Here, we describe the signaling system for tachykinin-related peptides (TKRPs) in a protostome, mollusk Aplysia. We cloned the Aplysia TKRP precursor, which encodes three TKRPs (apTKRP-1, apTKRP-2a, and apTKRP-2b) containing the FXGXR-amide motif. In situ hybridization and immunohistochemistry showed predominant expression of TKRP mRNA and peptide in the cerebral ganglia. TKRPs and their posttranslational modifications were observed in extracts of central nervous system ganglia using mass spectrometry. We identified two Aplysia TKRP receptors (apTKRPRs), named apTKRPR-A and apTKRPR-B. These receptors are two isoforms generated through alternative splicing of the same gene and differ only in their intracellular C termini. Structure-activity relationship analysis of apTKRP-2b revealed that both C-terminal amidation and conserved residues of the ligand are critical for receptor activation. C-terminal truncates and mutants of apTKRPRs suggested that there is a C-terminal phosphorylation-independent desensitization for both receptors. Moreover, apTKRPR-B also exhibits phosphorylation-dependent desensitization through the phosphorylation of C-terminal Ser/Thr residues. This comprehensive characterization of the Aplysia TKRP signaling system underscores the evolutionary conservation of the TKRP and TK signaling systems, while highlighting the intricacies of receptor regulation through alternative splicing and differential desensitization mechanisms.
Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.

A long-acting LEAP2 analog reduces hepatic steatosis and inflammation and causes marked weight loss in mice.

In Molecular Metabolism on 1 June 2024 by Shankar, K., Metzger, N. P., et al.

The number of individuals affected by metabolic dysfunction associated fatty liver disease [1] is on the rise, yet hormonal contributors to the condition remain incompletely described and only a single FDA-approved treatment is available. Some studies suggest that the hormones ghrelin and LEAP2, which act as agonist and antagonist/inverse agonist, respectively, for the G protein coupled receptor GHSR, may influence the development of MAFLD. For instance, ghrelin increases hepatic fat whereas synthetic GHSR antagonists do the opposite. Also, hepatic steatosis is less prominent in standard chow-fed ghrelin-KO mice but more prominent in 42% high-fat diet-fed female LEAP2-KO mice.
Here, we sought to determine the therapeutic potential of a long-acting LEAP2 analog (LA-LEAP2) to treat MAFLD in mice. LEAP2-KO and wild-type littermate mice were fed a Gubra-Amylin-NASH (GAN) diet for 10 or 40 wks, with some randomized to an additional 28 or 10 days of GAN diet, respectively, while treated with LA-LEAP2 vs Vehicle. Various metabolic parameters were followed and biochemical and histological assessments of MAFLD were made.
Among the most notable metabolic effects, daily LA-LEAP2 administration to both LEAP2-KO and wild-type littermates during the final 4 wks of a 14 wk-long GAN diet challenge markedly reduced liver weight, hepatic triglycerides, plasma ALT, hepatic microvesicular steatosis, hepatic lobular inflammation, NASH activity scores, and prevalence of higher-grade fibrosis. These changes were accompanied by prominent reductions in body weight, without effects on food intake, and reduced plasma total cholesterol. Daily LA-LEAP2 administration during the final 10 d of a 41.5 wk-long GAN diet challenge also reduced body weight, plasma ALT, and plasma total cholesterol in LEAP2-KO and wild-type littermates and prevalence of higher grade fibrosis in LEAP2-KO mice.
Administration of LA-LEAP2 to mice fed a MAFLD-prone diet markedly improves several facets of MAFLD, including hepatic steatosis, hepatic lobular inflammation, higher-grade hepatic fibrosis, and transaminitis. These changes are accompanied by prominent reductions in body weight and lowered plasma total cholesterol. Taken together, these data suggest that LEAP2 analogs such as LA-LEAP2 hold promise for the treatment of MAFLD and obesity.
Copyright © 2024 The Author(s). Published by Elsevier GmbH.. All rights reserved.

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