Aberrant androgen receptor (AR) signalling drives prostate cancer (PC) and is a key therapeutic target. Although initially effective, the generation of alternatively spliced AR variants (AR-Vs) compromises efficacy of treatments. In contrast to full-length AR (AR-FL), AR-Vs constitutively activate androgenic signalling and are refractory to the current repertoire of AR-targeting therapies, which together drives disease progression. There is an unmet clinical need therefore to develop more durable PC therapies that can attenuate AR-V function. Exploiting the requirement of co-regulatory proteins for AR-V function has the capacity to furnish tractable routes for attenuating persistent oncogenic AR signalling in advanced PC. DNA-PKcs regulates AR-FL transcriptional activity and is upregulated in both early and advanced PC. We hypothesised that DNA-PKcs is critical for AR-V function. Using a novel proximity biotinylation approach, we demonstrate that the DNA-PK holoenzyme is part of the AR-V7 interactome and is a key regulator of AR-V-mediated transcription and cell growth in models of advanced PC. Crucially, we provide evidence that DNA-PKcs controls global splicing, and via RBMX, regulates the maturation of AR-V and AR-FL transcripts. Ultimately, our data indicates that targeting DNA-PKcs attenuates AR-V signalling and provides evidence that DNA-PKcs blockade is an effective therapeutic option in advanced AR-V positive PC patients.
Beth Adamson, Nicholas Brittain, Laura Walker, Ruaridh Duncan, Sara Luzzi, Pasquale Rescigno, Graham R. Smith, Suzanne McGill, Richard J.S. Burchmore, Elaine Willmore, Ian Hickson, Craig N. Robson, Denisa Bogdan, Juan M. Jimenez-Vacas, Alec Paschalis, Jonathan Welti, Wei Yuan, Stuart R. McCracken, Rakesh Heer, Adam Sharp, Johann de Bono, Luke Gaughan
Background: Proglucagon can be processed to Glucagon-Like Peptide-1 (GLP-1) within the islet but its contribution to islet function in humans remains unknown. We sought to understand whether ‘pancreatic’ GLP-1 alters islet function in humans and whether this is affected by type 2 diabetes.Methods: We therefore studied individuals with and without type 2 diabetes on 2 occasions in random order. On one occasion exendin 9-39, a competitive antagonist of the GLP-1 Receptor (GLP1R), was infused, while on the other saline was infused. The tracer dilution technique ([3-3H] glucose) was used to measure glucose turnover during fasting and during a hyperglycemic clamp.Results: Exendin 9-39 increased fasting glucose concentrations; fasting islet hormone concentrations were unchanged, but inappropriate for the higher fasting glucose observed. In people with type 2 diabetes fasting glucagon concentrations were markedly elevated and persisted despite hyperglycemia. This impaired suppression of endogenous glucose production by hyperglycemia. These data show that GLP1R blockade impairs islet function, implying that intra-islet GLP1R activation alters islet responses to glucose and does so to a greater degree in people with type 2 diabetes.
Andrew A. Welch, Rahele A. Farahani, Aoife M. Egan, Marcello C. Laurenti, Maya Zeini, Max Vella, Kent R. Bailey, Claudio Cobelli, Chiara Dalla Dalla Man, Aleksey Matveyenko, Adrian Vella
Expanding β cell mass is a critical goal in the fight against diabetes. CDK4, an extensively characterized cell cycle activator, is required to establish and maintain β cell number. β cell failure in the IRS2-deletion mouse type 2 diabetes model is, in part, due to loss of CDK4 regulator cyclin D2. We set out to determine whether replacement of endogenous CDK4 with the inhibitor-resistant mutant CDK4-R24C rescued the loss of β cell mass in IRS2-deficient mice. Surprisingly, not only β cell mass but also β cell dedifferentiation was effectively rescued, despite no improvement in whole body insulin sensitivity. Ex vivo studies in primary islet cells revealed a mechanism in which CDK4 intervened downstream in the insulin signaling pathway to prevent FOXO1-mediated transcriptional repression of critical β cell transcription factor Pdx1. FOXO1 inhibition was not related to E2F1 activity, to FOXO1 phosphorylation, or even to FOXO1 subcellular localization, but rather was related to deacetylation and reduced FOXO1 abundance. Taken together, these results demonstrate a differentiation-promoting activity of the classical cell cycle activator CDK4 and support the concept that β cell mass can be expanded without compromising function.
Rachel E. Stamateris, Huguet V. Landa-Galvan, Rohit B. Sharma, Christine Darko, David Redmond, Sushil G. Rane, Laura C. Alonso
BACKGROUND Generally, clinical assessment of gonadal testosterone (T) in human physiology is determined using concentrations measured in peripheral blood. Prostatic T exposure is similarly thought to be determined from peripheral T exposure. Despite the fact that androgens drive prostate cancer, peripheral T has had no role in the clinical evaluation or treatment of men with localized prostate cancer.METHODS To assess the role of local androgen delivery in prostate cancer, we obtained blood from the (periprostatic) prostatic dorsal venous complex in 266 men undergoing radical prostatectomy from July 2014 to August 2021 and compared dorsal T (DT) levels with those in circulating peripheral blood (PT) and prostatic tissue. Comprehensive targeted steroid analysis and unbiased metabolomics analyses were performed. The association between the DT/PT ratio and progression-free survival after prostatectomy was assessed.RESULTS Surprisingly, in some men, DT levels were enriched several-fold compared with PT levels. For example, 20% of men had local T concentrations that were at least 2-fold higher than peripheral T concentrations. Isocaproic acid, a byproduct of androgen biosynthesis, and 17-OH-progesterone, a marker of intratesticular T, were also enriched in the dorsal vein of these men, consistent with testicular shunting. Men with enriched DT had higher rates of prostate cancer recurrence. DT/PT concentration ratios predicted worse outcomes even when accounting for known clinical predictors.CONCLUSIONS These data suggest that a large proportion of men have a previously unappreciated exposure to an undiluted and highly concentrated T supply. Elevated periprostatic T exposure was associated with worse clinical outcomes after radical prostatectomy.FUNDING National Cancer Institute (NCI), NIH grants R01CA172382, R01CA236780, R01CA261995, R01CA249279, and R50CA251961; US Army Medical Research and Development Command grants W81XWH2010137 and W81XWH-22-1-0082.
Mohammad Alyamani, Patrick Michael, Daniel Hettel, Lewis Thomas, Scott D. Lundy, Mike Berk, Mona Patel, Jianbo Li, Hooman Rashidi, Jesse K. McKenney, Eric A. Klein, Nima Sharifi
The adipose-derived hormone leptin acts via its receptor (LepRb) in the brain to control energy balance. A previously unidentified population of GABAergic hypothalamic LepRb neurons plays key roles in the restraint of food intake and body weight by leptin. To identify markers for candidate populations of LepRb neurons in an unbiased manner, we performed single-nucleus RNA-sequencing of enriched mouse hypothalamic LepRb cells, identifying several previously unrecognized populations of hypothalamic LepRb neurons. Many of these populations displayed strong conservation across species, including GABAergic Glp1r-expressing LepRb (LepRbGlp1r) neurons, which expressed more Lepr than other LepRb cell populations. Ablating Lepr from LepRbGlp1r cells provoked hyperphagic obesity without impairing energy expenditure. Similarly, improvements in energy balance by Lepr reactivation in GABA neurons of otherwise Lepr-null mice required Lepr expression in GABAergic Glp1r-expressing neurons. Furthermore, restoration of Glp1r expression in LepRbGlp1r neurons in otherwise Glp1r-null mice enabled food intake suppression by the GLP1R agonist, liraglutide. Thus, the conserved GABAergic LepRbGlp1r neuron population plays crucial roles in the suppression of food intake by leptin and GLP1R agonists.
Alan C Rupp, Abigail J. Tomlinson, Alison H. Affinati, Warren T. Yacawych, Allison M. Duensing, Cadence True, Sarah R. Lindsley, Melissa A. Kirigiti, Alexander J. MacKenzie, Joseph Polex-Wolf, Chien Li, Lotte B. Knudsen, Randy J. Seeley, David P. Olson, Paul Kievit, Martin G. Myers, Jr
Negative regulation of exocytosis from secretory cells is accomplished through inhibitory signals from Gi/o G-protein-coupled-receptors by Gβγ subunit inhibition of two mechanisms: decreased calcium entry and direct interaction of Gβγ with Soluble N-ethylmaleimide-sensitive factor Attachment Protein (SNAP) Receptor (SNARE) plasma membrane fusion machinery. Previously, we disabled the second mechanism with a SNAP25 truncation (SNAP25Δ3) decreasing Gβγ affinity for the SNARE complex, leaving exocytotic fusion and modulation of calcium entry intact, and removing GPCR-Gβγ inhibition of SNARE-mediated exocytosis. Here, we report substantial metabolic benefit in mice carrying this mutation. SNAP25Δ3/Δ3 mice exhibit enhanced insulin sensitivity and beiging of white fat. Metabolic protection was amplified in SNAP25Δ3/Δ3 mice challenged with high fat diet. Glucose homeostasis, whole body insulin action, and insulin-mediated glucose uptake into white adipose tissue were improved along with resistance to diet-induced obesity. Metabolic protection in SNAP25Δ3/Δ3 mice occurred without compromising the physiological response to fasting or cold. All metabolic phenotypes were reversed at thermoneutrality, suggesting basal autonomic activity is required. Direct electrode stimulation of sympathetic neuron exocytosis from SNAP25Δ3/Δ3 inguinal adipose depots resulted in enhanced and prolonged norepinephrine release. Thus, the Gβγ-SNARE interaction represents a cellular mechanism that deserves further exploration as an additional avenue for combatting metabolic disease.
Ryan P. Ceddia, Zack Zurawski, Analisa Thompson Gray, Feyisayo Adegboye, Ainsley McDonald-Boyer, Fubiao Shi, Dianxin Liu, Jose Maldonado, Jiesi Feng, Yulong Li, Simon Alford, Julio E. Ayala, Owen P. McGuinness, Sheila Collins, Heidi E. Hamm
Increasing studies have demonstrated that disease states of the endocrine or exocrine pancreas aggravate one another, which implies bi-directional blood flow between islets and exocrine cells. However, this is inconsistent with the current model of uni-directional blood flow, which is strictly from islets to exocrine tissues. This conventional model was first proposed in 1932 and it has never been revisited to date. Here, large-scale image capture was used to examine the spatial relationship between islets and blood vessels in the following species; human, monkey, pig, rabbit, ferret, and mouse. While some arterioles passed by or traveled through islets, the majority of islets had no association with them. Islets with direct contact with the arteriole were significantly larger in size and less in number than those without contact. Unique to the pancreas, capillaries directly branched out from the arterioles, and have been labeled as “small arterioles” in the past studies. Overall, the arterioles emerged to feed the pancreas regionally, not specifically targeting individual islets. Vascularizing the pancreas in this way may allow an entire downstream region of islets and acinar cells to be simultaneously exposed to changes in the blood levels of glucose, hormones and other circulating factors.
Adam A. Rizk, Michael P. Dybala, Khalil C. Rodriguez, Marjan Slak Rupnik, Manami Hara
Maintaining internal osmolality constancy is essential for life. Release of arginine vasopressin (AVP) responding to hyperosmolality is critical. Current hypotheses for osmolality sensors in circumventricular organs of the brain (CVOs) focus on mechanosensitive membrane proteins. The present study demonstrated that an intracellular protein kinase WNK1 was involved. Focusing on vascular-organ-of-lamina-terminalis (OVLT) nuclei, we showed that WNK1 kinase was activated by water restriction. Neuronal-specific knockout (cKO) of Wnk1 caused polyuria with decreased urine osmolality that persisted in water restriction and blunted water restriction-induced AVP release. Wnk1-cKO also blunted mannitol-indued AVP release but had no effect on osmotic thirst response. The role of WNK1 in the osmosensory neurons in CVOs was supported by neuronal pathway tracing. Hyperosmolality-induced increases in action potential firing in OVLT neurons was blunted by Wnk1 deletion or pharmacological WNK inhibitors. Knockdown of Kv3.1 channel in OVLT by shRNA reproduced the phenotypes. Thus, WNK1 in osmosensory neurons in CVOs detects extracellular hypertonicity and mediates the increase in AVP release by activating Kv3.1 and increasing action potential firing from osmosensory neurons.
Xin Jin, Jian Xie, Chia-Wei Yeh, Jen-Chi Chen, Chih-Jen Cheng, Cheng-Chang Lien, Chou-Long Huang
Cutaneous Skeletal Hypophosphatemia Syndrome (CSHS) is a mosaic RASopathy characterized by the association of dysplastic skeletal lesions, congenital skin nevi of epidermal and/or melanocytic origin, and fibroblast growth factor-23 (FGF23)-mediated hypophosphatemia. The primary physiological source of circulating FGF23 is bone cells. However, several reports have suggested skin lesions as the source of excess FGF23 in CSHS. Consequently, without convincing evidence of efficacy, many patients with CSHS have undergone painful removal of cutaneous lesions in an effort to normalize blood phosphate levels.This study aims to elucidate whether the source of FGF23 excess in CSHS is RAS mutation-bearing bone or skin lesions. Towards this end, we analyzed the expression and activity of Fgf23 in two mouse models expressing similar HRAS/Hras activating mutations in a mosaic-like fashion in either bone or epidermal tissue. We found that HRAS hyperactivity in bone, not skin, caused excess of bioactive intact FGF23, hypophosphatemia and osteomalacia. Our findings support RAS-mutated dysplastic bone as the primary source of physiologically active FGF23 excess in patients with CSHS. This evidence informs the care of patients with CSHS, arguing against the practice of nevi removal to decrease circulating, physiologically active FGF23.
Diana Ovejero, Zachary Michel, Christophe Cataisson, Amanda Saikali, Rebeca Galisteo, Stuart H. Yuspa, Michael T. Collins, Luis F. de Castro
BACKGROUND. Hepatic de novo lipogenesis (DNL) and β-oxidation are tightly coordinated, and their dysregulation is thought to contribute to the pathogenesis of non-alcoholic fatty liver (NAFL). Fasting normally relaxes DNL-mediated inhibition of hepatic β-oxidation, dramatically increasing ketogenesis and decreasing reliance on the TCA cycle. Thus, we tested whether aberrant oxidative metabolism in fasting NAFL subjects is related to the inability to halt fasting DNL. METHODS. Forty consecutive non-diabetic individuals with and without a history of NAFL were recruited for this observational study. After phenotyping, subjects fasted for 24-hr, and hepatic metabolism was interrogated using a combination of 2H2O and 13C tracers, magnetic resonance spectroscopy, and high-resolution mass spectrometry. RESULTS. Within a subset of subjects, DNL was detectable after a 24-hr fast and was more prominent in those with NAFL, though it was poorly correlated with steatosis. However, fasting DNL negatively correlated with hepatic β-oxidation and ketogenesis and positively correlated with citrate synthesis. Subjects with NAFL but undetectable fasting DNL (25th percentile) were comparatively normal. However, those with the highest fasting DNL (75th percentile) were intransigent to the effects of fasting on the concentration of insulin, NEFA, and ketones. Additionally, they sustained glycogenolysis and spared the loss of oxaloacetate to gluconeogenesis in favor of citrate synthesis, which correlated with DNL and diminished ketogenesis. CONCLUSION. Metabolic flux analysis in fasted subjects indicates that shared metabolic mechanisms link the dysregulations of hepatic DNL, ketogenesis, and the TCA cycle in NAFL. TRIAL REGISTRATION. Data obtained during the enrollment/non-intervention phase of Effect of Vitamin E on Non-Alcoholic Fatty Liver Disease; ClinicalTrials.gov NCT02690792.
Xiaorong Fu, Justin A. Fletcher, Stanisław Deja, Melissa Inigo-Vollmer, Shawn C. Burgess, Jeffrey D. Browning