Biallelic mutations of the gene encoding the transcription factor NEUROG3 are associated with a rare disorder that presents in neonates as generalized malabsorption – due to a complete absence of enteroendocrine cells – followed, in early childhood or beyond, by insulin-dependent diabetes mellitus (IDDM). The commonly delayed onset of IDDM suggests a differential requirement for NEUROG3 in endocrine cell generation in the human pancreas versus the intestine. However, previously identified human mutations were hypomorphic, and hence may have had residual function in pancreas. We report two patients with biallelic functionally null variants of the NEUROG3 gene who nonetheless did not present with IDDM during infancy, but instead developed permanent IDDM during middle childhood ages. The variants show no evidence of function in traditional promoter-based assays of NEUROG3 function and also fail to exhibit function in a variety of novel in vitro and in vivo molecular assays designed to discern residual NEUROG3 function. These findings imply that unlike in mice, pancreatic endocrine cell generation in humans is not entirely dependent on NEUROG3 expression, and hence suggests the presence of unidentified redundant in vivo pathways in human pancreas capable of yielding beta-cell mass sufficient to maintain euglycemia until early childhood.
R. Sergio Solorzano-Vargas, Matthew Bjerknes, Jiafang Wang, S. Vincent Wu, Manuel G. Garcia-Careaga, Duke Pisit, Hazel Cheng, Michael S. German, Senta Georgia, Martin G. Martín
Salt sensitivity of blood pressure (SSBP) and hypertension are common, but the underlying mechanisms remain unclear. Endoplasmic reticulum aminopeptidase 1 (ERAP1) degrades angiotensin II (ANGII). We hypothesized that decreasing ERAP1 increases BP via ANGII-mediated effects on aldosterone (ALDO) production and/or renovascular function. Compared with WT littermate mice, ERAP1-deficient (ERAP1+/–) mice had increased tissue ANGII, systolic and diastolic BP, and SSBP, indicating that ERAP1 deficiency leads to volume expansion. However, the mechanisms underlying the volume expansion differed according to sex. Male ERAP1+/– mice had increased ALDO levels and normal renovascular responses to volume expansion (decreased resistive and pulsatility indices and increased glomerular volume). In contrast, female ERAP1+/– mice had normal ALDO levels but lacked normal renovascular responses. In humans, ERAP1 rs30187, a loss-of-function gene variant that reduces ANGII degradation in vitro, is associated with hypertension. In our cohort from the Hypertensive Pathotype (HyperPATH) Consortium, there was a significant dose-response association between rs30187 risk alleles and systolic and diastolic BP as well as renal plasma flow in men, but not in women. Thus, lowering ERAP1 led to volume expansion and increased BP. In males, the volume expansion was due to elevated ALDO with normal renovascular function, whereas in females the volume expansion was due to impaired renovascular function with normal ALDO levels.
Sanjay Ranjit, Jian Yao Wong, Jia W. Tan, Chee Sin Tay, Jessica M. Lee, Kelly Yin Han Wong, Luminita H. Pojoga, Danielle L. Brooks, Amanda E. Garza, Stephen A. Maris, Isis Akemi Katayama, Jonathan S. Williams, Alicia Rivera, Gail K. Adler, Gordon H. Williams, Jose R. Romero
Islet transplantation is an effective therapy for achieving and maintaining normoglycemia in patients with type 1 diabetes mellitus. However, the supply of transplantable human islets is limited. Upon removal from the pancreas, islets rapidly disintegrate and lose function, resulting in a short interval for studies of islet biology and pretransplantation assessment. Here, we developed a biomimetic platform that can sustain human islet physiology for a prolonged period ex vivo. Our approach involved the creation of a multichannel perifusion system to monitor dynamic insulin secretion and intracellular calcium flux simultaneously, enabling the systematic evaluation of glucose-stimulated insulin secretion under multiple conditions. Using this tool, we developed a nanofibrillar cellulose hydrogel–based islet-preserving platform (iPreP) that can preserve islet viability, morphology, and function for nearly 12 weeks ex vivo, and with the ability to ameliorate glucose levels upon transplantation into diabetic hosts. Our platform has potential applications in the prolonged maintenance of human islets, providing an expanded time window for pretransplantation assessment and islet studies.
Yi-Ju Chen, Taiji Yamazoe, Karla F. Leavens, Fabian L. Cardenas-Diaz, Andrei Georgescu, Dongeun Huh, Paul Gadue, Ben Z. Stanger
The choroid plexus (ChP) is a highly vascularized tissue found in the brain ventricles, with an apical epithelial cell layer surrounding fenestrated capillaries. It is responsible for the production of most of the cerebrospinal fluid (CSF) in the ventricular system, subarachnoid space, and central canal of the spinal cord, while also constituting the blood-CSF barrier (BCSFB). In addition, epithelial cells of the choroid plexus (EChP) synthesize neurotrophic factors and other signaling molecules that are released into the CSF. Here we show that insulin is produced in EChP of mice and humans, and its expression and release are regulated by serotonin. Insulin mRNA and immune-reactive protein, including C-peptide, are present in EChP, as detected by several experimental approaches, and in much higher levels than any other brain region and non-pancreatic peripheral tissues. Moreover, insulin is produced in primary cultured mouse EChP, and its release, albeit Ca2+-sensitive, is not regulated by glucose. Instead, activation of the 5HT2C receptor by serotonin treatment led to activation of IP3-sensitive channels and Ca2+ mobilization from intracellular storage, leading to insulin secretion. In vivo depletion of brain serotonin in the dorsal raphe nucleus negatively affected insulin expression in the ChP, suggesting an endogenous modulation of ChP insulin by serotonin. Therefore, for the first time to our knowledge, here we show that insulin is produced by EChP in the brain, and its release is modulated at least by serotonin, and not glucose.
Caio Henrique Mazucanti, Qing-Rong Liu, Doyle Lang, Nicholas Huang, Jennifer F. O’Connell, Simonetta Camandola, Josephine M. Egan
During pregnancy the maternal pancreatic islets of Langerhans undergo adaptive changes to compensate for gestational insulin resistance. Kisspeptin has been shown to stimulate insulin release, through its receptor, GPR54. The placenta releases high levels of kisspeptin into the maternal circulation, suggesting a role in modulating the islet adaptation to pregnancy. In the present study we show that pharmacological blockade of endogenous kisspeptin in pregnant mice resulted in impaired glucose homeostasis. This glucose intolerance was due to a reduced insulin response to glucose as opposed to any effect on insulin sensitivity. A β cell–specific GPR54-knockdown mouse line was found to exhibit glucose intolerance during pregnancy, with no phenotype observed outside of pregnancy. Furthermore, in pregnant women circulating kisspeptin levels significantly correlated with insulin responses to oral glucose challenge and were significantly lower in women with gestational diabetes (GDM) compared with those without GDM. Thus, kisspeptin represents a placental signal that plays a physiological role in the islet adaptation to pregnancy, maintaining maternal glucose homeostasis by acting through the β cell GPR54 receptor. Our data suggest reduced placental kisspeptin production, with consequent impaired kisspeptin-dependent β cell compensation, may be a factor in the development of GDM in humans.
James E. Bowe, Thomas G. Hill, Katharine F. Hunt, Lorna I.F. Smith, Sian J.S. Simpson, Stephanie A. Amiel, Peter M. Jones
Intestinally derived glucagon-like peptide-1 (GLP-1), encoded by the preproglucagon (Gcg) gene, is believed to function as an incretin. However, our previous work questioned this dogma and demonstrated that pancreatic peptides rather than intestinal Gcg peptides, including GLP-1, are a primary regulator of glucose homeostasis in normal mice. The objective of these experiments was to determine whether changes in nutrition or alteration of gut hormone secretion by bariatric surgery would result in a larger role for intestinal GLP-1 in the regulation of insulin secretion and glucose homeostasis. Multiple transgenic models, including mouse models with intestine- or pancreas tissue–specific Gcg expression and a whole-body Gcg-null mouse model, were generated to study the role of organ-specific GLP-1 production on glucose homeostasis under dietary-induced obesity and after weight loss from bariatric surgery (vertical sleeve gastrectomy; VSG). Our findings indicated that the intestine is a major source of circulating GLP-1 after various nutrient and surgical stimuli. However, even with the 4-fold increase in intestinally derived GLP-1 with VSG, it is pancreatic peptides, not intestinal Gcg peptides, that are necessary for surgery-induced improvements in glucose homeostasis.
Ki-Suk Kim, Chelsea R. Hutch, Landon Wood, Irwin J. Magrisso, Randy J. Seeley, Darleen A. Sandoval
It is proposed that the impaired sympathoadrenal response to hypoglycemia induced by recurrent insulin-induced hypoglycemia (RH) is an adaptive phenomenon induced by specific changes in microRNA expression in the ventromedial hypothalamus (VMH). To test this hypothesis, genome-wide microRNAomic profiling of the VMH by RNA-sequencing was performed in control rats and rats treated for RH. Differential expression analysis identified microRNA-7a-5p and microRNA-665 as potential mediators of this phenomenon. To further test this hypothesis, experiments were conducted consisting of targeted lentiviral-mediated overexpression of microRNA-7a-5p and downregulation of microRNA-665 in the VMH. Hyperinsulinemic hypoglycemic clamp experiments demonstrated that targeted overexpression of microRNA-7a-5p (but not downregulation of microRNA-665) in the VMH of RH rats restored the epinephrine response to hypoglycemia. This restored response to hypoglycemia was associated with a restoration of GABAA receptor gene expression. Finally, a direct interaction of microRNA-7a-5p with the 3′-UTR of GABAA receptor α1-subunit (Gabra1) gene was demonstrated in a luciferase assay. These findings indicate that (a) the impaired sympathoadrenal response RH induces is associated with changes in VMH microRNA expression and (b) microRNA-7a-5p, possibly via direct downregulation of GABA receptor gene expression, may serve as a mediator of the altered sympathoadrenal response to hypoglycemia.
Rahul Agrawal, Griffin Durupt, Dinesh Verma, Michael Montgomery, Adriana Vieira-de Abreu, Casey Taylor, Sankar Swaminathan, Simon J. Fisher
The glucagon-like peptide 1 receptor agonist exenatide improves glycemic control by several and not completely understood mechanisms. Herein, we examined the effects of chronic intravenous exenatide infusion on insulin sensitivity, β- and α-cell function and relative volumes, islet cell apoptosis and replication in nondiabetic non-human primates (baboons). At baseline, baboons received a 2-step hyperglycemic clamp followed by an L-arginine bolus (HC/A). After HC/A, baboons underwent a partial pancreatectomy (tail removal) and received a continuous exenatide (n = 12) or saline (n = 12) infusion for 13 weeks. At the end of treatment, HC/A was repeated and the remnant pancreas (head-body) harvested. Insulin sensitivity increased dramatically after exenatide treatment and was accompanied by a decrease in insulin and C-peptide secretion, while the insulin secretion/insulin resistance (disposition) index increased by approximately 2-fold. β-, α-, and δ-cell relative volumes in exenatide-treated baboons were significantly increased compared to saline-treated controls, primarily as the result of increased islet cell replication. Features of cellular stress and secretory dysfunction were present in islets of saline-treated baboons and absent in islets of exenatide-treated baboons. In conclusion, chronic administration of exenatide exerts proliferative and cytoprotective effects on β-, α-, and δ-cells and produces a robust increase in insulin sensitivity in non-human primates.
Teresa Vanessa Fiorentino, Francesca Casiraghi, Alberto M. Davalli, Giovanna Finzi, Stefano La Rosa, Paul B. Higgins, Gregory A. Abrahamian, Alessandro Marando, Fausto Sessa, Carla Perego, Rodolfo Guardado- Mendoza, Subhash Kamath, Andrea Ricotti, Paolo Fiorina, Giuseppe Daniele, Ana M. Paez, Francesco Andreozzi, Raul A. Bastarrachea, Anthony G. Comuzzie, Amalia Gastaldelli, Alberto O. Chavez, Eliana S. Di Cairano, Patrice A. Frost, Livio Luzi, Edward J. Dick, Jr., Glenn A. Halff, Ralph A. DeFronzo, Franco Folli
Insulin resistance associates with increased risk for cognitive decline and dementia; however, the underpinning mechanisms for this increased risk remain to be fully defined. As insulin resistance impairs mitochondrial oxidative metabolism and increases ROS in skeletal muscle, we considered whether similar events occur in the brain, which — like muscle — is rich in insulin receptors and mitochondria. We show that high-fat diet–induced (HFD-induced) brain insulin resistance in mice decreased mitochondrial ATP production rate and oxidative enzyme activities in brain regions rich in insulin receptors. HFD increased ROS emission and reduced antioxidant enzyme activities, with the concurrent accumulation of oxidatively damaged mitochondrial proteins and increased mitochondrial fission. Improvement of insulin sensitivity by both aerobic exercise and metformin ameliorated HFD-induced abnormalities. Moreover, insulin-induced enhancement of ATP production in primary cortical neurons and astrocytes was counteracted by the insulin receptor antagonist S961, demonstrating a direct effect of insulin resistance on brain mitochondria. Further, intranasal S961 administration prevented exercise-induced improvements in ATP production and ROS emission during HFD, supporting that exercise enhances brain mitochondrial function by improving insulin action. These results support that insulin sensitizing by exercise and metformin restores brain mitochondrial function in insulin-resistant states.
Gregory N. Ruegsegger, Patrick M. Vanderboom, Surendra Dasari, Katherine A. Klaus, Parijat Kabiraj, Christina B. McCarthy, Claudia F. Lucchinetti, K. Sreekumaran Nair
Sustained therapeutic responses from traditional and next-generation antiandrogen therapies remain elusive in clinical practice due to inherent and/or acquired resistance resulting in persistent androgen receptor (AR) activity. Antisense oligonucleotides (ASO) have the ability to block target gene expression and associated protein products and provide an alternate treatment strategy for castration-resistant prostate cancer (CRPC). We demonstrate the efficacy and therapeutic potential of this approach with a Generation-2.5 ASO targeting the mouse AR in genetically engineered models of prostate cancer. Furthermore, reciprocal feedback between AR and PI3K/AKT signaling was circumvented using a combination approach of AR-ASO therapy with the potent pan-AKT inhibitor, AZD5363. This treatment strategy effectively improved treatment responses and prolonged survival in a clinically relevant mouse model of advanced CRPC. Thus, our data provide preclinical evidence to support a combination strategy of next-generation ASOs targeting AR in combination with AKT inhibition as a potentially beneficial treatment approach for CRPC.
Marco A. De Velasco, Yurie Kura, Kazuko Sakai, Yuji Hatanaka, Barry R. Davies, Hayley Campbell, Stephanie Klein, Youngsoo Kim, A. Robert MacLeod, Koichi Sugimoto, Kazuhiro Yoshikawa, Kazuto Nishio, Hirotsugu Uemura
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