The Glucagon-Like Adipokinetic Hormone in Drosophila melanogaster – Biosynthesis and Secretion

Metabolic homeostasis requires the precise regulation of circulating sugar titers. In mammals, homeostatic control of circulating sugar titers requires the coordinated secretion and systemic activities of glucagon and insulin. Metabolic homeostasis is similarly regulated in Drosophila melanogaster through the glucagon-like adipokinetic hormone (AKH) and the Drosophila insulin-like peptides (DILPs). In flies and mammals, glucagon and AKH are biosynthesized in and secreted from specialized endocrine cells. KATP channels borne on these cells respond to fluctuations in circulating glucose titers and thereby regulate glucagon secretion. The influence of glucagon in the pathogenesis of type 2 diabetes mellitus is now recognized, and a crucial mechanism that regulates glucagon secretion was reported nearly a decade ago. Ongoing efforts to develop D. melanogaster models for metabolic syndrome must build upon this seminal work. These efforts make a critical review of AKH physiology timely. This review focuses on AKH biosynthesis and the regulation of glucose-responsive AKH secretion through changes in CC cell electrical activity. Future directions for AKH research in flies are discussed, including the development of models for hyperglucagonemia and epigenetic inheritance of acquired metabolic traits. Many avenues of AKH physiology remain to be explored and thus present great potential for improving the utility of D. melanogaster in metabolic research.

Cytokine and Nitric Oxide-Dependent Gene Regulation in Islet Endocrine and Nonendocrine Cells

While exposure to inflammatory cytokines is thought to contribute to pancreatic β-cell damage during diabetes, primarily because cytokine-induced nitric oxide impairs β-cell function and causes cell death with prolonged exposure, we hypothesize that there is a physiological role for cytokine signaling that functions to protect β-cells from a number of environmental stresses. This hypothesis is derived from the knowledge that β-cells are essential for survival even though they have a limited capacity to replicate, yet they are exposed to high cytokine levels during infection as most of the pancreatic blood flow is directed to islets. Here, mouse islets were subjected to single-cell RNA sequencing following 18-hr cytokine exposure. Treatment with IL-1β and IFN-γ leads to expression of inducible nitric oxide synthase (iNOS) mRNA and antiviral and immune-associated genes as well as repression of islet identity factors in a subset of β- and non-β endocrine cells in a nitric oxide-independent manner. Nitric oxide-dependent expression of genes encoding heat shock proteins was observed in both β- and non-β endocrine cells. Interestingly, cells with high expression of heat shock proteins failed to increase antiviral and immune-associated genes, suggesting that nitric oxide may be an internal “off switch” to prevent the negative effects of prolonged cytokine signaling in islet endocrine cells. We found no evidence for pro-apoptotic gene expression following 18-hr cytokine exposure. Our findings suggest that the primary functions of cytokines and nitric oxide are to protect islet endocrine cells from damage, and only when regulation of cytokine signaling is lost does irreversible damage occur.

Increased expression of viral sensor MDA5 in pancreatic islets and in hormone-negative endocrine cells in recent onset type 1 diabetic donors

The interaction between genetic and environmental factors determines the development of type 1 diabetes (T1D). Some viruses are capable of infecting and damaging pancreatic β-cells, whose antiviral response could be modulated by specific viral RNA receptors and sensors such as Melanoma Differentiation Associated gene 5 (MDA5), encoded by the IFIH1 gene. MDA5 has been shown to be involved in pro-inflammatory and immunoregulatory outcomes, thus determining the response of pancreatic islets to viral infections. Although the function of MDA5 has been previously well explored, a detailed immunohistochemical characterization of MDA5 in pancreatic tissues of non-diabetic and T1D donors is still missing. In the present study we used multiplex immunofluorescence imaging analysis to characterize MDA5 expression and distribution in pancreatic tissues obtained from 22 organ donors (10 non-diabetic autoantibody-negative, 2 autoantibody-positive, 8 recent-onset and 2 long-standing T1D). In non-diabetic control donors, MDA5 was expressed both in α- and in β-cells. The colocalization rate imaging analysis showed that MDA5 was preferentially expressed in α-cells. In T1D donors, we observed an increased colocalization rate MDA5-glucagon respect to MDA5-insulin in comparison to non-diabetic controls; such increase was more pronounced in recent onset respect to long standing T1D donors. Of note, an increased colocalization rate MDA5-glucagon was found in insulin-deficient-islets (IDI) respect to insulin containing islets (ICI). Strikingly, in T1D donors we detected the presence of MDA5-positive/hormones-negative endocrine islet-like clusters, putatively deriving from dedifferentiation or neogenesis phoenomena. These clusters were exclusively identified in recent onset donors and not detected in autoantibody-positive non-diabetic or T1D long-standing ones. In conclusion, we showed that MDA5 is preferentially expressed in α-cells and its expression is increased in recent onset T1D donors. Finally, we observed that MDA5 may also characterize the phenotype of dedifferentiated or newly forming islet cells, thus opening to novel roles for MDA5 in pancreatic endocrine cells.

Claudin-9 constitutes tight junctions of folliculo-stellate cells in the anterior pituitary gland

The anterior pituitary gland regulates growth, metabolism, and reproduction by secreting hormones. Folliculo-stellate (FS) cells are non-endocrine cells located among hormone-producing cells in the anterior pituitary glands. They form follicular lumens, which are sealed by tight junctions (TJs). Although FS cells are hypothesized to contribute to fine-tuning of endocrine cells, little is known about the exact roles of FS cells. Here, we investigated the molecular composition of TJs in FS cells. We demonstrated that occludin is a good marker for TJs in the pituitary gland and examined the structure of the lumens surrounded by FS cells. We also found that claudin-9 is a major component of TJs in the FS cells. In immunoelectron microscopy, claudin-9 was specifically localized at TJs of the FS cells. The expression of claudin-9 was gradually increased in the pituitary gland after birth, suggesting that claudin-9 is developmentally regulated and performs some specific functions on the paracellular barrier of follicles in the pituitary gland. Furthermore, we found that angulin-1, angulin-2, and tricellulin are localized at the tricellular contacts of the FS cells. Our findings provide a first comprehensive molecular profile of TJs in the FS cells, and may lead us towards unveiling the FS cell functions.

Effective Perturbations on the Amplitude and Hysteresis of Erg-Mediated Potassium Current Caused by 1-Octylnonyl 8-[(2-hydroxyethyl)[6-oxo-6(undecyloxy)hexyl]amino]-octanoate (SM-102), a Cationic Lipid

SM-102 (1-octylnonyl 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoate) is an amino cationic lipid that has been tailored for the formation of lipid nanoparticles and it is one of the essential ingredients present in the ModernaTM COVID-19 vaccine. However, to what extent it may modify varying types of plasmalemmal ionic currents remains largely uncertain. In this study, we investigate the effects of SM-102 on ionic currents either in two types of endocrine cells (e.g., rat pituitary tumor (GH3) cells and mouse Leydig tumor (MA-10) cells) or in microglial (BV2) cells. Hyperpolarization-activated K+ currents in these cells bathed in high-K+, Ca2+-free extracellular solution were examined to assess the effects of SM-102 on the amplitude and hysteresis of the erg-mediated K+ current (IK(erg)). The SM-102 addition was effective at blocking IK(erg) in a concentration-dependent fashion with a half-maximal concentration (IC50) of 108 μM, a value which is similar to the KD value (i.e., 134 μM) required for its accentuation of deactivation time constant of the current. The hysteretic strength of IK(erg) in response to the long-lasting isosceles-triangular ramp pulse was effectively decreased in the presence of SM-102. Cell exposure to TurboFectinTM 8.0 (0.1%, v/v), a transfection reagent, was able to inhibit hyperpolarization-activated IK(erg) effectively with an increase in the deactivation time course of the current. Additionally, in GH3 cells dialyzed with spermine (30 μM), the IK(erg) amplitude progressively decreased; moreover, a further bath application of SM-102 (100 μM) or TurboFectin (0.1%) diminished the current magnitude further. In MA-10 Leydig cells, the IK(erg) was also blocked by the presence of SM-102 or TurboFectin. The IC50 value for SM-102-induced inhibition of IK(erg) in MA-10 cells was 98 μM. In BV2 microglial cells, the amplitude of the inwardly rectifying K+ current was inhibited by SM-102. Taken together, the presence of SM-102 concentration-dependently inhibited IK(erg) in endocrine cells (e.g., GH3 or MA-10 cells), and such action may contribute to their functional activities, assuming that similar in vivo findings exist.

Characterisation of Ppy-lineage cells clarifies the functional heterogeneity of pancreatic beta cells in mice

Aims/hypothesis Pancreatic polypeptide (PP) cells, which secrete PP (encoded by the Ppy gene), are a minor population of pancreatic endocrine cells. Although it has been reported that the loss of beta cell identity might be associated with beta-to-PP cell-fate conversion, at present, little is known regarding the characteristics of Ppy-lineage cells. Methods We used Ppy-Cre driver mice and a PP-specific monoclonal antibody to investigate the association between Ppy-lineage cells and beta cells. The molecular profiles of endocrine cells were investigated by single-cell transcriptome analysis and the glucose responsiveness of beta cells was assessed by Ca2+ imaging. Diabetic conditions were experimentally induced in mice by either streptozotocin or diphtheria toxin. Results Ppy-lineage cells were found to contribute to the four major types of endocrine cells, including beta cells. Ppy-lineage beta cells are a minor subpopulation, accounting for 12–15% of total beta cells, and are mostly (81.2%) localised at the islet periphery. Unbiased single-cell analysis with a Ppy-lineage tracer demonstrated that beta cells are composed of seven clusters, which are categorised into two groups (i.e. Ppy-lineage and non-Ppy-lineage beta cells). These subpopulations of beta cells demonstrated distinct characteristics regarding their functionality and gene expression profiles. Ppy-lineage beta cells had a reduced glucose-stimulated Ca2+ signalling response and were increased in number in experimental diabetes models. Conclusions/interpretation Our results indicate that an unexpected degree of beta cell heterogeneity is defined by Ppy gene activation, providing valuable insight into the homeostatic regulation of pancreatic islets and future therapeutic strategies against diabetes. Data availability The single-cell RNA sequence (scRNA-seq) analysis datasets generated in this study have been deposited in the Gene Expression Omnibus (GEO) under the accession number GSE166164 (www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE166164). Graphical abstract

Effects of iron, Vitamin A and the interaction between the two nutrients on intestinal development and cell differentiation in piglets

This study aimed to investigate the effects of iron, vitamin A (VA) and their interaction on intestinal development and differentiation of cells in suckling piglets. Therefore, 32 Duroc × Landrace × Yorkshire 0-day-old newborn boars with similar body weights were randomly divided into 4 groups, with 8 replicates in each group and 1 pig in each replicate. All the piglets were breastfed. In addition, the piglets were given normal saline (CON group) or ferrous sulfate (OAFe group) or VA (VA group) or ferrous sulfate and VA (OAFe + VA group) on the 2 nd, 7 th, 12 th and 17 th day, respectively. The piglets were then slaughtered on the 21 st day, and intestinal samples were collected. The results showed that: (1) iron (P < 0.001) significantly increased the length, weight, relative weight, and the length to weight ratio of the small intestine. On the other hand, VA had a significant effect on the weight to length ratio (P = 0.015) and relative weight (P < 0.001) of the small intestine; (2) with regard to intestinal morphology, supplementation with iron (P <0.05) had obvious effects on the villus height (VH), crypt depth (CD), villus width (VW), and surface area. Additionally, both VA and interaction of VA and iron increased the VH (P < 0.05) and surface area (P = 0.001). The results also showed that iron (P < 0.01) increased the number of crypt goblet cells, Ki67 positive cells, and endocrine cells. Moreover, both VA and the interaction between VA and iron increased the number of endocrine cells in the villi (P = 0.05); (3) With regard to the mRNA expression levels of stem cell differentiation marker genes, iron (P < 0.05) decreased the expression of trophinin 2 (Trop2), leucine rich repeat containing G protein-coupled receptor 5 positive (Lgr5+), male specific lethal 1(Msl1), BMI 1 proto-oncogene, polycomb ring finger (Bmi1), achaete-scute family bHLH transcription factor 2 (Ascl2). On the other hand, VA increased the expression of Ascl2 (P = 0.001) although the interaction of VA and iron (P < 0.05) had an effect on the expression of secreted phosphoprotein 1 (Spp1) and Bmi1. In addition, VA decreased the gene or mRNA expression of aconitase 1 (Aco1; P < 0.001), transferrin receptor (TFRC; P = 0.001), and solute carrier family 11 member 2 (DMT1; P = 0.003) in the Iron Reactive Element/Iron Regulatory Protein (IRE/IRP) signaling pathway although iron and the interaction of VA and iron had no effect on the genes’ expression. The results therefore showed that VA, iron, and their interaction can promote intestinal development and epithelial cell differentiation in piglets.

Insm1, Neurod1, and Pax6 promote murine pancreatic endocrine cell development through overlapping yet distinct RNA transcription and splicing programs

Insm1, Neurod1, and Pax6 are essential for the formation and function of pancreatic endocrine cells. Here, we report comparative immunohistochemical, transcriptomic, functional enrichment, and RNA splicing analyses of these genes using gene knock-out mice. Quantitative immunohistochemical analysis confirmed that elimination of each of these three factors variably impairs the proliferation, survival, and differentiation of endocrine cells. Transcriptomic analysis revealed that each factor contributes uniquely to the transcriptome although their effects were overlapping. Functional enrichment analysis revealed that genes downregulated by the elimination of Insm1, Neurod1, and Pax6 are commonly involved in mRNA metabolism, chromatin organization, secretion, and cell cycle regulation, and upregulated genes are associated with protein degradation, autophagy, and apoptotic process. Elimination of Insm1, Neurod1, and Pax6 impaired expression of many RNA-binding proteins thereby altering RNA splicing events, including for Syt14 and Snap25, two genes required for insulin secretion. All three factors are necessary for normal splicing of Syt14, and both Insm1 and Pax6 are necessary for the processing of Snap25. Collectively, these data provide new insights into how Insm1, Neurod1, and Pax6 contribute to the formation of functional pancreatic endocrine cells.

Motilin Comparative Study: Structure, Distribution, Receptors, and Gastrointestinal Motility

Motilin, produced in endocrine cells in the mucosa of the upper intestine, is an important regulator of gastrointestinal (GI) motility and mediates the phase III of interdigestive migrating motor complex (MMC) in the stomach of humans, dogs and house musk shrews through the specific motilin receptor (MLN-R). Motilin-induced MMC contributes to the maintenance of normal GI functions and transmits a hunger signal from the stomach to the brain. Motilin has been identified in various mammals, but the physiological roles of motilin in regulating GI motility in these mammals are well not understood due to inconsistencies between studies conducted on different species using a range of experimental conditions. Motilin orthologs have been identified in non-mammalian vertebrates, and the sequence of avian motilin is relatively close to that of mammals, but reptile, amphibian and fish motilins show distinctive different sequences. The MLN-R has also been identified in mammals and non-mammalian vertebrates, and can be divided into two main groups: mammal/bird/reptile/amphibian clade and fish clade. Almost 50 years have passed since discovery of motilin, here we reviewed the structure, distribution, receptor and the GI motility regulatory function of motilin in vertebrates from fish to mammals.