Mark O. Huising

Two divergent leptin paralogues in zebrafish (Danio rerio) that originate early in teleostean evolution.

We describe duplicate leptin genes in zebrafish (Danio rerio) that share merely 24% amino acid identity with each other and only 18% with human leptin. We were also able to retrieve a second leptin gene in medaka (Oryzias latipes). The presence of duplicate leptin genes in these two distantly related teleosts suggests that duplicate leptin genes are a common feature of teleostean fishes. Despite low primary sequence conservation, we are confident in assigning orthology between mammalian and zebrafish leptins for several reasons. First, both zebrafish leptins share their characteristic gene structure and display key features of conserved synteny with mammalian leptin genes. Secondly, the cysteine residues that make up leptins single disulphide bridge are equally spaced in mammalian and zebrafish leptins and are unique among all members of the class-I helical cytokine family. Thirdly, the zebrafish leptins cluster with other fish leptins and mammalian leptins in phylogenetic analysis, supported by high bootstrap values. Within the leptin cluster, leptin-b forms a separate clade with the leptin-b orthologue from medaka. Finally, our prediction of the tertiary structures shows that both leptins conform to the typical four alpha-helix bundle structure of the class-I alpha-helical cytokines. The zebrafish leptins are differentially expressed; the liver shows high leptin-a expression (in concordance with what we observed for carp leptins), while leptin-b is expressed at much lower levels, which are downregulated further upon fasting. The finding of duplicate leptin genes in teleosts adds to our understanding of the evolution of leptin physiology in the early vertebrate lineage.

Neuroendocrine-immune interactions in fish: a role for interleukin-1

Bi-directional communication between the hypothalamus-pituitary-adrenal (HPA)-axis and the sympathetic nervous system with the immune system is crucial to ensure homeostasis. Shared use of ligands and especially receptors forms a key component of this bi-directional interaction. Glucocorticoids (GC), the major end products of the HPA-axis differentially modulate immune function. Cytokines, especially interleukin-1 (IL-1), tumour necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6), ensure immune signalling to the neuroendocrine system. In addition, hormones from leukocyte origin such as corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH) and beta-endorphin, as well as centrally synthesised and secreted cytokines, contribute to the communication network. In teleost fish cortisol is the major product of the hypothalamus-pituitary-interrenal (HPI)-axis which is the teleost equivalent of the HPA-axis. Moderate and substantial increases in cortisol during stressful circumstances negatively affect B-lymphocytes, whereas rescue of neutrophilic granulocytes may support innate immunity. Recent elucidation of lower vertebrate cytokine sequences has facilitated research into neuroendocrine-immune interactions in teleosts and the first evidence for a significant function of interleukin-1 in the bi-directional communication is discussed.

The transcriptional landscape of mouse beta cells compared to human beta cells reveals notable species differences in long non-coding RNA and protein-coding gene expression

BackgroundInsulin producing beta cell and glucagon producing alpha cells are colocalized in pancreatic islets in an arrangement that facilitates the coordinated release of the two principal hormones that regulate glucose homeostasis and prevent both hypoglycemia and diabetes. However, this intricate organization has also complicated the determination of the cellular source(s) of the expression of genes that are detected in the islet. This reflects a significant gap in our understanding of mouse islet physiology, which reduces the effectiveness by which mice model human islet disease.ResultsTo overcome this challenge, we generated a bitransgenic reporter mouse that faithfully labels all beta and alpha cells in mouse islets to enable FACS-based purification and the generation of comprehensive transcriptomes of both populations. This facilitates systematic comparison across thousands of genes between the two major endocrine cell types of the islets of Langerhans whose principal hormones are of cardinal importance for glucose homeostasis. Our data leveraged against similar data for human beta cells reveal a core common beta cell transcriptome of 9900+ genes. Against the backdrop of overall similar beta cell transcriptomes, we describe marked differences in the repertoire of receptors and long non-coding RNAs between mouse and human beta cells.ConclusionsThe comprehensive mouse alpha and beta cell transcriptomes complemented by the comparison of the global (dis)similarities between mouse and human beta cells represent invaluable resources to boost the accuracy by which rodent models offer guidance in finding cures for human diabetes.

Molecular evolution of CXC chemokines: extant CXC chemokines originate from the CNS

The mammalian CXC chemokine system comprises 16 ligands and six receptors, and its actions stretch well beyond the immune system. Recent elucidation of the pufferfish genome, a representative of an evolutionary ancient vertebrate class, has enabled analysis of the mammalian CXC chemokine system in a phylogenetic context. Comparison of the phylogenies of vertebrate CXC chemokines revealed that fish and mammals have found different solutions to similar problems, grafted on the same basic structural motif. Phylogenetic analyses showed that the large, highly redundant CXC chemokine family is a very recent phenomenon that is exclusive to higher vertebrates. Moreover, its ancestral role is found within the central nervous system and not within the immune system.

CXC chemokines and leukocyte chemotaxis in common carp (Cyprinus carpio L.)

CXC chemokines, structurally recognizable by the position of four conserved cysteine residues, are prominent mediators of chemotaxis. Here we report a novel carp CXC chemokine obtained through homology cloning and compare it with fish orthologues genes and with a second, recently elucidated, carp CXC chemokine. Phylogenetic analyses clearly show that neither CXC chemokine resembles any of the mammalian CXC chemokines in particular. However, basal expression is most prominent in immune organs like anterior kidney and spleen, suggesting involvement in the immune response. Furthermore we show that anterior kidney phagocyte-enriched leukocyte suspensions express both chemokines and that this expression is upregulated by brief (4 h) stimulation with PMA, but not lipopolysaccharide. Neutrophilic granulocyte-enriched leukocytes display chemotaxis to human recombinant CXCL8 (hrCXCL8; interleukin-8), confirming CXC chemokine mediated chemotaxis of neutrophilic granulocytes in teleost fish. Factors secreted from carp phagocytes are also capable of inducing chemotaxis and secretion of these factors into culture supernatants is upregulated by PMA. Finally we demonstrate involvement of both CXC chemokines as well as CXCR1 and CXCR2 in acute Argulus japonicus infection. Collectively the data presented implicate the involvement of CXC chemokines in chemotaxis of fish neutrophils in a fashion that shares characteristics with the mammalian situation. However, the CXC chemokines involved differ enough from those involved in neutrophil chemotaxis in mammals to warrant their own nomenclature.

TRPV1 Pain Receptors Regulate Longevity and Metabolism by Neuropeptide Signaling

The sensation of pain is associated with increased mortality, but it is unknown whether pain perception can directly affect aging. We find that mice lacking TRPV1 pain receptors are long-lived, displaying a youthful metabolic profile at old age. Loss of TRPV1 inactivates a calcium-signaling cascade that ends in the nuclear exclusion of the CREB-regulated transcriptional coactivator CRTC1 within pain sensory neurons originating from the spinal cord. In long-lived TRPV1 knockout mice, CRTC1 nuclear exclusion decreases production of the neuropeptide CGRP from sensory endings innervating the pancreatic islets, subsequently promoting insulin secretion and metabolic health. In contrast, CGRP homeostasis is disrupted with age in wild-type mice, resulting in metabolic decline. We show that pharmacologic inactivation of CGRP receptors in old wild-type animals can restore metabolic health. These data suggest that ablation of select pain sensory receptors or the inhibition of CGRP are associated with increased metabolic health and control longevity.

Urocortin3 mediates somatostatin-dependent negative feedback control of insulin secretion

The peptide hormone urocortin3 (Ucn3) is abundantly expressed by mature beta cells, yet its physiological role is unknown. Here we demonstrate that Ucn3 is stored and co-released with insulin and potentiates glucose-stimulated somatostatin secretion via cognate receptors on delta cells. Further, we found that islets lacking endogenous Ucn3 have fewer delta cells, reduced somatostatin content, impaired somatostatin secretion, and exaggerated insulin release, and that these defects are rectified by treatment with synthetic Ucn3 in vitro. Our observations indicate that the paracrine actions of Ucn3 activate a negative feedback loop that promotes somatostatin release to ensure the timely reduction of insulin secretion upon normalization of plasma glucose. Moreover, Ucn3 is markedly depleted from beta cells in mouse and macaque models of diabetes and in human diabetic islets. This suggests that Ucn3 is a key contributor to stable glycemic control, whose reduction during diabetes aggravates glycemic volatility and contributes to the pathophysiology of this disease.

Comprehensive alpha, beta and delta cell transcriptomes reveal that ghrelin selectively activates delta cells and promotes somatostatin release from pancreatic islets

Objective Complex local crosstalk amongst endocrine cells within the islet ensures tight coordination of their endocrine output. This is illustrated by the recent demonstration that the negative feedback control by delta cells within pancreatic islets determines the homeostatic set-point for plasma glucose during mouse postnatal development. However, the close association of islet endocrine cells that facilitates paracrine crosstalk also complicates the distinction between effects mediated directly on beta cells from indirect effects mediated via local intermediates, such as somatostatin from delta cells. Methods To resolve this problem, we generated reporter mice that allow collection of pure pancreatic delta cells along with alpha and beta cells from the same islets and generated comprehensive transcriptomes for each islet endocrine cell type. These transcriptomes afford an unparalleled view of the receptors expressed by delta, alpha and beta cells, and allow the prediction of which signal targets which endocrine cell type with great accuracy. Results From these transcriptomes, we discovered that the ghrelin receptor is expressed exclusively by delta cells within the islet, which was confirmed by fluorescent in situ hybridization and qPCR. Indeed, ghrelin increases intracellular calcium in delta cells in intact mouse islets, measured by GCaMP6 and robustly potentiates glucose-stimulated somatostatin secretion on mouse and human islets in both static and perfusion assays. In contrast, des-acyl-ghrelin at the same dose had no effect on somatostatin secretion and did not block the actions of ghrelin. Conclusions These results offer a straightforward explanation for the well-known insulinostatic actions of ghrelin. Rather than engaging beta cells directly, ghrelin engages delta cells to promote local inhibitory feedback that attenuates insulin release. These findings illustrate the power of our approach to resolve some of the long-standing conundrums with regard to the rich feedback that occurs within the islet that is integral to islet physiology and therefore highly relevant to diabetes.

CRFR1 is expressed on pancreatic β cells, promotes β cell proliferation, and potentiates insulin secretion in a glucose-dependent manner

Corticotropin-releasing factor (CRF), originally characterized as the principal neuroregulator of the hypothalamus-pituitary-adrenal axis, has broad central and peripheral distribution and actions. We demonstrate the presence of CRF receptor type 1 (CRFR1) on primary β cells and show that activation of pancreatic CRFR1 promotes insulin secretion, thus contributing to the restoration of normoglycemic equilibrium. Stimulation of pancreatic CRFR1 initiates a cAMP response that promotes insulin secretion in vitro and in vivo and leads to the phosphorylation of cAMP response element binding and the induction of the expression of several immediate-early genes. Thus, the insulinotropic actions of pancreatic CRFR1 oppose the activation of CRFR1 on anterior pituitary corticotropes, leading to the release of glucocorticoids that functionally antagonize the actions of insulin. Stimulation of the MIN6 insulinoma line and primary rat islets with CRF also activates the MAPK signaling cascade leading to rapid phosphorylation of Erk1/2 in response to CRFR1-selective ligands, which induce proliferation in primary rat neonatal β cells. Importantly, CRFR1 stimulates insulin secretion only during conditions of intermediate to high ambient glucose, and the CRFR1-dependent phosphorylation of Erk1/2 is greater with elevated glucose concentrations. This response is reminiscent of the actions of the incretins, which potentiate insulin secretion only during elevated glucose conditions. The presence of CRFR1 on β cells adds another layer of complexity to the intricate network of paracrine and autocrine factors and their cognate receptors whose coordinated efforts can dictate islet hormone output and regulate β cell proliferation.

Recombinant human leptin attenuates stress axis activity in common carp (Cyprinus carpio L.)

Proper functioning of the endocrine stress axis requires communication between the stress axis and other regulatory mechanisms. We here describe an intimate interplay between the stress axis and recombinant human leptin (rhLeptin) in a teleostean fish, the common carp Cyprinus carpio. Restraint stress (by netting up to 96h) increased plasma cortisol but did not affect hepatic leptin expression. Perifusion of pituitary glands or head kidneys with rhLeptin revealed direct effects of rhLeptin on both tissues. RhLeptin suppresses basal and CRF-induced ACTH-secretion in a rapid and concentration-dependent manner. The rhLeptin effect persisted for over an hour after administration had been terminated. RhLeptin decreases basal interrenal cortisol secretion in vitro, and by doing so attenuates ACTH-stimulated cortisol production; rhLeptin does not affect interrenal ACTH-sensitivity. Our findings show that the endocrine stress axis activity and leptin are inseparably linked in a teleostean fish, a notion relevant to further our insights in the evolution of leptin physiology in vertebrates.