Characterization of Insulin and Glucagon Genes and Their Producing Endocrine Cells From Pygmy Sperm Whale (Kogia breviceps)

Insulin and glucagon are hormones secreted by pancreatic β and α cells, respectively, which together regulate glucose homeostasis. Dysregulation of insulin or glucagon can result in loss of blood glucose control, characterized by hyperglycemia or hypoglycemia. To better understand the endocrine physiology of cetaceans, we cloned and characterized the insulin and glucagon genes from pygmy sperm whale (Kogia breviceps). We obtained the complete coding sequences of the preproinsulin and preproglucagon genes, which encodes the preproinsulin protein of 110 amino acid (aa) residues and encodes the preproglucagon protein of 179 aa residues, respectively. Sequence comparison and phylogenetic analyses demonstrate that protein structures were similar to other mammalian orthologs. Immunohistochemistry and immunofluorescence staining using insulin, glucagon, and somatostatin antibodies allowed analysis of pygmy sperm whale islet distribution, architecture, and composition. Our results showed the pygmy sperm whale islet was irregularly shaped and randomly distributed throughout the pancreas. The architecture of α, β, and δ cells of the pygmy sperm whale was similar to that of artiodactyls species. This is the first report about insulin and glucagon genes in cetaceans, which provides new information about the structural conservation of the insulin and glucagon genes. Furthermore, offers novel information on the properties of endocrine cells in cetacean for further studies.


INTRODUCTION
The pancreatic islet is composed of five different types of endocrine cells, including insulin-secreting β cells and glucagon-secreting α cells, which together regulates glucose homeostasis (1). Insulin is secreted by β cells, primarily in response to elevated concentrations of blood glucose, which causes glucose uptake in peripheral tissues, and a conversion to glycogen (2). Glucagon is produced by α cells and acts to oppose the functions of insulin, which elevates the concentration of glucose in the blood by promoting gluconeogenesis and glycogenolysis in peripheral tissues, predominantly in the liver (3). Due to the clinical importance of their related diseases, such as diabetes, these two hormones have been studied extensively and in-depth, in both human and mammalian animal models.
In mammals, both insulin and glucagon are evolutionary wellconserved. However, although insulin and glucagon have also been widely studied in non-mammalian species, information on the genes, physiology, and evolution of cetaceans still remains unclear. Cetaceans, commonly known as whales, dolphins, and porpoises, are mammals that have secondarily adapted to the aquatic environment, and are important organisms for the understanding of genetic evolution (4,5). Upon aquatic transition cetaceans have acquired many characteristics, for example, a thick subcutaneous fatty deposit, or blubber, that is considered to be a primary energy store and is 10-fold greater than that of other artiodactyls species, the closest living relatives of cetaceans (6,7). It is poorly understood how insulin and glucagon contribute to this special energy store. Interestingly, in these aquatic mammals the glucose homeostasis are similar, in process, to humans. Studies have revealed that bottlenose dolphins (Tursiops truncatus) have sustained postprandial hyperglycemia and hyperinsulinemia, dyslipidemia, and fatty liver disease, which is similar to human diabetes (8)(9)(10).
In order to establish the molecular basis for further studies, we identified the preproinsulin and preproglucagon genes from a cetacean, pygmy sperm whale (Kogia breviceps). First, we performed sequence alignment and phylogenetic analysis of both genes. Second, we characterized the cetacean pancreatic endocrine α cells and β cells by immunohistochemistry and immunofluorescence.

Animal and Tissue Collection
On June 27th, 2019, a carcass of toothed whale was found in Pingtan Island, Fujian province in China, and taken to the Third Institute of Oceanography, Ministry of Natural Resources, China for further species identification and anatomy specimen making. External morphological assessment of the carcass was measured before the autopsy. And, sex was determined anatomically by the analysis of reproductive organs morphology. Since this toothed whale carcass showed fresh smell, normal appearance and with several scrapes in the skin, as well as eyes clear (Supplemental Figure 1A), it was classified as Code 2, <24 h post-mortem (11). During necropsy, all organ systems were examined macroscopically. Several tissues were collected and frozen at −20 • C. Dissections were performed using standard anatomical instruments at the Endangered Animal Center, Laboratory of Marine Biology and Ecology, Third Institute of Oceanography, Ministry of Natural Resources, Xiamen, China.
The pancreas of toothed whale was removed from the whale and fixed for 4 h in 4% paraformaldehyde-PBS at 4 • C. Followed equilibrated pancreas in 30% sucrose overnight until the tissue settles to bottom at 4 • C. Pancreas was then placed in cryomold containing OCT and Store at −80 • C for further tissues analysis. For the mouse pancreas experiments, the pancreas of a 19 weeks adult C57BL/6 mouse was used as control. The tissue collection protocol followed the study as previously reported (12). All procedures have been approved by the Xiamen University Institutional Animal Care and Use Committee (Protocol XMULAC20160089, 10 March 2016).

Molecular Cloning, Structural, and Phylogenetic Analysis of Insulin and Glucagon Genes
Based on the genomic comparison with several species insulin and glucagon loci, we designed the primers for both genes in the intron conserved region. We amplified insulin and glucagon genes from the genomic DNA sample. Primers were designed using the Primer Premier 5.   Sus scrofa, Ovis aries, Bos taurus, Ornithorhynchus anatinus), one Aves (Gallus gallus), one Reptilia (Alligator sinensis), one Amphibia (Xenopus tropicalis), and one Actinopterygii (Danio rerio) by neighbor-joining method using the Poisson model by MEGA 6.0 with 1,000 bootstraps (The Biodesign Institute, Tempe, AZ).

Immunofluorescence
For OCT frozen samples, 10-µm-thick sections were stained for β cells using guinea pig anti-insulin antibody (DAKO, A0564) and mouse anti-Urocortin 3 antibody (Santa, sc-517449), for α cells using mouse anti-glucagon antibody (Sigma G2654), and for δ cells using rat anti-somatostatin antibody (Abcam ab30788), followed by various Alexa Fluor-conjugated goat secondary antibodies (Jackson immunoresearch laboratories or molecular probes). We used confocal microscopy and Laser Scanning Microscope Software (Leica TCS SP8 STED) to survey colocalization and capture images.

Quantification of Islet Area, Statistics of the Composition of Endocrine Cells
Quantifications of stained areas were performed on digital images using the imageJ software. This software was programmed to automatically quantify stained areas within defined regions of interest. The insulin plus glucagon staining sections, insulin staining section, and glucagon staining section were measured for islet, β cells and α cells areas, respectively. Each measurement was average from 5 difference sections and expressed as the mean ± S.E.M. The percentage of the endocrine cell area were calculated as the specific endocrine cell area compared with the total pancreas area. For the proportions of endocrine cells, 11 islets were counted based on 5 difference immunofluorescence staining sections, pancreas regions were randomly selected and hormone positive endocrine cells with different fluorescent colors were counted.

Molecular Identification of the Specimen
The toothed whale specimen was measured 2.73 m and suggested to be a female pygmy sperm whale (K. breviceps) or a dwarf sperm whale (K. simus), based upon the external physical and morphological examination (Supplemental Figure 1A). To further identify the species, we successfully amplified and sequenced for the three mitochondrial regions from genomic DNA, the 5 ′ end of the cox1 gene (636 bp), the partial cytb gene (574 bp), and the complete D-loop sequence (938 bp). The cox1 sequence submitted to the BOLD Systems matched to pygmy sperm whale reference sequences with 100% similarity (Supplemental Figure 1B). The DNA Surveillance analysis results showed the present specimen sequences clustered with pygmy sperm whale reference sequences for both D-loop (Supplemental Figure 1C) and the cytb (Supplemental Figure 1D) with high bootstrap support (100%). According to literature (16) and our anatomical observation, as well as the molecular information, we identified that the whale was an adult female pygmy sperm whale (K. breviceps).
FIGURE 2 | Phylogenetic analysis of the preproinsulin gene family. Full-length sequences of preproinsulin were analyzed using the neighbor-joining method. Numbers on nodes represent the frequency with which the node is recovered per 100 bootstrap replications in a total of 1,000.
We next performed phylogenetic analysis using the sequences of 21 vertebrates preproinsulin obtained from GenBank. A phylogenetic tree generated by the neighbor-joining method revealed that the pygmy sperm whale preproinsulin forms a cluster with the cetacean predicted preproinsulin [Finless porpoise (N. asiaeorientalis) and Common bottlenose dolphin (T. truncatus)] with high bootstrap support value, suggesting that the protein was indeed the ortholog of the cetacean preproinsulin (Figure 2).
The phylogenetic tree generated by the neighbor-joining method using 21 vertebrates preproglucagon revealed that  the pygmy sperm whale preproglucagon forms a cluster with the cetacean predicted preproinsulins [Finless porpoise (N. asiaeorientalis) and Common bottlenose dolphin (T. truncatus)], The closest match was with the Common bottlenose dolphin with high bootstrap support value, suggesting that this protein is indeed the ortholog of the cetacean preproglucagon (Figure 4).

Pygmy Sperm Whale Islet Distribution
To determine the islet distribution in the pancreas, we first performed hematoxylin and eosin staining (H&E) on the pygmy sperm whale pancreas sections. We identified two different types of parenchymal tissue, the dark-stained acinar cells of the exocrine pancreas, and the light-stained islets of Langerhans (Figures 5B,D). Overall, islet shapes were irregular and scattered throughout the pancreatic lobules. Pygmy sperm whale islets were smaller in size and the border of acinar cell and islet was more obscure (Figure 5B), compared to mouse pancreatic islets (Figures 5A,C). Considering the difficulty in defining the islet area in the pygmy sperm whale pancreas by H&E, we also performed immunohistochemistry using both insulin and glucagon antibodies (Figures 5E,F). Insulin and glucagon co-staining confirmed the pancreatic islets in the pygmy sperm whale were irregular in shape, with only few oval in shape, unlike islets in both mouse and human. Furthermore, islet size was assorted, as demonstrated on both insulin and glucagon co-stained sections, the islet area of pygmy sperm whale was 20608 ± 1726 µm 2 , with 1.41 ± 0.35% of total pancreas area ( Figure 6G, blue bar).

Pygmy Sperm Whale Islet Architecture and Composition
To investigate pancreatic islets in the pygmy sperm whale further, we assessed the morphology of glucagon-secreting α cells and insulin-producing β cells. First, we performed immunohistochemistry using glucagon or insulin antibodies, separately. Pygmy sperm whale α and β cells were clustered randomly throughout the pancreatic lobes with various shapes (Figures 6A-F). Additionally, few glucagon-positive α cells or insulin-positive β cells without islet outlines were scattered throughout the exocrine pancreatic tissue (Figures 6A,B arrows). For α cells, as a percentage of total pancreas area was 0.47 ± 0.07%, with the area of 7,085 ± 589.9 µm 2 ( Figure 6G, green bar). The insulin positive β cells, as a percentage of total pancreas was 0.99 ± 0.22%, with the area of 15,073 ± 1,725 µm 2 (Figure 6G, red bar).
To obtain further information on the distribution and composition of endocrine cells in the islet, we performed multicolor immunostaining using antibodies against glucagon and insulin. As shown in Figure 7A, the majority of islets displayed β cells in the islet core, with glucagon positive α cells in the islet periphery. However, we also observed a small number of α cells scattered throughout the islets, including the center of the islet core (Figure 7A, bottom panel). Considering the urocortin 3 (UCN3) transcription factor regulates both human and mouse β-cell maturation, we also examined UCN3 immunostaining in pygmy sperm whale (Figure 7B). This mature β-cell marker showed a high degree of uniformity when co-stained with insulin in the pygmy sperm whale islets, with the subcellular location in the cytoplasm of the β cells, as described in human and mouse (17,18).
Lastly, we analyzed somatostatin-positive δ cells, by immunostaining, shown in Figure 8. The δ cells were very few in number in the pancreatic islets, surrounding the β cells ( Figure 8A, middle panel). However, there were also a few δ cells scattered in pancreas lobes alone (Figure 8A, lower panel). The percentage of α cells, β cells, and δ cells in the pancreatic islets were ∼33.07%, 65.94%, and 0.99%, respectively (Figure 8B).

DISCUSSION
The islets of Langerhans are comprised of highly specialized endocrine cell populations, including α cells, β cells, and δ cells (19). Both insulin and glucagon have been of significant focus, due to the key roles in glucose metabolism, diabetes and other disorders (20). Importantly, the genetic sequences of both hormones found in cetaceans have yet to be identified, however we now, for the first time, report both insulin and glucagon cetacean sequences.
In this study, we cloned and characterized both insulin and glucagon genes from the pygmy sperm whale. To our knowledge, this is the first time the full-length structure of preproinsulin and preproglucagon have been determined in a cetacean species. By phylogenetic analysis, we confirmed that the gene we cloned was pygmy sperm whale preproinsulin (Figure 2). The preproinsulin protein was highly conserved in pygmy sperm whale, compared with other vertebrates. The mature insulin peptide (B-chain and A-chain) was 100% identical in the cetacean species, also observed in the pig (Sus scrofa), belonging to artiodactyla group (21), and only have a few amino acid differences with other mammalian insulin sequences (Figure 1B).
For the pygmy sperm whale, the amino acid sequence of preproglucagon was highly similar to the other mammalian orthologs, and exhibited a highly conserved region which encodes mature glucagon peptide and GLP-1 peptide and showed 100% identity with human, mouse, pig and bovine sequences ( Figure 3B). The insulin and glucagon molecular network regulates many key biological processes of organisms, including reproduction, development, metabolism, and lifespan (19,22,23). Our results showed that both preproinsulin and preproglucagon proteins of pygmy sperm whale were highly similar to mammals and may suggest that the function of both hormones is also conserved in cetaceans, including pygmy sperm whale. However, further investigation is needed to determine differences in pre and post-translational modifications and the epigenetic regulation of both proteins in pygmy sperm whale, compared to other mammals.
So far, histomorphology of endocrine pancreas has been analyzed in several different species of cetaceans. Such as, area and architecture of endocrine cells were evaluated from 22 bottlenose dolphin (Tursiops truncatus), and the islet area was positive linear association with dolphin age (24). Using Peroxidase-antiperoxidase (PAP) techniques, the location of α cell and β cell were showed in the pygmy sperm whale (K. breviceps) and dwarf sperm whale (Kogia simus) (25). Moreover, the pancreas weight, islet size, and distribution of α cells and β cells were also investigated in the beluga whale (Delphinapterus leucas) (26). However, more detailed analyses are still required to understand the physiological study in cetaceans. Therefore, we combined H&E, immunohistochemistry and immunofluorescence techniques to study pygmy sperm whale islet distribution and architecture. Our results showed that the pygmy sperm whale islets are randomly distributed, but clustered within the pancreatic lobes in various shapes and sizes ( Figure 5). The mean islet size was smaller than that of the mouse (Figure 5), however the islet volume was similar to other mammals, particularly to the bottlenose dolphin and pig (24,27,28). Surprisingly, the architecture was variable across the different species analyzed (29). Specifically, mice islets have a β-cell-rich core which is surrounded by few α cells and δ cells. While in human islets, the α cells, β cells, and δ cells appear to be randomly distributed throughout the islet (28,29). In pygmy sperm whale islet, β cells were in the central core, the majority of α cells in the periphery and a few α cells dispersed throughout the islets (Figures 6, 7). This architecture is somehow between mice and human, but more close to mice than humans. In bottlenose dolphin, β cells were found clustered in the cords, while α cells were found both dispersed or in the periphery (24). These arrangements of α cell and β cell in pygmy sperm whale and bottlenose dolphin are highly similar to their closest evolutionary relatives, animals in the Artiodactyla. The islet architectures of pigs (Sus domesticus), sheep (Ovis aries), and cow (Bos taurus) all display β cells centrally located, and most α cell reside in the periphery, along with some α cells are located in the center of islets (27,(29)(30)(31)(32)(33). Taken together, these studies may reveal that the animals in the Artiodactyla have preserve highly similar islet architecture during evolution. However, in previous studies of beluga whale and pygmy sperm whales suggested that β cells centrally located in the islets and the α cells are peripherally. These difference from early studies may due to the individual difference or low resolution of previous staining techniques (25,26).
In addition, individual endocrine cells such as α, β, and δ cells can be observed outside the islets (Figures 6-8). This phenomenon was also detected in and many other mammalian species, including rat, cat, dog, pig, non-human primate, bottlenose dolphin, and beluga whale (24,26,34,35). Although why this kind of endocrine cell arrangements in these species are still largely unknown, some studies suggested that these alone endocrine cells may be an indication of proliferation or neogenesis (34)(35)(36)(37). The composition of α and β cells were 33.07% and 65.94%, respectively, in pygmy sperm whale islet, resembling the human islet arrangement (20-40% α-cells and 50-70% β-cells) (1). In summary, we have identified insulin and glucagon genes from the pygmy sperm whale, which is the first report about these two genes in cetaceans. We have further characterized α cells and β cells, including their distribution, architecture, and composition in pygmy sperm whale pancreas. These results provide new information about the structural conservation of the insulin and glucagon genes, and new information of the properties on endocrine cells in cetacean for further studies. Although the lifestyle and nutrition state were changed during evolution, he highly conserved genes sequences and islet location of insulin and glucagon hormones, suggested that cetaceans preserve the insulin and glucagon physiological function during the adaption from terrestrial to fully aquatic environment.

DATA AVAILABILITY STATEMENT
The datasets generated for this study can be found in the Genbank (MN581742 and MN581743).

ETHICS STATEMENT
This animal study was reviewed and approved by Xiamen University Institutional Animal Care and Use Committee.

AUTHOR CONTRIBUTIONS
ML and XW was the guarantor of this work and as such, had full access to all of the data in the study, takes responsibility for the integrity of the data, and the accuracy of the data analysis. ML, LZ, and XW designed the study. LZ, LW, and RA performed the key experiments and drafted the manuscript. LZ, LW, RA, WW, XW, and ML participated in the planning of the work and the interpretation of the results. ML, XW, and WW have participated in the revising of the paper.

ACKNOWLEDGMENTS
We thank Joanne Boldison (Cardiff University, UK) for the editing and proof reading this manuscript. We thank Yuhong Chen, Yun Hong, and Zhen Li from School of Pharmaceutical Sciences, Xiamen University, and members of the Li lab for constructive discussions.

SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fendo. 2020.00174/full#supplementary-material Based on global database of cox1 sequences, the BOLD Systems gave a NJ tree showing the relationship of cox1 sequence from the sequences we obtained in this study (present sequences with red arrow) to its 100 nearest neighbors. The present sequences showed 100% sequence similarity with the pygmy sperm whale reference sequences. (C) NJ tree based on D-loop query and reference sequences of all recognized cetaceans. (D) NJ tree based on cytochrome b query and reference sequences of all recognized cetaceans. Bootstrap values based on 1,000 replicates. Both DNA surveillance trees showing high bootstrap support (100%) grouping "present sequence" we obtained in this study (with red arrow) with the reference sequences of pygmy sperm whale.