Development of endocrine pancreatic islets in embryos of the grass snake Natrix natrix (Lepidosauria,Serpentes)

Differentiation of the pancreatic islets in grass snake Natrix natrix embryos, was analyzed using light, transmission electron microscopy, and immuno-gold labeling. The study focuses on the origin of islets, mode of islet formation, and cell arrangement within islets. Two waves of pancreatic islet formation in grass snake embryos were described. The first wave begins just after egg laying when precursors of endocrine cells located within large cell agglomerates in the dorsal pancreatic bud differentiate. The large cell agglomerates were divided by mesenchymal cells thus forming the first islets. This mode of islet formation is described as fission. During the second wave of pancreatic islet formation which is related to the formation of the duct mantle, we observed four phases of islet formation: (a) differentiation of individual endocrine cells from the progenitor layer of duct walls (budding) and their incomplete delamination; (b) formation of two types of small groups of endocrine cells (A/D and B) in the wall of pancreatic ducts; (c) joining groups of cells emerging from neighboring ducts (fusion) and rearrangement of cells within islets; (d) differentiated pancreatic islets with characteristic arrangement of endocrine cells. Mature pancreatic islets of the grass snake contained mainly A endocrine cells. Single B and D or PP–cells were present at the periphery of the islets. This arrangement of endocrine cells within pancreatic islets of the grass snake differs from that reported from most others vertebrate species. Endocrine cells in the pancreas of grass snake embryos were also present in the walls of intralobular and intercalated ducts. At hatching, some endocrine cells were in contact with the lumen of the pancreatic ducts.     

Human embryonic stem cell differentiation into insulin secreting β‐cells for diabetes

hESC (human embryonic stem cells), when differentiated into pancreatic β ILC (islet‐like clusters), have enormous potential for the cell transplantation therapy for Type 1 diabetes. We have developed a five‐step protocol in which the EBs (embryoid bodies) were first differentiated into definitive endoderm and subsequently into pancreatic lineage followed by formation of functional endocrine β islets, which were finally matured efficiently under 3D conditions. The conventional cytokines activin A and RA (retinoic acid) were used initially to obtain definitive endoderm. In the last step, ILC were further matured under 3D conditions using amino acid rich media (CMRL media) supplemented with anti‐hyperglycaemic hormone‐Glp1 (glucagon‐like peptide 1) analogue Liraglutide with prolonged t_½ and Exendin 4. The differentiated islet‐like 3D clusters expressed bonafide mature and functional β‐cell markers‐PDX1 (pancreatic and duodenal homoeobox‐1), C‐peptide, insulin and MafA. Insulin synthesis de novo was confirmed by C‐peptide ELISA of culture supernatant in response to varying concentrations of glucose as well as agonist and antagonist of functional 3D β islet cells in vitro. Our results indicate the presence of almost 65% of insulin producing cells in 3D clusters. The cells were also found to ameliorate hyperglycaemia in STZ (streptozotocin) induced diabetic NOD/SCID (non‐obese diabetic/severe combined immunodeficiency) mouse up to 96 days of transplantation. This protocol provides a basis for 3D in vitro generation of long‐term in vivo functionally viable islets from hESC.     

Method for isolation of pancreatic blood vessels,their culture and coculture with islets of langerhans

The only cure available for Type 1 diabetes involves the transplantation of islets of Langerhans isolated from donor organs. However, success rates are relatively low. Disconnection from vasculature upon isolation and insufficient rate of revascularization upon transplantation are thought to be a major cause, as islet survival and function depend on extensive vascularization. Research has thus turned toward the development of pretransplantation culture techniques to enhance revascularization of islets, so far with limited success. With the aim to develop a technique to enhance islet revascularization, this work proposes a method to isolate and culture pancreas-derived blood vessels. Using a mild multistep digestion method, pancreatic blood vessels were retrieved from whole murine pancreata and cultured in collagen Type 1. After 8 days, 50% of tissue explants had formed anastomosed microvessels which extended up to 300 μm from the explant tissue and expressed endothelial cell marker CD31 but not ductal marker CK19. Cocultures with islets of Langerhans revealed survival of both tissues and insulin expression by islets up to 8 days post-embedding. Microvessels were frequently found to encapsulate islets, however no islet penetration could be detected. This study reports for the first time the isolation and culture of pancreatic blood vessels. The methods and results presented in this work provide a novel explant culture model for angiogenesis and tissue engineering research with relevance to islet biology. It opens the door for in vivo validation of the potential of these pancreatic blood vessel explants to improve islet transplantation therapies. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2745, 2019.     

Oestrogen regulates proliferation and differentiation of human islet‐derived precursor cells through oestrogen receptor alpha

E2 (oestradiol‐17β) is an important hormone that regulates various cell functions including insulin production. hIPCs (human islet‐derived precursor cells) are capable of proliferating and differentiating into cells that secrete insulin in response to glucose in vivo and in vitro. However, the effect of E2 on hIPCs is currently unclear. In this study, we found that ERα (oestrogen receptor alpha), but not ERβ, was expressed on hIPCs, and E2 promoted the proliferation and inhibited the differentiation of adult hIPCs. Although fetal hIPCs also express ERα, no effect of E2 on the fetal hIPCs was observed, suggesting differing roles of E2 at different stages of pancreatic development. This study indicates that E2 may be one of the key factors that control the turnover of adult pancreatic β cells by regulating the proliferation and differentiation of adult hIPCs through ERα.     

Controlled aggregation of primary human pancreatic islet cells leads to glucose‐responsive pseudoislets comparable to native islets

Clinical islet transplantation is a promising treatment for patients with type 1 diabetes. However, pancreatic islets vary in size and shape affecting their survival and function after transplantation because of mass transport limitations. To reduce diffusion restrictions and improve islet cell survival, the generation of islets with optimal dimensions by dispersion followed by reassembly of islet cells, can help limit the length of diffusion pathways. This study describes a microwell platform that supports the controlled and reproducible production of three‐dimensional pancreatic cell clusters of human donor islets. We observed that primary human islet cell aggregates with a diameter of 100–150 μm consisting of about 1000 cells best resembled intact pancreatic islets as they showed low apoptotic cell death (<2%), comparable glucose‐responsiveness and increasing PDX1, MAFA and INSULIN gene expression with increasing aggregate size. The re‐associated human islet cells showed an a‐typical core shell configuration with beta cells predominantly on the outside unlike human islets, which became more randomized after implantation similar to native human islets. After transplantation of these islet cell aggregates under the kidney capsule of immunodeficient mice, human C‐peptide was detected in the serum indicating that beta cells retained their endocrine function similar to human islets. The agarose microwell platform was shown to be an easy and very reproducible method to aggregate pancreatic islet cells with high accuracy providing a reliable tool to study cell–cell interactions between insuloma and/or primary islet cells.     

Generation of living color transgenic zebrafish to trace somatostatin-expressing cells and endocrine pancreas organization

In the present study, both gfp and rfp transgenic zebrafish lines using a 2.5-kb zebrafish somatostain2 (sst2) promoter were generated. During embryonic development, expression of GFP/RFP in the endocrine pancreas of transgenic embryos was initiated at ∼20 hpf and the number of GFP/RFP positive cells in the pancreas increased in subsequent stages; thus, our newly generated Tg(sst2:gfp) and Tg(sst2:rfp) lines faithfully recapitulated sst2 expression in endocrine pancreatic cells and provided a useful tool in analyzing the development of Sst2-producing δ-cells in the pancreas. By crossing these new transgenic lines with previously available transgenic lines targeted in insulin (Ins)-producing β-cells, Tg(ins:gfp) and Tg(ins:rfp), in combination with immunodetection of glucagon (Gcg)-producing α-cells and pancreatic polypeptide (PP)-producing PP-cells, the organization and composition of endocrine islets were investigated in both embryonic and adult pancreas. We found that there was always a big cluster of endocrine cells (principal islet) in the anterior-dorsal pancreas, followed by numerous smaller clusters (variable in size) of endocrine cells (secondary islets) along the anterior–posterior axis of the pancreas. All four types of endocrine cells were found in the principal islet, but secondary islets may or may not contain PP-cells. In addition, there were also discrete endocrine cells throughout the pancreas. In all co-localization experiments, we did not find any endocrine cells positive for more than one hormone markers, suggesting that these endocrine cells produce only a single hormone. In both principal and secondary islets, we found that β-cells were generally located in the center and non-β cells in the periphery; reminiscent of the “mantel–core” organization of islets of Langerhans in mammals where β-cells form the core and non-β-cells the mantel. In zebrafish primary islet, β-cells constitute most of the mass (∼50%), followed by δ-cells and α-cells (20–25% each), and PP-cells (1–2%); this is also similar to the composition of mammalian islets.     

Insulin secretion kinetics from single islets reveals distinct subpopulations

Type II diabetes progresses with inadequate insulin secretion and prolonged elevated circulating glucose levels. Also, pancreatic islets isolated for transplantation or tissue engineering can be exposed to glucose over extended timeframe. We hypothesized that isolated pancreatic islets can secrete insulin over a prolonged period of time when incubated in glucose solution and that not all islets release insulin in unison. Insulin secretion kinetics was examined and modeled from single mouse islets in response to chronic glucose exposure (2.8‐20 mM). Results with single islets were compared to those from pools of islets. Kinetic analysis of 58 single islets over 72 h in response to elevated glucose revealed distinct insulin secretion profiles: slow‐, fast‐, and constant‐rate secretors, with slow‐secretors being most prominent (ca., 50%). Variations in the temporal response to glucose therefore exist. During short‐term (<4 h) exposure to elevated glucose few islets are responding with sustained insulin release. The model allowed studying the influence of islet size, revealing no clear effect. At high‐glucose concentrations, when secretion is normalized to islet volume, the tendency is that smaller islets secrete more insulin. At high‐glucose concentrations, insulin secretion from single islets is representative of islet populations, while under low‐glucose conditions pooled islets did not behave as single ones. The characterization of insulin secretion over prolonged periods complements studies on insulin secretion performed over short timeframe. Further investigation of these differences in secretion profiles may resolve open‐ended questions on pre‐diabetic conditions and transplanted islets performance. This study deliberates the importance of size of islets in insulin secretion. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1059–1068, 2018     

Formation of pancreatic islets involves coordinated expansion of small islets and fission of large interconnected islet-like structures

The islets of Langerhans, micro-organs for maintaining glucose homeostasis, range in size from small clusters of <10 cells to large islets consisting of several thousand endocrine cells. Islet size distributions among various species are similar and independent of body size, suggesting an intrinsic limit to islet size. Little is known about the mechanisms regulating islet size. We have carried out a comprehensive analysis of changes of islet size distribution in the intact mouse pancreas from birth to eight months, including mathematical modeling to quantify this dynamic biological process. Islet growth was size-dependent during development, with preferential expansion of smaller islets and fission of large interconnected islet-like structures occurring most actively at approximately three weeks of age at the time of weaning. The process of islet formation was complete by four weeks with little or no new islet formation thereafter, and all the β-cells had low proliferation potential in the adult, regardless of islet size. Similarly, in insulinoma-bearing mice, the early postnatal developmental process including fission followed the same time course with no new islet formation in adults. However, tumor progression led to uncontrolled islet growth with accelerated expansion of larger islets. Thus, islet formation and growth is a tightly regulated process involving preferential expansion of small islets and fission of large interconnected islet-like structures.     

The nucleosome binding protein HMGN3 is expressed in pancreatic α‐cells and affects plasma glucagon levels in mice

Glucose homeostasis requires the coordinated actions of various organs and is critically dependent on the proper functioning of the various cell types present in the pancreatic Langerhans islets. Here we report that chromatin architectural protein HMGN3 is highly expressed in all pancreatic endocrine islet cells, and that Hmgn3?/? mice which have a mild diabetic phenotype, have reduced glucagon levels in their blood. To elucidate the mechanism leading to altered glucagon secretion of Hmgn3?/? mice, we tested whether HMGN3 affect glucagon synthesis and secretion in αTC1‐9 cells, a glucagon secreting cell line that is used to study pancreatic α‐cell function. We find that in these cells deletion of either HMGN3 or other HMGN variants, does not significantly affect glucagon gene expression or glucagon secretion. Our studies demonstrate a link between HMGN3 and glucagon blood levels that is not directly dependent of the function of pancreatic α‐cells. J. Cell. Biochem. 109: 49–57, 2010. Published 2009 Wiley‐Liss, Inc.     

5α‐Androst‐16‐en‐3α‐ol β‐D‐Glucuronide,Precursor of 5α‐Androst‐16‐en‐3α‐ol in Human Sweat

5α‐Androst‐16‐en‐3α‐ol (α‐androstenol) is an important contributor to human axilla sweat odor. It is assumed that α‐andostenol is excreted from the apocrine glands via a H₂O‐soluble conjugate, and this precursor was formally characterized in this study for the first time in human sweat. The possible H₂O‐soluble precursors, sulfate and glucuronide derivatives, were synthesized as analytical standards, i.e., α‐androstenol, β‐androstenol sulfates, 5α‐androsta‐5,16‐dien‐3β‐ol (β‐androstadienol) sulfate, α‐androstenol β‐glucuronide, α‐androstenol α‐glucuronide, β‐androstadienol β‐glucuronide, and α‐androstenol β‐glucuronide furanose. The occurrence of α‐androstenol β‐glucuronide was established by ultra performance liquid chromatography (UPLC)/MS (heated electrospray ionization (HESI)) in negative‐ion mode in pooled human sweat, containing eccrine and apocrine secretions and collected from 25 female and 24 male underarms. Its concentration was of 79 ng/ml in female secretions and 241 ng/ml in male secretions. The release of α‐androstenol was observed after incubation of the sterile human sweat or α‐androstenol β‐glucuronide with a commercial glucuronidase enzyme, the urine‐isolated bacteria Streptococcus agalactiae, and the skin bacteria Staphylococcus warneri DSM 20316, Staphylococcus haemolyticus DSM 20263, and Propionibacterium acnes ATCC 6919, reported to have β‐glucuronidase activities. We demonstrated that if α‐ and β‐androstenols and androstadienol sulfates were present in human sweat, their concentrations would be too low to be considered as potential precursors of malodors; therefore, the H₂O‐soluble precursor of α‐androstenol in apocrine secretion should be a β‐glucuronide.     

Beta cells within single human islets originate from multiple progenitors

Background

In both humans and rodents, glucose homeostasis is controlled by micro-organs called islets of Langerhans composed of beta cells, associated with other endocrine cell types. Most of our understanding of islet cell differentiation and morphogenesis is derived from rodent developmental studies. However, little is known about human islet formation. The lack of adequate experimental models has restricted the study of human pancreatic development to the histological analysis of different stages of pancreatic development. Our objective was to develop a new experimental model to (i) transfer genes into developing human pancreatic cells and (ii) validate gene transfer by defining the clonality of developing human islets.

Methods and Findings

In this study, a unique model was developed combining ex vivo organogenesis from human fetal pancreatic tissue and cell type-specific lentivirus-mediated gene transfer. Human pancreatic progenitors were transduced with lentiviruses expressing GFP under the control of an insulin promoter and grafted to severe combined immunodeficient mice, allowing human beta cell differentiation and islet morphogenesis. By performing gene transfer at low multiplicity of infection, we created a chimeric graft with a subpopulation of human beta cells expressing GFP and found both GFP-positive and GFP-negative beta cells within single islets.

Conclusion

The detection of both labeled and unlabeled beta cells in single islets demonstrates that beta cells present in a human islet are derived from multiple progenitors thus providing the first dynamic analysis of human islet formation during development. This human transgenic-like tool can be widely used to elucidate dynamic genetic processes in human tissue formation.     

Development of the islets, exocrine pancreas, and related ducts in the Nile tilapia, Oreochromis niloticus (Pisces: Cichlidae)

Pancreatic development and the relationship of the islets with the pancreatic, hepatic, and bile ducts were studied in the Nile tilapia, Oreochromis niloticus, from hatching to the onset of maturity at 7 months. The number of islets formed during development was counted, using either serial sections or dithizone staining of isolated islets. There was a general increase in islet number with both age and size. Tilapia housed in individual tanks grew more quickly and had more islets than siblings of the same age left in crowded conditions. The pancreas is a compact organ in early development, and at 1 day posthatch (dph) a single principal islet, positive for all hormones tested (insulin, SST-14, SST-28, glucagon, and PYY), is partially surrounded by exocrine pancreas. However, the exocrine pancreas becomes more disseminated in older fish, following blood vessels along the mesenteries and entering the liver to form a hepatopancreas. The epithelium of the pancreatic duct system from the intercalated ducts to the main duct entering the duodenum was positive for glucagon and SST-14 in 8 and 16 dph tilapia. Individual insulin-immunopositive cells were found in one specimen. At this early stage in development, therefore, the pancreatic duct epithelial cells appear to be pluripotent and may give rise to the small islets found near the pancreatic ducts in 16-37 dph tilapia. Glucagon, SST-14, and some PPY-positive enteroendocrine cells were present in the intestine of the 8 dph larva and in the first part of the intestine of the 16 dph juvenile. Glucagon and SST-14-positive inclusions were found in the apical cytoplasm of the mid-gut epithelium of the 16 dph tilapia. These hormones may have been absorbed from the gut lumen, since they are produced in both the pancreatic ducts and the enteroendocrine cells. At least three hepatic ducts join the cystic duct to form the bile duct, which runs alongside the pancreatic duct to the duodenum.     

Arginase expression and modulation of IL-1β-induced nitric oxide generation in rat and human islets of Langerhans

Proinflammatory cytokine induction of NO synthesis may contribute to the destruction of pancreatic beta cells leading to type 1 diabetes. The NO synthase substrate arginine can also be metabolized to ornithine and urea in a reaction catalyzed by cytosolic (AI) or mitochondrial (AII) isoforms of arginase. Recent evidence suggests that the rate of NO generation is dependent on the relative activities of NO synthase and arginase. The objectives of this study were (i) to identify the arginase isoforms expressed in rat and human islets of Langerhans and a rat beta cell line, RINm5F and (ii) to investigate the competition for arginine between NO synthase and arginase in IL-1β-treated rat islets. Arginase activity was detected in rat islets (fresh tissue, 346 mU/mg protein; cultured, 587 mU/mg), cultured human islets (56 mU/mg), RINm5F cells (376 mU/mg), rat kidney (238 mU/mg), and rat liver (6119 mU/mg). Using Western blots, AI was shown to be the predominant isoform expressed in rat islets and in RINm5F cells while human islets expressed far more AII than AI. Rat islets were cultured in medium containing 1.14, 0.1, and 0.01 mM arginine and treated with IL-1β and the arginase inhibitor 2(S)-amino-6-boronohexanoic acid (ABH). IL-1β-induced NO generation was unaffected by ABH at 1.14 mM arginine, but significantly increased at 0.1 and 0.01 mM arginine. These findings suggest that the level of islet arginase activity can regulate the rate of induced NO generation and this may be relevant to the insulitis process leading to beta cell destruction in type 1 diabetes.     

LIGHT/IFN‐γ triggers β cells apoptosis via NF‐κB/Bcl2‐dependent mitochondrial pathway

LIGHT recruits and activates naive T cells in the islets at the onset of diabetes. IFN‐γ secreted by activated T lymphocytes is involved in beta cell apoptosis. However, whether LIGHT sensitizes IFNγ‐induced beta cells destruction remains unclear. In this study, we used the murine beta cell line MIN6 and primary islet cells as models for investigating the underlying cellular mechanisms involved in LIGHT/IFNγ – induced pancreatic beta cell destruction. LIGHT and IFN‐γ synergistically reduced MIN6 and primary islet cells viability; decreased cell viability was due to apoptosis, as demonstrated by a significant increase in Annexin V⁺ cell percentage, detected by flow cytometry. In addition to marked increases in cytochrome c release and NF‐κB activation, the combination of LIGHT and IFN‐γ caused an obvious decrease in expression of the anti‐apoptotic proteins Bcl‐2 and Bcl‐xL, but an increase in expression of the pro‐apoptotic proteins Bak and Bax in MIN6 cells. Accordingly, LIGHT deficiency led to a decrease in NF‐κB activation and Bak expression, and peri‐insulitis in non‐obese diabetes mice. Inhibition of NF‐κB activation with the specific NF‐κB inhibitor, PDTC (pyrrolidine dithiocarbamate), reversed Bcl‐xL down‐regulation and Bax up‐regulation, and led to a significant increase in LIGHT‐ and IFN‐γ‐treated cell viability. Moreover, cleaved caspase‐9, ‐3, and PARP (poly (ADP‐ribose) polymerase) were observed after LIGHT and IFN‐γ treatment. Pretreatment with caspase inhibitors remarkably attenuated LIGHT‐ and IFNγ‐induced cell apoptosis. Taken together, our results indicate that LIGHT signalling pathway combined with IFN‐γ induces beta cells apoptosis via an NF‐κB/Bcl2‐dependent mitochondrial pathway.     

MALE ALTERNATIVE REPRODUCTIVE STRATEGIES IN A MARINE ISOPOD CRUSTACEAN (PARACERCEIS SCULPTA): THE USE OF GENETIC MARKERS TO MEASURE DIFFERENCES IN FERTILIZATION SUCCESS AMONG α-, β-, AND γ-MALES

Three discrete male morphs coexist in Paracerceis sculpta, a marine isopod crustacean inhabiting the northern Gulf of California. Ornamented α-males establish themselves in the spongocoels of intertidal sponges, where females congregate to breed. Smaller β-males, resembling sexually mature females, enter spongocoels by deception, while tiny γ-males invade spongocoels by stealth. Isopods breed year-round, and the operational sex ratio fluctuates widely over short durations. When females are abundant, receptive females accumulate in spongocoels, and these spongocoels are preferentially invaded by β- and γ-males. To test the hypothesis that the density of receptive females affects relative fertilization success among male morphs, individual β- and γ-males, heterozygous for a dominant cuticular pigmentation allele, were placed in artificial spongocoels with an unmarked α-male and densities of one, two, and three unmarked, receptive females. The fertilization success of each male was determined by counting the number of marked and unmarked progeny each female produced. Alpha-males guard females effectively and sire nearly all young when one female is in a spongocoel. The success of β- and γ-males increases, however, and may even exceed the success of α-males when two or three females are present. The regular occurrence of more than one receptive female in the harems of α-males may contribute to the persistence of β- and γ-males in this species.     

Expression and regulation of α1β1 integrin in Schwann cells

The interaction of cells with the extracellular matrix plays a critical role in morphogenesis and cell differentiation. To define how Schwann cells might interact with the extracellular matrix, we chose to study the expression of the laminin/collagen receptor α1β1 integrin during nerve development in the rat from embryonic day 14 to maturity. We found that this integrin is expressed predominantly on mature non-myelin-forming cells and only at very low levels on myelin-forming cells. Significant levels of this integrin were not detected on Schwann cell precursors or embryonic Schwann cells in vivo. Experiments using transected and crushed sciatic nerve showed that α1β1 integrin expression is regulated at least in part by axonal contact. Furthermore, Schwann cell culture experiments showed that α1β1 integrin levels are strongly upregulated by transforming growth factor-βs and phorbol esters. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 914–928, 1997     

Synthesis,Structure, and Biological Applications of α‐Fluorinated β‐Amino Acids and Derivatives

This review gives a broad overview of the state of play with respect to the synthesis, conformational properties, and biological activity of α‐fluorinated β‐amino acids and derivatives. General methods are described for the preparation of monosubstituted α‐fluoro‐β‐amino acids (Scheme 1). Nucleophilic methods for the introduction of fluorine predominantly involve the reaction of DAST with alcohols derived from α‐amino acids, whereas electrophilic sources of fluorine such as NFSI have been used in conjunction with Arndt? Eistert homologation, conjugate addition or organocatalyzed Mannich reactions. α,α‐Difluoro‐β‐amino acids have also been prepared using DAST; however, this area of synthesis is largely dominated by the use of difluorinated Reformatsky reagents to introduce the difluoro ester functionality (Scheme 9). α‐Fluoro‐β‐amino acids and derivatives analyzed by X‐ray crystal and NMR solution techniques are found to adopt preferred conformations which are thought to result from stereoelectronic effects associated with F located close to amines, amides, and esters (Figs. 2–6). α‐Fluoro amide and β‐fluoro ethylamide/amine effects can influence the secondary structure of α‐fluoro‐β‐amino acid‐containing derivatives including peptides and peptidomimetics (Figs. 7–9). α‐Fluoro‐β‐amino acids are also components of a diverse range of bioactive anticancer (e.g., 5‐fluorouracil), antifungal, and antiinsomnia agents as well as protease inhibitors where such fluorinated analogs have shown increased potency and spectrum of activity.     

Progenitor cells and TNF-alpha involvement during morphological changes in pancreatic islets of obese mice

Overnutrition during pregnancy and lactation lend increasing support to the development of obesity and several chronic diseases in adulthood such as type 2 diabetes mellitus, which leads to beta-cell dysfunction and insulin resistance. In this work, we aimed to study the effects of early life overnutrition on the development of obesity, analyzing the morphological changes, expression of TNF-α, and also the stem cell marker CD133 in the pancreatic islets of young and adult mice. Overnutrition during lactation phase was used as an experimental model to induce obesity. The animals were analyzed at 28 and 150 days of age, when pancreata were collected for histological, ultrastructural and western blotting analysis. The results showed that islet hypertrophy is established in obese groups at day 28 and remained until adulthood. CD133⁺ cells were observed as small cells within pancreatic islets in both control and obese young mice. However, at day 150, these cells were observed only in the islet peripheries and near ducts of the obese group. Furthermore, TNF-α expression in pancreatic islets was increased in both young and adult obese groups when compared to control groups. This work shows interesting data about CD133 receptor and TNF-α roles in the pancreas during obesity development.     

RAC1mutation is not a predictive biomarker for PI3’‐kinase‐β‐selective pathway‐targeted therapy

Mutational activation of RAC1 is detected in ~7% of cutaneous melanoma, with the most frequent mutation (RAC1^C85T) encoding for RAC1^P29S. RAC1^P29S is a fast‐cycling GTPase that leads to accumulation of RAC1^P29S‐GTP, which has potentially pleiotropic regulatory functions in melanoma cell signaling and biology. However, the precise mechanism by which mutationally activated RAC1^P29S propagates its pro‐tumorigenic effects remains unclear. RAC1‐GTP is reported to activate the beta isoform of PI3’‐kinase (PIK3CB/PI3Kβ) leading to downstream activation of PI3’‐lipid signaling. Hence, we employed both genetic and isoform‐selective pharmacological inhibitors to test if RAC1^P29S propagates its oncogenic signaling in melanoma through PI3Kβ. We observed that RAC1^P29S‐expressing melanoma cells were largely insensitive to inhibitors of PI3Kβ. Furthermore, RAC1^P29S melanoma cell lines showed variable sensitivity to pan‐class 1 (α/β/γ/δ) PI3’‐kinase inhibitors, suggesting that RAC1‐mutated melanoma cells may not rely on PI3’‐lipid signaling for their proliferation. Lastly, we observed that RAC1^P29S‐expressing cell lines also showed variable sensitivity to pharmacological inhibition of the RAC1 → PAK1 signaling pathway, questioning the relevance of inhibitors of this pathway for the treatment of patients with RAC1‐mutated melanoma.