Fluorescent protein vectors for pancreatic islet cell identification in live-cell imaging
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The islets of Langerhans contain different types of endocrine cells, which are crucial for glucose homeostasis. β- and α-cells that release insulin and glucagon, respectively, are most abundant, whereas somatostatin-producing δ-cells and particularly pancreatic polypeptide-releasing PP-cells are more scarce. Studies of islet cell function are hampered by difficulties to identify the different cell types, especially in live-cell imaging experiments when immunostaining is unsuitable. The aim of the present study was to create a set of vectors for fluorescent protein expression with cell-type-specific promoters and evaluate their applicability in functional islet imaging. We constructed six adenoviral vectors for expression of red and green fluorescent proteins controlled by the insulin, preproglucagon, somatostatin, or pancreatic polypeptide promoters. After transduction of mouse and human islets or dispersed islet cells, a majority of the fluorescent cells also immunostained for the appropriate hormone. Recordings of the sub-plasma membrane Ca2+ and cAMP concentrations with a fluorescent indicator and a protein biosensor, respectively, showed that labeled cells respond to glucose and other modulators of secretion and revealed a striking variability in Ca2+ signaling among α-cells. The measurements allowed comparison of the phase relationship of Ca2+ oscillations between different types of cells within intact islets. We conclude that the fluorescent protein vectors allow easy identification of specific islet cell types and can be used in live-cell imaging together with organic dyes and genetically encoded biosensors. This approach will facilitate studies of normal islet physiology and help to clarify molecular defects and disturbed cell interactions in diabetic islets.
KeywordsIslets α-cell β-cell δ-cell PP-cell Insulin Glucagon Somatostatin Pancreatic polypeptide Ca2+ cAMP
The pancreatic islets of Langerhans are key regulators of glucose homeostasis. They contain four major types of endocrine cells: α-, β-, δ-, and PP-cells, secreting glucagon, insulin, somatostatin, and pancreatic polypeptide (PP), respectively [36, 45]. The β-cells are most abundant and constitute 60–80 % of the endocrine cells in islets from mice and ~50 % in human islets. α-cells make up ~20 % in mouse and ~40 % in man, while δ- and PP-cells are more scarce constituting <10 and <1 % of the islet cells, respectively. There is also a small fraction of ε-cells secreting ghrelin . Insulin decreases blood glucose levels and insufficient insulin release from the β-cells causes most forms of diabetes mellitus. Glucagon, on the contrary, increases blood glucose. Diabetic patients often show exaggerated glucagon secretion that contributes to hyperglycemia, but also a reduced glucagon response to hypoglycemia, which may become fatal due to the brain’s strong glucose dependence [14, 50]. All major islet hormones are released in a pulsatile manner [15, 21, 24], which promotes hormone sensitivity in the target cells [37, 42]. Indeed, insulin and glucagon pulsatility is typically disturbed in early stages of diabetes [27, 34, 40] but the molecular mechanisms that underlie the dysregulated secretion are unclear. The physiological relevance of islet somatostatin and PP is not well understood. PP was recently found to suppress glucagon secretion by direct effects on α-cells  and somatostatin inhibits both insulin and glucagon secretion . Somatostatin pulsatility is in phase with that of insulin [20, 21], and somatostatin’s stronger inhibitory effect on glucagon than on insulin secretion  may help to shape pulsatile glucagon secretion in opposite phase .
Studies of cell functions and interactions in situ within pancreatic islets depend on unambiguous cell identification, which is far from trivial. Immunostaining of dispersed islet cells has successfully been used to identify all major islet cell types after studying their physiological Ca2+ responses [6, 19, 30, 31], but the process is time-consuming and cells may be lost during fixation and staining. The immunostaining approach is even more difficult when applied to cells within pancreatic islets, since it requires that the cells remain in exact position within the high magnification view field throughout the identification procedure. Electrophysiological techniques have also been used to identify α-, β-, and δ-cells [3, 8, 26] but this approach is difficult to apply to more than one cell at a time. The difference in expression of adrenergic receptors can also be utilized to discriminate α- and β-cells. α-cells mainly express α1- and β-receptors , which trigger elevations of the cytoplasmic Ca2+ and cAMP concentrations in response to adrenaline [49, 52]. In contrast, β-cells express mainly α2-receptors , and adrenaline therefore lowers cAMP  and suppresses Ca2+ signaling . Similarly, the Ca2+ response to glutamate has been used for identifying α- and β-cells [9, 29] based on different expression of ionotropic glutamate receptors of AMPA/kainate type. These pharmacological tools depend on recordings of intracellular Ca2+ and cAMP. Moreover, they do not allow unambiguous separation of α- and β-cells from the less common δ- and PP-cells.
Cell identification by genetic labeling is an attractive alternative, where tissue-specific promoters are used to control expression of fluorescent markers. This approach has been applied to generate transgenic mice with selective expression of fluorescent proteins in α-, β-, and δ-cells under the promoters for preproglucagon, insulin, and somatostatin, respectively [1, 17, 38]. A drawback with this approach is that it is restricted to cell identification in specific transgenic mouse strains and not practically applicable to other species.
In the present study, we tested cell-type-specific promoter-driven expression of fluorescent proteins for identification of mouse and human islet cells in live-cell fluorescence microscopy applications. To ascertain sufficient expression levels, we constructed adenoviral vectors based on the Tet-On 3G conditional expression system [56, 32]. The preproglucagon promoter (Pppg), the rat insulin 2 promoter (Rip2), the somatostatin promoter (Psst), and the pancreatic polypeptide promoter (Pppy) were used to drive expression of the reverse tetracycline transactivator protein (rtTA), while the genes for the fluorescent proteins mCherry or enhanced green fluorescent protein (GFP) were placed downstream of the tetracycline response element. We demonstrate that this method results in mCherry or GFP expression in β-, α-, δ-, or PP-cells with a high degree of specificity and that appropriately labeled cells in mouse and human islets were amenable for on-line recordings of the sub-plasma membrane Ca2+ and cAMP concentrations ([Ca2+]pm and [cAMP]pm).
Materials and methods
Isolation of cell-type-specific promoters
cDNA for Rip2 was a kind gift from Dr. Lena Stenson-Holst, Lund University. Pppg, Psst, and Pppy were isolated from genomic DNA using PCR. DNA was purified from MIN6 β-cells with PureLink® Genomic DNA Mini Kit (Thermo-Fisher Scientific, Waltham, MA, USA) and used as PCR template. Primer design was based on published promoter sequence data [11, 55, 22, 33, 25, 46, 47]. An XhoI restriction site (underlined) was added to the sense primer (5′-ATCGTACTCGAGGACACTCGCAATCATAAAGAGCAATC-3′ for Pppg; 5′-TATACTCGAGCAACCACTCCAAGTGGAG-3′ for Rip2; 5′-ATATATCTCGAGAGCCTAGAGGCAGAGCAAGCGCTG-3′ for Psst; 5′-CATGAGGCTCGAGTCGGAACTAGCCACTGGTTTTG-3′ for Pppy) and an AscI site to the antisense primer (5′-AAATGGCGCGCCTGAGCTGCGAACAGGTGTAG-3′ for Pppg; 5′-ATTTAAATGGCGCGCCTTACTGAATCCCCAC-3′ for Rip2; 5′-AATTAAGGCGCGCCGGAGACCGTGGAGAGCTCCATAGCG-3′ for Psst; 5′-GACATATGGCGCGCCTGTGCTGAGCTAGTGAGTG-3′ for Pppy). Standard PCR reactions were performed with PfuTurbo high-fidelity DNA polymerase (Agilent Technoliges, Santa Clara, CA, USA). The 1659-bp Pppg, 684-bp Rip2, 1986-bp Psst, and 1420-bp Pppy PCR products were purified by gel extraction, ligated into the pCR®II-TOPO vector (Thermo-Fisher Scientific, Waltham, MA, USA) and verified by sequencing (Eurofins Genomics, Ebergberg, Germany). Pppg, Rip2, Psst, and Pppy were subsequently released by digestion with XhoI and AscI.
Construction of Tet-On 3G plasmids with cell-type-specific promoters
To avoid the potential problem that the promoters are too weak to drive expression of sufficient amounts of fluorescent protein for cell identification, we took advantage of the Tet-On conditional expression system. In this approach, a cell-type-specific promoter could drive expression of a small amount of rtTA, which is sufficient to strongly activate the tetracycline response element (TRE) and downstream genes in the presence of doxycycline.
To insert the cell-type-specific promoters in plasmids encoding rtTA, the pBR322-E3-Tet-On-rev plasmid  (kind gift from Prof Göran Akusjärvi, Uppsala University) was cleaved with XhoI and XbaI to release the cytomegalovirus promoter with a 5′UTR extension of the rtTA gene. This CMV-5′UTR fragment was subsequently ligated into an in-house made cloning vector denoted pCFH to get pCFH-CMV-5′UTR. After cleavage with XhoI and Eco53kI, the CMV promoter was released and replaced by the Rip2 promoter carrying a AscI site at the 3′ end. Eventually, the Rip2-5′UTR fragment was cut out with XhoI and XbaI and ligated into the cleaved pBR322-E3-rtTA-∆CMV fragment to get pBR322-E3-Rip2-rtTA, which now included the cell-type-specific promoter instead of the original CMV promoter. All other promoter constructs were completed by replacing Rip2 with Pppg, Psst, or Pppy, respectively, after XhoI-AscI double digestion. Finally, rtTA was replaced with Tet-On 3G transactivator [32, 56] by isolating the Tet-On 3G from pCMV-Tet3G plasmid (Clontech, USA) after digestion with XhaI and HpaI.
Construction of plasmids for TRE-regulated mCherry and EGFP expression
In the next step, the pTRE3G-IRES plasmid (Clontech, Palo Alto, CA, USA) was cleaved with BglII and BamHI, to release the IRES component and insert either of the fluorescent proteins mCherry or GFP.
Virus recombination and amplification
Islet isolation, culture, and transduction
All procedures for animal handling and islet isolation were approved by the Uppsala animal ethics committee. Islets of Langerhans were isolated from C57B16J female mice as described previously . After isolation, the islets were cultured for 1 to 2 days in RPMI-1640 medium containing 5.5 mM glucose, 10 % (v/v) fetal calf serum, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a 5 % CO2 humidified air atmosphere. Human islets were obtained from four normoglycemic cadaveric organ donors (two males, two females aged 57–65 years) via the Nordic Network for Clinical Islet Transplantation in Uppsala. All experiments with human islets were approved by the Uppsala human ethics committee. The isolated islets were cultured up to 7 days at 37 °C in an atmosphere of 5 % CO2 in CMRL 1066 culture medium containing 5.5 mM glucose, 100 U/mL penicillin and 100 μg/mL streptomycin, 2 mM glutamine, and 10 % FBS. For some experiments, the islets were dispersed into single cells and small clusters by transferring them to 2 mL cell dissociation buffer (Thermo Fisher Scientific) containing 10 % (v/v) trypsin-EDTA (0.05 %, Life Technologies), followed by pipetting up and down 20–30 times until all islets were disintegrated. The trypsin digestion was interrupted by addition of 8 ml serum-containing RPMI-1640 medium, followed by centrifugation for 5 min at 160g and resuspension of the cells in islet culture medium. The cell suspension was subsequently added onto poly-lysine-coated 25-mm coverslips and cultured overnight. The islets or cells were infected with adenovirus by 3 to 4 h exposure to a concentration of 105 fluorescence forming units (FFU)/islet , followed by addition of regular medium with 4 μM doxycycline and further culture for 16 to 20 h before use.
The infected islets were washed three times with PBS, fixed with 4 % (w/v) paraformaldehyde for 10 min in room temperature and permeabilized with 0.2 % (v/v) TrionX-100 for 10 min on ice. The reaction was blocked by adding PBS containing 5 % FBS in room temperature. After 30 min incubation, the primary antibody (polyclonal rabbit anti-insulin or polyclonal rabbit anti-glucagon from Invitrogen, Carlsbad, CA; polyclonal rabbit anti-somatostatin from Dako, Stockholm, Sweden; polyclonal goat anti-pancreatic polypeptide from Bio-Techne, Abingdon, UK) was added (1:200) for 2 h, followed by thorough rinsing with PBS. The secondary antibody, Alexa Fluor® 488 goat anti-rabbit IgG (Invitrogen, Carlsbad, CA) or Alexa Fluor® 488-AffiniPure F(ab’)2 fraction donkey anti-goat IgG (H + L) (Jackson ImmunoResearch Europe Ltd., Suffolk, UK), was then applied (1:200) for 1 h in darkness. After rinsing with PBS, the islets or cells were used for confocal microscopy imaging.
The islets or cells were imaged in a spinning-disk confocal system (Yokogawa CSU-10, Andor Technology, Belfast, Northern Ireland) attached to an Eclipse TE2000 microscope (Nikon, Kawasaki, Japan) equipped with a 60×, 1.40-NA objective (Nikon, Kawasaki, Japan). Diode-pumped solid-state lasers (Cobolt, Stockholm, Sweden) were used for excitation of mCherry (561 nm) and GFP or Alexa Fluor® 488 (491 nm). Fluorescence was selected with interference filters (520 with 35 nm half-bandwidth for GFP and Alexa Fluor® 488, and 586/20 nm for mCherry) and images were acquired with a back-illuminated EMCCD camera (DU888, Andor Technology) under MetaFluor software control (Molecular Devices Corp., Downington, PA). The confocal imaging experiments were performed at room temperature.
Imaging of [cAMP]pm and [Ca2+]pm
For imaging of [cAMP]pm, the islets were transduced with a cyan and yellow fluorescent protein (CFP and YFP)-based cAMP translocation biosensor  together with the fluorescent labeling vector and cultured over night. The islets or cells were then pre-incubated for 30 min at 37 °C in experimental buffer containing 125 mM NaCl, 4.8 mM KCl, 1.3 mM CaCl2, 1.2 mM MgCl2, and 25 mM HEPES (pH 7.40 set with NaOH) prior to imaging. For [Ca2+]pm recordings, the islets were loaded with 1.3 μM of the Ca2+ indicator Fluo-4 during the pre-incubation period. After incubation, the islets were attached to poly-lysine-coated 25-mm coverslips and the coverslips with cells or islets were mounted in an open 50-μl chamber and superfused with experimental buffer at a rate of 0.3 ml/min. The chamber was attached to the stage of an objective-based total internal reflection fluorescence (TIRF) microscopy system consisting of an Eclipse Ti microscope (Nikon) with a 60×, 1.45-NA objective and a TIRF illuminator (Nikon) . The chamber and superfusion medium was thermostated to 37 °C. The 457, 491, 515, and 561 nm lines of diode-pumped solid-state lasers (Cobolt) were used to excite CFP, Fluo-4, YFP, and mCherry, respectively. Fluorescence was detected with a back-illuminated EMCCD camera (DU-897, Andor Technology) controlled by MetaFluor. Emission wavelengths were selected with filters [485 nm/25 nm half-bandwidth for CFP, 527/27 nm for Fluo-4, 560/40 nm for YFP (Semrock Rochester, NY) and 620 nm long-pass for mCherry (Melles Griot, Didam, The Netherlands)] mounted in a filter wheel (Sutter Instruments). For time-lapse recordings, images or image pairs were acquired every 5 s with a shutter (Sutter Instruments) blocking the light between image captures. Image analysis was performed with MetaFluor. Ca2+ indicator fluorescence intensities under the plasma membrane were expressed as changes in relation to initial fluorescence intensity F0 after subtraction of background (F/F0). [cAMP]pm was expressed as the ratio of CFP over YFP fluorescence after subtraction of background.
Chi-square analysis with Yates correction was used to compare the proportion of cells that immunostained for a hormone in relation those with hormone promoter-controlled fluorescent protein expression.
Results and discussion
Fluorescence labeling of β-cells in mouse and human pancreatic islets
Fluorescence labeling of α-cells in mouse and human pancreatic islets
Fluorescence labeling of mouse and human δ- and PP-cells
TIRF recordings of [Ca2+]pm in islet cells identified by cell-type-specific fluorescent protein expression
We next tested whether the cell identification strategy could be used in live-cell imaging applications. Islets transduced with the respective cell-type-specific vector expressing mCherry were loaded with the green fluorescent Ca2+ indicator fluo-4, which does not show spectral overlap with the red label. The islets were imaged with a total internal reflection fluorescence microscope, which excites a small volume within <100 nm of the plasma membrane of the cells in contact with the coverslip. Although this technique does not enable analysis of cells located deeper in the islet, it is advantageous in allowing recordings from small cells with weak fluorescence without contamination from cells located in a different focal plane.
Variability of [Ca2+]pm responses to glucose in α-cells
Recordings from α-cells identified by Pppg-mCherry fluorescence were largely consistent with a previous study of α-cell [Ca2+]pm signaling . Accordingly, most cells showed irregular brief [Ca2+]pm oscillations in the presence of 3 mM glucose, and increases of the glucose concentration to 7 and 11 mM did not induce sustained suppression of [Ca2+]pm signaling (Fig. 5b) but there was sometimes a transient interruption of [Ca2+]pm oscillations or somewhat lower amplitude of the oscillations at 11 mM compared to 3 mM glucose. Seven out of 10 α-cells showed no or weak response to KATP channel activation with diazoxide (Fig. 5b), supporting the view that this channel may not be required for glucose regulation of glucagon secretion . In accordance with previous observations , the mouse α-cells responded to 1 mM glutamate with a distinct, stable [Ca2+]pm elevation (Fig. 5b). Also, 10 μM adrenaline induced [Ca2+]pm elevation but this response was usually weaker and/or more transient than that to glutamate. mCherry-negative cells from the same islets showed β-cell-like responses with low and stable [Ca2+]pm at 3 mM glucose, a distinct increase of [Ca2+]pm at 11 mM, which was reversed by diazoxide, and no responses to glutamate or adrenaline (Fig. 5b). Our genetic labeling strategy thus mostly identifies α- and β-cells with characteristic [Ca2+]pm responses to low and high glucose concentrations as well as to glutamate and adrenaline.
A few mCherry-positive α-cells showed somewhat more β-cell-like response with absence of prominent [Ca2+]pm signaling activity at 3 mM glucose and distinct [Ca2+]pm increases after elevation of glucose to 7 mM (Fig. 5c). The two types of α-cell responses were sometimes observed in different cells within the same islet. In the cell shown in Fig. 5c, the glucose-induced [Ca2+]pm elevation was reversed by diazoxide, like in β-cells. One may speculate that this type of response represents “mislabeled” cells with Pppg-controlled mCherry expression and positive immunostaining for insulin. However, the cell shown in Fig. 5c responded to glutamate and adrenaline like a typical α-cell. Simultaneous recording from an adjacent mCherry negative cell exhibited the typical β-cell behavior with glucose-induced [Ca2+]pm increases and lack of glutamate and adrenaline responses. The results indicate possible presence of sub-populations of α-cells that respond differently to glucose. Although glucose is generally an inhibitor of glucagon release, secretion from purified rodent α-cells is paradoxically stimulated by the sugar [23, 28, 35]. One possibility is that purification by FACS sorting somehow selects for the α-cells with β-cell-like Ca2+ responses and glucose-stimulated glucagon secretion, although the α-cells with glucose-inhibited secretion dominate within the islet. However, there is also evidence that Ca2+ signaling may differ between isolated α-cells and those within islets [29, 53] and that glucagon release is controlled by factors that may override a stimulatory [Ca2+]pm signal [28, 29]. It is pertinent to note that the dose-response relationship for glucose-regulated glucagon secretion is U-shaped with maximal inhibition at 7 mM glucose. At higher concentrations of the sugar, inhibition is gradually diminished and secretion even stimulated [28, 41]. It remains to establish if the population of α-cells with [Ca2+]pm increase in response to glucose elevation may perhaps explain the stimulatory glucose component. We conclude that our labeling approach dominatingly selects for α-cells with expected [Ca2+]pm responses to glutamate and adrenaline and reveals a considerable variability in the α-cell reactivity to glucose.
[Ca2+]pm signaling in mouse δ-cells
Another example of δ-cell [Ca2+]pm signaling is shown in Fig. 6b. Apart from the mCherry-expressing δ-cell, the recording includes a non-expressing β-cell. When the glucose concentration was increased from 3 to 20 mM, the β-cell shows the typical transition from silence at low glucose to [Ca2+]pm elevation followed by quite regular, slow oscillations and [Ca2+]pm reaches baseline within 10 min after restoration of glucose to 3 mM. In contrast, the δ-cell is active at 3 mM glucose with no major change after introduction of 20 mM glucose. After approximately 10 min, the [Ca2+]pm oscillations in the δ-cell become well synchronized with those in the β-cell (Fig. 6b). The temporal relationship between [Ca2+]pm oscillations in β- and δ-cells within intact islets have previously not been clarified, but since pulsatile release of insulin and somatostatin is synchronized in phase , the prediction is that [Ca2+]pm oscillations are synchronized in the same phase. Similar synchronization was recently found also between β- and α-cells, although insulin and glucagon pulses are synchronized in opposite phase . This apparent paradox may be explained by release of somatostatin that suppresses glucagon secretion with little influence on α-cell [Ca2+]pm.
[Ca2+]pm signaling in identified PP-cells
PP-cells identified by Pppy-mediated expression of mCherry were also amenable for [Ca2+]pm recordings. Figure 6c shows an example of a PP-cell exhibiting [Ca2+]pm oscillations in the presence of 3 mM glucose, which did not change appearance when glucose is increased to 7 or 11 mM. However, diazoxide promptly interrupted the signaling. There is only a weak response to glutamate and no consistent response to adrenaline (Fig. 6c).
Imaging cells co-expressing genetically encoded biosensors and an identity marker
We have created a set of viral vectors that allow pancreatic islet cell-specific expression of fluorescent proteins for their identification in various live-cell imaging applications. These vectors provide a valuable addition to the toolbox for live-cell imaging applications. In such settings, fluorescent cells are easily identified and simultaneous recordings can often be made on several cells. We first applied the tools with [Ca2+]pm recordings to verify that cells responded normally to well-known stimuli and previously described functional identification criteria. These initial measurements highlighted a previously overlooked [Ca2+]pm response pattern in certain α-cells and indicated that [Ca2+]pm oscillations in β-cells are synchronized with those in δ-cells. We also demonstrated that the labeling strategy can be combined with genetically encoded fluorescence biosensors as exemplified by intracellular cAMP recordings. This approach is considerably more flexible than cell-type-specific expression of biosensors. The potential of this system could be further improved using an infrared fluorescent protein for identification, and leave the visual spectral channels for functional biosensors.
We thank Prof. Göran Akusjärvi, Uppsala University, for providing the pBR322-E3-Tet-On-rev plasmid and generous advice on the vector design and Mrs. Helene Dansk for skillful help with islet isolations. This study was supported by grants from the Swedish Research Council (325-2012-6778, 55X-06240), the Swedish Diabetes Foundation, Diabetes Wellness Foundation (670_2015PG), Family Ernfors Foundation, European Foundation for the Study of Diabetes/MSD, Novo-Nordisk Foundation and the Swedish national strategic grant initiative EXODIAB (Excellence of diabetes research in Sweden). The Nordic Network for Clinical Islet transplantation supported by EXODIAB and the Juvenile Diabetes Research Foundation generously provided human islets.
HS generated expression vectors, performed labeling and immunostaining experiments, [Ca2+]pm recordings, and data analysis, and wrote the manuscript; YX designed and generated expression vectors; QY performed [Ca2+]pm recordings; EG analyzed data and wrote the manuscript; and AT conceived the study, analyzed data, and wrote the manuscript. All authors have approved the final version of the manuscript.
Compliance with ethical standards
All procedures for animal handling and islet isolation were approved by the Uppsala animal ethics committee, and all experiments with human islets were approved by the Uppsala human ethics committee.
Conflict of interest
The authors declare that they have no conflict of interest.
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