Evidence indicates that subtle abnormalities in GC (glucocorticoid) plasma concentrations and/or in tissue sensitivity to GCs are important in the metabolic syndrome, and it is generally agreed that GCs induce insulin resistance. In addition, it was recently reported that short-term exposure to GCs reduced the insulinotropic effects of the incretin GLP-1 (glucagon-like peptide 1). However, although defective GLP-1 secretion has been correlated with insulin resistance, potential direct effects of GCs on GLP-1-producing L-cell function in terms of GLP-1 secretion and apoptosis have not been studied in any greater detail. In the present study, we sought to determine whether GCs could exert direct effects on GLP-1-producing L-cells in terms of GLP-1 secretion and cell viability. We demonstrate that the GR (glucocorticoid receptor) is expressed in GLP-1-producing cells, where GR activation in response to dexamethasone induces SGK1 (serum- and glucocorticoid-inducible kinase 1) expression, but did not influence preproglucagon expression or cell viability. In addition, dexamethasone treatment of enteroendocrine GLUTag cells reduced GLP-1 secretion induced by glucose, 2-deoxy-D-glucose, fructose and potassium, whereas the secretory response to a phorbol ester was unaltered. Furthermore, in vivo administration of dexamethasone to rats reduced the circulating levels of GLP-1 concurrent with induction of insulin resistance and glucose intolerance. We can conclude that GR activation in GLP-1-producing cells will diminish the secretory responsiveness of these cells to subsequent carbohydrate stimulation. These effects may not only elucidate the pathogenesis of steroid diabetes, but could ultimately contribute to the identification of novel molecular targets for controlling incretin secretion.
It is generally agreed that GCs induce insulin resistance. In addition, it was recently reported that short-term exposure to GCs reduced the insulinotropic effects of GLP-1. However, potential direct effects of GCs on GLP-1-producing L-cell function in terms of GLP-1 secretion and apoptosis have not been studied in any greater detail.
We report that GR activation in GLP-1-producing cells will diminish the secretory responsiveness to subsequent carbohydrate stimulation.
These effects not only may elucidate pathogenesis of steroid diabetes, the natural unfolding of polygenic T2DM, but also could contribute to the identification of novel molecular targets for controlling incretin secretion.
Evidence is accumulating in support of an important role for abnormalities in plasma GC (glucocorticoid) concentrations and/or tissue sensitivity to GC in the metabolic syndrome . Furthermore, chronic stress and side effects of GC treatment involve an increased risk of developing insulin resistance and diabetes.
In the human body, the main endogenous GC is cortisol, an integral component of the response to stress . GC synthesis and release are regulated by the HPA (hypothalamo–pituitary–adrenal) axis . Neuroendocrine neurons in the hypothalamus synthesize and secrete CRH (corticotropin-releasing hormone), which acts on the pituitary gland and causes release of ACTH (adrenocorticotropic hormone) [3,4]. ACTH is transported to the adrenal glands, where it stimulates secretion of GCs. GCs act by binding to the GR (glucocorticoid receptor). Binding of GC agonists to the cytoplasmic GR induces the release of Hsp90 (heat-shock protein 90), resulting in a conformational change that unmasks the receptor nuclear localization signal. Subsequently, the receptor translocates to the nucleus, where it either activates or represses gene expression .
The name ‘glucocorticoid’ derives from the fact that these hormones, in addition to exerting anti-inflammatory actions, are involved in glucose metabolism. Blood glucose levels are determined mainly through the balance between insulin-dependent processes such as hepatic gluconeogenesis and peripheral glucose utilization. As regulators of glycaemia and components of the stress response, GCs stimulate several processes that collectively serve to increase or maintain blood glucose concentrations . Specifically, GCs stimulate hepatic gluconeogenesis through increased expression of rate-limiting enzymes [7,8]. Moreover, GCs decrease key mediators of insulin action in peripheral tissues (insulin receptor substrate 1, phosphoinositide 3-kinase and protein kinase B) , inhibiting glucose uptake in muscle and adipose tissue and stimulating lipolysis. Insulin resistance may thus be a direct effect of GCs on the insulin receptor signalling pathway and/or an indirect effect of GCs resulting from changes in lipid metabolism . These insulin-opposing metabolic actions on individual organs induced by GCs contribute to GC-induced insulin resistance and steroid diabetes, side effects of GC treatment . Although it is generally agreed that GCs induce insulin resistance, it has also been reported that short-term exposure to GCs reduced the insulinotropic effects of the incretin GLP-1 (glucagon-like peptide 1) .
GLP-1 is synthesized from the preproglucagon gene in intestinal enteroendocrine L-cells and secreted in response to nutrient intake . Increased glucose metabolism and closure of KATP (ATP-dependent K+) channels (found in GLP-1-producing cells and many other glucose-responsive tissues) results in membrane depolarization, entry of Ca2+ through voltage-gated Ca2+ channels and GLP-1 release . Furthermore, non-metabolizable, as well as metabolizable, sugars can stimulate GLP-1 release in the presence of sodium, through the activity of SGLTs (sodium glucose transporters) . In fact, the EC50 of glucose-induced GLP-1 secretion from GLUTag cells (0.2–0.5 mM) is close to the half-maximally effective substrate concentration of SGLT, therefore SGLT activity has been postulated to determine the concentration-dependence of glucose-stimulated GLP-1 release .
In addition to its insulinotropic effects, GLP-1 also stimulates β-cell proliferative and anti-apoptotic pathways and reduces insulin resistance, while also inhibiting glucagon release, gastric emptying and food intake .
Decreased GLP-1 levels have been observed with increased BMI (body mass index) and obesity in patients with or without T2DM (Type 2 diabetes mellitus) , where defective GLP-1 secretion has also been correlated to insulin resistance . Administration of GLP-1 to T2DM patients restores glucose-induced insulin secretion and normalizes glycaemia . However, native GLP-1 is rapidly degraded by DPP-4 (dipeptidyl peptidase 4) present in plasma, therefore stable analogues of GLP-1 such as exendin-4 and liraglutide, as well as DPP-4 inhibitors such as sitagliptin, are available as treatments for T2DM. Enhancing endogenous GLP-1 production may be superior to current incretin therapy, as GLP-1 would be released by its native route directly into the portal vein, containing regulatory GLP-1-sensitive glucose sensors, before hepatic passage . Furthermore, endogenous GLP-1 secretion has a pulsatile secretory pattern that is not mimicked by current incretin therapy . However, to modulate endogenous secretion, it is necessary to first gain an understanding of L-cell function and factors that influence its regulation. The association of increased GC signalling and insulin resistance, as well as reports of reduced plasma GLP-1 in conditions characterized by insulin resistance, provokes the question of whether GCs can modulate GLP-1 secretion. However, whether GCs can directly modulate GLP-1-producing L-cell function in terms of GLP-1 secretion and cell viability have not previously been studied. Consequently, in the present study, we sought to determine whether GC signalling could exert direct effects on GLP-1-producing L-cells in terms of GLP-1 secretion and viability, aiming to not only further characterize effects of increased GC signalling, but also further elucidate the regulation of GLP-1-producing cells.
MATERIALS AND METHODS
Cell culture and
in vitro exposure
The GLP-1-secreting GLUTag cell line (source: glucagon-producing enteroendocrine cell tumour that arose in transgenic mice generated on an outbred CD-1 background) , graciously donated by Dr Neil Portwood (Karolinska Institutet, Solna, Sweden), and originally from Dr Daniel J. Drucker (Mount Sinai Hospital, Samuel Lunenfeld Research Institute, University of Toronto, Toronto, Canada), was cultured in DMEM (Dulbecco's modified Eagle's medium) (Invitrogen) supplemented with 10% FBS (Sigma–Aldrich), 5.5 mM glucose, 10000 units/ml penicillin and 10 mg/ml streptomycin sulfate (Invitrogen) under 5% CO2. Culture of MIN6 cells was performed as described previously . Dexamethasone and RU486 were from Sigma–Aldrich. SGK (serum- and glucocorticoid-inducible kinase) inhibitors were purchased from Tocris Bioscience (GSK 650394) and ChemBo Pharma (EMD638683). The PKC (protein kinase C) activator PMA and the adenylate cyclase activator forskolin were purchased from Sigma–Aldrich.
Male Wistar rats were housed in pairs in solid-bottomed plastic cages with wood shavings and paper as bedding. All rats had food and water available ad libitum. At 10 weeks of age, animals were divided into two groups, receiving once daily intraperitoneal injections of either dexamethasone (dexamethasone 21-phosphate disodium salt, 1 mg/kg) or vehicle (saline) for 7 consecutive days between 09:00 and 10:00 h. The use of laboratory animals was performed according to the guidelines of the Karolinska Institutet, Sweden, in accordance with national law and approved by the local animal ethics committee (Stockholm South Animal Ethics Committee, permit # S-201-10).
OGTT (oral glucose tolerance test), blood glucose, serum insulin and GLP-1 determinations
On the seventh day of treatment, after the final injection of dexamethasone or saline, food was removed 6 h before oral gavage administration of glucose (2 g/kg of body weight). Blood samples were collected from the saphenous vein before glucose administration and at different time points (0–120 min) following glucose administration. Blood glucose concentrations were determined directly by a Bayer's Elite® Glucometer. Blood was also collected in tubes in which 10 μl/ml DPP-4 inhibitor (Millipore) was added. The blood samples were spun to obtain serum. The serum was stored in a freezer at −70°C for later analysis of insulin and active GLP-1-(7–36). Serum insulin concentrations were determined using a mouse ultrasensitive ELISA (Mercodia), according to the manufacturer's instructions. Briefly, this ELISA uses a sandwich technique in which two monoclonal antibodies are directed against separate antigenic determinants on the insulin molecule. Insulin in the sample reacts with horseradish peroxidase-conjugated anti-insulin antibodies and anti-insulin antibodies immobilized in the wells of the microwell plate. The bound conjugate is detected by reaction with 3,3′,5,5′-tetramethylbenzidine. The reaction is stopped by adding acid to give a colorimetric endpoint that is read with a spectrophoto-meter. Serum (5 μl) was used for analysis. Serum GLP-1-(7–36) was determined using a specific ELISA (Millipore) according to the manufacturer's instructions. Briefly, this assay is based on the capture of active GLP-1 [i.e. GLP-1-(7–36)-amide and GLP-1-(7–37)] by a monoclonal antibody, immobilized in the wells of a microwell plate, which binds to the N-terminal region of GLP-1, binding of an anti-GLP-1–alkaline phosphatase detection conjugate to immobilized GLP-1, and quantification of bound detection conjugate by adding MUP (methylumbelliferyl phosphate), which in the presence of alkaline phosphatase forms the fluorescent product umbelliferone. A 33 μl serum sample was used at a 1:3 dilution.
Cell viability assay
GLUTag cells were plated and cultured in 96-well plates at a density of 136000 cells/ml for 24 h, followed by 48 h of exposure to culture medium with or without indicated doses of dexamethasone. Palmitate (0.125 mM) exposure was used as positive control for cell toxicity . Viable cell densities were determined by metabolic conversion of the dye MTT. A 15 μl volume of the supplied MTT solution was added to each well and the plates were then incubated for an additional 4 h. The MTT reaction was terminated by the addition of 100 μl of acidified propan-2-ol, dissolving the formazon product formed. After 1–2 h at 4°C, MTT assay results were read by measuring the absorption at 540 nm. Each experiment was performed in six duplicates and repeated at least three times to assess consistency of results.
GLUTag cells were washed twice with PBS and lysed on ice in RIPA lysis buffer containing 150 mM NaCl, 20 mM Tris/HCl, 0.1% SDS, 1% Triton X-100, 0.25% sodium deoxycholate, 1 mM Na3VO4, 50 mM NaF, 2 mM EDTA and protease-inhibitory cocktail (Sigma–Aldrich) for 30 min. Samples were clarified by centrifugation, supernatants were transferred to new tubes, and the total protein concentration was determined using the Bio-Rad DC protein assay (method of Lowry et al. ), using BSA as a standard.
GLUTag cells were plated at a density of 180000 cells/ml and grown in 24-well plates for 24–48 h. Cells were then treated with dexamethasone at the indicated doses for an additional 48 h. Immediately after the 48 h incubation, medium was discarded and the cells were washed with pre-warmed glucose-free KRBH (Krebs–Ringer bicarbonate Hepes) buffer/0.2% BSA, followed by a 30 min pre-incubation with the same buffer. Cells were then treated with or without indicated doses of glucose/PMA in the same buffer. Immediately thereafter, DPP-4 inhibitor was added and the buffer was collected. GLP-1 content in medium/buffer was analysed using the active GLP-1-(7–36)-amide and GLP-1-(7–37) ELISA (Millipore; catalogue number EGLP-35K) as described above. The GLP-1 determinations were performed according to the manufacturer's instructions for this ELISA. All experiments were performed in triplicates and repeated at least three times to assess consistency of results.
RNA extraction, cDNA synthesis and quantitative RT (reverse transcription)–PCR
GLUTag and MIN6 cells were lysed and RNA was extracted using Aurum total RNA mini kit (Bio-Rad Laboratories; catalogue number 7326820) according to the manufacturer's instructions. cDNA was synthesized for qPCR using iScript™ cDNA synthesis kit (Bio-Rad Laboratories) according to the manufacturer's instructions. For qPCR, 5 ng/μl cDNA was added and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) mRNA expression was used as an internal control. An iScript™ one-step RT–PCR kit with SYBR® Green (Bio-Rad Laboratories) was used for real-time quantitative RT–PCR. This kit utilizes iScript RNase H+ reverse transcriptase and hot-start iTaq DNA polymerase.
For each sample, the mRNA level of each target gene relative to GAPDH was estimated by calculating the ΔCT (CT,target gene−CT,GAPDH) and then converting to 2−ΔCT. To compare mRNA levels between experimental groups, the ratio of the average 2−ΔCT for each treatment group relative to the control group (2−ΔΔCT) was determined for each gene.
A set of ten relevant genes was analysed. Primers were designed using Invitrogen custom primer design software. The primer list and specifications are given in Supplementary Table S1.
Western blot analysis
GLUTag and MIN6 cellular protein was extracted using RIPA lysis buffer as above for 30 minutes on ice. Samples were clarified by centrifugation, supernatants were transferred to new tubes and the total protein concentration was determined using the Bio-Rad DC protein assay using BSA as a standard. Equal amounts of protein were then mixed with reducing SDS/PAGE sample buffer and boiled for 5 min, and proteins were separated by SDS/PAGE using a 10% polyacrylamide gel under denaturing conditions, followed by transfer on to a PVDF membrane (Bio-Rad Laboratories). Membranes were blocked with 5% (w/v) milk solids in PBS-T (PBS containing Tween 20); primary (overnight) and secondary (1 h) antibody incubations were performed in the same buffer, with three 10-min washes in PBS-T intervening. The anti-GR antibody was from Abcam (ab52190) . Horseradish peroxidase-conjugated secondary antibodies (1:5000 dilution) (Santa Cruz Biotechnology) and ECL (GE Healthcare) reagents were used to detect proteins. Images and quantifications were obtained using Molecular Imager ChemiDoc XRS with Quantity One Software version 4.6.5 (Bio-Rad Laboratories).
Comparisons between multiple groups were made by a one-way ANOVA. Student–Newman–Keuls post hoc test was used. Comparisons between control and single treatment groups were made using two-tailed Student's t test. P<0.05 was deemed statistically significant.
Dexamethasone induces glucose intolerance, hyperinsulinaemia and reduces circulating levels of GLP-1 in rats
To determine the effect of GC treatment on GLP-1 secretion in vivo, male Wistar rats were subjected to daily i.p. (intraperitoneal) injections of dexamethasone (1 mg/kg) or vehicle for 7 consecutive days. Administration of the steroid hormone resulted in a decrease in body weight (Figure 1A). An OGTT was performed on day 7 of treatment. In line with previous findings , glucose tolerance was significantly impaired in rats receiving this dose of dexamethasone, as indicated by elevated blood glucose levels following oral administration of glucose on day 7 of treatment (Figure 1B). Furthermore, the reduced glucose tolerance was observed in conjunction with increased circulating levels of insulin (Figure 1C), indicating the manifestation of insulin resistance after 7 days of dexamethasone injections. In addition, compared with control rats receiving i.p. injections of saline, dexamethasone-treated Wistar rats displayed significantly lower levels of circulating GLP-1-(7–36) following oral administration of glucose (Figure 1D).
Daily injections of dexamethasone to Wistar rats for 7 consecutive days induce glucose intolerance and hyperinsulinaemia and reduce circulating levels of GLP-1
The GR is expressed in GLP-1-producing cells, where receptor activation results in increased expression of SGK1
To investigate whether GCs could exert direct effects on the intestinal GLP-1-producing cells, we analysed GR expression in this cell type using GLP-1-producing GLUTag cells  as a model. For comparison, we used insulin-producing MIN6 cells that are sensitive towards GCs . Our results demonstrate the presence of GR mRNA and protein (Figures 2A and 2B) in these cells, rendering the possibility of direct actions exerted by GCs. Dexamethasone is a GR agonist, 25 times more potent than cortisol in its GC effect. To confirm dexamethasone-induced activation of GR signalling in this cell type, we analysed the expression of SGK1, a known target of GR signalling . Our data demonstrate that 48 h of treatment with dexamethasone significantly and dose-dependently induces SGK1 mRNA expression in GLUTag cells (Figure 2C). In addition, co-incubation with the GR antagonist RU486 reversed dexamethasone-induced effects on SGK1 mRNA expression (Figure 2C).
The GR is expressed in GLP-1-producing cells, where receptor activation results in increased expression of SGK1
Dexamethasone reduces GLP-1 secretion, but not preproglucagon mRNA expression, in GLP-1-producing cells
A 48 h exposure to dexamethasone significantly reduced the amount of GLP-1 secreted into the incubation medium by GLUTag cells (Figure 3A). To determine whether the reduced GLP-1 secretion observed in response to a 48 h treatment with dexamethasone was the result of induced cytotoxicity, GLUTag cell viability was assessed following exposure to increasing concentrations of dexamethasone, using the MTT assay. Our results demonstrate no induction of cytotoxicity following exposure to dexamethasone (0–1000 nM) (Figure 3B), nor did the 48 h treatment with dexamethasone alter protein content in the wells (results not shown). Furthermore, our data indicate no effect of dexamethasone on glucagon gene expression, as measured by preproglucagon mRNA expression (Figure 3C), collectively arguing against GC toxicity in this cell type as opposed to insulin-producing β-cells [22,28].
Dexamethasone reduces GLP-1 secretion, but not viability or proglucagon expression, in GLP-1-producing cells
Dexamethasone reduces glucose-induced GLP-1 secretion from GLP-1-producing cells
GLUTag cells used in the present study demonstrated GLP-1 secretion in response to a range of known secretagogues, including the adenylate cyclase activator forskolin and the PKC activator PMA (Figure 4A). Notably, and analogous to its effect in insulin-producing β-cells, simultaneous activation of adenylate cyclase and PKC evoked an additive secretory response. To determine whether long-term treatment with GCs could alter the secretory response to subsequent exposure to secretagogues, GLUTag cells were cultured in the presence of 0–500 nM dexamethasone for 48 h followed by acute (2 h) exposure to glucose or PMA. Basal secretion did not differ between cells pre-treated with vehicle or dexamethasone for 48 h (152±16.0 compared with 151±22.2 pM, n=4). In response to 1 μM PMA, untreated GLUTag cells increased their secretory rate and after 2 h of incubation, the GLP-1 concentration in the well was 472±54.0 pM, a secretion rate not affected by a 48 h pre-exposure to dexamethasone (449±51.0 pM, n=4). In contrast, a 48 h dexamethasone pre-treatment reduced subsequent GLP-1 secretion evoked by glucose from GLUTag cells (Figures 4B and 4C). This GC effect on glucose stimulated GLP-1 secretion evoked by 20 mM glucose was significantly attenuated by co-incubation with the GR antagonist RU486 (Figure 4D), whereas inhibition of SGK1 activity was without effect on the attenuated glucose (20 mM) response following dexamethasone (Figure 4E).
Dexamethasone specifically reduces glucose-induced GLP-1 secretion from GLP-1-producing cells
Dexamethasone reduces fructose-induced GLP-1 secretion from GLP-1-producing cells, and K+ channel activity/expression is indicated in the defective response to sugars
Glucose is known to stimulate secretion of GLP-1 through mechanisms involving closure of KATP channels . However, non-metabolizable sugars have also been shown to stimulate GLP-1 secretion in a sodium-dependent manner, involving SGLTs . Consequently, we used 2-deoxy-D-glucose to further characterize the defective GLP-1 secretion following administration of dexamethasone. Our results demonstrate a significant attenuation of the response to 2-deoxy-D-glucose following a 48 h dexamethasone pre-treatment (Figure 4F). Glucose and 2-deoxy-D-glucose alike are substrates for SGLTs . Fructose, on the other hand, stimulates GLP-1 release through closure of KATP channels without involving SGLTs . Following a 48 h exposure to dexamethasone, defective fructose-induced GLP-1 release was observed (Figure 4G) and unaltered expression of SGLTs (Figure 4H, and Supplementary Table S1).
To determine whether dexamethasone could modulate K+ channel activity/expression, thereby providing a possible mechanism for the induction of a defective glucose response, we analysed the secretory potential of extracellular KCl and the mRNA expression of a number of different K+ channel subunits. In control experiments, GLUTag cells exposed to 10.6 mM extracellular K+ released significantly more GLP-1 than cells exposed to basal (3.6 mM) K+ (Figure 4I). In contrast, increasing the extracellular K+ concentration up to 16.6 mM was without any effect in terms of GLP-1 secretion from cells pre-treated with dexamethasone (Figure 4I). However, analysis of mRNA expression for seven different K+ channel subunits reveals no significant difference in response to dexamethasone (Figure 4H, and Supplementary Table S1). Neither was the mRNA expression of glucokinase altered following treatment with dexamethasone (results not shown).
The present study demonstrates glucose intolerance, insulin resistance and reduced circulating levels of GLP-1 following oral glucose load in dexamethasone-treated rats. Our novel data also provide support of direct inhibitory effects of GCs on GLP-1-producing L-cells, suppressing the secretion of GLP-1. Specifically, our data show that long-term treatment with dexamethasone significantly reduces subsequent sugar-induced secretion of GLP-1 through direct GR-dependent effects on GLP-1-producing cells in vitro. The GLUTag cells used as a model of enteroendocrine L-cells in the present study is a stable immortalized murine enteroendocrine cell line that expresses the proglucagon gene and secretes the glucagon-like peptides . GLUTag cells appear quite well differentiated, and recapitulate the responsiveness of primary intestinal cell cultures to physiological and pharmacological GLP-1 secretagogues, as demonstrated previously [29,30], and thus constitute one of the best available models of the L-cell.
The impact of dexamethasone on GLP-1-producing cells demonstrated in the present study suggests that direct effects of the steroid on intestinal L-cells may underlie the reduced GLP-1 plasma levels that we observed following oral glucose administration in dexamethasone-treated rats. It is unlikely that the hyperglycaemia and hyperinsulinaemia in these animals contributed to the decreased GLP-1 secretion in vivo, as both glucose and insulin are stimulators of this process . Interestingly, GLP-1 is also synthesized in parts of the CNS (central nervous system) [31,32], and CNS preproglucagon and GLP-1 immunoreactivity have been indicated to be down-regulated by exogenous GCs in vivo , effects that may result from decreased stability of preproglucagon mRNA by binding to the AU-rich elements in the 3′-UTR by the GR . However, in the present study, we were unable to detect a significant effect of dexamethasone on the expression of preproglucagon mRNA in GLP-1-producing GLUTag cells.
Importantly, the present study shows that the direct effects of dexamethasone on GLP-1-producing cells modulate secretory function, whereas no induction of cytotoxicity or altered viability is observed. The reduced glucose response of GLP-1-producing cells following pre-treatment with dexamethasone is indicated to be independent of SGK1 signalling. Furthermore, the finding that no alteration of PMA-induced GLP-1 secretion could be observed following dexamethasone pre-treatment argues against a generalized defect in the secretory mechanisms. Rather, our data indicate that dexamethasone pre-treatment alters the activity/expression of transporters and/or proteins involved in signalling pathways mediating sugar-induced GLP-1 secretion. GLUTag cells have been reported to express relatively low levels of the glucose transporter GLUT2 , an observation corroborated by our quantitative PCR data. Instead, these cells express higher levels of, especially, SGLT1 (Supplementary Table S1). However, the demonstrated impairment of GLP-1 secretion evoked not only by glucose and 2-deoxy-D-glucose, but also by fructose, following dexamethasone pre-treatment indicates that the effect is not specific for substrates of the SGLTs. In agreement with this assumption and previous studies , we failed to detect any effect of dexamethasone treatment on SGLT mRNA expression. Fructose is known to evoke GLP-1 secretion through closure of KATP channels, and dexamethasone regulates K+ channel expression and activity in other cell types . The fact that the response to 2-deoxy-D-glucose is impaired in response to dexamethasone could result from the inward current generated by SGLT activity being relatively very small, rendering its ability to depolarize the membrane and cause Ca2+ entry and GLP-1 release dependent on the magnitude of background currents regulated by K+ channel activity . Altered responsiveness to extracellular KCl following dexamethasone treatment is indicated in the present study, which could imply the potential involvement of K+ channel activity/expression in the defective glucose response observed. Although we found that dexamethasone does not alter the mRNA expression of any of the seven K+ channel subunits analysed, protein expression/activity of the different K+ channels may be altered. However, dexamethasone could potentially also regulate other mediators of the glucose response downstream of membrane depolarization. For instance, dexamethasone may reduce production of cAMP ; cAMP production potentiates glucose-induced GLP-1 secretion through activation of protein kinase A, Ca2+ release from the endoplasmic reticulum and a subsequent increase in intracellular Ca2+. Importantly, further studies are needed to elucidate the exact mechanisms mediating the defective glucose response of GLP-1-producing cells following GC treatment.
Direct effects of GR signalling on intestinal GLP-1-producing cells are strongly indicated by the in vitro data from GLUTag cells. However, evaluation of the reduced circulating levels of GLP-1 observed in vivo, i.e. the possible involvement of altered DPP-4 activity and clearance of the circulating peptide, should be undertaken. Furthermore, as we and others have shown increased GLP-1 secretion following insulin receptor signalling , it is possible that increased insulin resistance in response to GCs contributes to the reduced GLP-1 levels observed in vivo. The effect of GR signalling on GLUTag cells found in the present study can be put in contrast with the stimulatory action recently shown in response to another steroid hormone, progesterone , and could contribute to increased understanding of the factors that positively or negatively regulate this cell type. In the future, studies designed to determine whether the effects observed in the present study in response to dexamethasone are reproduced by the endogenous hormone corticosterone should be undertaken. Furthermore, it would be of interest to determine possible effects of dexamethasone on expression of the GC-metabolizing enzyme 11β-HSD1 (11β-hydroxysteroid dehydrogenase 1), as dexamethasone has previously been indicated to induce the expression of 11β-HSD1 through activation of GR , and induction of 11β-HSD1 expression may produce more biologically active corticosterone from inert 11-dehydrocorticosterone, and thus amplify the effects of GCs.
In conclusion, the present study provides novel data in support of negative regulation of glucose-stimulated GLP-1 secretion, and the incretin effect in response to long-term GC treatment. These effects are indicated to be mediated, at least in part, by direct inhibitory actions of GCs on GLP-1-producing cells. Such effects could potentially contribute to the well-known development of steroid diabetes in response to GCs, and would imply that GCs not only induce insulin resistance, but also, through attenuation of incretin tone, indirectly impede the ability of the β-cell to compensate through increased insulin secretion. Furthermore, potential direct effects of GCs on intestinal L-cells could lead to the identification of novel signalling pathways involved in the regulation of this cell type and potential targets for enhancing endogenous GLP-1 secretion.
Camilla Kappe contributed to the research plan, designed and performed in vitro experiments, performed animal experiments, analysed data, participated in discussions and wrote the paper. Liselotte Fransson contributed to the research plan, performed animal experiments and participated in discussions. Petra Wolbert performed animal experiments and analysis of insulin in plasma samples, and participated in discussions. Henrik Ortsäter conceived of the research plan, performed and designed animal experiments, and participated in preparation of the paper and discussions.
We thank the personnel at the animal facility of Södersjukhuset for excellent animal care and the research centre at Södersjukhuset for providing laboratory facilities.
This work was generously supported financially by Folksam Research Foundation, Fredrik and Inger Thuring's Foundation, Lars Hiertas memorial foundation, Tore Nilson's Foundation, and by Golje Foundation.
central nervous system
dipeptidyl peptidase 4
glucagon-like peptide 1
11β-hydroxysteroid dehydrogenase 1
channel. ATP-dependent K+ channel
oral glucose tolerance test
protein kinase C
serum- and glucocorticoid-inducible kinase
sodium glucose transporter
Type 2 diabetes mellitus
Present address: Department of Medical Cell Biology, Uppsala Biomedical Centre, Uppsala University, Sweden.