Dapagliflozin Does Not Directly Affect Human α or β Cells

Abstract

Selective inhibitors of sodium glucose cotransporter-2 (SGLT2) are widely used for the treatment of type 2 diabetes and act primarily to lower blood glucose by preventing glucose reabsorption in the kidney. However, it is controversial whether these agents also act on the pancreatic islet, specifically the α cell, to increase glucagon secretion. To determine the effects of SGLT2 on human islets, we analyzed SGLT2 expression and hormone secretion by human islets treated with the SGLT2 inhibitor dapagliflozin (DAPA) in vitro and in vivo. Compared to the human kidney, SLC5A2 transcript expression was 1600-fold lower in human islets and SGLT2 protein was not detected. In vitro, DAPA treatment had no effect on glucagon or insulin secretion by human islets at either high or low glucose concentrations. In mice bearing transplanted human islets, 1 and 4 weeks of DAPA treatment did not alter fasting blood glucose, human insulin, and total glucagon levels. Upon glucose stimulation, DAPA treatment led to lower blood glucose levels and proportionally lower human insulin levels, irrespective of treatment duration. In contrast, after glucose stimulation, total glucagon was increased after 1 week of DAPA treatment but normalized after 4 weeks of treatment. Furthermore, the human islet grafts showed no effects of DAPA treatment on hormone content, endocrine cell proliferation or apoptosis, or amyloid deposition. These data indicate that DAPA does not directly affect the human pancreatic islet, but rather suggest an indirect effect where lower blood glucose leads to reduced insulin secretion and a transient increase in glucagon secretion.

Blood glucose levels, which are normally tightly controlled through the coordination of multiple organ systems, are elevated in type 2 diabetes (T2D). Sodium glucose cotransporter-2 (SGLT2) inhibitors such as dapagliflozin (DAPA) are important medications in the treatment of T2D and lower blood glucose by preventing glucose reabsorption by the kidney, thereby promoting glucosuria. Dapagliflozin treatment also decreases insulin secretion from pancreatic islet β cells and improves muscle insulin sensitivity, which is thought to be a consequence of lower blood glucose (1, 2). Interestingly, initiation of DAPA treatment is also associated with a transient rise in glucagon from pancreatic islet α cells, which may partially counteract the glucose-lowering benefits (1, 2).

While the metabolic benefits of DAPA are clear, it remains controversial whether the effects of DAPA on glucagon secretion are due to direct effects on α cells. Previous studies of DAPA on cultured human islets have been conflicting. This is partly because the DAPA concentrations used have been outside the therapeutic window (100–200 ng/ml; 250–500 nM) (3–5), calling into question the clinical relevance of the findings. For example, very low concentrations of DAPA (10 nM, below therapeutic range) increased glucagon secretion under high glucose (11 mM), but decreased glucagon secretion under low glucose (1 mM) (6). In contrast, high concentrations of DAPA (12.5 µM, above therapeutic range) increased glucagon secretion in a subset of donor human islets under low and moderate glucose (1 and 6 mM) but had no effect under high glucose (15 mM) (7, 8). Moreover, recent studies have contended that SGLT2 is not expressed in islet cells and argued that SGLT2 inhibitors do not directly affect islet hormone secretion in rodent models (9, 10).

Importantly, human islets show key differences from rodent islets, including their endocrine cell composition and arrangement, gene expression (including glucose transporters), glucose set-point, and both basal and stimulated insulin and glucagon secretion (11–15). We therefore sought to clarify the effects of clinically relevant levels of DAPA on human islets both in vitro and in an in vivo transplant model that mimics the clinical setting and drug exposure. Here, we show that SGLT2 expression in human islets is extremely low and that DAPA does not directly affect insulin or glucagon secretion from the human islet.

Materials and Methods

Human tissue and islet transplantation

Human islets (n = 22 donor preparations, Table 1) were obtained from Integrated Islet Distribution Network (http://iidp.coh.org/), Alberta Diabetes Institute Islet Core (https://www.epicore.ualberta.ca/IsletCore/), or isolated at the Institute of Cellular Therapeutics of the Allegheny Health Network (Pittsburgh, PA). Assessment of human islet function was performed by islet perifusion assay on the day of arrival, as previously described (16, 17). Male NOD.Cg-Prkdc^scidIl2rg^tm1Wjl/Sz (NSG) mice (18) ages 12 to 18 weeks were used for transplantation. All mice were maintained on standard rodent chow under a 12-hour light/dark cycle. Human islets were transplanted under the kidney capsule, as previously described (13, 16). Each mouse received 1500 IEQ, and islets from a given donor were divided evenly among 2 treatment groups. Human kidney sample was provided by Dr Agnes Fogo, Vanderbilt University Medical Center.

Table 1.

Human islet donor information

Islet Preparation	1	2	3	4	5	6	7	8
Unique identifier	8769130	8768699	Don120	8617638	R237	8768998	8611143	8774468
Donor age (years)	37	24	19	49	61	35	32	44
Donor sex (M/F)	M	F	M	M	M	M	M	F
Donor BMI (kg/m2)	27.6	32.2	20.1	34	19.7	24.3	26.2	23.8
Donor HbA1c	5.6	N/A	5.1	N/A	N/A	5.4	N/A	N/A
Origin/source of islets	IIDP	IIDP	AHN	IIDP	Alberta IsletCore	IIDP	IIDP	IIDP
Islet isolation center	U Wisconsin	U Penn	AHN	S California	Alberta IsletCore	S California	U Wisconsin	U Penn
Donor history of diabetes?	No	No	No	No	No	No	No	No
Donor cause of death	CVA	Anoxia	Head Trauma	CVA	N/A	CVA	Anoxia	CVA
Warm ischaemia time (h)	N/A	N/A	N/A	N/A	N/A	N/A	N/A	N/A
Cold ischaemia time (h)	11	9	N/A	8	N/A	N/A	8	6.2
Estimated purity (%)	90	95	90	80	95	80	98	95
Estimated viability (%)	98	90	N/A	97	N/A	98	99	95
Total culture time (h)	18	87	48	23	N/A	52	34	46
Glucose-stimulated insulin secretion	perifusion	perifusion	perifusion	Perifusion	Perifusion	Perifusion	Perifusion	perifusion
Handpicked to purity?	Yes	No	No	No	Yes	Yes	Yes	Yes
Experiments used for	Dapa experiments	Dapa experiments	Dapa experiments	Dapa experiments	Dapa experiments	Dapa experiments	Dapa experiments	RNA
Islet Preparation	9	10	11	12	13	14	15	16
Unique identifier	8776514	8776508	8784601	8768702	8775095	Don39	Don77	Don84
Donor age (years)	48	53	51	59	61	47	59	51
Donor sex (M/F)	F	F	F	F	M	M	M	M
Donor BMI (kg/m2)	29.2	25.4	21.2	22	42.1	31.3	27.5	31.1
Donor HbA1c	6.4	N/A	N/A	5.2	N/A	10.2	6.2	N/A
Origin/source of islets	IIDP	IIDP	IIDP	IIDP	IIDP	AHN	AHN	AHN
Islet isolation centre	U Penn	U Penn	U Penn	U Wisconsin	S California	AHN	AHN	AHN
Donor history of diabetes?	No	No	No	No	Yes	Yes	Yes	Yes
Diabetes duration (years)					8	3	6	3
Glucose-lowering therapy at time of death					N/A	insulin	“Oral agents”	Sitagliptin
Donor cause of death	CVA	N/A	CVA	CVA	CVA	CVA	CVA	Head Trauma
Warm ischaemia time (h)	N/A	N/A	N/A	N/A	N/A	N/A	N/A	N/A
Cold ischaemia time (h)	4	6.5	10	6.5	N/A	N/A	15.6	14
Estimated purity (%)	85	97	85	95	80	55	70	70
Estimated viability (%)	94	97	92	96	95	N/A	85	90
Total culture time (h)	16	19	48	36	27	22	36	21
Glucose-stimulated insulin secretion	perifusion	perifusion	perifusion	perifusion	Perifusion	perifusion	perifusion	Perifusion
Handpicked to purity?	Yes	Yes	Yes	Yes	Yes	Yes	Yes	Yes
Experiments used for	RNA	RNA	RNA	RNA	RNA	RNA	RNA	RNA
Islet Preparation	17	18	19	20	21	22
Unique identifier	Don100	Don75	Don78	8774213	8769829	8930762
Donor age (years)	60	16	19	20	35	68
Donor sex (M/F)	M	M	F	M	M	M
Donor BMI (kg/m2)	38.3	23	23.8	21.7	28.5	24.9
Donor HbA1c	7.2	5	N/A	N/A	N/A	4.8
Origin/source of islets	AHN	AHN	AHN	AHN	AHN	IIDP
Islet isolation centre	AHN	AHN	AHN	AHN	AHN	U Illinois
Donor history of diabetes?	Yes	No	No	No	No	No
Diabetes duration (years)	1
Glucose-lowering therapy at time of death	none
Donor cause of death	Head Trauma	Overdose	CVA	Head Trauma	Head Trauma	CVA
Warm ischaemia time (h)	N/A	N/A	N/A	N/A	N/A	N/A
Cold ischaemia time (h)	12.9	20.9	N/A	11.3	6.5	7
Estimated purity (%)	75	N/A	N/A	90	95	95
Estimated viability (%)	90	N/A	N/A	90	95	98
Total culture time (h)	<24	N/A	N/A	24	65	48
Glucose-stimulated insulin secretion	Perifusion	perifusion	perifusion	perifusion	perifusion	no
Handpicked to purity?	Yes	Yes	Yes	Yes	Yes	Yes
Experiments used for	RNA	RNA	RNA	RNA	RNA	RNA

Checklist for reporting human islet preparations used in research. Format adapted from (30-32).

This study used data from the Organ Procurement and Transplantation Network (OPTN). The OPTN data system includes data on all donors, wait-listed candidates, and transplant recipients in the United States, submitted by members of the OPTN. The Health Resources and Services Administration, US Department of Health and Human Services provides oversight to the activities of the OPTN contractor. The data reported here have been supplied by the United Network of Organ Sharing as the contractor for the OPTN. The interpretation and reporting of these data are the responsibility of the author(s) and in no way should be seen as an official policy of or interpretation by the OPTN or the US Government.

Immunohistochemistry

Five-µm-thick frozen human islet graft sections and mouse pancreas were cut and stained, as described (13, 16). Frozen tissue sections were permeabilized with 0.2% Triton-X-100/PBS for 10’ and blocked with 5% normal donkey serum for 1 hour. Primary antibodies to insulin (Dako, Santa Clara, CA, #A0564, 1:1000) (19), glucagon (abcam, ab-10988, 1:500; Cell Signaling, Beverly, MA, #2760, 1:500) (20, 21), Ki67 (abcam, ab-15580, 1:1000) (22), and SGLT2 (Novus, Littleton, CO, NBP1-92384, 1:100; abcam, ab-58298, 1:100) (23, 24) were diluted in 0.1% Triton-X-100/PBS, then added to the sections and incubated for overnight at 4^oC. After 3 washes with 0.1% Triton-X-100/PBS for 20’, secondary antibodies (donkey antiguinea pig-Cy2, Jackson ImmunoResearch, West Grove, PA, 706-225-148, 1:500; donkey antirabbit-Cy3, Jackson ImmunoResearch, 711-165-152, 1:500; donkey antimouse-Cy5, Jackson ImmunoResearch, 715-175-151, 1:200; donkey antirabbit-Cy5, JacksonImmunoResearch, 711-175-152, 1:200) (25–28) diluted in 0.1% Triton-X-100/PBS were added and incubated for 1 hour at room temperature and then followed by another 3 washes for 20 minutes. Apoptosis was assessed by TUNEL (Millipore, Burlington, MA, S7165) following the manufacturer’s instructions. To assess amyloid deposits, sections from human grafts were first stained with the insulin antibody and then incubated with 0.5% concentration Thioflavin S (#T-1892, Sigma, St. Louis, MO) in PBS. Images were acquired with an Olympus BX41 fluorescence microscope or a confocal laser-scanning microscope. Images were quantified with MetaMorph software.

Electron microscopy

The ultrastructure of β and α cells was studied by transmission electron microscopy, as previously described (16, 29). The mice were perfused with PBS first for 5 minutes and then followed by a fixing solution (2.5% gluteraldehyde in 0.1M cacodylate buffer) for 20 minutes. Mouse kidney with transplanted human islet grafts were removed and continued to fix for 1 hour at room temperature. Human grafts were removed and kept in the fixing solution at 4^oC overnight. The samples then were delivered to Vanderbilt electron microscopy core for embedding and sectioning and were subsequently imaged on the Philips/FEI Tecnai T12 microscope at various magnifications.

In vitro human culture experiments

Human islets were cultured overnight in Connaught Medical Research Laboratories media at 37°C with 5% CO₂ (14). The next day, 15 size-matched islets were precultured for 1 hour with or without 500 nM DAPA in 5.6 mM glucose containing Dulbecco’s Modified Eagle Medium (DMEM) media with 0.1% Bovine Serum Albumin (BSA). Islets were then cultured for 1 additional hour in DMEM media with 0.1% BSA and 3.3 or 16.7 mM glucose with or without 500 nM DAPA, and the media and islets were collected for analyses. Glucagon and insulin levels were measured in the culture media by glucagon (Mercodia, Uppsala, Sweden, 10-1271-01) (33) and human insulin (Alpco, Salem, NH, 80-ISNHU-E01.1) (34) ELISA and normalized to acid-ethanol total hormone extracts of islets recovered from the culture.

Ribonucleic acid isolation, cDNA synthesis, and quantitative RT-PCR

Total Ribonucleic acid (RNA) was extracted from human islets or kidney tissue using an RNAqueous RNA isolation kit (Ambion, Austin, TX). Ribonucleic acid quality control and quantity assessment (QC/QA) was performed using Bioanalyzer instrument in the Vanderbilt Technologies for Advanced Genomics core lab. Complementary DNA (cDNA) was synthesized using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, 4368814) according to the manufacturer’s instructions. Quantitative PCR (qPCR) was performed using TaqMan assays and reagents from Applied Biosystems (Foster City, CA), as described (14, 16, 17). Primers used in this project were: SLC5A2 (Hs00894642_m1), 18S (Hs99999901_s1), and ACTB (Hs99999903_m1). 18S and ACTB were used in parallel for control genes. Relative changes in mRNA expression was calculated by the comparative ∆Ct method using Applied Biosystems Step one Plus System.

DAPA measurements

Dapagliflozin was measured in mouse serum by in the Vanderbilt University Mass Spectrometry Core Facility. Sample analyses were carried out using an Acquity UPLC system (Waters Corp., Milford, MA) interfaced with a TSQ Quantum triple-stage quadrupole mass spectrometer (Thermo, San Jose, CA) equipped with a standard APCI ion source. Quantitation was based on SRM in negative ion mode (dapagliflozin: m/z 408 → 330, CE 14 V; tolbutamide internal standard [IS]: m/z 269 → 106, CE 21 V). Calibration curves were constructed by plotting peak area ratios (dapagliflozin/tolbutamide) against analyte concentrations for a series of 8 spiked plasma standards, ranging from 0.05 to 50 µM dapagliflozin. A weighting factor of 1/C2 was applied in the linear least-squares regression analysis to maintain homogeneity of variance across the concentration. A Waters Atlantis dC18 analytical column (2.1 mm x 50 mm, 3 µ m) was used for all chromatographic separations. Mobile phases were made up of 0.2% propionic acid in (A) H2O and in (B) CH3CN. Gradient conditions were as follows: 0 to 1 minute, B = 5%; 1 to 5 minutes, B = 5%–100%; 5 to 6.5 minutes, B = 100%; 6.5 to 7.0 minutes, B = 100%–5%; 7 to 11 minutes, B = 5%. Plasma samples (50 µ L) were spiked with tolbutamide (5 µ L), lightly vortexed, allowed to stand at room temperature for 15 to 20 minutes, and deproteinized with 150 µ L of cold acetonitrile. Samples were then vortexed and placed on ice for 10 to 15 minutes. Precipitated proteins were removed by centrifugation (10 000 x g, 20 minutes, 5°C). The clear supernatant (150 µ L) of each sample was transferred to a clean microcentrifuge tube and evaporated to dryness under a gentle stream of nitrogen gas. The residue was reconstituted in 100 µ L H₂O/ CH₃CN (3:1), vigorously vortexed, and transferred to a 200- µ L silanized autosampler vials equipped with Teflon-lined bonded rubber septa.

Serum hormone analyses

Mice were fasted for 6 hours and then challenged with a 2 g/kg glucose bolus administered intraperitoneally, as previously described (13, 16). Blood glucose levels were measured and blood samples were collected before (0’) and 15’ after glucose challenge. Total glucagon (Mercodia, 10-1271-01) (33), mouse insulin (Alpco, Salem, NH, 80-INSMSU-E01) (35), and human insulin (Alpco, 80-ISNHU-E01.1) (31) were analyzed by ELISA, as described above for in vitro secretion experiments and also expressed as a ratio to blood glucose levels.

Measurement of total graft insulin or glucagon content

Transplanted human islet graft were carefully separated from host mouse kidney, as previously described (13, 29). The graft was transferred to 1.5 ml eppendorf tube and cut in 2 to 3 pieces using a sharp scalpel and 0.25 ml acid alcohol (0.1ml of concentrated HCl into 11ml of 95% ethanol) was added to the eppendorf. After a 48-hour incubation period at 4^oC, the supernatant was collected and stored at -80^oC. Insulin and glucagon content in graft extracts were diluted at least 1:1000 and measured by ELISA, as above (33, 34).

Statistics

Data were expressed as mean ± standard error of mean. A P-value less than 0.05 was considered significant. Statistical comparisons were performed using Prism v8 software (GraphPad, San Diego, CA). Statistical details of experiments are described in figure legends and results.

Data availability

All data generated or analyzed during this study are included in this published article or in the data repositories listed in the References section.

Study approval

The Vanderbilt University Institutional Review Board does not classify deidentified human pancreatic specimens as human subject research. All animal studies were approved by the Vanderbilt Institutional Animal Care and Use Committee.

Results

SGLT2 expression in the human islet is extremely low

To characterize SGLT2 (gene name SLC5A2) expression, we first performed immunofluorescence analyses of human and mouse kidney sections, finding robust SGLT2 staining in the kidney cortex (Fig. 1A and 1B). However, protein expression in both human and mouse islets was undetectable using the same antibody under the same conditions (Fig. 1C and 1D). In order to directly compare staining, we analyzed sections from human islets transplanted under the renal capsule of immunodeficient mice. While staining in the mouse kidney was robust, we could not detect any signal from the adjacent human islet graft (Fig. 1E). At the transcript level, SLC5A2 expression in whole islets was detectable but at levels 1600-fold lower than in human kidney (Fig. 1G). Since SGLT2 expression may change in the diabetic state where inhibitors are used clinically, we also analyzed T2D islet grafts for SGLT2 staining and again could not detect signal within the graft (Fig. 1F). Further, there was no difference in SLC5A2 expression in T2D islets compared to nondiabetic islets (Fig. 1H). Together, these data suggest that SGLT2/SLC5A2 is not expressed in human islet cells to a significant degree, if at all.

Figure 1.

SGLT2/SLC5A2 expression is extremely low in mouse and human islets. A–F: Representative images of immunostaining for SGLT2 expression in human (A) and mouse (B) kidney, human (C) and mouse (D) pancreatic islets, and transplanted normal (E) or T2D (F) human islet grafts under the mouse kidney capsule, allowing us to visualize mouse kidney and human islet tissue on the same section. C’, D’, E’, and F’ show only immunostaining for SGLT2. Scale bar in A, B, C, C’, D, and D’: 50 µm. Scale bar in E, E’, F, F’: 100 µm. Green: insulin (guinea pig anti-insulin, Dako); blue: glucagon (mouse antiglucagon, abcam); red: SGLT2 (rabbit anti-SGLT2, Novus). SGLT2 staining was confirmed with a second independent antibody (rabbit anti-SGLT2, abcam; images not shown). See methods for detailed antibody information. G: Expression of SLC5A2 mRNA (encodes SGLT2) in human pancreatic islets (n = 5 donors) and human kidney cortex (n = 1) measured by qRT-PCR. H: Expression of SLC5A2 mRNA in age-matched normal (ND, 44–59 years old, n = 5) and T2D human pancreatic islets (47–61 years old, n = 5) measured by qRT-PCR. P = 0.7377. Student’s t-test was used for the analysis of statistical significance.

DAPA treatment does not directly affect glucagon or insulin secretion in vitro

We next sought to clarify direct effects of therapeutic levels of DAPA on human islets in vitro using an acute exposure model (Fig. 2A). We selected 500 nM DAPA (within the therapeutic range of total DAPA) and performed experiments in albumin-containing media to mimic the clinical situation where a majority of DAPA is bound by albumin (3–5). We cultured human islets from 4 different islet preparations at basal glucose (5.6 mM) for 1 hour before transferring islets to low (3.3 mM) or high (16.7 mM) glucose, chosen to be reflective of biological fasted and fed states. In this system, insulin secretion from β cells was unchanged at either high or low glucose by DAPA treatment (Fig. 2B and 2C). Similarly, glucagon secretion from α cells was also unchanged at either low or high glucose (Fig. 2D and 2E). Thus, these data suggest acute DAPA exposure does not directly affect human islet cell function.

Figure 2.

DAPA treatment does not affect α cell or β cell function in human islets in vitro. A: Schematic design of acute DAPA treatment of human islets in vitro. After 1 hour with or without DAPA treatment in basal (5.6 mM) glucose, islets were transferred to low (3.3 mM) or high (16.7 mM) glucose for 1 hour. Low and high glucose media and islet extract was collected and analyzed for insulin and glucagon. B–E: Insulin and glucagon secretion in low or high glucose with or without DAPA. Each data point corresponds to 1 donor (with symbols matched across panels; n = 4 donors total) that is an average of multiple technical replicates. Student’s t-test was used for analysis of statistical significance; P > 0.05.

Acute DAPA treatment in vivo mildly increased glucagon secretion

To investigate the effects of DAPA on human islets in a dynamic in vivo environment, we used a human islet transplant model in the immunocompromised NOD.Cg-Prkdc^scidIl2rg^tm1WjlSz (NSG) mouse (18). First, we defined DAPA pharmacokinetics in the NSG mouse without transplanted human islets and found that 2 mg/kg daily oral dosing resulted in DAPA blood levels within the therapeutic window (Fig. 3A). Dapagliflozin was cleared from the blood within 24 hours, as similarly reported in human and did not accumulate in blood over several days of treatment (Fig. 3B and 3C). Further, after a 1-week treatment, blood glucose levels were reduced and mouse glucagon levels were elevated after stimulation, mirroring clinical data and indicating effective treatment (Fig. 3D–3H).

Figure 3.

Effect of DAPA in NSG mice without transplanted human islets. A–C: Pharmacokinetics of DAPA treatment by oral gavage in the NSG mice. A: Dose-dependent plasma C_max, with the dotted lines representing the clinical therapeutic dose range. B: Time course of DAPA absorption and clearance. C: Accumulation across repeated dosing. Red arrow in (A) indicates the dose we used in the study. D–J: NSG mice without human islets transplanted received DAPA treatment for 1 week. The mice were fasted for 6 hours and blood glucose (D, F) and mouse glucagon (E, G) were measured at 0’ or 15’ after the glucose challenge (2 g/kg glucose delivered i.p., stimulated, 15’). Y-axes have been set to match Fig. 4 and allow for comparison. Data also expressed as a ratio of stimulated and mouse glucagon to blood glucose (H). * P < 0.05, ** P < 0.01. Student’s t-test was used for the analysis of statistical significance.

Having established the appropriate dosing, we then transplanted human islets under the kidney capsule of NSG mice and repeated this across 4 different donor islet preparations. Following 2 weeks to allow engraftment, mice began treatment with DAPA (Fig. 4A). After 1 week of DAPA treatment, we assessed graft function by measuring serum human insulin levels using a human-specific insulin assay and also measured total serum glucagon in the fasted state and after glucose stimulation. We found that mice treated with DAPA had similar fasting blood glucose and fasted levels of human insulin and total glucagon (Fig. 4B–4D). While mouse and human glucagon cannot be distinguished due to 100% sequence identity, mouse glucagon levels from experiments without transplanted human islets are about one-third of that seen in those with transplants; thus, the majority of glucagon measured comes from the human islet grafts (Fig. 3E and 4D). After glucose stimulation, blood glucose was lower in the DAPA-treated group, indicating effective treatment (Fig. 4E). Human insulin was also lower in the DAPA-treated group as expected given the lower blood glucose (Fig. 4F). When normalized to the glucose level of the mouse, human insulin levels were unchanged with DAPA treatment (Fig. 4G). Additionally, total glucagon was elevated in the DAPA group (Fig. 4H and 4I), demonstrating that this human islet transplant model recreates the transient elevations in glucagon secretion seen when initiating DAPA treatment in patients.

Figure 4.

Effects of DAPA treatment on insulin and glucagon secretion in vivo. A: Schematic design of DAPA treatment on human islets in vivo. The mice were given DAPA (2 mg/kg/day) daily by oral gavage for 4 weeks, with graft function measured after 1 week of treatment (B–I) and 4 weeks of treatment (J–Q). Basal (6-hour fasted, 0’) and stimulated blood glucose (2 g/kg glucose delivered i.p., stimulated, 15’) (B, E), human insulin (C, F), and total glucagon (D, H) from the mice treated for 1 week with DAPA. Basal and stimulated blood glucose (J, M), human insulin (K, N), and total glucagon (L, P) after 4 weeks of treatment. Data expressed as a ratio of stimulated human insulin (G, O) and total glucagon-to-blood glucose (I, Q). Each dot represents a serum sample from a single mouse with transplanted human islets. These studies involve transplanted human islets from 4 independent donors to achieve the full data set (each human islet preparation was transplanted into multiple mice). * P < 0.05, *** P < 0.001. Student’s t-test was used for the analysis of statistical significance.

Chronic DAPA treatment in vivo did not affect the human islet graft function

To assess for longer-term effects of DAPA treatment, we continued treatment with DAPA for an additional 3 weeks (Fig. 4A). After 4 total weeks of DAPA treatment, there were no differences in fasted blood glucose, fasting total glucagon levels, or fasting human insulin levels (Fig. 4J–4L). After glucose stimulation, DAPA-treated mice again had lower blood glucose (Fig. 4M). Human insulin levels were lower in DAPA-treated animals but not different when corrected for the lower blood glucose (Fig. 4N–4O). Interestingly, glucagon levels were no longer elevated in DAPA-treated mice (Fig. 4P and 4Q). In total, these data suggest that prolonged DAPA treatment with clinically relevant dosing does not alter glucagon secretion from human α cells.

Human islet grafts show no effects of DAPA treatment

With an in vivo model, which recreates the clinical situation, we next analyzed the human islet grafts to investigate for direct effects of DAPA on human islets (Fig. 4A). The ability to harvest the graft and perform detailed molecular studies on human islets highlights the additional value provided by this model. We extracted total hormone from the grafts and found no difference in insulin or glucagon content with DAPA treatment (Fig. 5A and 5B), suggesting that glucagon and insulin synthesis and processing were not affected by DAPA treatment. To assess for ultrastructural alterations in the human α cell and β cell, we analyzed control and DAPA-treated grafts by electron microscopy but found equal numbers of both α cell and β cell granules (Fig. 5C–5F). Furthermore, α cell and β cell proliferation (Fig. 5G and 5H) and apoptosis (Fig. 5I and 5J) were very low and unchanged in both DAPA-treated and control groups, suggesting that DAPA does not impact endocrine cell mass. Finally, islet amyloid deposition, a marker of islet stress and dysfunction, was not changed with DAPA treatment (Fig. 5K and 5L). Together, these data do not support a direct role for clinically relevant levels of DAPA in islet cell function or health.

Figure 5.

DAPA does not alter insulin or glucagon content, human α cell or β cell granule number, proliferation, apoptosis, or amyloid deposition in human grafts. A and B: Insulin and glucagon content of human islet grafts (n = 5 grafts from 2 donor transplantations). In panels A and B, G–J, and L, unique symbols are used to distinguish grafts from different donors. C-F: Representative electron microscopy images of human transplanted β (C) and α cells (D) (scale bar: 1 µm), with or without DAPA treatment and quantification of granules in β cells (E) and α cells (F); each dot represents a section of a β or α cell from 2 islet grafts for each treatment group, which came from 1 donor. G and H: Percentage of Ki67-positive α cells and β cells (antibodies used: guinea pig ani-insulin, Dako; mouse antiglucagon, abcam; rabbit anti-Ki67, abcam). I and J: Percentage of TUNEL-positive α cells and β cells (antibodies used: guinea pig anti-insulin, Dako; rabbit anti-glucagon, cell signaling; TUNEL assay, Millipore-S7165). See methods for detailed antibody information. K: Representative images of amyloid deposits in human grafts labeled with insulin (green) and thioflavin S (amyloid, red) and quantification of the amyloid area (L). Scale bar: 50 µm; n = 8 grafts from 3 donor transplantations (G–J, L). Student’s t-test was used for analysis of statistical significance; P > 0.05.

Discussion

Inhibitors of SGLT2, including DAPA, are common medications in the treatment of T2D, with numerous benefits on whole body metabolism, but it has been unclear if these inhibitors act directly on the pancreatic islet, particularly α cells. To study this, we investigated the expression of SGLT2/SLC5A2 and the effects of DAPA on hormone secretion in human islets in vitro and in vivo after transplantation into mice. SLC5A2 transcript was multiple orders of magnitude lower than levels detected in the human kidney, and SGLT2 was undetectable in islets using the same antibody that readily detected expression in the kidney and has been used previously (7, 8). Further, neither SGLT2/SLC5A2 were altered in the T2D state. Functionally, acute DAPA treatment at therapeutic levels in vitro did not alter insulin or glucagon secretion at low or high glucose. In vivo, DAPA treatment led to lower blood glucose and proportionally lower human insulin levels. Total glucagon levels were elevated after 1 week of treatment but returned to normal levels after 4 weeks of treatment. Finally, molecular analysis of the human islet graft demonstrated that insulin and glucagon content, α cell and β cell granules, α cell and β cell proliferation and apoptosis, and amyloid deposition in human islet grafts were not changed with DAPA treatment.

This study differs from prior reports on DAPA’s effect on the islet in several ways. For in vitro studies, we treated human islets with 500 nM DAPA to remain within the therapeutic window. Use of different DAPA concentrations may explain the dissimilarities seen from other studies where effects could be due to off-target effects on ion channels or other glucose transporters (10). Consistent with this notion, a recently published dose-response curve for DAPA reported that stimulation on glucagon secretion in vitro is not seen until DAPA levels are significantly above the range of levels seen in humans treated with DAPA (8).

Furthermore, while static in vitro studies (including ours) can be helpful to isolate direct islet effects, they are not fully reflective of the clinical situation where islets are vascularized and dynamically respond to glucose and other nutrients, as well as endogenous hormones, which modulate both α cell and β cell function. In this study, investigating human islet function in a context where DAPA’s primary actions on the kidney change blood glucose allows for study in the appropriate context and appreciation of both direct and indirect actions. We found that DAPA had no effect in the fasted state when glucose levels are lower, and thus SGLT2 is less active in the kidney. After glucose stimulation, blood glucose levels become elevated, but mice with DAPA treatment showed lower blood glucose, reflective of the reduced ability of the kidney to resorb glucose. At this lower blood glucose level, we observed proportionally lower insulin and an elevation in glucagon with acute DAPA treatment. Because the effects of DAPA on insulin and glucagon secretion were not observed in vitro or in vivo in the fasted state, but only in vivo with glucose stimulation, these effects are likely indirect and related to the dynamic in vivo environment. This is consistent with a recent multivariate analysis that concluded that glucose changes were the main determinant of the changes in glucagon secretion seen with SGLT2 inhibition (36).

Finally, a major advantage of our model is the ability to harvest the human islet graft and perform molecular analyses to assess for effects of DAPA on the islet, studies that cannot be performed in clinical research. For example, a recent study in mice showed that luseoglifozin, also an SGLT2 inhibitor, can stimulate mouse β cell proliferation in certain contexts (37); however, our data indicates that this is unlikely to be the case for human β cells. Additionally, islet stressors, such as dyslipidemia, or treatment with immunosuppressive drugs can lead to evidence of poor islet health through the formation of amyloid (16, 29), but this was not observed with DAPA treatment. Overall, the lack of discernible evidence of these effects on the islet graft further argues that DAPA does not directly act on the human islet.

There are limitations to our study. Principally, in vivo one is unable to differentiate glucagon that originates from endogenous mouse α cells from that which comes from engrafted human α cells due to 100% sequence identity. To address this, we performed parallel studies on mice without engrafted human islets and found that glucagon levels were about one-third what they were in mice with engrafted human islets, suggesting that a majority of the detected glucagon originates from human α cells. Further, our in vitro studies are not hindered by this limitation and also show no direct effects on the human islet. Additionally, a recent study suggested SGLT2 inhibitors may act on δ cells within the islet if exogenous insulin is added (38). While our data does not suggest that SGLT2 is expressed in δ cells, we did not measure somatostatin secretion and thus do not know if DAPA has effects on δ cells in our system that did not include exogenous insulin.

In sum, we conclude that DAPA does not have direct effects on human islet function but, rather, the transient effects on human α cell function are secondary to the acute glucose lowering of DAPA through increased renal glucose excursion. Future studies should focus on how α cells sense acute glycemic changes and establish chronic glycemic set points as well as how these systems may be altered in the diabetic state.

Additional Information

Disclosure Summary: D.L.G. is a consultant and receives research support from The Jackson Laboratory. L.D.S. is employed by The Jackson Laboratory, the source of mice used in this study. All other authors have nothing to disclose.

Data Availability. All data generated or analyzed during this study are included in this published article or in the data repositories listed in References.