The N-Methyl-D-Aspartate Receptor Antagonist Dextromethorphan Improves Glucose Homeostasis and Preserves Pancreatic Islets in NOD Mice

Abstract

For treatment of type 1 diabetes mellitus, a combination of immune-based interventions and medication to promote beta-cell survival and proliferation has been proposed. Dextromethorphan (DXM) is an N-methyl-D-aspartate receptor antagonist with a good safety profile, and to date, preclinical and clinical evidence for blood glucose-lowering and islet-cell-protective effects of DXM have only been provided for animals and individuals with type 2 diabetes mellitus. Here, we assessed the potential anti-diabetic effects of DXM in the non-obese diabetic mouse model of type 1 diabetes. More specifically, we showed that DXM treatment led to five-fold higher numbers of pancreatic islets and more than two-fold larger alpha- and beta-cell areas compared to untreated mice. Further, DXM treatment improved glucose homeostasis and reduced diabetes incidence by 50%. Our data highlight DXM as a novel candidate for adjunct treatment of preclinical or recent-onset type 1 diabetes.

Fig. 1

In the type 1 diabetes mouse model NOD, treatment with dextromethorphan delays diabetes onset and improves glucose tolerance. a : Schematic timeline of the experimental setup: 4 week-old female NOD mice continuously received either normal drinking water (control group) or 3 g/l dextromethorphan via drinking water (DXM group); mice were euthanized at the age of 10 weeks (prediabetic mice: green), at diabetes onset, defined as a random blood glucose concentration ≥250 mg/dl on two consecutive days (diabetic mice: red), or following a maximum of 26 weeks of DXM treatment at the age of 30 weeks (non-diabetic mice: blue). b : Kaplan–Meier curve indicating the incidence of diabetes in DXM-treated NOD mice compared to control NOD mice (n=12 mice per treatment group). c : Blood glucose concentrations of 10–11 week-old female NOD mice during an intraperitoneal glucose tolerance test (1.5 mg g ^-1 body weight) either treated or untreated with DXM via the drinking water starting at the age of 4 weeks (n=11 control and n=12 DXM-treated mice). d : Random blood glucose concentrations of either DXM-treated or control NOD mice at diabetes onset (n=8 control and n=4 DXM-treated mice). Statistical significance determined by Mantel–Cox log-rank test, p=0.0915 ( b ), multiple Student’s t -test ( c ), and unpaired Student’s t -test ( d ). Data in ( c ) and ( d ) are presented as mean ± SD ( c ) and mean ± SEM ( d ).

Fig. 2

DXM treatment increases islet numbers, as well as beta- and alpha-cell areas in NOD mice. a : Representative fluorescence microscopy images of pancreatic sections from 30 week-old non-diabetic NOD mice, either untreated (control) or continuously treated with DXM over 26 weeks (DXM); blue: DAPI; red: insulin; scale bar: 1000 μm (≥16 cross-sections from n ≥4 mice per treatment group). ( b ) Islet numbers, ( c ) beta- and ( d ) alpha-cell areas within pancreatic sections of 30 week-old non-diabetic NOD mice treated as described in ( a ) (≥16 sections from n ≥4 mice per treatment group). Statistical significance determined by unpaired Student’s t -test. Data are shown as mean ± SEM.

Fig. 3

DXM treatment numerically reduces the number of apoptotic islet-cells but has no effect on islet-cell proliferation in NOD mice. a : Islet numbers per total pancreatic nuclei area in 10 week-old prediabetic, 12–30 week-old diabetic and 30 week-old non-diabetic NOD mice, either untreated (control) or continuously treated with DXM since 4 weeks old (DXM), dashed lines indicate separate cohorts (n ≥4 mice per treatment group). b : Representative LSM images of pancreatic islets from 10 week-old prediabetic NOD mice treated as described in ( a ); blue: DAPI; red: insulin; grey: cleaved caspase-3 (≥47 islets per mouse from n=6 mice per treatment group). c : Cleaved caspase-3-positive area per islet area (≥47 islets per mouse from n=6 mice per treatment group). d : Representative LSM images of pancreatic islets from mice treated as described in ( b ); blue: DAPI; red: insulin; green: Ki67; arrow heads: Ki67-positive cell nuclei within pancreatic islet (≥38 islets per mouse from n=6 mice per treatment group). e : Number of Ki67-positive cells per islet area (≥38 islets per mouse from n=6 mice per treatment group). Scale bars: 50 μm. Statistical significance determined by unpaired Student’s t -test comparing treatment groups. Data are shown as mean ± SEM.

Fig. 4

DXM treatment reduces the number of infiltrating T-cells, particularly CD8-positive T-lymphocytes. a : Representative fluorescence microscopy images of pancreatic islets from 10 week-old prediabetic NOD mice either untreated (control) or DXM-treated for 6 weeks (DXM); blue: DAPI; red: insulin; green: CD45; scale bar: 100 μm (≥60 islets per mouse from n=6 mice per treatment group). b : Insulitis score and ( c ) CD45-positive area per islet area of the same mice (≥60 islets per mouse from n=6 mice per treatment group). d : Representative LSM images of pancreatic islets from mice treated as described in ( a ); blue: DAPI; green: insulin; red: CD8; grey: CD4; scale bar: 50 μm (≥ 40 islets per mouse from n=6 mice per treatment group). e : CD4- and ( f ) CD8-positive area per total pancreatic nuclei area of the same mice (≥40 islets per mouse from n=6 mice per treatment group). Statistical significance determined by unpaired Student’s t -test comparing treatment groups. Data in ( c ), ( e ) and ( f ) are shown as mean±SEM.

Fig. 5

DXO treatment decreases the expression of chemokines in pancreatic islets. a , b : Relative mRNA expression of Cx3cl1 , Cxcl1 , Cxcl2 , Cxcl16 , and Ccl2 normalized to the expression of Rplp0 of either untreated islets compared to islets treated for 24 hours with 1 μM DXO ( a ) or of islets treated for 24 hours with cytokines (IFNγ, IL-1β, TNFα) in the presence or absence of 1 μM DXO ( b ) (n=5 independent experiments). c : Concentration of CCL2 in supernatants of islets treated as described in ( b ) (n=5 per condition). Statistical significance determined by paired ( a , b ) or unpaired ( c ) Student’s t -test comparing treatments with or without DXO.