PART I. Early Neurological Deterioration
D. GLP-1 immunohistochemistry in ischemic rat brain tissue
Immunohistochemistry of GLP-1 receptors in rat brain shows that it localizes to the neurons when compared to DAPI staining and NeuN staining, confirming previous reports of GLP-1 receptor expression in neurons (Figure 4). Quantitative analysis of GLP-1 receptor density in penumbra region of cortex shows significantly higher values in evogliptin 1mg/kg, evogliptin 10mg/kg and metformin 200mg/kg + evogliptin 1mg/kg group compared to normal rats, but do not show difference when compared to vehicle group, or intergroup difference between medication groups.
GLP-1 receptor density in core region of cortex showed no statistical difference between groups (Figure 5). When GLP-1 staining density was compared at the striatum level, both core and penumbra show no difference between groups (Figure 6).
Figure 4. The NeuN and DAPI staining in brain cortex tissue at 1day after tMCAO localizes the GLP-1R staining to the neurons. (a,b,c) DAPI (blue) staining to localizing to nucleus. (d,e,f) NeuN (red) staining visualizing neuronal nucleus. (g,h,i) merged image of dapi and NeuN staining. (j,k,l) GLP1R (brown) staining shows co-localization to only neurons. (a,d,g,j) normal. (b,e,h,k) DM vehicle.
(c,f,i,l) Metformin 200mg/kg + evogliptin 1mg/kg. Magnification, 400x. Met,
metformin; Evo, evogliptin; tMCAO, transient middle cerebral artery occlusion;
GLP-1R, glycogen related peptide-1 receptor; DM, diabetes mellitus.
Figure 5. The GLP-1R expression in brain cortical tissue at 1day after tMCAO.
A. A MR image showing the histological localization in which 1-core at cortex level;
and 2-penumbra at cortex level is located. B. Cortex of normal rat. C. Cortex level, core of vehicle. D. Cortex level, core of co-administration group. E. Cortex level, penumbra of vehicle. F. Cortex level, core of co-administration group. G.
Quantitative analysis of GLP-1 receptor density in core region of cortex shows no statistical difference between groups. H. Quantitative analysis of GLP-1 receptor density in penumbra region of cortex shows significantly higher values in evogliptin 1mg/kg, evogliptin 10mg/kg and metformin 200mg/kg + evogliptin 1mg/kg group compared to normal rats, but do not show difference when compared to vehicle group (one-way ANOVA, Dunnett’s multiple comparison for host hoc tests, *p<0.05).
Magnification, 400x. GLP-1R, glycogen related peptide-1 receptor; Met, metformin;
Evo, evogliptin; DM, diabetes mellitus; tMCAO, transient middle cerebral artery occlusion.
Figure 6. The GLP-1R expression in striatal brain tissue at 1 day after tMCAO.
A. A MR image showing the histological localization in which 1-core at striatal level;
and 2-penumbra at striatal level is located. B. Striatum of normal rat. C. Striatal level, core of vehicle. D. Striatal level, core of co-administration group. E. Striatal level, penumbra of vehicle. F. Striatal level, penumbra of co-administration group. G.
Quantitative analysis of GLP-1 receptor density in core region of striatum shows no statistical difference between groups. H. Quantitative analysis of GLP-1 receptor density in penumbra region of striatum shows no significant difference between groups (one-way ANOVA, Dunnett’s multiple comparison for host hoc tests,
*p<0.05). Magnification, 400x. . GLP-1R, glycogen related peptide-1 receptor; Met, metformin; Evo, evogliptin; DM, diabetes mellitus; tMCAO, transient middle cerebral artery occlusion.
IV. DISCUSSION
The aim of this study was to access the protective effect of anti-diabetic medication in diabetic rats according to its main mechanism of glucose lowering effects; amelioration of insulin resistance or improvement in beta cell function. In clinical practice, neuroprotection against stroke in diabetic individuals could be practically achieved through optimal antidiabetic medication. In this regard, the results of our study show that in a STZ induced hyperglycemic rat model, co-administration of metformin and evogliptin pretreatment significantly reduced infarct volume 24hours after tMCAO. This was accompanied by lower blood glucose, lower HbA1c levels, and higher plasma insulin levels.
The primary mechanism for the observed reduction in infarct volume seems to be the maximized glycemic control. This was shown by the improvement in fasting glucose levels throughout the drug administration period. While the glucose levels were also significantly lower in the metformin group, glycated hemoglobin levels were only reduced in the co-administration group, suggesting further control of post-prandial glucose, and overall variations in glucose levels. Furthermore, the co-administration resulted in higher levels of plasma insulin. Clinical studies have consistently proven the efficacy of this combination therapy (Liu and Hong, 2014).
Cohort data show that combination of DPP-4 inhibitors and metformin tend to reduce major cardiovascular events and all-cause mortality compared with sulfonylurea-metformin combination (Yu et al., 2015). There is evidence that sulfonylurea-metformin increases GLP-1 secretion, suppresses activity of DPP-4 and upregulates the expression of GLP-1 receptor in pancreatic β-cells, while DPP-4 inhibitors have a favorable effect on insulin sensitivity in patients with T2DM, resulting in this synergistic effect (Liu and Hong, 2014). Further, there is preclinical evidence that evogliptin increases beta-cell replication and neogenesis in streptozotocin-induced diabetes (Cho et al., 2011),
and co-administration of metformin may have strengthened such protective effects.
Such synergy most likely resulted in reduction of infarct volume in our study.
Another important issue is the direct neuroprotective mechanisms of drugs.
While previous reports of neuroprotection against stroke have been reported for both DPP-4 inhibitors and metformin, a single agent did not result in reducing infarct volume in our study. The neuroprotective roles of metformin pretreatment have been reported in permanent MCAO models (Jiang et al., 2014) and global ischemia models (Ashabi et al., 2014) of mostly non-diabetic mice. While the neuroprotective role of metformin seems to be mediated by the AMPK pathway, its results have been conflicting (Li et al., 2010; Jiang et al., 2014).
Anti-stroke properties of GLP-1 analogs and DPP-4 inhibitors have been reported in non-diabetic (Briyal et al., 2012; Darsalia et al., 2013; Zhu et al., 2016) and type 2 diabetic (Darsalia et al., 2012; Darsalia et al., 2013) rat models of MCAO.
The reports of neuroprotection of GLP-1 analogs have been consistent (Briyal et al., 2012; Darsalia et al., 2012), and its action seems to be modulated through PI3K/Akt and MAPK pathways (Zhu et al., 2016). The anti-apoptotic role of insulin signaling is also known to be mediated by the PI3K (Ryu et al., 1999). In neurodegenerative disease such as Parkinson’s disease, the neuroprotective roles of GLP-1 analogues may lie in promotion of downstream insulin signaling (Santiago and Potashkin, 2013). It remains to be proven that co-administration of metformin and evogliptin results in improvement of insulin signaling in the brain, and results in reduced infarct volume.
As seen in previous studies (Darsalia et al., 2013), The GLP-1 receptor immunohistochemistry localized mostly to the neurons. While its signal density was not significantly higher in the evogliptin administration groups compared to vehicle, its density in penumbra region of cortex shows significantly higher values in evogliptin administration groups compared to normal rats. While this finding is
limited in interpretation, there may be compensatory upregulation of GLP-1 receptor signaling in ischemia. Further studies may be needed to address this issue.
In conclusion, in STZ induced diabetic rats, combined treatment of metformin and evogliptin improved beta cell function and offered neuroprotective effects against ischemic stroke. Anti-diabetic selection to address both insulin resistance and beta cell dysfunction may be a reasonable approach against acute ischemic stroke in DM.
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