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. 2023 Jun 2:14:1156952.
doi: 10.3389/fendo.2023.1156952. eCollection 2023.

Association of epilepsy, anti-epileptic drugs (AEDs), and type 2 diabetes mellitus (T2DM): a population-based cohort retrospective study, impact of AEDs on T2DM-related molecular pathway, and via peroxisome proliferator-activated receptor γ transactivation

Ni Tien  1   2 , Tien-Yuan Wu  3   4 , Cheng-Li Lin  5   6 , Fang-Yi Chu  7 , Charles C N Wang  8   9 , Chung Y Hsu  10 , Fuu-Jen Tsai  11   12   13   14 , Yi-Jen Fang  15   16   17   18   19 , Yun-Ping Lim  7   12   20
Affiliations

Association of epilepsy, anti-epileptic drugs (AEDs), and type 2 diabetes mellitus (T2DM): a population-based cohort retrospective study, impact of AEDs on T2DM-related molecular pathway, and via peroxisome proliferator-activated receptor γ transactivation

Ni Tien et al. Front Endocrinol (Lausanne). .

Abstract

Introduction: A potential association between epilepsy and subsequent type 2 diabetes mellitus (T2DM) has emerged in recent studies. However, the association between epilepsy, anti-epileptic drugs (AEDs), and the risk of T2DM development remains controversial. We aimed to conduct a nationwide, population-based, retrospective, cohort study to evaluate this relationship.

Methods: We extracted data from the Taiwan Longitudinal Generation Tracking Database of patients with new-onset epilepsy and compared it with that of a comparison cohort of patients without epilepsy. A Cox proportional hazards regression model was used to analyze the difference in the risk of developing T2DM between the two cohorts. Next-generation RNA sequencing was used to characterize T2DM-related molecularchanges induced by AEDs and the T2DM-associated pathways they alter. The potential of AEDs to induce peroxisome proliferator-activated receptor γ (PPARγ) transactivation was also evaluated.

Results: After adjusting for comorbidities and confounding factors, the case group (N = 14,089) had a higher risk for T2DM than the control group (N = 14,089) [adjusted hazards ratio (aHR), 1.27]. Patients with epilepsy not treated with AEDs exhibited a significantly higher risk of T2DM (aHR, 1.70) than non-epileptic controls. In those treated with AEDs, the risk of developing T2DM was significantly lower than in those not treated (all aHR ≤ 0.60). However, an increase in the defined daily dose of phenytoin (PHE), but not of valproate (VPA), increased the risk of T2DM development (aHR, 2.28). Functional enrichment analysis of differentially expressed genes showed that compared to PHE, VPA induced multiple beneficial genes associated with glucose homeostasis. Among AEDs, VPA induced the specific transactivation of PPARγ.

Discussion: Our study shows epilepsy increases the risk of T2DM development, however, some AEDs such as VPA might yield a protective effect against it. Thus, screening blood glucose levels in patients with epilepsy is required to explore the specific role and impact of AEDs in the development of T2DM. Future in depth research on the possibility to repurpose VPA for the treatment of T2DM, will offer valuable insight regarding the relationship between epilepsy and T2DM.

Keywords: anti-epileptic drugs; epilepsy; next-generation RNA sequencing; peroxisome proliferator-activated receptor γ; type 2 diabetes mellitus.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Cumulative incidence of type 2 diabetes mellitus (T2DM) compared between with and without epilepsy using the Kaplan-Meier method.
Figure 2
Figure 2
Differential gene expression in control, phenytoin (P)-, and valproate (V)-treated cells. Heatmap of all differentially expressed genes in the control, phenytoin (PHE)-, and valproate (VPA)-treated samples.
Figure 3
Figure 3
Functional enrichment analysis of differential gene expressions (DEGs). (A) Top representatives of gene ontology biological process terms and (B) KEGG analysis of T2DM-related DEGs obtained using the samples treated with phenytoin (P) and valproate (V). The red box indicates gene expression: VPA > PHE; the green box indicates gene expression: PHE > VPA, P < 0.05 in (B).
Figure 4
Figure 4
Gene family expression (presented by FPKM) of T2DM-related pathways from RD cells treated with DMSO, PHE, and VPA. Cells were treated with DMSO, PHE (79 μM), and VPA (693.4 μM) for 72 h; mRNA was extracted; and RNA-Seq and DEG analyses were performed. (A) KEGG adipocytokine signaling pathway; (B) KEGG T2DM pathway; (C) BIOCARTA PPARA pathway; (D) Reactome regulation of lipid metabolism by PPARα; (E) KEGG PPAR signaling pathway. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 5
Figure 5
Viability of C2C12 and RD cells after exposure to anti-epileptic drugs (AEDs). (Left) C2C12 and (Right) RD cells were exposed to valproate (VPA, trough: 346.7 μM, peak: 693.4 μM), phenytoin (PHE, trough: 40 μM, peak: 79 μM), carbamazepine (CBZ, trough: 17 μM, peak: 51 μM), phenobarbital (PB, trough: 43 μM, peak: 172 μM), clonazepam (CLZ, trough: 0.06 μM, peak: 0.22 μM), and gabapentin (GBP, trough: 12 μM, peak: 117 μM) for 24 h. Cell viability was monitored using cellular acid phosphatase activity by using 4-nitrophenyl phosphate disodium salt as a substrate. The data are shown as the mean ± SE (error bars; n = 3).
Figure 6
Figure 6
Gene and protein expression of T2DM-related genes after treatment with AEDs. (A) C2C12 and (B, D, F) RD cells were treated for 24 h with AEDs (each with trough and peak concentrations); after treatments, total RNA was extracted, and the expression levels of GLUT4, IR, and PPARγ and β-actin as a control were analyzed using qRT-PCR. Values were normalized to the expression of β-actin, with the levels of DMSO-treated cells set at 1. Results are expressed as means ± SE (n = 3). ***P < 0.001 compared with control cells as indicated. (C, E) RD cells were treated for 24 h with VPA, CBZ, and PHE. Whole-cell extract was harvested, and the expression levels of (C) GLUT4 and (E) IR and the internal control (β-actin) were analyzed using western blot analysis. A representative blot is shown.
Figure 7
Figure 7
Intracellular glucose content in AEDs-treated cells. RD cells were treated with VPA, CBZ, and PHE for 24 h; cell lysates were extracted; and glucose contents were analyzed using a glucose colorimetric assay kit, according to manufacturer’s instructions. Values were normalized to individual protein contents, with the levels of DMSO-treated cells set at 1. Results are expressed as means ± SE (n = 3). ***P < 0.001 compared with control cells as indicated.
Figure 8
Figure 8
Transactivation of PPARγ–PPRE promoter activity and GLUT4 expression after AEDs treatment. Transient transcriptional assays of 3 × PPRE reporter activity were performed in HepG2 cells to determine the effects of (A) AEDs- and (B) VPA-, VPA + T0070907-mediated activation of PPARγ. HepG2 cells were co-transfected with a control vector (pcDNA3) or a PPARγ expression plasmid (pcDNA3-PPARγ) and a 3 × PPRE reporter plasmid. Transfected cells were subsequently exposed to AEDs for 24 h. Results are expressed as means ± SE (n = 4), with the levels of DMSO-treated cells set at 1. ***P < 0.001 compared with control cells as indicated. (C) RD cells were treated with VPA alone or in combination with T0070907; after treatments, total RNA was extracted, and the expression levels of GLUT4 and β-actin were analyzed using qRT-PCR. Values were normalized to the expression of β-actin, with the levels of DMSO-treated cells set at 1. Results are expressed as means ± SE (n = 3). ***P < 0.001 compared with control cells as indicated.
Figure 9
Figure 9
Knockdown of PPARs and mRNA expression of PPARs and GLUT4. (A, B) RD cells were transfected with 30 nM siControl or siPPARs for 48 h or treated with (C) VPA for additional 24 h. Total RNA was extracted, and the expression levels of PPARs, GLUT4, and β-actin were analyzed using qRT-PCR. Values were normalized to the expression of β-actin, with the levels of DMSO-treated cells set at 1. Results are expressed as means ± SE (n = 3). ***P < 0.001 compared with control cells as indicated.
Figure 10
Figure 10
Molecular docking analysis of valproate on PPARγ. Superimposition of docked compounds in the PPARγ-binding pocket of the 3D structure.
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This study was supported by the Ministry of Science and Technology, Taiwan, R.O.C. (MOST110-2320-B-039-016-MY3), China Medical University, Taichung, Taiwan (CMU111-MF-34), China Medical University Hospital, Taichung, Taiwan (DMR-111-105), partially supported by the Taiwan Ministry of Health and Welfare Clinical Trial Center (MOHW110-TDU-B-212-124004), Taichung Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation (TTCRD112-28), Show Chwan Memorial Hospital, Changhua, Taiwan (SRD-111024).