SGLT2 and DPP4 inhibitors improve Alzheimer's disease–like pathology and cognitive function through distinct mechanisms in a T2D–AD mouse model

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Biomedicine & Pharmacotherapy 168 (2023) 115755

Available online 21 October 2023

SGLT2 and DPP4 inhibitors improve Alzheimer ’ s disease – like pathology and cognitive function through distinct mechanisms in a T2D – AD

mouse model

A Young Sim

^a^,^b^,¹

, Da Hyun Choi

^c^,^d^,^e^,¹

, Jong Youl Kim

^a

, Eun Ran Kim

^c

, A-ra Goh

^a^,^b

, Yong-ho Lee

^c^,^d^,^e^,^*

, Jong Eun Lee

^a^,^b^,^f^,^**

aDepartment of Anatomy, Yonsei University College of Medicine, Seoul, Republic of Korea

bGraduate School of Medical Science, Brain Korea 21 Project, Yonsei University College of Medicine, Seoul, Republic of Korea

cDepartment of Internal Medicine, Yonsei University College of Medicine, Seoul, Republic of Korea

dDepartment of Systems Biology, Glycosylation Network Research Center, Yonsei University, Seoul, Republic of Korea

eInterdisciplinary Program of Integrated OMICS for Biomedical Science, Yonsei University, Seoul, Republic of Korea

fBrain Research Institute, Yonsei University College of Medicine, Seoul, Republic of Korea

A R T I C L E I N F O Keywords:

Alzheimer’s disease Type 2 diabetes mellitus

Sodium–glucose cotransporter-2 inhibitor Dipeptidyl peptidase-4 inhibitor Hyperphosphorylated tau Amyloid β

A B S T R A C T

Alzheimer’s disease (AD) and type 2 diabetes mellitus (T2D) share common features, including insulin resistance.

Brain insulin resistance has been implicated as a key factor in the pathogenesis of AD. Recent studies have demonstrated that anti-diabetic drugs sodium–glucose cotransporter-2 inhibitor (SGLT2-i) and dipeptidyl peptidase-4 inhibitor (DPP4-i) improve insulin sensitivity and provide neuroprotection. However, the effects of these two inhibitors on the brain metabolism and insulin resistance remain uninvestigated. We developed a T2D–AD mouse model using a high-fat diet (HFD) for 19 weeks along with a single dose of streptozotocin (100 mg/kg, intraperitoneally) at the fourth week of HFD initiation. Subsequently, the animals were treated with SGLT2-i (empagliflozin, 25 mg/kg/day orally [p.o.]) and DPP4-i (sitagliptin, 100 mg/kg/day p.o.) for 7 weeks.

Subsequently, behavioral tests were performed, and the expression of insulin signaling, AD-related, and other signaling pathway proteins in the brain were examined. T2D–AD mice not only showed increased blood glucose levels and body weight but also insulin resistance. SGLT2-i and DPP4-i effectively ameliorated insulin sensitivity and reduced body weight in these mice. Furthermore, SGLT2-i and DPP4-i significantly improved hippocampal- dependent learning, memory, and cognitive functions in the T2D–AD mouse model. Interestingly, SGLT2-i and DPP4-i reduced the hyperphosphorylated tau (pTau) levels and amyloid β (Aβ) accumulation and enhanced brain insulin signaling. SGLT2-i reduced pTau accumulation through the angiotensin converting enzyme-2/angiotensin (1−7)/ mitochondrial assembly receptor axis, whereas DPP4-i reduced Aβ accumulation by increasing insulin-

Abbreviations: AD, Alzheimer’s disease; Aβ, Amyloid β; pTau, Hyperphosphorylated tau; NFTs, Neurofibrillary tangles; T2D, Type 2 diabetes mellitus; SGLT2-i, Sodium–glucose cotransporter-2 inhibitor; BG, Body glucose; DPP4-i, Dipeptidyl peptidase-4 inhibitor; GLP-1, Glucagon-like peptide-1; HFD, High-fat diet; STZ, Streptozotocin; ND, Normal chow diet; i.p., Intraperitoneally; BW, Body weight; p.o., Per os (orally); ITT, Insulin tolerance test; GTT, Glucose tolerance test; HOMA- IR, Homeostatic model assessment of insulin resistance; NORT, Novel object recognition test; MWM, Morris water maze; WB, Western blotting; IHC, Immunohis- tochemistry; PBS, Phosphate-buffered saline; CTX, Cortex; HIPP, Hippocampus; PFA, Paraformaldehyde; BSA, Bovine serum albumin; IRS1, Insulin receptor substrate 1; pIRS1, phosphorylated insulin receptor substrate 1; Akt, Protein kinase B; pAkt, phosphorylated protein kinase B; GSK3β, Glycogen synthase kinase 3β; pGSK3β, phosphorylated glycogen synthase kinase 3β; ACE2, Angiotensin-converting enzyme 2; Ang1–7, Angiotensin (1−7); MasR, Mitochondrial assembly receptor; IDE, Insulin-degrading enzyme; TBS, Tris-buffered saline; H&E, Hematoxylin and eosin; qPCR, Quantitative polymerase chain reaction; RAS, Renin–angiotensin system;

ACE1, Angiotensin-converting enzyme 1; Ang I, Angiotensin I (1−10); Ang II, Angiotensin II (1−8); AT1R, Angiotensin II type I receptor; AT2R, Angiotensin II type II receptor; fAD, Familial AD; sAD, Sporadic AD.

* Corresponding author at: Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Republic of Korea.

** Corresponding author at: Department of Anatomy, Yonsei University College of Medicine, Seoul, Republic of Korea.

E-mail addresses: [email protected] (A.Y. Sim), [email protected] (D.H. Choi), [email protected] (J.Y. Kim), [email protected] (A.-r. Goh), [email protected] (Y.-h. Lee), [email protected] (J.E. Lee).

1 These authors contributed equally to this work.

Contents lists available at ScienceDirect

Biomedicine & Pharmacotherapy

journal homepage: www.elsevier.com/locate/biopha

https://doi.org/10.1016/j.biopha.2023.115755

Received 16 August 2023; Received in revised form 17 October 2023; Accepted 17 October 2023

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Biomedicine & Pharmacotherapy 168 (2023) 115755 degrading enzyme levels. These findings suggest that SGLT2-i and DPP4-i prevent AD-like pathology and cognitive dysfunction in T2D mice potentially through affecting brain insulin signaling via different mechanisms.

1. Introduction

Alzheimer’s disease (AD) is the leading cause of dementia, and its prevalence is expected to increase as the population ages [1]. The hallmark clinical phenotype of AD is a progressive decline in two or more cognitive domains [2]. Also, the common pathological features of AD are abnormal accumulation of amyloid β (Aβ) plaques, hyperphosphorylated tau (pTau) protein, and neurofibrillary tangles (NFTs) in the brain [3].

Type 2 diabetes mellitus (T2D) is a chronic metabolic disorder characterized by elevated blood glucose levels and caused by insulin resistance in peripheral organs and relative insulin deficiency in pancreatic β cells. Recently, ample evidence suggests that mechanisms such as insulin resistance, oxidative stress, mitochondrial dysfunction, and neuroinflammation, exist in both AD and T2D [4,5]. Therefore, there has been a growing interest in the link between AD and T2D, and AD may represent a brain-specific form of diabetes known as type 3 diabetes [6].

Accordingly, several reports have shown that medications for T2D improve brain function when metabolic dementia occurs [7]. In addition to metformin and thiazolidinedione, which have been targets of numerous studies on dementia, there is ongoing research into the neuroprotective effects of sodium–glucose cotransporter-2 inhibitors (SGLT2-i) and dipeptidyl peptidase-4 inhibitors (DPP4-i), which are widely prescribed in patients with T2D [8].

SGLT2-i reduces blood glucose levels in an insulin independent manner [9]. SGLT2-i improves insulin resistance with reduction of weights and blood pressure by increasing the excretion of glucose and sodium in the urine and it contributes to the preservation of β-cell function by reducing glucose toxicity [10]. As SGLT2-i can alleviate inflammatory conditions through the elevation of ketone production in humans [11] and restore energy utilization and autophagy flux [12,13], these pleiotropic actions may lead to beneficial effects of SGLT2-i on dementia.

DPP4-i improves glucose metabolism by enhancing the blood con- centration of active glucagon-like peptide-1 (GLP-1), a potent insulino- tropic hormone, by inhibiting DPP4 enzyme activities [14]. DPP4-i has a low risk of hypoglycemia and weight gain and can improve glucose fluctuations which is linked to various chronic vascular complications [15]. Besides its glucose-lowering effects, GLP-1 is known to enhance cerebral glucose metabolism in humans with AD [16], and its agonists are currently undergoing clinical trials in individuals with AD [17].

Furthermore, considering that DPP4 is a cell surface glycoprotein extensively expressed in various cells including vascular epithelial cells, immune cells, and resident cells in the brain such as microglia, neurons, and astrocytes [18], its inhibition by DPP4-i may exert a protective function against dementia. Several studies have reported that SGLT2-i and DPP4-i possess neuroprotective properties, as they can modulate apoptosis, inhibit the phosphorylation of toxic proteins and neuroinflammation, and enhance synaptic plasticity in AD models [19,20].

However, their therapeutic roles in AD and their mechanisms have not been fully elucidated yet.

Previously, we and other researchers have established a T2D–AD mouse model using a high-fat diet (HFD) and streptozotocin (STZ), demonstrating the deterioration of both insulin resistance and cognitive decline in this model [21–25]. However, effects of SGLT2-i and DPP4-i and their mechanisms regulating insulin resistance and the accumulation of Aβ and pTau in the brain of the T2D–AD mouse model have not yet been investigated. Herein, we aimed to elucidate these mechanisms, hypothesizing that SGLT2-i or DPP4-i treatment may confer protective effects against Aβ and pTau accumulation via different pathways in

T2D–AD mice.

2. Materials and methods 2.1. Animals

Male C57BL/6 mice (8 weeks old) were purchased from Central Lab Animal Inc. (Seocho, Seoul, Republic of Korea) and housed under a 12-h light/dark cycle with food and water ad libitum. After a week of accli- matization, the mice were randomly divided into two groups that were fed two different diets for 19 weeks: normal chow diet (ND; 13.1 % kcal fat) and a HFD (60 % kcal fat, Research Diet, New Brunswick, NJ, USA).

The mice fed a HFD were injected once with a low dose of STZ (100 mg/

kg, intraperitoneally [i.p.], Sigma-Aldrich, St. Louis, MO, USA) dissolved in citrate buffer (pH 4.4) at week 4 to establish T2D by inducing partial insulin deficiency [26]. All animal experiments were performed in accordance with the guidelines and approved by the Institutional Animal Care and Yonsei Laboratory Animal Research Center (IACUC approval no. 20220045).

2.2. Treatment

At week 12, the mice with fasting blood glucose level (BG) >

250–300 mg/dL, body weight (BW) >45 g, and impaired insulin and glucose tolerance were classified as T2D mice and randomly assigned to one of the following three groups: vehicle (0.5 % carboxymethylcellu- lose, Sigma-Aldrich), SGLT2-i (empagliflozin; 25 mg/kg/day, orally [p.

o.]), or DPP4-i (sitagliptin; 100 mg/kg/day, p.o.) (Fig. S1).

2.3. Measurement of blood glucose level and body weight

The mice fasted for 4 h before their BG levels and BW were recorded every week. Blood samples were collected by cutting off the tail tip, and BG was measured using a glucometer (Accu-Chek, Roche Diagnostics Corp., Indianapolis, IN, USA).

2.4. Insulin tolerance test (ITT) and glucose tolerance test (GTT) For the ITT, the mice were moved to an empty cage and fasted for 4 h before the test, following which insulin (0.75 U/kg) dissolved in saline was injected i.p. Blood was collected from the tail tip, and BG levels were measured using a glucometer (Accu-Chek) at 0, 15, 30, 60, 90, and 120 min after the injection. The GTT was performed 2 days after the ITT.

Mice were moved to another empty cage and fasted for 6 h before the test, following which glucose (2 g/kg) dissolved in saline was injected i.

p. Blood was collected from the tail tip, and BG levels were measured using a glucometer (Accu-Chek) at 0, 15, 30, 60, 90, and 120 min after the injection.

2.5. Measurement of serum insulin levels and insulin resistance

Fasting serum insulin levels were measured using a mouse insulin enzyme-linked immunosorbent assay kit (Morinaga Institute of Biolog- ical Science, Yokohama, Japan), according to the manufacturer’s in- structions. Insulin sensitivity was evaluated using the following index:

homeostatic model assessment of insulin resistance (HOMA-IR) = [(fasting serum insulin [µU/mL] ×fasting serum glucose [mmol/L])/

22.5].

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2.6. Novel object recognition test (NORT)

The NORT is a commonly used behavioral test for evaluating learning and memory functions in mice. The NORT was performed in a square arena (40 × 40 × 40 cm) over 3 days and consisted of habituation, training, and testing phases. During the habituation, the mice were removed from their home cages and placed in an empty rectan- gular box for 5 min. During the training session, the mice were allowed to explore two identical objects placed in opposite quadrants of the arena for 5 min. On the test day, one of the objects used during the training was replaced with a novel object. The mice were allowed to explore the two objects freely for 10 min. The arena and the objects were washed with 70 % ethanol to minimize olfactory cues.

2.7. Morris water maze (MWM)

The MWM was performed to evaluate hippocampal-dependent learning, including the acquisition of spatial memory. The MWM was conducted in a circular tank (100 cm, diameter and 35 cm, depth) filled with water (22–25 ^◦C) within a soundproof and dimly lit room. On the first day, the mice were habituated to the water. Four positions were artificially specified within the pool: north (N), south (S), east (E), and west (W). The mice were trained four times per day for 4 consecutive days. The mice were placed in the pool facing the wall with the starting location at N, S, E, and W each day, and the trial was completed as soon as mice found the platform or when 60 s had elapsed. On the sixth day, the mice were allowed to swim freely in the pool without the platform for 90 s to conduct a probe test in the same environment. The latency to find the platform and the path taken by the animals were analyzed.

2.8. Brain sample preparation

The mice were randomly selected for either western blotting (WB) or immunohistochemistry (IHC). For WB, the mice were transcardially perfused with 0.1 mol/L of phosphate-buffered saline (PBS). The whole brain was dissected, and the cortex (CTX) and hippocampus (HIPP) were isolated and frozen in liquid nitrogen, followed by storage at − 80 ^◦C. For IHC, the mice were transcardially perfused with PBS and 4 % paraformaldehyde (PFA) in PBS. The brains were removed and incubated in 4 % PFA for 24 h at 4 ^◦C. The fixed brain tissues were placed in 30 % sucrose for 1–3 days and stored at − 80 ^◦C.

2.9. Western blotting (WB)

The CTX and HIPP tissues were lysed in ice-cold radio- immunoprecipitation assay buffer (Sigma-Aldrich) containing 1 mM phenylmethylsulfonyl fluoride using a homogenizer. Protein concen- tration was determined using the Bicinchoninic acid method. The isolated proteins (50 µg) were separated using 10 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted onto a polyvinylidene difluoride membrane (Millipore, Burlington, MA, USA).

The membrane was incubated for 1 h with 5 % bovine serum albumin (BSA; GenDEPOT, Barker, TX, USA) at room temperature and then incubated overnight with primary antibodies at 4 ^◦C. The following antibodies were used: anti-insulin receptor substrate 1 (IRS1; Abcam, Boston, MA, USA; Cell Signaling Technology, Danvers, MA, USA), anti- phospho-IRS1 (pIRS1; Tyr632; Santa Cruz Biotechnology, Dallas, TX, USA and Ser307; Cell Signaling Technology), anti-protein kinase B (Akt;

Cell Signaling Technology), anti-phospho-Akt (pAkt; Ser473; Invitrogen, Waltham, MA, USA and Cell Signaling Technology), anti-glycogen synthase kinase 3β (GSK3β; Abcam), anti-phospho-GSK3β (pGSK3β; Ser9;

Abcam), anti-Tau (Abcam), anti-pTau (Thr231; Abcam), anti-Aβ (Thermo Fisher Scientific, Waltham, MA, USA), anti-angiotensin- converting enzyme 2 (ACE2; Abcam), anti-angiotensin (1− 7)/mitochondrial assembly receptor [Ang(1− 7)/MasR; Alomone Labs, Jerusa- lem, Israel], anti–insulin-degrading enzyme (IDE; Abcam), and anti-β

actin (Abcam and Sigma-Aldrich). After washing thrice with 0.5 % tris- buffered saline (TBS), the membrane was incubated with goat anti- mouse (Invitrogen), goat anti-rabbit (Invitrogen), or rabbit anti-goat (Invitrogen) HPR-conjugated IgG (H+L) at room temperature for 2 h.

After washing thrice with 0.5 % TBS, signals were detected using the ECL reagent (GenDEPOT) and visualized using LAS-4000 (Fujifilm Life Science USA, Stamford, CT, USA).

2.10. Immunohistochemistry (IHC)

Frozen tissues were sliced into 14-μm thick sections which were then air-dried at room temperature. The sections were fixed in cold methanol for 15 min at − 20 ^◦C and blocked using 5 % BSA (GenDEPOT) for 1 h at room temperature to block nonspecific binding sites. The sections were incubated with the following primary antibodies overnight at 4 ^◦C: anti- IRS1 (Abcam), anti-pIRS1 (Tyr632; Santa Cruz Biotechnology), anti-Akt (Cell Signaling Technology), anti-pAkt (Ser473; Invitrogen), anti-GSK3β (Abcam), anti-pGSK3β (Ser9; Abcam), anti-Tau (Thermo Fisher Scien- tific), anti-pTau (Thr231; Abcam), anti-Aβ (Thermo Fisher Scientific), anti-ACE2 (Abcam), anti-Ang(1− 7)/MasR (Alomone Labs), anti-IDE (Abcam), and anti-microtubule-associated protein 2 (MAP2; Abcam).

After incubation, the slides were washed in PBS thrice and incubated with the appropriate secondary antibodies for 2 h at room temperature in a dark chamber. The following secondary antibodies were used: Alexa Fluor 488-conjugated IgG (Abcam) and rhodamine-conjugated IgG (Millipore). After staining with DAPI (Thermo Fisher Scientific), images were acquired using the LSM710 microscope (Carl Zeiss, Thornwood, NY, USA).

2.11. Hematoxylin and eosin (H&E) staining of the liver and pancreas The mice liver and pancreas were collected, washed in PBS, and fixed in 10 % PFA for 24 h. The tissues were then dehydrated and embedded in paraffin. The samples were sectioned into 4-μm thick slices and stained using H&E. For a morphometric analysis, the pancreas was sectioned as previously described, and islet morphology was analyzed using the ImageJ Fiji software (National Institutes of Health, Bethesda, MD, USA).

The islet size (μm²) was measured by dividing the total islet area by the total number of islets in at least three sections per animal. The number of islets per square micrometer of the section was counted.

2.12. Quantitative polymerase chain reaction (qPCR)

Total RNA was isolated from frozen liver tissues using the TRIzol reagent (Invitrogen) and reverse transcribed into cDNA using the High- Capacity cDNA Reverse Transcription kit (Applied Biosystems, Wal- tham, MA, USA), according to the manufacturer’s protocol. The qPCR was conducted using the Power SYBR® Green PCR Master Mix (Applied Biosystems) with specific primers (Supplementary Table S1). Relative mRNA expression levels were analyzed using the ΔΔCt method and the Step One software version 2.2.2 (Applied Biosystems). We used 18 S rRNA as an internal normalizer.

2.13. Statistical analysis

All statistical tests were performed using GraphPad Prism version 8 (Dotmatics, San Diego, CA, USA). All values are expressed as the mean

±standard error of the mean. Data were compared using a one-way analysis of variance with Bonferroni’s multiple comparison test. Stu- dent’s t-test was used to compare the means of the two groups. P <0.05 was considered statistically significant (*P <0.05, **P <0.01, ***P <

0.001, and ****P <0.0001).

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Biomedicine & Pharmacotherapy 168 (2023) 115755

3. Results

3.1. SGLT2-i and DPP4-i exert metabolic benefits in C57BL/6 mice with insulin resistance and T2D

The T2D–AD mouse model developed using a HFD and STZ is shown in Fig. 1A. During the 12 weeks of HFD, BG and BW increased;

conversely, no change was observed in the ND group. SGLT2-i and DPP4-i treatment for 7 weeks reduced BG and BW of the T2D–AD mouse model (Fig. 1B, C). Furthermore, the ITT results demonstrated the development of insulin resistance in the T2D–AD mouse model, with SGLT2-i and DPP4-i treatment improving insulin resistance in these mice. Similarly, impaired glucose tolerance was observed at week 12 of

a HFD, with SGLT2-i and DPP4-i treatment improving glucose tolerance in these mice (Fig. 1D, E). Consistent with our ITT results, the HOMA-IR, which reflects systemic insulin resistance, improved in the SGLT2-i and DPP4-i groups (Fig. 1F). Hyperinsulinemia was observed in the vehicle group, whereas lower insulin levels were detected in the SGLT2-i and DPP4-i groups (Fig. 1G). Regarding pancreatic islet morphology, the area of the islet mass was significantly decreased in all groups except for the ND group, implying a late T2D stage. However, no significant change was observed in the islet area in the SGLT2-i and DPP4-i groups (Fig. S2).

Fig. 1. Effects of SGLT2-i and DPP4-i ↱on peripheral insulin resistance. (A) Comparison of ND and T2D group mice at 12 weeks. (B) BG and (C) BW changes were monitored weekly. (D) ITT and (E) GTT were performed at weeks 12 and 19. n=8/group, ^#,$P<0.05, ^##,$$P<0.01, ^###,$$$P<0.001, ^####,$$$$P<0.0001 compared with the vehicle and the SGLT2-i or DPP4-I groups. (F) HOMA-IR represents insulin sensitivity index. (G) Serum insulin levels. n=3/group, *P<0.05,

**P<0.005, ***P<0.001.

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3.2. SGLT2-i and DPP4-i restore brain insulin signaling via the IRS1/Akt/

GSK3β pathway

Insulin signaling regulates the downstream IRS1/Akt/GSK3β [27].

We isolated the CTX and HIPP and performed the WB and IHC analyses.

We confirmed that pIRS1 is associated with insulin resistance, whereas pAkt and pGSK3β are associated with insulin sensitivity. The signal intensity of pIRS1 (Tyr632) associated with insulin resistance was distinct and more intense in the vehicle group than in the SGLT2-i and DPP4-i groups (Fig. 2A). In addition, the expression level of pIRS1 and the pIRS1/total IRS1 ratio was significantly higher in the CTX and HIPP of the vehicle group than that for the SGLT2-i and DPP4-i groups (Fig. 2B, C). pAkt (Ser473) levels associated with insulin sensitivity in the CTX and HIPP of the vehicle group were significantly lower than those in the CTX and HIPP of the other groups (Fig. 2D). In the CTX and HIPP of the SGLT2-i and DPP4-i groups, pAkt expression level and the pAkt/total Akt ratio were significantly higher than those in the CTX and HIPP of the vehicle group (Fig. 2E, F). Furthermore, compared with pGSK3β (Ser9) associated with insulin sensitivity, GSK3β signal was more intense in the vehicle group than in the other groups (Fig. 2G). Especially, the pGSK3β/total GSK3β ratio in the HIPP of the SGLT2-i group was significantly higher than that in the HIPP of the vehicle group (Fig. 2H, I).

3.3. Improving brain insulin signaling reduces pTau and Aβ accumulation in the brain

Regulation of insulin signaling pathway through IRS1/Akt/GSK3β can affect tau phosphorylation in the brain. GSK3β phosphorylates tau at most serine and threonine residues and phosphorylation at Thr231 is

particularly increased in AD [28,29]. Furthermore, GSK3β regulates Aβ accumulation and promotes Aβ-mediated neuronal cell death [30]. To explore the neuroprotective effects of SGLT2-i and DPP4-i, we evaluated tau hyperphosphorylation and Aβ accumulation in the CTX and HIPP of the mice from all groups. A weaker signal of pTau (Thr231) was observed in the SGLT2-i and DPP4-i groups than in the vehicle group (Fig. 3A). Moreover, the levels of pTau were higher in the CTX and HIPP of the vehicle group than that for the other groups. pTau expression was significantly lower in the HIPP of the SGLT2-i and DPP4-i groups than in the HIPP of the vehicle group (Fig. 3B). The pTau/total tau ratio was significantly lower in the SGLT2-i group than in the DPP4-i group (Fig. 3C). Furthermore, Aβ fluorescence intensity was highest in the CTX and HIPP of the vehicle group than in the other groups. The expression of MAP2, a neuron-specific marker, was lower in the vehicle group than in the other groups. Aβ accumulation was lower whereas MAP2 expression was higher in the ND, SGLT2-i, and DPP4-i groups than in the vehicle group (Fig. 3D). The WB findings were congruent with the IHC findings (Fig. 3E). Particularly, the Aβ/β actin ratio was significantly more reduced in the CTX and HIPP of the DPP4-i group than in those of the vehicle group, and this decrease was more pronounced compared with that in the SGLT2-i group (Fig. 3F). Taken together, these results suggest that SGLT2-i and DPP4-i regulate tau phosphorylation and Aβ accumulation by different mechanisms in the brain.

3.4. SGLT2-i reduces pTau levels by activating ACE2/MasR, whereas DPP4-i decreases Aβ accumulation by increasing IDE levels

In the renin–angiotensin system (RAS), angiotensin-converting enzyme 1 (ACE1) converts angiotensin I (1− 10) [Ang I] to angiotensin II (1− 8) [Ang II], while ACE2 cleaves Ang I and Ang II into Ang(1− 7), Fig. 2. Improved brain insulin signaling following SGLT2-i and DPP4-i treatment. (A) IHC and (B) WB analyses for pIRS1 (Tyr632) and IRS1 expression in the CTX and HIPP. Scale bar =20µm. (C) Densitometric evaluation of the WB. (D) IHC and (E) WB analyses of pAkt (Ser473) and Akt expression in the CTX and HIPP. Scale bar =20µm. (F) Densitometric evaluation of the WB. (G) IHC and (H) WB analyses of pGSK3β (Ser9) and GSK3β expression in the CTX and HIPP. Scale bar =20µm.

(I) Densitometric evaluation of the WB. β actin served as a loading control. n=3/group, *P<0.05, **P<0.005.

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which interacts with MasR to activate insulin signaling (Fig. 4A) [31]. It is known that SGLT2-i modulates RAS system and activates MasR [32].

In the kidney of the T2D–AD mouse model, ACE1 and Ang II type I receptor (AT1R) expression were significantly decreased in the SGLT2-i and DPP4-i groups while ACE2 expression was marginally higher in the SGLT2-i group (Fig. 4B-D). The expression of Ang II type II receptor (AT2R) and MasR in the renal were significantly increased in the SGLT2-i group (Fig. 4E, F).

The IHC analysis confirmed that MasR expression was significantly higher in the CTX and HIPP of the ND and SGLT2-i groups than in those of the vehicle group (Fig. 4G). Conversely, ACE2 expression was lower in the vehicle group (Fig. 4H). The ACE2/β actin and MasR/β actin ratios in the CTX and HIPP of the ND and SGLT2-i groups were higher than the vehicle group, especially in the CTX, where the increase was statistically significant (Fig. 4I).

IDE is known to degrade not only insulin but also Aβ [33]. We examined the expression of IDE and MAP2 by IHC and WB to analyze whether DPP4-i affects IDE expression. The results showed that the

expression of IDE was significantly higher in the CTX and HIPP of the ND and DPP4-i groups than in those of the vehicle group (Fig. 4J, K). The WB quantification showed that the IDE/β actin ratio was significantly higher in the ND and DPP4-i groups, especially in the CTX (Fig. 4L).

3.5. SGLT2-i and DPP4-i improve hippocampal-dependent cognitive functions

The NORT and MWM test were performed to investigate the effects of SGLT2-i and DPP4-i on spatial perception and memory in the mice. A novel object was placed in Q4 (red area) and the familial object was placed in Q1 (green area) (Fig. 5A), and the behavior of the mice was observed for 5 min. The results showed that the ND group mice spent more time in Q4 than did the vehicle group mic. Although there was no significant difference, compared with the vehicle group, the SGLT2-i and DPP4-i group spent more time in Q4 than in Q1 (Fig. 5B, C). In addition, compared with the vehicle group, the ND, SGLT2-i, and DPP4-i groups showed significant differences in the exploration time for the familial Fig. 3. Reduction in pTau and Aβ levels following SGLT2-i and DPP4-i treatment. (A) IHC and (B) WB analyses for pTau (Thr231) expression in the CTX and HIPP.

Scale bar =20µm. (C) Densitometric evaluation of the WB. (D) IHC and (E) WB analyses of Aβ in the CTX and HIPP. Scale bar =50µm. (F) Densitometric evaluation of the WB. β actin served as a loading control. n=3/group, *P<0.05, **P<0.005, ***P<0.001.

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and novel objects (Fig. 5D). The platform was positioned as shown in Fig. 5E and the mice tracking path during probe testing is shown in Fig. 5F. During the MWM training period, the time taken by the mice to find the platform gradually decreased in the ND, SGLT2-i, and DPP4-i groups but not in the vehicle group (Fig. 5G). On day 6, during the probe test, we observed that the time spent on the platform and the number of platform crossings in the vehicle group was significantly lower than the other groups (Fig. 5H, I). In addition, the time taken by the mice to locate the platform was significantly higher in the vehicle group than the other groups (Fig. 5J).

3.6. SGLT2-i and DPP4-i reduce hepatic lipid accumulation and inflammation and promote glucose homeostasis

Next, we investigated the effects of SGLT2-i and DPP4-i on metabolism and inflammation in the liver and adipose tissue. The H&E staining revealed significantly reduced the fat droplets in the liver of the SGLT2-i and DPP4-i groups than in that of the vehicle group (Fig. 6A).

The qPCR analysis confirmed that the expression of various pro- inflammatory cytokines, such as Mcp1, Tnfα, and Il6 was significantly lower in the SGLT2-i and DPP4-i groups than in the vehicle group. The expression of genes related to endoplasmic reticulum stress (Grp78), gluconeogenesis (G6pc and Pepck), and lipogenesis (Srebp1, Fas) was Fig. 4.Expression of Ace2/At2r/Masr and Ace1/At1r in the SGLT2-i and DPP4-i groups in the kidney and ACE2/MasR and IDE in the SGLT2-i and DPP4-i groups. (A) Simplified view of RAS. Created using Biorender.com. qPCR evaluating (B) Ace 1, (C) Ace 2, (D) At1r, (E) At2r and (F) Masr transcript levels in the kidney. (G) IHC for evaluating MasR and MAP2 expression. Scale bar =20µm (MasR), 100µm (MERGE). (H) WB analysis for ACE2 and MasR expression in the ND, vehicle, and SGLT2-i groups. (I) Densitometric evaluation of the WB. (J) IHC for evaluating IDE expression in the CTX and HIPP. Scale bar =200µm. (K) WB analysis of IDE and (L) densitometric evaluation of the WB. β actin served as a loading control. n=3/group, *P<0.05, **P<0.005, ****P<0.0001.

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Fig. 5. Effects of SGLT2-i and DPP4-i on learning and memory. (A) Schematics showing the NORT design. (B) The path taken by the mouse during the 5 min of probe test. (C) Comparison of time spent by the mice in Q1 and Q4. (D) Comparison of familial and novel object exploration times during the probe test. (E) Schematics showing the MWM test design. (F) The path taken by the mouse during the 1 min of probe test. (G) Changes in escape latency on the training day. (H) Time spent in locating the platform and (I) the number of times in the area where the platform was located. (J) Time taken to reach the area where the platform was located. ^#,

$P<0.05, ^##,$$P<0.01 compared with the vehicle and SGLT2-i or DPP4-i groups. n=8/group, *P<0.05, **P<0.005, ***P<0.001.

A.Y. Sim et al.

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lower in the SGLT2-i and DPP4-i groups than in the vehicle group.

Furthermore, the expression of genes related to fatty acid oxidation, such as Ampk and Pparα was increased in the liver of the SGLT2-i and DPP4-i groups. However, Cpt1α was lower in the SGLT2-i and DPP4-i groups than in the vehicle group (Fig. 6B). Regarding the expression of insulin signaling pathway proteins in the adipose tissue [34], pIRS1 (Ser307) and pAkt (Ser473) expression in the epididymal fat was significantly lower in the vehicle group. However, SGLT2-i and DPP4-i

marginally restored the expression of insulin signaling pathway proteins in the epididymal fat (Fig. 6C, D). Overall, these observations suggest that SGLT2-i and DPP4-i reduce inflammation and improve glucose metabolism in the liver and fat of the T2D mouse model.

4. Discussion

We showed that HFD and STZ-induced T2D mice are associated with Fig. 6. Effects of SGLT2-i and DPP4-i on the liver and adipose tissue. (A) Lipid accumulation in the liver was evaluated using the H&E staining. Scale bar =500µm (left), 200µm (right). (B) qPCR evaluating Mcp1, Tnfα, Il6, Grp78, G6pc, Pepck, Srebp1, Fas, Ampk, Pparα_{, and}_Cpt1α transcript levels in the liver. (C, D) The epididymal fat tissue lysates were subjected to WB analysis for pIRS1 (Ser307) and pAkt (Ser473) expression. β actin served as a loading control. n=3/group,

*P<0.05, **P<0.005, ***P<0.001, ****P<0.0001.

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Biomedicine & Pharmacotherapy 168 (2023) 115755 AD. The 12–15 weeks HFD feeding mouse model is widely known as a

model for inducing insulin resistance in T2D [22,23]. Additionally, HFD and STZ-induced T2D mouse models are supported by various experiments [26,35–38]. In this study, we showed that SGLT2-i and DPP4-i improve peripheral insulin resistance while ameliorating inflammation and promoting the expression of metabolic genes in the liver of T2D mice. Furthermore, SGLT2-i and DPP4-i restore brain insulin signaling by inducing IRS1/Akt/GSK3β pathway, thereby improving the learning and memory behaviors of the T2D–AD mouse model. We also demonstrated that SGLT2-i regulates tau phosphorylation via RAS, whereas DPP4-i regulates Aβ accumulation by increasing IDE levels in the brain.

Our results suggest that SGLT2-i or DPP4-i treatment can reduce pTau expression and Aβ accumulation via different mechanisms, thereby enhancing brain functions in animals with dementia due to metabolic dysfunction.

Dementia is not a specific disease but a general term used to describe a reduced ability to remember, think, or make decisions. Dementia in- terferes with activities of daily living and is generally due to various causes that affect the brain. AD, the most common type of dementia, can be divided into two clinical subtypes: familial AD (fAD) and sporadic AD (sAD) [39]. Although they exhibit similar pathological phenotypes, such as the development of Aβ plaques and NFTs, synaptic damage, and neuronal loss, the factors that trigger the neurodegenerative process are completely different. Different perspectives have emerged to explain late-onset sAD, which affects more than 95 % of AD patients. Moreover, T2D-associated insulin resistance is considered a risk factor for AD [40].

Individuals with T2D are approximately twice as likely to develop AD than those without, and approximately 80 % of people with dementia have impaired glucose tolerance [41]. However, the mechanism through which systemic and brain insulin resistance leads to AD remains unclear.

In our previous study, we confirmed that insulin resistance and dementia symptoms, such as decreased cognitive functions and the accumulation of Aβ and pTau, are prevalent in a T2D mouse model developed using an HFD with STZ. Previous studies have also shown that drugs used for T2D improve brain function in cases of metabolic dementia [21]. However, to date, very few studies have investigated the rela- tionship between T2D drug use and AD symptoms.

RAS is a key regulator of blood pressure and electrolyte homeostasis and is associated with the regulation of brain functions, such as cognition, memory, and depression [42]. Changes in RAS contribute to the development of insulin resistance [43]. Among RAS components, Ang is a hormone that causes vasoconstriction and elevated blood pressure.

Ang II is considered a regulator of cardiovascular, renal, and adrenal functions [44]. ACE1 directly converts Ang I to Ang II, which binds to AT1R to induce vasoconstriction, hypertension, inflammatory re- sponses, and insulin resistance. Ang I produces Ang(1− 7) indirectly via ACE2, which binds to MasR to stimulate proteins belonging to the insulin signaling pathway and ameliorates the negative effects of Ang II [45]. Recent studies suggest that drugs which decrease the formation or action of Ang II may also reduce the incidence of diabetes [46] and ACE2/Ang(1− 7)/MasR signaling pathway is vasoprotective and neuroprotective by regulating ACE/Ang II/AT1R axis not only in the peripheral but also within the central nervous system. We hypothesized that brain insulin signaling pathway could be restored in SGLT2-i groups by modulating ACE2/Ang(1− 7)/MasR axis. Additionally, we speculated that SGLT2-i activates RAS [47] and confirmed a positive correlation between SGLT2-i levels and RAS activity. Our results suggest that SGLT2-i treatment ameliorates metabolic dementia and improves insulin resistance via RAS. Furthermore, SGLT2-i modulates the activity of GSK3β and consequently affects the expression of pTau.

We found that DPP4-i improves brain insulin resistance and activates IDE, an enzyme that degrades Aβ. IDE reportedly plays important roles in insulin degradation and clearance in vivo [48]. Besides, several studies have shown that of all proteases secreted by cells, only IDE specifically binds to and degrade Aβ [49]. IDE also plays an important role in sAD. In cases of insulin resistance, the amount of insulin

clearance through IDE is higher than that of Aβ leading to Aβ accumulation [50]. DPP4-i inhibits DPP4, an enzyme that degrades GLP-1, thereby increasing GLP-1. GLP-1 is an incretin hormone that lowers BG, and its actions vary depending on the BG [51]. When BG is high, GLP-1 increase. Upon treatment with DPP4-i, GLP-1 receptor stimula- tion is increased, leading to improved insulin signaling and increased IDE, thereby enhancing Aβ clearance. In this study, we confirmed that IDE expression was decreased in the T2D–AD mouse model and DPP4-i administration increased IDE expression, thereby reducing Aβ accumulation in these mice. This suggests that IDE influences Aβ levels and DPP4-i treatment potentially modulates Aβ levels in AD-associated metabolic disorders.

Several retrospective studies were conducted to investigate therapeutic effects of DPP4-i and SGLT2-i on dementia in people with T2D [52]. A nested case-control study using Danish National Diabetes Reg- istry demonstrated that ever users of DPP4-i or SGLT2-i had significantly lower risk of dementia [53]. DPP4-i use was associated with a slower rate of memory decline [54] and longitudinal decrease in Mini-Mental State Examination score in individuals with diabetes and AD–related cognitive impairment [14]. In addition, patients treated with DPP4-i had lower global amyloid burden by 18F-florbetaben amyloid PET scan. A recent population-based study from Canada demonstrated that SGLT2-i was associated with 20 % lower dementia risk in elderly individuals with T2D compared to DPP4-i [55]. Further well-designed randomized controlled clinical trials are required to evaluate the neuroprotective effects of DPP4-i and SGLT2-i.

Our study had a few limitations. We did not assess the direct mechanisms of action of SGLT2-i and DPP4-i in the brain. Furthermore, we did not evaluate the combined effects of SGLT2-i and DPP4-i on AD.

Due to the nature of diabetogenic effects of STZ on female mice [56], therapeutic effects of these agents were not assessed in female gender.

Although limited, the NORT and MWM test results of this study confirmed that SGLT2-i or DPP4-i improve learning and memory functions in T2D mice via distinct mechanisms.

Taken together, our results suggest that SGLT2-i and DPP4-i used in T2D patients are potential prophylactic and therapeutic agents against T2D–AD by restoring peripheral and brain insulin signals and protecting neurons and synapses to improve cognitive function. In addition, our study lays the groundwork for follow-up studies of T2D drugs to prevent and treat cognitive dysfunction in patients with T2D.

5. Conclusion

We identified that SGLT2-i and DPP4-i improve cognitive function potentially through restoring brain insulin signaling, protecting neurons and synapses, and counteracting the accumulation of pathogenic AD proteins, such as Aβ and pTau in a T2D–AD mouse model. Interestingly, we found that SGLT2-i and DPP4-i are associated with different AD- related proteins. In conclusion, our findings may not only offer new insights into the regulatory mechanism of the brain signaling effects of SGLT2-i and DPP4-i, but also suggest the potential utility in AD.

Funding

This study was supported by the National Research Foundation of Korea funded by the Korea government (NRF-2021R1A2C2008034 to JEL), Faculty Research Grant from Yonsei University College of Medi- cine (6-2020-0229 to JEL), the Ministry of Science and ICT (NRF- 2016R1A5A1010764 to Y-HL) and the National Research Foundation of Korea funded by the Ministry of Education (NRF- 2018R1D1A1B07050005 to Y-HL).

CRediT authorship contribution statement

J.E.L contributed to the study design and supervised the study. Y-H.L co-designed the experiments. A.Y.S and D.H.C performed experiments, A.Y. Sim et al.

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analyzed the results, and drafted the manuscript. J.Y.K interpreted the results. E.R.K performed and collected data. A-r.G collected data. Y-H.L and J.E.L revised and edited the final draft of the manuscript. All the authors have read and approved the final version of the manuscript.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

All the data used to support the findings of this study are included within the article and results presented were carried out by authors.

Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.biopha.2023.115755.

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