GRAPHICAL ABSTRACT

Highlights

• SGLT2i and acarbose reduced glucose levels and improved β-cell function in db/db mice.

• The combination also decreased senescent β-cells in the pancreatic islets.

• This therapy shows potential as a senotherapeutic approach for T2DM.

INTRODUCTION

During the development of type 2 diabetes mellitus (T2DM), insulin sensitivity declines with age, and there is an insufficient compensatory increase in insulin secretion due to reduced function and/or mass of pancreatic β-cells [1-4]. This age-related decline mirrors cellular senescence, a state characterized by irreversible growth arrest and altered secretory profiles. Cellular senescence of β-cells has been characterized in multiple mouse models related to diabetes, such as in high-fat diet-treated [5], S961-induced insulin-resistant [5,6], and T-cell-mediated autoimmune mice [7]. These metabolic stresses have been shown to induce senescence of pancreatic β-cells, causing them to lose their ability to respond to proliferative signals while remaining metabolically active. Markers of senescence, including cyclin-dependent kinase inhibitors (p16^INK4a, p21^CIP1/WAF1) and the senescence-associated secretory phenotype, have consistently been found in the islets of T2DM and are commonly shared in both humans and animal models [5-15].

Drugs approved for use in T2DM have been shown to attenuate major hallmarks of aging in animal models. Their potential in slowing the aging process is currently being examined in human trials (ClinicalTrials.gov, number NCT03498001, NCT04511416, NCT05891496, NCT01864096), along with their senotherapeutic potentials, as the accumulation of senescent cells in various tissues has been reported to contribute to pathological consequences of aging, such as metabolic disease, cardiovascular disease, and bone loss [16-21]. For example, canagliflozin, a sodium-glucose cotransporter 2 (SGLT2) inhibitor, was reported to extend lifespan in genetically heterogeneous mice [22]. Canagliflozin-treated mice showed a significantly improved response during glucose tolerance tests, but their weight, body composition, and glycosylated hemoglobin were comparable to the control group. Another study reported that canagliflozin increased circulating 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside in mice, which enhanced the immune-mediated clearance of senescent cells in adipose tissue, thereby improving insulin sensitivity [23]. We also reported that dapagliflozin reduced cellular senescence and oxidative stress in the kidney through β-hydroxybutyrate (β-HB)-induced nuclear factor erythroid 2-related factor 2 (NRF2) activation in db/db mice [24]. However, whether SGLT2 inhibitors could also delay cellular senescence of pancreatic β-cells remains largely unknown.

Another drug that has shown potential as a senotherapeutic agent is acarbose, which is an α-glucosidase inhibitor blocking the enzymes responsible for breaking down complex carbohydrates. In addition to its glucose-lowering effect, acarbose has demonstrated significant lifespan extension in mice [25]. Acarbose also ameliorated age-related pathologic changes in mice, such as loss of balance, cardiac hypertrophy, and hepatic steatosis [26]. This increase in longevity in preclinical models was explained through mechanisms such as reduced postprandial glucose excursions and modification of gut microbiota, in addition to the mild negative energy balance induced by the drug [27,28]. However, its effect on pancreatic β-cell senescence still requires further investigation.

In this study, we hypothesized that treatment with enavogliflozin (an SGLT2 inhibitor) and/or acarbose (an α-glucosidase inhibitor) to db/db mice, a murine model of obese T2DM, would not only ameliorate hyperglycemia but also reduce cellular senescence. We report the comparison of β-cell function and senescent phenotypes in the pancreatic islets and kidney glomeruli after 6 weeks of treatment with enavogliflozin, acarbose, or both, as well as the possible link between their senotherapeutic effect and β-HB-induced NRF2 activation in the pancreatic islets.

METHODS

Animals

Six-week-old male db/db mice and lean db/m+ mice were procured from Japan SLC (Shizuoka, Japan). All the animals were provided with unrestricted access to a standard chow diet and water. Upon reaching 8 weeks of age, the mice were categorized into five groups: (1) db/m+ (as normal control), (2) db/db+V (10% dimethyl sulfoxide in corn oil, 0.5% carboxymethyl cellulose sodium salt), (3) db/db+S (enavogliflozin, 0.3 mg/kg), (4) db/db+A (acarbose, 10 mg/kg), and (5) db/db+S+A (enavogliflozin, 0.3 mg/kg, acarbose, 10 mg/kg). Enavogliflozin (Daewoong pharmaceutical Co., Seoul, Korea), acarbose (PHR1253, Sigma-Aldrich, St. Louis, MO, USA) or vehicle (0.5% carboxymethyl cellulose sodium salt) were orally administered daily for a duration of 6 weeks. Measurements of body weight, blood glucose levels, and daily food intake were taken twice a week throughout the treatment period. After 6 weeks, all animals were anesthetized using 2% isoflurane inhalation, and tissues were promptly collected before euthanization. All experimental procedures were conducted in accordance with institutional guidelines for animal research. The study protocol received approval from the Animal Care and Use Committee at Seoul National University Hospital (IACUC number: 22-0006-S1A1).

Oral glucose tolerance test

After 4 weeks of treatment, an oral glucose tolerance test (OGTT) was conducted following an overnight fast of 16 hours. Prior to the test, fasted mice were weighed, and then they were orally administered a 20% glucose solution at a dosage of 1 g/kg relative to their body weight. Glucose levels were monitored by obtaining blood samples from the tail vein at intervals of 0, 15, 30, 60, and 120 minutes using a glucometer (Accu-Chek, Roche Diagnostics, Mannheim, Germany). To measure plasma insulin levels, blood was collected from the orbital area into heparinized tubes, followed by the separation of plasma through centrifugation at 1,500 g for 10 minutes. The quantification of plasma insulin levels was performed using a mouse insulin enzyme-linked immunosorbent assay (ELISA) Kit (80-INSMSUE01, Alpco, Salem, NH, USA), following the manufacturer’s instructions. The insulinogenic index was calculated using the following formula: Δ serum insulin (pmol/L) (15 min–0 min)/Δ serum glucose (mmol/L) (15 min–0 min). Homeostasis model assessment of β-cell function (HOMA-β) was calculated using the following formula: (360×insulin [mU/L])/(glucose [mg/dL]–63). Homeostasis model assessment of insulin resistance (HOMA-IR) was calculated using the following formula: (glucose [mg/dL]×insulin [mU/L])/405.

Histology and immunofluorescence staining

To prepare frozen pancreas tissue sections for immunofluorescence staining, 10 μm-thick sections of the pancreas were obtained. Blocking was done with either goat or donkey serum (5%) diluted with Dako REAL antibody diluent (S2022, Dako, Carpinteria, CA, USA), and the sections were incubated at room temperature for 1 hour. After blocking and gently removing the 5% dilute serum, the sections were then incubated overnight at 4°C with an insulin polyclonal antibody (1:200; PA1-26938, Invitrogen, Waltham, MA, USA) and anti-γ H2A. X variant histone (γH2AX) antibody (1:500; 2577S, Cell Signaling Technology, Danvers, MA, USA). After washing, the sections were incubated with Alexa Fluor 488 goat anti-guinea pig secondary antibody (1:1,000; A11073, Invitrogen), Alexa Fluor 647 goat anti-rabbit secondary antibody (1:1,000; A21245, Invitrogen), Alexa Fluor 647 CDKN2A/p16-INK4a polyclonal antibody (1:1,000; BS-23797R-A647, Thermo Fisher Scientific, Waltham, MA, USA), or Alexa Fluor 647 anti-p21 antibody (1:1,000; ab237265, Abcam, Cambridge, UK) overnight at 4°C. After rinsing with tris-buffered saline with Tween 20 (TBS-T) (TR2007-100-74, Biosesang, Yongin, Korea), the sections were mounted with 4´,6-diamidino-2-phenylindole (DAPI) (H-1500, VECTOR Laboratories, Burlingame, CA, USA). Images were captured using a confocal microscope (Leica TCS STED CW, Leica Microsystems, Wetzlar, Germany). For the kidney section, tissues were collected and immediately fixed with 4% paraformaldehyde at 4°C overnight. Then, sections were cut with a thickness of 4 μm. Slides were subjected to staining using periodic acid-Schiff’s (PAS) reagent to assess mesangial expansion and the glomerular area, and analysis was preformed using MetaMorph Image Analysis (Molecular Devices, San Jose, CA, USA).

Immunohistochemistry

Kidney tissues were fixed in 4% paraformaldehyde for 24 hours and subsequently embedded in paraffin. Paraffin-embedded sections were cut to a thickness of 5 μm, then deparaffinized and rehydrated. Antigen retrieval was performed using sodium citrate buffer (10×, pH 6.0; S1700, Dako). Non-specific binding was blocked using 5% bovine serum albumin in goat serum. The sections were then incubated with anti-γH2AX antibody (ab11174, 1:1,000; Abcam) overnight at 4°C. The following day, the sections were incubated with anti-rabbit immunoglobulin G (IgG) H&L (horseradish peroxidase [HRP] polymer) (ab214880, Abcam) at 37°C for 20 minutes. Images were captured using a Nikon microscope (Tokyo, Japan).

Isolation of pancreatic islets

Isoflurane was administered to induce comprehensive anesthesia in C57BL/6 mice at a concentration ranging from 2% to 5%. Mice were positioned with the abdominal side facing upward, and the skin was sterilized with 70% ethanol. An incision was made around the upper abdomen, and the ampulla’s position was identified. Surgical clamps were employed on the duodenal wall to obstruct the biliary tract. A needle was then inserted into the common bile duct through the joint site of the hepatic duct and the cystic duct, and 3 mL of Solution I (collagenase XI, Sigma-Aldrich; dissolved in Hanks’ balanced salt solution [HBSS], LB 003-02, Welgene, Gyeongsan, Korea) was slowly injected to inflate the pancreas. The tube containing the inflated pancreas was placed in a water bath at 37.5°C for 15 minutes, with gentle manual shaking two to three times during the incubation. Following incubation, the tube was manually shaken for homogenization, placed on ice, and 25 mL of Solution II (1 mM CaCl2 added to HBSS) was added. Centrifugation at 290 g for 30 seconds at 4°C was performed, and the supernatant was discarded. The pellet was washed once again with 20 mL of Solution II, and the obtained pellet was resuspended in 15 mL of Solution II. The resuspended solution was transferred onto a pre-wetted 70 μm cell strainer, rinsed with 20 mL of Solution II, and the strainer was washed into a fresh petri dish using 15 mL of Solution III (Roswell Park Memorial Institute [RPMI] 1640 medium supplemented with 20 mM L-glutamine, 0.5% penicillin, 0.5% streptomycin, and 10% fetal bovine serum [FBS]). This procedure was adapted from previous studies for islet isolation [29].

Quantitative real-time polymerase chain reaction

Total RNA was extracted from tissue samples using TRIzol Reagent (15596018, Invitrogen) according to the manufacturer’s instructions. cDNA was synthesized through reverse transcription of 1 μg of total RNA using avian myeloblastosis virus (AMV) reverse transcriptase (M1701, Promega, Madison, WI, USA), following the manufacturer’s protocol. Quantitative real-time polymerase chain reaction (PCR) was performed using a TB Green Master Mix (RR420, Takara, Shiga, Japan) with an Applied Biosystems 7500 Real-Time PCR System (Thermo Fisher Scientific) to evaluate the mRNA expression of mouse p16, p21, and p53. Primer sequences are presented in Supplementary Table 1.

Western blot analysis

Proteins were extracted from tissue samples using RIPA lysis and extraction buffer (89900, Thermo Fisher Scientific). The lysate was centrifuged at 14,000 rpm for 20 minutes at 4°C and the supernatants were boiled for 5 minutes at 95°C. The supernatants were subjected to electrophoresis using 8%–16% precast sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gels (Bio-Rad, Hercules, CA, USA). Subsequently, the separated proteins from the pre-cast SDS-PAGE gels were transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% skim milk for 1 hour and then incubated overnight at 4°C with the following primary antibodies: anti-p53BP1 (1:1,000; A300-272A, Bethyl Laboratories, Montgomery, TX, USA), anti-p21 (1:1,000; 28248-1-AP, Proteintech, Rosemont, IL, USA), anti-p16 (1:1,000; ab211542, Abcam), anti-heat shock protein 90 (1:1,000; 4874S, Cell Signaling Technology), and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:1,000; 5174S, Cell Signaling Technology). The membranes underwent three washes with TBS-T and were subsequently incubated with HRP-conjugated secondary antibodies at room temperature for 1 hour. They were then visualized by employing a chemiluminescent substrate (34580, Thermo Fisher Scientific) and the images were captured with an Amersham Imager 680 (GE Healthcare, Chicago, IL, USA).

Biochemical analysis

The measurement of plasma β-HB was conducted using a commercially available colorimetric assay kit (MAK041, Sigma-Aldrich).

Cell culture

For in vitro experiments, mouse pancreatic islet cells were isolated from C57BL/6 mice. Pancreatic islets were grown in a 5% CO2 incubator at 37°C using Dulbecco’s Modified Eagle Medium/high glucose supplemented with 10% FBS (Hyclone, Logan, UT, USA) and 1% of penicillin and streptomycin. After isolation, the islets were first exposed to H₂O₂ (500 μmol/L; H325-500, Thermo Fisher Scientific) for a duration of 6 hours. Subsequently, they were incubated with or without β-HB (3 mmol/L; GK-2020, Glentham Life Sciences, London, UK) for 18 hours.

Statistical analysis

The data were presented as mean±standard error of the mean. Statistical comparisons between groups were conducted using one-way analysis of variance (ANOVA) or Tukey’s post hoc test, which was performed with GraphPad Prism version 9.5 (GraphPad Software, San Diego, CA, USA). A P value of <0.05 was considered to indicate statistical significance.

RESULTS

Enavogliflozin and acarbose improved glucose tolerance in db/db mice

To assess the metabolic effects of enavogliflozin and/or acarbose, 8-week-old db/db mice underwent treatment for a duration of 6 weeks. Treatments included a vehicle control group (db/db+V), enavogliflozin alone (db/db+S), acarbose alone (db/db+A), and a combination of enavogliflozin and acarbose (db/db+S+A). The body weights across the five groups were similar, with the db/db+V group having slightly lower values (Fig. 1A). The groups treated with enavogliflozin (db/db+S and db/db+S+A) exhibited lower random blood glucose levels compared to the other groups, with the db/db+S+A group showing similar blood glucose levels to the db/db+S group (Fig. 1B). Interestingly, the groups treated with enavogliflozin showed increased food intake compared to the other groups (Fig. 1C). During the OGTT conducted in the 4th week of treatment (Fig 1D-F), the db/db+V group showed a significant increase in the glucose area under the curve (AUC) compared to db/m+. The groups treated with enavogliflozin displayed significantly lower blood glucose AUC compared to db/db+V, whereas the db/db+A group did not. Of note, the glucose AUC was similar between the db/db+S+A and db/db+S groups (Fig. 1D and E).

The combination of enavogliflozin and acarbose improved β-cell function in db/db mice

To evaluate β-cell function and insulin resistance, insulinogenic index at 15 minutes, HOMA-β and HOMA-IR were measured. The insulinogenic index showed a significant increase in the db/db+S+A group compared to db/db+V (Fig. 2A). A similar trend was observed in the HOMA-β analysis, with significant increases observed in db/db+S and db/db+S+A compared to db/db+V. Regarding insulin resistance, while there was a significant increase of HOMA-IR in the db/db+V group, there were no significant differences in the remaining groups compared to db/m+ (Fig. 2B and C). To assess whether this functional change was observed in immunocytochemical examination, pancreatic tissues were stained with an anti-insulin antibody to measure the insulin-positive β-cell area. The insulinpositive β-cell area was significantly increased in the groups treated with enavogliflozin compared to db/db+V, similar to the extent of the db/m+ group (Fig. 2D and E). Direct comparison of the phenotypes between db/db+S and db/db+S+A group showed a significantly higher insulinogenic index in the combination group.

The combination of enavogliflozin and acarbose reduced cellular senescence in the pancreatic islets of db/db mice

To determine the anti-senescence effect of the combination of enavogliflozin and acarbose, islets were isolated from the pancreas and the expression of senescence markers were examined. The expression of p16, p21, and p53 in the islets of the db/db+V group was increased compared to db/m+, with statistically significant differences observed in p16 and p53. The expression of these senescence markers tended to decrease to levels comparable to db/m+ with treatment, with a significant decrease in p16 observed especially in the db/db+S+A group (Fig. 3A). To further validate the anti-senescence effects of enavogliflozin and acarbose, we conducted immunofluorescence staining for senescence markers (p16, p21, and γH2AX) on pancreatic sections. In comparison with the db/m+ group, the expression of p16 was significantly elevated in the db/db+V group, accompanied by faint insulin expression. However, in the groups treated with enavogliflozin and/or acarbose, the expression of p16 was not observed, and there was a restoration of insulin staining (Fig. 3B, Supplementary Fig. 1). The decreased expression of β-cell markers (paired box 6 [Pax6], pancreatic and duodenal homeobox 1 [Pdx1], and insulin 1 [Ins1]) in the whole pancreas specimen of db/db mouse were also increased by enavogliflozin and/or acarbose treatment (Supplementary Fig. 2). Similar findings were observed in p21 and γH2AX staining for the enavogliflozin and/or acarbose treated groups (Fig. 3C and D). While the direct comparison of senescent marker expression between db/db+S and db/db+S+A group did not achieve statistical significance, a trend for reduced senescence markers in the combination group was observed during a relatively short 6-week course of treatment.

β-HB increased NRF2 in the H₂O₂ induced-senescent pancreatic islets

After 6 weeks of drug administration, the plasma concentration of β-HB tended to increase in all treatment groups (Fig. 4A). To investigate whether the decrease in senescent β-cells in the db/db+S+A group was due to the increased β-HB levels, we performed an in vitro assay with the pancreatic islets isolated from C57BL/6 mice. Isolated islets treated with H₂O₂ showed increased expression levels of senescence markers (p16, p21, and p53) compared to those treated with vehicle. Interestingly, these elevated senescence markers were reduced to a similar extent to control when treated with β-HB (Fig. 4B). As it has been reported that ketone bodies could activate NRF2 in the liver, retina and the kidney glomerulus [24,30], we checked for NRF2 levels in H₂O₂-induced senescent pancreatic islets treated with β-HB. We observed an increase in NRF2 levels with β-HB treatment only in the H₂O₂-induced senescent islets, and not in the control islets (Fig. 4C). Similar findings were observed in the whole pancreas of the db/db mouse, where a trend toward lower levels of senescence markers and higher levels of NRF2 and adenosine monophosphate-activated protein kinase (AMPK) activation was observed in the treatment group (Fig. 4D).

The combination of enavogliflozin and acarbose reduced cellular senescence in the kidney of db/db mice

In our previous work, we found that dapagliflozin delayed the development of diabetic kidney disease in db/db mice by reducing cellular senescence [24]. We further investigated the effect of combined treatment of enavogliflozin and/or acarbose on the progression of diabetic kidney disease. Although there was no significant change in mesangial expansion, the combination significantly attenuated the increase in glomerular size compared to the db/db+V group (Fig. 5A-C). To assess the anti-senescence effects of enavogliflozin and/or acarbose, we examined the protein expression related to senescence markers in the kidney by Western blot. The band intensity of senescence markers (p53BP1 and p16) significantly increased in db/db+V compared to db/m+, while only the combined treatement significantly mitigated the increase of senescence markers (Fig. 5D). Similar findings were observed in γH2AX staining, particularly in the glomeruli, for the enavogliflozin and/or acarbose treated groups (Fig. 5E).

DISCUSSION

In this study, we investigated the potential senotherapeutic effect of enavogliflozin and/or acarbose on β-cells of db/db mice. We found that, although body weight and food intake did not significantly differ between the groups treated with enavogliflozin and/or acarbose, glucose tolerance was significantly improved in those treated with enavogliflozin, either alone or in combination with acarbose. This improvement in glucose tolerance could be explained by the improvement in β-cell function or mass, as insulin resistance measured with HOMA-IR did not differ significantly between treatment groups. Notably, only the group treated with the combination of enavogliflozin and acarbose showed a significant improvement in the insulinogenic index compared to the vehicle-treated group. The greatest increase in HOMA-β index and insulin-positive area was also observed in the group treated with the combination of enavogliflozin and acarbose, consistent with the change observed in the insulinogenic index. Senescence marker (p16) was significantly reduced only in the group treated with the combination of enavogliflozin and acarbose, suggesting a potential additive effect of the two drugs in reducing the cellular senescence of β-cells.

The analysis of β-cell function indicated an added benefit of acarbose in combination with enavogliflozin, while the OGTT results demonstrated similar responses between the db/db+S and db/db+S+A group. In random blood glucose measurements, a reduction in blood glucose was primarily observed in the groups treated with enavogliflozin (db/db+S and db/db+S+A), with similar levels of reductions observed in the two groups (Fig. 1B). These results suggest that the improvement in glucose tolerance was mainly attributable to enavogliflozin, and the added benefit of acarbose may have acted through pathways other than its glucose-lowering effect. One possible mechanism is the change in microbiota and the subsequent increase in circulating butyrate levels by acarbose. Acarbose increases the flow of polysaccharide substrates to the lower digestive system, leading to higher levels of circulating short-chain fatty acids, such as butyrate, which are produced through fermentation by the gut microbiota [27]. Previous studies have reported that butyrate improved age-associated sarcopenia [31] and neuroinflammation [32] in mice and reversed senescence-associated changes in human fibroblasts [33]. These alternative pathways may have contributed to the added benefit of acarbose in reducing cellular senescence and improving β-cell function of db/db mice.

The pancreatic islets and kidneys from the group treated with the combination of enavogliflozin and acarbose demonstrated a significant reduction in senescence markers, a finding not observed in other treatment groups. Canagliflozin and acarbose were shown to expand lifespan and preserve organ function in mice [22,23,25,26]. The two drugs lower circulating glucose levels by increasing glucose excretion through urine and inhibiting absorption of monosaccharide from the intestine, which may induce a systemic metabolic state similar to that observed during moderate caloric restriction. Specifically, canagliflozin enhanced fat and ketone body metabolism through transcriptional changes in the liver, mimicking the effects seen with caloric restriction [34,35]. Acarbose impairs monosaccharide absorption from the intestine, further reducing glucose availability. Caloric restriction has been reported to be associated with reduced accumulation of senescent cells in the kidney, ovary, and heart [9] and has been shown to improve β-cell function in mice by recovering gap-junction connectivity [36] and restoring β-cell dedifferentiation [37]. Our study reported the reduction of cellular senescence and improved β-cell function by enavogliflozin and acarbose, possibly mediated through the systemic metabolism similar to that induced by caloric restriction, with the effect being particularly pronounced when both drugs are combined.

We observed a possible additive effect of enavogliflozin and acarbose, which was not robustly observed with either drug used alone. Given that T2DM is a progressive disease, early initiation of combination therapy has been proposed to accelerate the normalization of hyperglycemia and potentially delay the deterioration of glycemic control, with the possibility of enhanced preservation of β-cell function [38,39]. In this study, the group treated with enavogliflozin and not only showed a significant reduction in blood glucose levels but also a notable increase in the insulinogenic index compared to the db/db vehicle group. Moreover, assessment of β-cell function using fasting blood glucose and fasting insulin concentrations showed a significant increase in HOMA-β compared to the db/db vehicle group. While this combination did not further reduce insulin resistance, it exhibited an additive effect on β-cell mass and function when enavogliflozin and acarbose were used together. These findings could partly be explained by the reduced senescence in the pancreatic islets. Our group previously demonstrated that β-HB, which was increased by enavogliflozin and/or acarbose treatment, induced nuclear translocation of NRF2 and mediated anti-senescent effects by upregulating antioxidant-related gene such as NAD(P)H quinone dehydrogenase 1 (NQO1) and heme oxygenase 1 (HO-1) in the kidney [24]. In vitro analysis also showed increased expression of Nrf2 by β-HB in H₂O₂-induced senescent islets, which may explain the reduced senescence markers observed in the pancreatic islets of the combinatorially treated db/db mice.

In conclusion, we demonstrated that the combined administration of enavogliflozin and acarbose improved glucose tolerance by improving β-cell function and reducing cellular senescence in the pancreatic islets of a T2DM mouse model. Our findings suggest the potential repurposing of previously approved antidiabetics drugs as senotherapeutic agents to mitigate the accumulation of senescent pancreatic β-cells induced by metabolic stress. These results provide a foundation for targeting the progressive β-cell dysfunction in T2DM within the context of cellular senescence.

SUPPLEMENTARY MATERIALS

NOTES

CONFLICTS OF INTEREST

Young Min Cho is an outside director of Daewoong Pharmaceutical and received consultation fees from LG Chemicals.

AUTHOR CONTRIBUTIONS

Conception or design: S.H., B.S.K.

Acquisition, analysis, or interpretation of data: all authors.

Drafting the work or revising: all authors.

Final approval of the manuscript: all authors.

FUNDING

The experiments conducted in this study were supported by funding from Daewoong Pharmaceutical. Their financial support facilitated the progression of the research and played a pivotal role in conducting the experiments. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Hyunsuk Lee is supported by the MD-PhD/Medical Scientist Training Program through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea.

Byung Soo Kong was supported by Basic Science Research program through the National Research Foundation of Korea (NRF) funded by the Korea government (MSIT) (NRF-2022R1C1-C2003297) and Seoul National University Hospital Research Fund (042022-4080).

ACKNOWLEDGMENTS

None

Fig. 1.

The effect of enavogliflozin and/or acarbose on body weight, blood glucose, cumulative food intake, oral glucose tolerance test and plasma insulin levels. (A) Body weight, (B) blood glucose, (C) cumulative food intake of mice in db/m+ (n=10), db/db+ vehicle (db/db+V), db/db+enavogliflozin (db/db+S), db/db+acarbose (db/db+A) (n=10), and db/db+S+A (n=10) groups during 6 weeks of treatment. (D) Blood glucose concentrations and (E) the corresponding area under the curve (AUC) during the oral glucose tolerance test after 4 weeks of treatment (n=10 per group). (F) Plasma insulin levels during oral glucose tolerance test. Measured at 0 and 15 minutes after administration of glucose (n=10–11 per group). ELISA, enzyme-linked immunosorbent assay. ^aP<0.01 vs. db/db+V, ^bP<0.0001 vs. db/m+ (one-way analysis of variance [ANOVA]).

Fig. 2.

The effect of enavogliflozin and/or acarbose on insulinogenic index, homeostasis model assessment of β-cell function (HOMA-β), homeostasis model assessment of insulin resistance (HOMA-IR), and the relative insulin-positive β-cell area of the pancreas sections. (A) Insulinogenic index in an oral glucose tolerance test (OGTT) (n=7–8 per group). (B) HOMA-β% indexes. (C) HOMA-IR (n=5 per group). (D) Relative insulin-positive β-cell area of immunofluorescence staining in the pancreas sections. Scale bars=500 μm. (E) Insulin-positive β-cell area. SGLT2i, sodium-glucose cotransporter 2 inhibitor; AGI, α-glucosidase inhibitor. ^aP<0.05 db/db+enavogliflozin (db/db+S) vs. db/db+enavogliflozin+acarbose (db/db+S+A) (t-test), ^bP<0.05, ^cP<0.01, ^dP<0.001 vs. db/db+vehicle (db/db+V) (analysis of variance [ANOVA]), ^eP<0.05 vs. db/m+, ^fP<0.01 vs. db/m+ (ANOVA).

Fig. 3.

Relative mRNA expression of p16, p21, and p53 in pancreatic islets and the immunofluorescence staining of insulin, p16 and p21 in the pancreas sections by treatment groups. (A) Relative mRNA expression of p16, p21, and p53 in pancreatic islet cells. (B) Immunofluorescence staining of insulin (green) and p16 (red). Scale bars=150 μm. (C) Immunofluorescence staining of insulin (green), p21 (red) in the pancreas sections. Scale bars=150 μm. (D) Immunofluorescence staining of insulin (green), γ H2A. X variant histone (γH2AX) (red) in the pancreas sections. Scale bars=150 μm. SGLT2i, sodium-glucose cotransporter 2 inhibitor; AGI, α-glucosidase inhibitor; S, enavogliflozin; A, acarbose. ^aP<0.05, ^bP<0.01 vs. db/db+vehicle (analysis of variance [ANOVA]).

Fig. 4.

Plasma β-hydroxybutyrate (β-HB) concentrations by treatment groups, the relative mRNA expression of p16, p21, and p53 in pancreatic islets and the protein level of nuclear factor erythroid 2-related factor 2 (NRF2) in the pancreatic islets after treatment with H₂O₂ followed by β-HB. (A) Plasma β-HB concentrations (n=9–10 per group) (analysis of variance [ANOVA]). (B) Relative expression of p16, p21, and p53 in the H₂O₂ treated pancreatic islet cells. (C) Western blot and band intensity for NRF2 in the pancreatic islet cells. (D) Western blot analysis of p53BP1, p16, p21, phosphor-adenosine monophosphate-activated protein kinase (p-AMPK), and NRF2 in whole pancreas tissue. V, vehicle; S, enavogliflozin; A, acarbose; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; SGLT2i, sodium-glucose cotransporter 2 inhibitor; AGI, α-glucosidase inhibitor; HSP90, heat shock protein 90. ^aP<0.05, ^bP<0.01, ^cP<0.001 (ANOVA).

Fig. 5.

The effect of enavogliflozin and/or acarbose on mesangial expansion, glomerulus size and cellular senescence of the kidney. (A) Representative images of periodic acid-Schiff (PAS)-stained section of the kidney. (B) The mesangial area (scale bars=50 μm) and (C) glomerular area after treatment. (D) Western blot for p53BP1, p16, phosphor-adenosine monophosphate-activated protein kinase (p-AMPK), and nuclear factor erythroid 2-related factor 2 (NRF2) in the kidney. (E) Immunohistochemical staining of γ H2A.X variant histone (γH2AX) in the kidney. Scale bars=50 μm. S, enavogliflozin; A, acarbose; SGLT2i, sodium-glucose cotransporter 2 inhibitor; AGI, α-glucosidase inhibitor; HSP90, heat shock protein 90. ^aP<0.05 vs. db/db+vehicle, ^bP<0.01 vs. db/m+ (analysis of variance [ANOVA]), ^cP<0.01 (ANOVA).

REFERENCES

• 1. Tabak AG, Jokela M, Akbaraly TN, Brunner EJ, Kivimaki M, Witte DR. Trajectories of glycaemia, insulin sensitivity, and insulin secretion before diagnosis of type 2 diabetes: an analysis from the Whitehall II study. Lancet 2009;373:2215-21.ArticlePubMedPMC
• 2. Ohn JH, Kwak SH, Cho YM, Lim S, Jang HC, Park KS, et al. 10- Year trajectory of β-cell function and insulin sensitivity in the development of type 2 diabetes: a community-based prospective cohort study. Lancet Diabetes Endocrinol 2016;4:27-34.ArticlePubMed
• 3. Weyer C, Bogardus C, Mott DM, Pratley RE. The natural history of insulin secretory dysfunction and insulin resistance in the pathogenesis of type 2 diabetes mellitus. J Clin Invest 1999;104:787-94.ArticlePubMedPMC
• 4. Cerf ME. Beta cell dysfunction and insulin resistance. Front Endocrinol (Lausanne) 2013;4:37.ArticlePubMedPMC
• 5. Aguayo-Mazzucato C, Andle J, Lee TB, Midha A, Talemal L, Chipashvili V, et al. Acceleration of β cell aging determines diabetes and senolysis improves disease outcomes. Cell Metab 2019;30:129-42.e4.ArticlePubMedPMC
• 6. Aguayo-Mazzucato C, van Haaren M, Mruk M, Lee TB, Crawford C, Hollister-Lock J, et al. β Cell aging markers have heterogeneous distribution and are induced by insulin resistance. Cell Metab 2017;25:898-910.e5.ArticlePubMedPMC
• 7. Thompson PJ, Shah A, Ntranos V, Van Gool F, Atkinson M, Bhushan A. Targeted elimination of senescent beta cells prevents type 1 diabetes. Cell Metab 2019;29:1045-60.e10.ArticlePubMed
• 8. Krishnamurthy J, Ramsey MR, Ligon KL, Torrice C, Koh A, Bonner-Weir S, et al. p16INK4a induces an age-dependent decline in islet regenerative potential. Nature 2006;443:453-7.ArticlePubMedPDF
• 9. Krishnamurthy J, Torrice C, Ramsey MR, Kovalev GI, Al-Regaiey K, Su L, et al. Ink4a/Arf expression is a biomarker of aging. J Clin Invest 2004;114:1299-307.ArticlePubMedPMC
• 10. Helman A, Klochendler A, Azazmeh N, Gabai Y, Horwitz E, Anzi S, et al. p16(Ink4a)-induced senescence of pancreatic beta cells enhances insulin secretion. Nat Med 2016;22:412-20.ArticlePubMedPMCPDF
• 11. Schafer MJ, Zhang X, Kumar A, Atkinson EJ, Zhu Y, Jachim S, et al. The senescence-associated secretome as an indicator of age and medical risk. JCI Insight 2020;5:e133668.ArticlePubMedPMC
• 12. Basisty N, Kale A, Jeon OH, Kuehnemann C, Payne T, Rao C, et al. A proteomic atlas of senescence-associated secretomes for aging biomarker development. PLoS Biol 2020;18:e3000599.ArticlePubMedPMC
• 13. Midha A, Pan H, Abarca C, Andle J, Carapeto P, Bonner-Weir S, et al. Unique human and mouse β-cell senescence-associated secretory phenotype (SASP) reveal conserved signaling pathways and heterogeneous factors. Diabetes 2021;70:1098-116.ArticlePubMedPMCPDF
• 14. Yu Z, Zhai G, Singmann P, He Y, Xu T, Prehn C, et al. Human serum metabolic profiles are age dependent. Aging Cell 2012;11:960-7.ArticlePubMedPMCPDF
• 15. Iwasaki K, Abarca C, Aguayo-Mazzucato C. Regulation of cellular senescence in type 2 diabetes mellitus: from mechanisms to clinical applications. Diabetes Metab J 2023;47:441-53.ArticlePubMedPMCPDF
• 16. Lopez-Otin C, Blasco MA, Partridge L, Serrano M, Kroemer G. Hallmarks of aging: an expanding universe. Cell 2023;186:243-78.ArticlePubMed
• 17. Munoz-Espin D, Serrano M. Cellular senescence: from physiology to pathology. Nat Rev Mol Cell Biol 2014;15:482-96.ArticlePubMedPDF
• 18. Chaib S, Tchkonia T, Kirkland JL. Cellular senescence and senolytics: the path to the clinic. Nat Med 2022;28:1556-68.ArticlePubMedPMCPDF
• 19. Palmer AK, Xu M, Zhu Y, Pirtskhalava T, Weivoda MM, Hachfeld CM, et al. Targeting senescent cells alleviates obesity-induced metabolic dysfunction. Aging Cell 2019;18:e12950.PubMedPMC
• 20. Baker DJ, Childs BG, Durik M, Wijers ME, Sieben CJ, Zhong J, et al. Naturally occurring p16(Ink4a)-positive cells shorten healthy lifespan. Nature 2016;530:184-9.PubMedPMC
• 21. Childs BG, Baker DJ, Wijshake T, Conover CA, Campisi J, van Deursen JM. Senescent intimal foam cells are deleterious at all stages of atherosclerosis. Science 2016;354:472-7.ArticlePubMedPMC
• 22. Miller RA, Harrison DE, Allison DB, Bogue M, Debarba L, Diaz V, et al. Canagliflozin extends life span in genetically heterogeneous male but not female mice. JCI Insight 2020;5:e140019.ArticlePubMedPMC
• 23. Katsuumi G, Shimizu I, Suda M, Yoshida Y, Furihata T, Joki Y, et al. SGLT2 inhibition eliminates senescent cells and alleviates pathological aging. Nat Aging 2024;4:926-38.ArticlePubMedPMCPDF
• 24. Kim MN, Moon JH, Cho YM. Sodium-glucose cotransporter-2 inhibition reduces cellular senescence in the diabetic kidney by promoting ketone body-induced NRF2 activation. Diabetes Obes Metab 2021;23:2561-71.PubMed
• 25. Harrison DE, Strong R, Alavez S, Astle CM, DiGiovanni J, Fernandez E, et al. Acarbose improves health and lifespan in aging HET3 mice. Aging Cell 2019;18:e12898.ArticlePubMedPMCPDF
• 26. Herrera JJ, Louzon S, Pifer K, Leander D, Merrihew GE, Park JH, et al. Acarbose has sex-dependent and -independent effects on age-related physical function, cardiac health, and lipid biology. JCI Insight 2020;5:e137474.ArticlePubMedPMC
• 27. Smith BJ, Miller RA, Ericsson AC, Harrison DC, Strong R, Schmidt TM. Changes in the gut microbiome and fermentation products concurrent with enhanced longevity in acarbose-treated mice. BMC Microbiol 2019;19:130.ArticlePubMedPMCPDF
• 28. Wu B, Yan J, Yang J, Xia Y, Li D, Zhang F, et al. Extension of the life span by acarbose: is it mediated by the gut microbiota? Aging Dis 2022;13:1005-14.ArticlePubMedPMC
• 29. Li DS, Yuan YH, Tu HJ, Liang QL, Dai LJ. A protocol for islet isolation from mouse pancreas. Nat Protoc 2009;4:1649-52.ArticlePubMedPDF
• 30. Izuta Y, Imada T, Hisamura R, Oonishi E, Nakamura S, Inagaki E, et al. Ketone body 3-hydroxybutyrate mimics calorie restriction via the Nrf2 activator, fumarate, in the retina. Aging Cell 2018;17:e12699.ArticlePubMedPMCPDF
• 31. Walsh ME, Bhattacharya A, Sataranatarajan K, Qaisar R, Sloane L, Rahman MM, et al. The histone deacetylase inhibitor butyrate improves metabolism and reduces muscle atrophy during aging. Aging Cell 2015;14:957-70.ArticlePubMedPMCPDF
• 32. Matt SM, Allen JM, Lawson MA, Mailing LJ, Woods JA, Johnson RW. Butyrate and dietary soluble fiber improve neuroinflammation associated with aging in mice. Front Immunol 2018;9:1832.ArticlePubMedPMC
• 33. Yang JH, Petty CA, Dixon-McDougall T, Lopez MV, Tyshkovskiy A, Maybury-Lewis S, et al. Chemically induced reprogramming to reverse cellular aging. Aging (Albany NY) 2023;15:5966-89.ArticlePubMedPMC
• 34. Osataphan S, Macchi C, Singhal G, Chimene-Weiss J, Sales V, Kozuka C, et al. SGLT2 inhibition reprograms systemic metabolism via FGF21-dependent and -independent mechanisms. JCI Insight 2019;4:e123130.ArticlePubMedPMC
• 35. Anton SD, Moehl K, Donahoo WT, Marosi K, Lee SA, Mainous AG, et al. Flipping the metabolic switch: understanding and applying the health benefits of fasting. Obesity (Silver Spring) 2018;26:254-68.ArticlePubMedPMCPDF
• 36. Corezola do Amaral ME, Kravets V, Dwulet JM, Farnsworth NL, Piscopio R, Schleicher WE, et al. Caloric restriction recovers impaired β-cell-β-cell gap junction coupling, calcium oscillation coordination, and insulin secretion in prediabetic mice. Am J Physiol Endocrinol Metab 2020;319:E709-20.ArticlePubMedPMC
• 37. Ishida E, Lei X, Horiguchi K, Matsumoto S, Yoshino S, Nakajima Y, et al. Efficient restoration of beta cell dedifferentiation by calorie restriction with high fat/low carbohydrate diet in obese diabetes model and the possible role of GLP-1. J Endocrine Soc 2021;5(Suppl 1):A439.ArticlePMCPDF
• 38. Cahn A, Cefalu WT. Clinical considerations for use of initial combination therapy in type 2 diabetes. Diabetes Care 2016;39 Suppl 2:S137-45.ArticlePubMedPMCPDF
• 39. Kim JY, Kim NH. Initial combination therapy in type 2 diabetes. Endocrinol Metab (Seoul) 2024;39:23-32.ArticlePubMedPMCPDF

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Hong S, Kong BS, Lee H, Cho YM. Anti-Senescence Effect of Inhibiting Sodium-Glucose Cotransporter 2 and α-Glucosidase in a Type 2 Diabetes Mellitus Animal Model. Diabetes Metab J. 2025;49(6):1229-1241.

Received: Jun 28, 2024; Accepted: Jan 23, 2025

DOI: https://doi.org/10.4093/dmj.2024.0339.

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