A Sodium Glucose Cotransporter 2 Inhibitor Fails to Improve Perivascular Adipose Tissue-Mediated Modulation of Vasodilation and Cardiac Function in Rats With Metabolic Syndrome

Abstract

Arterial perivascular adipose tissue (PVAT) can elicit vasodilator signals complementary to those elicited by the endothelium in SHRSP.Z-Lepr^fa /IzmDmcr (SHRSP.ZF) rats, an animal model of metabolic syndrome (MetS). Here, we tested whether a glucose cotransporter 2 inhibitor (SGLT2-i; tofogliflozin) increased this PVAT effect to prevent the deterioration of cardiac function in aging SHRSP.ZF rats. Tofogliflozin treatments (1 or 10 mg/kg/day) or vehicle (control) were administered for 10 weeks by oral gavage to SHRSP.ZF rats, starting at 13 weeks of age. At 23 weeks of age, glucose levels in the serum and urine (24 h after the last administration) were determined using commercial kits. Vasodilator responsiveness of PVAT-surrounded or PVAT-free superior mesenteric arteries was determined using acetylcholine with organ-bath methods. Cardiac ventricular function and coronary flow were determined using Langendorff heart preparations. Serum and urine glucose levels in SGLT2-i treatment groups did not differ from those in the controls, but the ratios of glycated to non-glycated albumin were lower than those in the controls. Tofogliflozin treatments did not alter relaxations in the presence of PVAT or affect relaxations of PVAT-free arteries. Left ventricular systolic pressures, maximum rate of pressure decline, and coronary flow in ex vivo hearts did not differ among the treatment groups. PVAT effects and cardiac dysfunction were not altered by tofogliflozin treatment in SHRSP.ZF rats with MetS. These results do not provide strong evidence to support the use of SGLT2-i as a cardiovascular protective therapy in MetS, which occurs prior to the onset of type 2 diabetes.

Keywords

adipose tissue metabolic syndrome sodium glucose cotransporter-2 inhibitors tofogliflozin vasodilation

Introduction

Metabolic syndrome (MetS) is a cluster condition that increases the risk of developing coronary heart disease, stroke, and type 2 diabetes. The initiating sequence may begin with obesity, which leads to insulin resistance. The latter is followed by high blood glucose, abnormal lipid metabolism, and increased blood pressure resulting in increased atherogenic risk.¹ Pharmacological interventions and lifestyle changes for patients (regular exercise and low-calorie diets) are common treatments for dyslipidemia, hypertension, and hyperglycemia.^2
-4 With respect to pharmacological agents, sodium glucose cotransporter-2 inhibitors (SGLT2-i) effectively lower blood glucose levels by inhibiting glucose reabsorption in the proximal renal tubules, and, according to a large international study, protect against the development of cardiovascular events in patients with type 2 diabetes.⁵ Importantly, studies in animal models suggest that these inhibitors may also exert favorable effects in MetS. For example, tofogliflozin induced body weight reduction by increasing calorie loss through urinary glucose excretion and prevented fat accumulation and inflammation in the adipose tissue and liver of rats with diet-induced obesity.⁶ Another example is luseogliflozin, which increased the urinary excretion of glucose, decreased the blood pressure via an increase in the urinary excretion of sodium,⁷ and improved the circadian rhythm of sympathetic nervous function⁸ in SHR/NDmcr-cp rats, an animal MetS model. These findings support the hypothesis that SGLT2-i increases the urinary excretion of glucose and exerts a therapeutic effect against MetS-related health complications, such as cardiovascular events, coronary heart disease, and stroke.

Perivascular adipose tissue (PVAT), which surrounds the blood vessels, regulates vascular homeostasis by releasing vasoactive molecules.^9,10 For example, the PVAT surrounding coronary arteries is associated with coronary inflammation and atherosclerosis, processes that elevate the risk of cardiovascular events.¹¹ As PVAT dysfunction may lead to the development of vascular complications, the amelioration of PVAT functions is a valid strategy for disease prevention.^12,13 Because epicardial adipose tissue can mediate the adverse effects of metabolic disorders on the heart, it is also an important target for therapeutic interventions aimed at improving cardiac function.¹⁴ Our research using SHRSP.ZF rats, an animal model of MetS,¹⁵ also provides strong support for the idea that PVAT dysfunction is an important symptom of MetS. We have previously shown that the PVAT around the mesenteric arteries compensates for the effects of impaired nitric oxide (NO)-dependent vasodilation on the circulatory system, a condition that develops when these rats reach 17 and 20 weeks of age, but this compensatory function disappears as MetS progresses (around 23 and 30 weeks of age).^16,17 We have also reported that aged SHRSP.ZF rats exhibit ventricular diastolic dysfunction^18,19 and investigated the potential mechanisms underlying the deterioration of PVAT compensatory function associated with the development of cardiac dysfunction in MetS. We also reported that angiotensin II type I receptor antagonists (ARBs), including azilsartan, telmisartan, and olmesartan, protect against MetS-associated vascular and cardiac dysfunction in SHRSP.ZF rats.^18,20
-22 Surprisingly, azilsartan does not prevent PVAT dysfunction,²⁰ which suggests that there remain gaps in our understanding of these new therapeutic strategies.

The cardiovascular benefits of glycemic control with SGLT2-i in patients with type 2 diabetes were demonstrated in several large clinical outcome studies. In particular, the EMPA-REG OUTCOME trial provided evidence that empagliflozin reduces cardiovascular death, non-fatal myocardial infarction, non-fatal stroke, and hospitalization for heart failure in people with type 2 diabetes.²³ Animal models of type 2 diabetes have been used to investigate the putative mechanisms that mediate the cardiovascular benefits of SGLT2-i.^24

-29 Recent studies reported that empagliflozin improved vascular endothelial function in rats with MetS³⁰ and ameliorated both cardiac remodeling and function in nondiabetic animals with metabolic syndrome and heart failure.^30
-32 In light of these positive outcomes, further study on the effects of SGLT2-i in MetS, particularly their potential as therapeutics to be used before the onset of type 2 diabetes, appears to be warranted.

In the current study, we performed in vivo SGLT2-i treatment to assess whether the use of tofogliflozin prevents the disappearance of the ameliorating effect of PVAT on vasodilation and cardiac dysfunction that occurs in aging SHRSP.ZF rats. Specifically, we examined whether the administration of tofogliflozin preserves the functional changes in PVAT surrounding the mesenteric arteries and prevents cardiac dysfunction that occurs in SHRSP.ZF rats as aging progresses.

Methods and Materials

Drugs

Tofogliflozin was provided by Kowa Company, Ltd. (Aichi, Japan). Purchased reagents and chemicals included L-phenylephrine hydrochloride (Sigma-Aldrich Co., LLC., St. Louis, USA), acetylcholine chloride (Daiichi Pharmaceutical Co., Ltd., Tokyo, Japan), and sodium nitroprusside (Nacalai Tesque Inc., Kyoto, Japan). Other chemical regents of analytical reagent grade were purchased from Nacalai Tesque Inc.

Experimental Animals

All protocols involving the care and use of animals were approved by the Animal Ethics Committee and performed in accordance with the Guidelines for the Care and Use of Laboratory Animals at Mukogawa Women’s University (Protocol number: P-12-2018-01-A).

The SHRSP.ZF rat model of MetS was established by crossing stroke-prone spontaneously hypertensive rats with Zucker obese rats (Disease Model Cooperative Research Association, Kyoto, Japan). They exhibit major biochemical features of MetS in that they spontaneously develop obesity and severe hypertension and exhibit hyperlipidemia and abnormal glucose tolerance.¹⁵ Eighteen male SHRSP.ZF rats at 12 weeks of age were purchased from Japan SLC, Inc. (Hamamatsu, Japan). After an acclimatization period of 1 week, the systolic blood pressure and body weight of each rat were measured. The 13-week-old rats were randomly divided among the following 3 groups (n = 6 per group): Low-dose group, in which the rats were administered 1 mg/kg body weight of tofogliflozin by oral gavage once daily; High-dose group, in which the rats received a higher dose of tofogliflozin (10 mg/kg body weight); and Control group, in which the rats were administered the vehicle (0.5% methylcellulose solution; 0.1 mL/100 g body weight). The treatment started at 13 weeks of age (T₀ ) and continued for 10 consecutive weeks. The age range of animals was chosen based on the studies showing that the “breakdown” of the PVAT compensatory system occurs at 23 weeks of age,^16,17 while the specific doses of tofogliflozin were previously used in Zucker diabetic rats and db/db mice.⁶ Rats were provided a standard chow diet (CE-2; Clea Japan Inc., Tokyo, Japan) and water ad libitum during the experimental period. Food intake of each group and body weights were measured daily and weekly during the treatment period, respectively.

Determination of Metabolic Parameters

Systolic arterial blood pressure without heating was assessed in each animal at T₀+9 weeks by the tail-cuff method using an MK-2000 meter (Muromachi, Tokyo, Japan) as described previously.³³ The systolic blood pressure values for each rat were averaged from at least 3 consecutive readings under resting conditions. The rats were familiarized with the procedure by being exposed to it once every 4 weeks before measurement at T₀+9 weeks. At T₀+10 weeks (23 weeks of age), body weight, waist circumference, and body length were measured. The waist circumference-to-body length ratio was calculated as an index of abdominal obesity. Non-fasting blood and urine samples were collected from each rat by needle puncture from the abdominal aorta and bladder, respectively, after anesthesia with an intraperitoneal injection of ketamine/xylazine (90 and 10 mg per kg of body weight, respectively). Blood was also collected from the tail vein after 16 h-fasting at T₀+9 weeks. Blood was centrifuged at 3,000 × g for 10 min at 4°C to separate the serum, to determine the levels of glucose, insulin, and thiobarbituric acid reactive substances (TBARS), an index of oxidative stress using commercial kits from Wako Pure Chemical Ind. Ltd. (Osaka, Japan), Morinaga Institute of Biological Science (Tokyo, Japan), and Cayman Chemical Co. (Ann Arbor, MI, USA), respectively. Total and glycated serum albumin concentrations were measured using commercial kits, Albumin II HA-test Wako and Lucica GA-L, from Wako Pure Chemical Ind. Ltd. and Asahi Kasei Pharma Co. (Tokyo, Japan), respectively. Glycated albumin values were calculated as glycated albumin concentration divided by total albumin concentration and were considered to be zero when the assayed glycated albumin levels were below the limit of detection of 0.010 g/dL. Urine glucose levels were measured using colorimetry with the Uropaper III urinalysis test strip (E-UR80; Eiken Chemical Co., Ltd., Tochigi, Japan) according to the manufacturer’s instructions. Results were recorded using a 6-point scale as follows: negative (none detected) = 0; 50 mg/100 mL = 1; 100 mg/100 mL = 2; 250 mg/100 mL = 3; 500 mg/100 mL = 4; 2000 mg/100 mL = 5.

Determination of Vasodilation

Differences in vasodilation of arteries with or without surrounding PVAT were assessed using organ-bath methods as described previously.¹⁶ Briefly, the superior mesenteric arteries were removed from each rat and immediately placed in Krebs-Henseleit (Krebs) buffer (pH 7.4; 118.4 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO₄, 2.5 mM CaCl₂, 25 mM NaHCO₃, 1.2 mM KH₂PO₄, and 11.1 mM glucose), saturated by bubbling with a 95% O₂/5% CO₂ gas mixture. Arteries with intact or removed PVAT were cut into 3-mm rings, which were then mounted isometrically at an optimal resting tension (0.3 g) in 10 mL organ baths filled with Krebs solution. Drugs were added cumulatively into these baths containing the arterial rings. Drug concentration-response curves were determined (concentration ranges indicated) for phenylephrine (0.1 nM-30 µM), acetylcholine (0.1 nM-1 µM), and sodium nitroprusside (0.1 nM-1 µM). Isometric tension changes were measured with a Model t-7 force-displacement transducer (NEC San-Ei, Tokyo, Japan) coupled to a Model 8K21 dual channel chart recorder (NEC San-Ei). Stock solutions of the tested compounds were prepared in distilled water. The relaxation data were calculated as 100% relaxation when the contraction induced by phenylephrine was completely inhibited by acetylcholine or sodium nitroprusside. The contractile responses triggered by noradrenaline (the active isometric force in g) were normalized to the wet weight of the respective arterial preparation in mg and expressed as g/mg. Individual concentration-response curves were analyzed using nonlinear regression curve fitting of relaxation-drug concentration associations to determine the negative log of EC₅₀, the E_max, and the area under the curve (AUC) using GraphPad Prism ver. 5.0 (GraphPad Software, Inc., San Diego, CA, USA).

Determination of Cardiac Function

Cardiac function was assessed using an Isolated Perfused Heart, Working Heart & Langendorff system (IPH-L2A; Primetech Co., Tokyo, Japan) as described previously.¹⁸ Briefly, the heart of each rat was excised and placed immediately in Krebs buffer (described above). After cannulation of the aorta, the coronary circulation was perfused with Krebs buffer saturated with a 95% O₂/ 5% CO₂ gas mixture at 37°C. Left ventricular systolic pressure (LVSP), left ventricular pressure decline (−dP/dt), and coronary flow rate (CFR) were measured at a constant perfusion pressure of 80 mmHg.

Statistical Analysis

Values are expressed as mean ± standard error of the mean (SEM). N = number of animals. Statistical comparisons of the mean metabolic parameters between groups were performed using 1-way ANOVA followed by Bonferroni post-hoc test. The main effects of treatment (3 levels) and PVAT (2 levels with sample matching) plus their interaction with arterial relaxation responses were determined using 2-way ANOVA. If the main or interaction effects were found to be significant in the ANOVA, then multiple comparison testing between the means of different groups was performed using the Bonferroni post-hoc test. P < .05 was considered significant for all tests.

Results

Effects of SGLT2-i Treatment on the Metabolic Parameters of SHRSP.ZF Rats

As demonstrated by the measurements acquired from the 23-week-old SHRSP.ZF rats (Figure 1), daily administration of tofogliflozin for 10 weeks starting at 13 weeks of age did not significantly affect either food intake or body weight. Moreover, tofogliflozin did not significantly affect the body weight or waist circumference-to-body length ratio (an index of abdominal obesity) in SHRSP.ZF rats at 23 weeks (Table 1); this also applies to the systolic arterial blood pressures and heart rates. Serum glucose levels, insulin levels, and glucose excretion in the urine were not significantly different among the groups. Serum glucose levels under fasting conditions were also not altered by tofogliflozin (Table 1). In contrast, in the tofogliflozin treatment group, the glycated albumin values were one-half to one-fifth lower than those in the controls. TBARS levels, an index of systemic oxidative stress, did not differ among the groups.

Figure 1.

Changes in food intake (A) and body weight (B) in SHRSP.Z-Lepr^fa /IzmDmcr (SHRSP.ZF) rats. SHRSP.ZF rats were administered vehicle (Control, n = 6), 1 mg/kg (Low-dose, n = 6), or 10 mg/kg (High-dose, n = 6) tofogliflozin by oral gavage daily for 10 weeks starting at 13 weeks of age.

Table 1.

Effects of Tofogliflozin Treatment on Metabolic and Cardiac Parameters of SHRSP.Z-Lepr^fa /IzmDmcr (SHRSP.ZF) Rats.

Group (n)	Control (6)	Low-dose (6)	High-dose (6)
Body weight (g)	477 ± 18	473 ± 14	459 ± 4
Waist circumference-to-body length ratio (cm/cm)	0.963 ± 0.02	0.955 ± 0.022	0.952 ± 0.025
Systolic blood pressure (mmHg)	265 ± 14	264 ± 5	281 ± 13
Heart rate (beats/min)	384 ± 11	352 ± 15	414 ± 16
Serum analysis (non-fasting condition)
Glucose (mg/100 mL)	253 ± 27	266 ± 20	236 ± 6
Insulin (ng/mL)	90.3 ± 15.4	82.5 ± 13.7	68.9 ± 5.7
TBARS (µM)	18.8 ± 2.0	23.0 ± 3.9	20.3 ± 1.6
Glycated albumin value (%)	1.18 ± 0.256	0.417 ± 0.134*	0.117 ± 0.075*
Serum glucose (mg/100 mL) (fasting condition)	102 ± 11	127 ± 16	121 ± 10
Urine analysis (non-fasting condition)
Glucose (score)	0.333 ± 0.211	0.667 ± 0.307	0.583 ± 0.301
Heart weight
Atrium (mg)	100 ± 5	104 ± 5	94.1 ± 4.9
Ventricle (mg)	1434 ± 22	1448 ± 28	1479 ± 25
Total heart weight-to-body weight ratio (mg/g)	3.28 ± 0.12	3.33 ± 0.11	3.43 ± 0.06

Results are expressed as the mean ± SEM. SHRSP.ZF rats were administered vehicle (Control group), 1 mg/kg tofogliflozin (Low-dose group), or 10 mg/kg tofogliflozin (High-dose group) by oral gavage daily for 10 weeks starting at 13 weeks of age. *P < .05, statistical comparisons of means between the groups were performed using 1-way ANOVA followed by Bonferroni post-hoc test.

Effects of SGLT2-i Treatment on the Vascular Functions of SHRSP.ZF Rats

Figure 2 and Table 2 show the contractions and relaxations in mesenteric arteries with and without PVAT. The same data are shown in Figure 3 for simpler comparison of plots with and without PVAT in each treatment group. SGLT2-i in vivo treatment did not alter the contractions or relaxations in mesenteric arteries without PVAT (Figure 2A-C). Similarly, there were no significant differences in these responses in arteries with PVAT (Figure 2D-F). As shown in Figure 2, the presence of PVAT significantly increased vasodilation in response to acetylcholine in all groups (2-way ANOVA, P < .01), but tofogliflozin had no impact on this effect (Figure 3D-F and Table 2). The phenylephrine-induced contractions (Figure 3A-C) and sodium nitroprusside-induced relaxations (Figure 3G-I) were unchanged with and without PVAT.

Figure 2.

Vascular contraction by phenylephrine (A and D) and relaxation in response to acetylcholine (B and E) and sodium nitroprusside (C and F) in perivascular adipose tissue (PVAT)-surrounded [PVAT(+)] or PVAT-free [PVAT(−)] superior mesenteric arteries from SHRSP.Z-Lepr^fa /IzmDmcr (SHRSP.ZF) rats. Rats were administered vehicle (Control, n = 6), 1 mg/kg (Low-dose, n = 6), or 10 mg/kg (High-dose, n = 6) tofogliflozin by oral gavage daily for 10 weeks starting at 13 weeks of age.

Figure 3.

Vascular contraction by phenylephrine (A to C) and relaxation by acetylcholine (D to F), and sodium nitroprusside (G to I) in perivascular adipose tissue (PVAT)-surrounded [PVAT(+)] or PVAT-free [PVAT(−)] superior mesenteric arteries from SHRSP.Z-Lepr^fa /IzmDmcr (SHRSP.ZF) rats. Rats were administered vehicle (Control, n = 6), 1 mg/kg (Low-dose, n = 6), or 10 mg/kg (High-dose, n = 6) tofogliflozin by oral gavage daily for 10 weeks starting at 13 weeks of age.

Table 2.

Effects of Tofogliflozin Treatment on Phenylephrine-Induced Contraction and Acetylcholine- and Sodium Nitroprusside-Induced Relaxation Responses in Isolated Superior Mesenteric Arteries With (+) or Without (−) Perivascular Adipose Tissue (PVAT) From SHRSP.Z-Lepr^fa /IzmDmcr (SHRSP.ZF) Rats.

Treatment	Control (6)		Low-dose (6)		High-dose (6)
Presence of PVAT	(−)	(+)	(−)	(+)	(−)	(+)	Two-way ANOVA
Phenylephrine
−Log EC₅₀	6.21 ± 0.30	6.12 ± 0.17	6.04 ± 0.19	6.16 ± 0.14	6.10 ± 0.20	6.00 ± 0.20
E_max	0.28 ± 0.07	0.25 ± 0.03	0.28 ± 0.04	0.25 ± 0.03	0.33 ± 0.07	0.34 ± 0.04
AUC	75.6 ± 19.9	67.9 ± 9.2	72.4 ± 10.6	67.9 ± 8.9	80.5 ± 17.3	76.6 ± 10.4
Acetylcholine
−Log EC₅₀	7.43 ± 0.14	7.65 ± 0.09	7.40 ± 0.11	7.76 ± 0.09	7.45 ± 0.07	7.67 ± 0.15	* PVAT
E_max	72.9 ± 9.9	89.0 ± 2.7	79.5 ± 7.1	89.8 ± 2.3	81.2 ± 10.1	94.5 ± 1.8	* PVAT
AUC	105 ± 17	143 ± 10	112 ± 17	154 ± 10	120 ± 17	156 ± 14	* PVAT
Nitroprusside
−Log EC₅₀	7.20 ± 0.19	7.11 ± 0.18	7.09 ± 0.08	7.08 ± 0.08	7.17 ± 0.11	7.08 ± 0.11
E_max	84.5 ± 5.4	80.6 ± 4.4	85.5 ± 6.4	89.5 ± 2.3	93.8 ± 3.1	93.1 ± 2.7
AUC	144 ± 25	152 ± 18	146 ± 16	156 ± 9	154 ± 14	153 ± 12

SHRSP.ZF rats were administered vehicle (Control group), 1 mg/kg tofogliflozin (Low-dose group), or 10 mg/kg tofogliflozin (High-dose group) by oral gavage daily for 10 weeks starting at 13 weeks of age. Results are expressed as the mean ± SEM. Numbers in parentheses indicate the treatment group sample size. *P < .05, significant effect of PVAT on response to treatment with acetylcholine as determined by 2-way ANOVA. Treatment and interaction effects on Emax, EC_50, and AUC in all drugs were not statistically significant, as evaluated by 2-way ANOVA.

Effects of SGLT2-i Treatment on the Cardiac Function of SHRSP.ZF Rats

The SGLT2-i did not alter the weight of the atria or the ventricles of the hearts of SHRSP.ZF rats (Table 1). The total heart weight-to-body weight ratio, an index of cardiac hypertrophy, was also not altered by tofogliflozin (Table 1). Moreover, tofogliflozin did not affect LVSP or −dP/dt, indices of cardiac contractile and diastolic functions, respectively. Similarly, no changes in CFR or heart rate were observed (Figure 4).

Figure 4.

Left ventricular cardiac function (A and B), coronary flow rate (C), and heart beats (D) in isolated heart from SHRSP.Z-Lepr^fa /IzmDmcr (SHRSP.ZF) rats. Rats were administered vehicle (Control, n = 6), 1 mg/kg (Low-dose, n = 6), or 10 mg/kg (High-dose, n = 6) tofogliflozin by oral gavage daily for 10 weeks starting at 13 weeks of age.

Discussion

PVAT assists the regulation of vascular tone by compensating for impaired vasodilation in the mesenteric artery of SHRSP.ZF rats, a murine MetS model, but this compensatory PVAT function disappears as MetS progresses.¹⁶ The present study demonstrates that administering the SGLT2-i tofogliflozin did not enhance the modulation of vascular function by PVAT in the mesenteric arteries of SHRSP.ZF rats. Indeed, tofogliflozin treatment failed to ameliorate the cardiac dysfunction and increased heart size observed in aged SHRSP.ZF rats. These findings suggest that SGLT2-i treatment does not significantly alter the supplementary effects of PVAT or the cardiac dysfunction in MetS, which occurs prior to the onset of type 2 diabetes.

In the current study, chronic treatment with tofogliflozin, administered by oral gavage to SHRSP.ZF rats, showed no lasting effect on blood glucose or glycosuria after the treatment ended (24 h after the last dose). These doses of tofogliflozin (1 or 10 mg/kg) were based on the study by Suzuki et al,⁶ which reported that a single oral administration of 1 or 10 mg/kg of tofogliflozin decreased the serum glucose level and increased renal glucose clearance in several rodent models of type 2 diabetes, including Zucker diabetic fatty rats, Goto-Kakizaki rats, and db/db mice. In our study, the higher dose, 10 mg/kg of tofogliflozin, was the maximum rat dose recommended by the supplier. The discrepancy in observations between our study and the study by Suzuki et al⁶ may be explained by the study designs using chronic and acute treatment, respectively, different animal models, and the timing of blood collection. It was noted that even in the study conducted by Suzuki et al,⁶ the serum glucose levels of Zucker diabetic fatty rats returned to pretreatment levels 24 h after a single oral administration of 10 mg/kg tofogliflozin. Other researchers using chronic treatments in their study design have reported observations similar to ours, specifically that long-term administration of tofogliflozin (6-month treatment starting at 4 weeks of age) failed to alter glucose metabolism in mice with obesity induced by a high-fat diet.³⁴ An interesting observation in experimental studies is that tofogliflozin did not reduce renal glucose reabsorption under hypo- or euglycemic conditions in rats.³⁵ This characteristic has been recognized in other SGLT2-selective inhibitors, which increased safety by lowering the risk of causing hypoglycemia. Because SHRSP.ZF rats were not hyperglycemic based on the fasting glucose data, it is not surprising that these values did not differ between the treatment groups. Glycated hemoglobin, which is clinically used to diagnose prediabetes in humans, is also affected by SGLT2-i. Indeed, a 4-week administration of 1 or 10 mg/kg tofogliflozin decreased the glycated hemoglobin levels in db/db mice; and serum glucose level was also decreased at 4 h post-SGLT2-i treatment.⁶ We found that tofogliflozin treatment lowered the glycated albumin levels in the serum of SHRSP.ZF rats with MetS compared to those in the control groups. The serum glycated albumin level reflects the glycemic balance over a period of 3 weeks, and it has recently received attention as an alternative glycemic marker for cases in which HbA1c cannot be assessed or is deficient, such as in patients with hemoglobinopathies, chronic kidney disease, or pregnancy.^36,37 Considering that glycated albumin, like glycated hemoglobin, reflects long-term glycemic balance, as it changes depending on the glucose concentration, we interpret these data to indicate that tofogliflozin treatments must have been sufficient to alter blood glucose levels over the course of the study to reduce the production of glycated albumin during the treatment period in SHRSP.ZF rats.

In our study, the 10-week period of tofogliflozin treatment did not prevent dysfunction of vascular relaxations in mesenteric arteries of aged SHRSP.ZF rats with MetS. This differs from the results reported for other SGLT2-i treatments in diabetic rodents. For example, ipragliflozin ameliorated vascular relaxation in response to acetylcholine in the aortas of streptozotocin-induced type 1 diabetic mice²⁴ and rats.²⁶ Empagliflozin has been reported to improve acetylcholine-induced endothelium-dependent relaxations in the aorta in a type 2 diabetes model (Zucker diabetic fatty rats)²⁷; it also improved the endothelium-independent relaxations induced by the NO donor, nitroso-N-acetylpenicillamine, in the aortas of db/db mice.²⁵ The findings from those animal models of type 1 and 2 diabetes suggest that oxidative stress may counter vascular dysfunction under hyperglycemic conditions.^24

-27 In a previous study using the SHRSP.ZF rats, we demonstrated that elevated levels of oxidative stress under MetS are correlated with impaired vascular relaxations, resulting from the decreased expression of soluble guanylyl cyclase, a key enzyme for NO-mediated vasodilation.³⁸ Surprisingly, in the current study, SGLT2 inhibition by tofogliflozin did not avert an increase in TBARS levels, which we have commonly used as an index of systemic oxidative stress. We have shown that ARBs reduce this oxidative stress and concomitantly improve vascular function. Our new finding that tofogliflozin also failed to ameliorate the dysfunction of vasorelaxations in aged SHRSP.ZF rats suggests that its inability to reduce oxidative stress underlies, at least partly, its inability to ameliorate vascular dysfunction in these rats. An alternative explanation of our results is the selection of vascular tissues for our study. Interestingly, canagliflozin was found to improve the endothelium-independent (sodium nitroprusside-induced) vascular relaxations in the pulmonary artery, but not the coronary artery, of mice with high-fat diet-induced obesity,²⁸ suggesting that the effects of SGLT2-i on vascular functions may be dependent on artery type. However, another study on empagliflozin has shown that it prevents a decrease in ACh-induced endothelium-dependent relaxations in the mesenteric artery of Zucker diabetic fatty/spontaneously hypertensive heart failure F1 hybrid rats, an experimental model of MetS. Thus, further studies are necessary to fully assess the complex effects of SGLT2-i on vascular functions that differ according to the metabolic disorder conditions and individual drugs. Such studies may provide further fundamental understanding of the effects of SGLT2-i in different patient populations.

The results of the present study suggest that the 10-week treatment with tofogliflozin cannot enhance the complementary effects of PVAT on mesenteric artery vasorelaxations under the condition of impaired vasorelaxation in SHRSP.ZF rats with MetS. As pointed out by the authors of a study on the effects of tofogliflozin on the inflammation of visceral adipose tissue in mice with obesity induced by a high-fat diet,³⁴ the observation of organ-protective effects of SGLT2-i therapy is hampered by the absence of an animal model in which the SGLT2-i significantly increases urinary glucose excretion and decreases blood glucose levels. In SHRSP.ZF rats with MetS, SGLT2-i had no effect on the basal levels of cardiac dysfunction. Along with the cardiac dysfunction, increased heart weights and fibrosis have been observed in aged SHRSP.ZF rats.¹⁹ In our study, tofogliflozin was unable to avert the changes in heart weight. This could be linked to its inability to reduce oxidative stress, as shown by the high levels of TBARS, resulting from high blood pressure. This hypothesis is based on the fact that the cardioprotective role of another SGLT2-i, dapagliflozin, in high-carbohydrate diet-induced MetS rats was associated with the amelioration of mitochondrial dysfunction, resulting in lower oxidative stress.²⁹ The discrepancies between the results in these 2 MetS rat models could be strain- or species-dependent. The fact that a different SGLT2-i was used in each study may also play a role. Clearly, further studies with many different models of metabolic and cardiovascular dysfunction are needed to evaluate the full potential of using SGLT2-i in therapy.

The limitations of this study are as follows. First, the treatment conditions used in the present study were based on previous reports from other groups using MetS rats.⁶ However, a higher dose or earlier treatment with tofogliflozin could have beneficial effects on cardiovascular functions and PVAT modifications in MetS. Second, the waist circumference-body length ratio was calculated as an index of abdominal obesity. However, fat mass, levels of adipokines, cytokines, and other tissue biomarkers of inflammation were not assessed. Similarly, the absolute amounts of glycated rat albumin could not be precisely determined using the commercial kits; hence, our interpretations of efficacy are based on relative glycated albumin ratios. Secondary effects arising from the loss of fat mass or direct effects regulating adipose tissue function could affect the inflammatory state of the animals. It has been reported that treatment with canagliflozin improves the serum levels of leptin, adiponectin, and IL-6³⁹ and dapagliflozin reduces epicardial adipose tissue volume in type 2 diabetes patients.⁴⁰ Third, the relationship between changes in coronary flow and peripheral vascular effects, such as mesenteric vascular functions and PVAT-mediated responses, was not directly tested. Lastly, this study examined only the basal levels of cardiac functions and coronary flow in the Langendorff working heart assay system. Further investigation of the effects on the responsiveness of the heart and coronary flow are warranted to fully determine the potential benefits of SGLT2-i treatment.

Conclusion

SGLT2-i did not preserve vasodilation capacity, as it was unable to ameliorate the deterioration of vasorelaxation either directly or by enhancing PVAT-dependent vascular response in SHRSP.ZF rats with MetS. This failure could probably lead to its inability to prevent the development of cardiac dysfunction at a later stage of MetS. Overall, our results show that, unlike in the case of diabetes, SGLT2-i is not effective in preventing the vascular dysfunctions that accompany MetS.

Footnotes

Authors’ Note

Acknowledgments

Tofogliflozin was generously provided by Kowa Company, Ltd. (Aichi, Japan). The authors sincerely thank Ms. Miho Shimari, Ms. Maho Mizuno, Ms. Fuuka Tarutani, Ms. Rina Hayashi, Ms. Tomoe Ishibashi, and Ms. Yayoi Shiokawa at Mukogawa Women’s University for their technical support.

Author Contributions

Satomi Kagota conceptualized the study and designed the experiments. Satomi Kagota and Kana Maruyama-Fumoto performed the experiments and analyzed the data. Satomi Kagota and John J. McGuire interpreted the data and prepared the manuscript. Satomi Kagota and Kazumasa Shinozuka acquired funding and supervised the study. All authors read and approved the final manuscript.

Declaration of Conflicting Interests

The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Kowa Company, Ltd. provided the tofogliflozin used in this study. Moreover, the company received a copy of this manuscript for review purposes prior to submission. The authors state that the company did not influence their decision to publish the data.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iDs

Satomi Kagota

John J. McGuire

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