**Cardiovascular and Metabolic Disease: New Treatment and Future Directions**

Editors **Alfredo Caturano Raffaele Galiero**

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*Editors* Alfredo Caturano Department of Advanced Medical and Surgical Sciences University of Campania Luigi Vanvitelli Naples Italy

Raffaele Galiero Department of Advanced Medical and Surgical Sciences University of Campania Luigi Vanvitelli Naples Italy

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This is a reprint of articles from the Special Issue published online in the open access journal *Biomedicines* (ISSN 2227-9059) (available at: www.mdpi.com/journal/biomedicines/special issues/ Cardiovascular Metabolic).

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## **About the Editors**

## **Alfredo Caturano**

Alfredo Caturano, MD, graduated in Medicine and Surgery from Second University of Naples (SUN) in 2016. He achieved the Specialization Degree in Internal Medicine in 2021 at University of Campania "Luigi Vanvitelli". His specialization thesis, concerning the impact of glycaemic control on cardiovascular outcomes and mortality in a cohort of patients with type 2 diabetes mellitus in a multifactorial randomized controlled trial, was awarded the Annapaola Braggio prize by the Italian Society of Diabetology as the best specialization thesis on topics related to insulin resistance discussed in 2021 in Italy. He presently serves as PhD attendant at University of Campania "Luigi Vanvitelli" and has been supported with multiple grants from both Italian and European scientific societies.

### **Raffaele Galiero**

Raffaele Galiero, MD, graduated in Medicine and Surgery at the University of Campania "Luigi Vanvitelli", Naples, in 2013. He achieved the Specialization Degree in Internal Medicine in 2020 at the University of Campania "Luigi Vanvitelli", Naples. At this time, he serves as PhD attendant at University of Campania "Luigi Vanvitelli", Naples.

## **Preface to "Cardiovascular and Metabolic Disease: New Treatment and Future Directions"**

Cardiovascular diseases (CVDs) are the leading cause of death worldwide. In 2019, it has been estimated that 17.9 million people died from CVDs (32% of all global deaths). Of these deaths, 85% were due to heart attack and stroke. These data are usually powered by the coexistence of metabolic diseases, in particular diabetes. In fact, about 422 million people worldwide have diabetes, the majority living in low- and middle-income countries, and 1.5 million deaths are directly attributed to diabetes each year. It is vital to detect CVDs and metabolic disease as early as possible, as most cases can be prevented by addressing behavioral risk factors, such as tobacco use, unhealthy diet and obesity, physical inactivity, and harmful use of alcohol. Moreover, we are the main actors and observers of the innovations in every field of CVDs and metabolic disease treatment, from the pharmacological approach, with the advent of sodium glucose cotransporter 2 inhibitors for both heart failure and diabetes, to the improvement of new less-invasive and invasive techniques, such as immediate revascularization in both heart and brain infarction and new methods of mechanical cardiac support and multi-organ transplantation for the most advanced forms of heart failure. Given the complexity of this topic and its impact on clinical practice and public health, *Biomedicines* is launching a Special Issue entitled "Cardiovascular and Metabolic Disease: New Treatments and Future Directions" with the aim of gathering accurate and up-to-date scientific information on all aspects of new and upcoming treatment opportunities for CVDs and metabolic diseases.

The published articles not only report on the pathophysiological mechanisms underpinning this association, but also describe the results of latest clinical research about cardiovascular and metabolic diseases and offer a comprehensive overview of the recent advances in understanding pharmacological interventions to prevent cardiovascular events.

> **Alfredo Caturano and Raffaele Galiero** *Editors*

## *Article Ldlr***-Deficient Mice with an Atherosclerosis-Resistant Background Develop Severe Hyperglycemia and Type 2 Diabetes on a Western-Type Diet**

**Weibin Shi 1,\* , Jing Li 1 , Kelly Bao 1 , Mei-Hua Chen 1 and Zhenqi Liu 2**

> 1 Department of Radiology and Medical Imaging, University of Virginia, Charlottesville, VA 22908, USA<EMAIL_ADDRESS>(J.L<EMAIL_ADDRESS>(K.B<EMAIL_ADDRESS>(M.-H.C.)

2 Department of Medicine, University of Virginia, Charlottesville, VA 22908, USA<EMAIL_ADDRESS>
**\*** Correspondence<EMAIL_ADDRESS>Tel.: +1-(434)-243-9420; Fax: +1-(434)-982-5680

**Abstract:** *Apoe*-/- and *Ldlr*-/- mice are two animal models extensively used for atherosclerosis research. We previously reported that *Apoe*-/- mice on certain genetic backgrounds, including C3H/HeJ (C3H), develop type 2 diabetes when fed a Western diet. We sought to characterize diabetes-related traits in C3H-*Ldlr*-/- mice through comparing with C3H-*Apoe*-/- mice. On a chow diet, *Ldlr*-/- mice had lower plasma total and non-HDL cholesterol levels but higher HDL levels than *Apoe*-/- mice. Fasting plasma glucose was much lower in *Ldlr*-/- than *Apoe*-/- mice (male: 122.5 ± 5.9 vs. 229.4 ± 17.5 mg/dL; female: 144.1 ± 12.4 vs. 232.7 ± 6.4 mg/dL). When fed a Western diet, *Ldlr*-/- and *Apoe*-/- mice developed severe hypercholesterolemia and also hyperglycemia with fasting plasma glucose levels exceeding 250 mg/dL. Both knockouts had similar non-HDL cholesterol and triglyceride levels, and their fasting glucose levels were also similar. Male *Ldlr*-/- mice exhibited greater glucose tolerance and insulin sensitivity compared to their *Apoe*-/- counterpart. Female mice showed similar glucose tolerance and insulin sensitivity though *Ldlr*-/- mice had higher non-fasting glucose levels. Male *Ldlr*-/- and *Apoe*-/- mice developed moderate obesity on the Western diet, but female mice did not. These results indicate that the Western diet and ensuing hyperlipidemia lead to the development of type 2 diabetes, irrespective of underlying genetic causes.

**Keywords:** low-density lipoprotein receptor; apolipoprotein E; hyperglycemia; type 2 diabetes; Western diet

#### **1. Introduction**

Diabetes is a chronic health condition featured by fasting hyperglycemia (elevations in blood sugar). Type 2 diabetes (T2D) is the major form of diabetes accounting for 90–95% of all diabetes and constitutes one of the major public health problems in the U.S. and globally. Thirty-five million (13.5%) Americans aged 18 years or older are believed to have T2D [1]. Globally, 463 million adults are estimated to be living with T2D [2]. Additionally, 92 million American adults (37.6%) have prediabetes [1], a condition that carries the risk of contracting T2D. Diabetic patients experience increased rates of macrovascular complications, including more than twice the rates of coronary artery disease and stroke [3,4], and microvascular retinopathy and chronic kidney disease [5,6].

Dyslipidemia, which comprises elevated triglyceride and LDL cholesterol levels and reduced HDL cholesterol levels, often occurs together with hyperglycemia as integral components of T2D and the metabolic syndrome, with the latter also including abdominal obesity and hypertension. Accumulating evidence suggests that dyslipidemia may play a causal role in the pathogenesis of T2D. Prospective studies show that individuals with high triglyceride and LDL cholesterol levels have an increased risk of developing T2D [7–10].

**Citation:** Shi, W.; Li, J.; Bao, K.; Chen, M.-H.; Liu, Z. *Ldlr*-Deficient Mice with an Atherosclerosis-Resistant Background Develop Severe Hyperglycemia and Type 2 Diabetes on a Western-Type Diet. *Biomedicines* **2022**, *10*, 1429. https://doi.org/ 10.3390/biomedicines10061429

Academic Editors: Alfredo Caturano and Raffaele Galiero

Received: 9 May 2022 Accepted: 14 June 2022 Published: 16 June 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Strong correlations between plasma lipid and glucose levels have been observed in segregating F2 populations derived from *Apoe-/-* mouse strains [11–13]. The strong evidence supporting a causal role for dyslipidemia in diabetes comes from genetic studies of rare mutations in *ABCA1* [14], LIPE [15], *LPL* [16], and *LRP6* [17], showing that these lipid genes are also linked to hyperglycemia or diabetes. However, contradictory findings concerning the relationship between dyslipidemia and diabetes are also noted: Hyperlipidemic patients receiving lipid-lowering therapy with PCSK9 inhibitory antibodies or statins show an increased incidence of new-onset diabetes [18–20]. Patients with heterozygous familial hypercholesterolemia caused by mutations in *LDLR*, *APOB*, *APOE,* and *PCSK9* are less vulnerable to diabetes [21,22].

*Apoe*-/- and *Ldlr*-/- mice are two widely used rodent models of dyslipidemia and atherosclerosis. On a chow diet, *Apoe*-/- mice have total plasma cholesterol levels of 300 to 500 mg/dL mainly from elevations of VLDL and chylomicron remnants, and *Ldlr*-/- mice have cholesterol levels of 200 to 300 mg/mL due to an accumulation of LDL [23–26]. When fed a Western diet, both *Apoe*-/- and *Ldlr*-/- mice develop severe hypercholesterolemia, with total cholesterol levels of ~1000 mg/dL [23,25,26]. We found that *Apoe*-/- mice on certain genetic backgrounds such as C57BL/6 (B6), C3H/HeJ (C3H), and SWR/J develop significant hyperglycemia and T2D with fasting plasma glucose exceeding 250 mg/dL when fed a Western-type diet [27,28]. Thus, we reasoned that *Ldlr*-/- mice would develop hyperglycemia and diabetes on a Western diet due to the ensuing severe dyslipidemia. To test this hypothesis, we characterized diabetes-related phenotypes in *Ldlr*-/- mice by comparing with *Apoe*-/- mice.

#### **2. Materials and Methods**

#### *2.1. Mice*

C3H-*Apoe*-/- mice and C3H-*Ldlr*-/- mice at N10 or more backcrossed generations were generated in our laboratory using the classical congenic breeding strategy [28]. Mice of both sexes were weaned at 3 weeks of age onto a chow diet containing 19% protein, 5% fat, 5% crude fiber, and 58% of the calories from carbohydrates (Teklad LM-485, Envigo, Indianapolis, IN, USA). At 6 weeks of age, one group was switched onto a Western diet containing 21% fat, 34.1% sucrose, 0.15% cholesterol, and 19.5% casein by weight (TD 88137, Envigo) and maintained on the diet for 12 weeks. The other group remained on a rodent chow diet throughout the entire experimental period. The animals were housed in a pathogen-free facility with a 12 h light–12 h dark cycle, consistent temperature (22 ◦C), and 50% humidity. Animal care and experimentation were carried out according to the current National Institutes of Health guidelines and approved by the University of Virginia Animal Care and Use Committee (Protocol # 3109). The reporting in this study follows the recommendations in the ARRIVE guidelines.

#### *2.2. Measurements of Plasma Lipids, Glucose and Insulin*

Mice were bled twice: once before and once at the end of the Western diet feeding. The animals were fasted overnight before retro-orbital blood was collected under isoflurane anesthesia. Plasma levels of total cholesterol, HDL cholesterol, and triglyceride were measured using cholesterol reagent from Stanbio Laboratory, HDL precipitating reagent from FUJIFILM Wako Diagnostics, and triglyceride reagent from Thermo DMA (Louisville, CO, USA) as reported in [11]. Plasma was diluted 3x with H2O for measurement of total cholesterol concentrations in mice fed the Western diet. Plasma glucose concentrations were determined using a Sigma assay kit (Cat. # GAHK20) as reported in [11]. Plasma was diluted 3x for mice fed the Western diet but not diluted for chow-fed mice. Plasma insulin concentrations were measured with an ultra-sensitive ELISA kit from Crystal Chem INC (Elk Grove Village, IL; Cat. # 90080) according to the manufacturer's instructions.

#### *2.3. Glucose Tolerance Test (GTT) and Insulin Tolerance Test (ITT) 2.3. Glucose Tolerance Test (GTT) and Insulin Tolerance Test (ITT)*  GTT and ITT were performed as we described [27,28]. Briefly, overnight-fasted mice

*Biomedicines* **2022**, *10*, x FOR PEER REVIEW 3 of 13

GTT and ITT were performed as we described [27,28]. Briefly, overnight-fasted mice were injected intraperitoneally (IP) with 1 mg of glucose in 0.9% saline per gram of body weight in a volume of <0.3 mL. Blood glucose levels were measured with an UltraTouch glucometer using blood taken from cut tail tip at 0, 10, 20, 30, 60, 90, and 120 min after injection. ITT was performed under a non-fasting condition by IP injection with 0.75 U insulin in 0.9% saline per kg of body weight. Blood glucose was measured as above at 0, 15, 30, 45, and 60 min after insulin injection. were injected intraperitoneally (IP) with 1 mg of glucose in 0.9% saline per gram of body weight in a volume of <0.3 mL. Blood glucose levels were measured with an UltraTouch glucometer using blood taken from cut tail tip at 0, 10, 20, 30, 60, 90, and 120 min after injection. ITT was performed under a non-fasting condition by IP injection with 0.75 U insulin in 0.9% saline per kg of body weight. Blood glucose was measured as above at 0, 15, 30, 45, and 60 min after insulin injection.

#### *2.4. Statistical Analysis 2.4. Statistical Analysis*

Values were expressed as means ± SE, with *n* indicating the number of animals. The distributions of trait values were assessed for normality by examining skewness, kurtosis, and Q–Q plot as reported in [29]. Student's t test and ANOVA (Analysis of Variance) were used for determining statistical significance between two or more groups. When the *p*-value for ANOVA was statistically significant, Dunnett's correction was applied. Differences were considered statistically significant at *p* < 0.05. Values were expressed as means ± SE, with *n* indicating the number of animals. The distributions of trait values were assessed for normality by examining skewness, kurtosis, and Q–Q plot as reported in [29]. Student's t test and ANOVA (Analysis of Variance) were used for determining statistical significance between two or more groups. When the *p*value for ANOVA was statistically significant, Dunnett's correction was applied. Differences were considered statistically significant at *p* < 0.05.

#### **3. Results 3. Results**

#### *3.1. Fasting Plasma Lipid Levels 3.1. Fasting Plasma Lipid Levels*

Both C3H-*Apoe*-/- and C3H-*Ldlr*-/- mice developed spontaneous hypercholesterolemia on a chow diet, and it was more severe in the former (Figure 1). Total cholesterol levels of C3H-*Ldlr*-/- mice were significantly lower than those of C3H-*Apoe*-/- mice (male: 285.8 ± 10.1 vs. 344.1 ± 24.6 mg/dL; female: 302.4 ± 14.6 vs. 419.2 ± 11.8 mg/dL; *p* ≤ 0.013; *n* = 11 to 29). On the Western diet, both genotypes developed severe hypercholesterolemia. The total cholesterol level was significantly lower in male C3H-*Ldlr*-/- mice than in the C3H-*Apoe*-/- counterparts (719.1 ± 46.2 vs. 917.0 ± 43.1 mg/dL; *p* = 0.035; *n* = 4 to 12). Female C3H-*Ldlr*-/- mice did not differ significantly in total cholesterol level from the C3H-*Apoe*-/- counterparts (904.2 ± 41.3 vs. 1075.3 ±106.5 mg/dL; *p* = 0.09; *n* = 6 to 11). Both C3H-*Apoe*-/- and C3H-*Ldlr*-/- mice developed spontaneous hypercholesterolemia on a chow diet, and it was more severe in the former (Figure 1). Total cholesterol levels of C3H-*Ldlr*-/- mice were significantly lower than those of C3H-*Apoe*-/- mice (male: 285.8 ± 10.1 vs. 344.1 ± 24.6 mg/dL; female: 302.4 ± 14.6 vs. 419.2 ± 11.8 mg/dL; *p* ≤ 0.013; *n* = 11 to 29). On the Western diet, both genotypes developed severe hypercholesterolemia. The total cholesterol level was significantly lower in male C3H-*Ldlr*-/- mice than in the C3H-*Apoe*-/ counterparts (719.1 ± 46.2 vs. 917.0 ± 43.1 mg/dL; *p* = 0.035; *n* = 4 to 12). Female C3H-*Ldlr*-/ mice did not differ significantly in total cholesterol level from the C3H-*Apoe*-/- counterparts (904.2 ± 41.3 vs. 1075.3 ±106.5 mg/dL; *p* = 0.09; *n* = 6 to 11).

Male C3H-*Ldlr*-/- mice had significantly higher HDL cholesterol levels than the C3H-*Apoe*-/- counterparts on both chow (99.1 ± 5.6 vs. 57.0 ± 2.1 mg/dL; *p* = 0.0023; *n* = 11 to 29) and Western diets (94.7 ± 5.4 vs. 50.1 ± 4.3 mg/dL; *p* = 0.00057; *n* = 4 to 12) (Figure 2). Though statistically insignificant, female C3H-*Ldlr*-/- mice also had higher HDL cholesterol Male C3H-*Ldlr*-/- mice had significantly higher HDL cholesterol levels than the C3H-*Apoe*-/ counterparts on both chow (99.1 ± 5.6 vs. 57.0 ± 2.1 mg/dL; *p* = 0.0023; *n* = 11 to 29) and Western diets (94.7 ± 5.4 vs. 50.1 ± 4.3 mg/dL; *p* = 0.00057; *n* = 4 to 12) (Figure 2). Though statistically insignificant, female C3H-*Ldlr*-/- mice also had higher HDL cholesterol levels than female C3H-*Apoe*-/- mice on either chow (76.9 ± 2.1 vs. 53.0 ± 7.1 mg/dL; *p* = 0.058; *n* = 11 to 29) or the Western diet (44.5 ± 6.6 vs. 31.6 ± 2.1 mg/dL; *p* = 0.10; *n* = 6 to 11).

ence was found between the two knockouts (*p* > 0.2).

**Figure 2.** Fasting plasma levels of non-HDL, HDL cholesterol, and triglyceride in male and female C3H-*Apoe*-/- and C3H-*Ldlr*-/- mice fed a chow (**A**,**B**) or Western diet (**C**,**D**). Results are means ± SE of 4 to 29 mice per group. \* *p* < 0.05 vs. *Apoe*-/- mice; + *p* < 0.05 vs. chow diet. **Figure 2.** Fasting plasma levels of non-HDL, HDL cholesterol, and triglyceride in male and female C3H-*Apoe*-/- and C3H-*Ldlr*-/- mice fed a chow (**A**,**B**) or Western diet (**C**,**D**). Results are means ± SE of 4 to 29 mice per group. \* *p* < 0.05 vs. *Apoe*-/- mice; + *p* < 0.05 vs. chow diet.

levels than female C3H-*Apoe*-/- mice on either chow (76.9 ± 2.1 vs. 53.0 ± 7.1 mg/dL; *p* = 0.058; *n* = 11 to 29) or the Western diet (44.5 ± 6.6 vs. 31.6 ± 2.1 mg/dL; *p* = 0.10; *n* = 6 to 11). Non-HDL cholesterol levels of *Ldlr*-/- mice were significantly lower than those of the *Apoe*-/- counterparts on the chow diet for both males (119.2 ± 8.7 vs. 420.1 ± 50.1 mg/dL; *p* = 0.0096; *n* = 4 to 18) and females (102.5 ± 8.7 vs. 303.6 ± 24.4 mg/dL; *p* = 0.0002; *n* = 4–9) (Figure 2 A,B). On the Western diet, non-HDL levels were dramatically elevated in *Ldlr*-/ and *Apoe*-/- mice of both sexes, exceeding 800 mg/dL (Figure 2 C,D). No significant differ-

C3H-*Ldlr*-/- and C3H-*Apoe*-/- mice had similar plasma triglyceride levels except for males on the chow diet, with the former being significantly lower (100.1 ± 4.2 vs. 150.4 ± 15.3 mg/dL; *p* = 0.00018; *n* = 4 to 29). *3.2. Fasting Plasma Glucose Levels*  On the chow diet, both male and female C3H-*Ldlr*-/- mice had 40~50% lower fasting Non-HDL cholesterol levels of *Ldlr*-/- mice were significantly lower than those of the *Apoe*-/- counterparts on the chow diet for both males (119.2 ± 8.7 vs. 420.1 ± 50.1 mg/dL; *p* = 0.0096; *n* = 4 to 18) and females (102.5 ± 8.7 vs. 303.6 ± 24.4 mg/dL; *p* = 0.0002; *n* = 4–9) (Figure 2A,B). On the Western diet, non-HDL levels were dramatically elevated in *Ldlr*-/- and *Apoe*-/- mice of both sexes, exceeding 800 mg/dL (Figure 2C,D). No significant difference was found between the two knockouts (*p* > 0.2).

glucose levels than the C3H-*Apoe*-/- counterparts (male: 122.5 ± 5.9 vs. 229.4 ± 17.5 mg/dL; *p* = 2.0 × 10-8; female: 144.1 ± 12.4 vs. 232.7 ± 6.4 mg/dL; *p* = 1.6 × 10-6; *n* = 11 to 29) (Figure 3). On the Western diet, both C3H-*Ldlr*-/- and C3H-*Apoe*-/- mice developed significant hy-C3H-*Ldlr*-/- and C3H-*Apoe*-/- mice had similar plasma triglyceride levels except for males on the chow diet, with the former being significantly lower (100.1 ± 4.2 vs. 150.4 ± 15.3 mg/dL; *p* = 0.00018; *n* = 4 to 29).

#### perglycemia, with fasting glucose levels exceeding 250 mg/dL. Male C3H-*Ldlr*-/- mice had *3.2. Fasting Plasma Glucose Levels*

a lower plasma glucose level (304.7 ± 21.8 vs. 398.1 ± 46.4 mg/dL; *p* = 0.061; *n* = 4 to 13), while female C3H-*Ldlr*-/- mice had a higher plasma glucose level when compared to the On the chow diet, both male and female C3H-*Ldlr*-/- mice had 40~50% lower fasting glucose levels than the C3H-*Apoe*-/- counterparts (male: 122.5 ± 5.9 vs. 229.4 ± 17.5 mg/dL; *p* = 2.0 × 10−8 ; female: 144.1 ± 12.4 vs. 232.7 ± 6.4 mg/dL; *p* = 1.6 × 10−6 ; *n* = 11 to 29) (Figure 3). On the Western diet, both C3H-*Ldlr*-/- and C3H-*Apoe*-/- mice developed significant hyperglycemia, with fasting glucose levels exceeding 250 mg/dL. Male C3H-*Ldlr*-/- mice had a lower plasma glucose level (304.7 ± 21.8 vs. 398.1 ± 46.4 mg/dL; *p* = 0.061; *n* = 4 to 13), while female C3H-*Ldlr*-/- mice had a higher plasma glucose level when compared to the C3H-*Apoe*-/- counterparts (381.7 ± 25.4 vs. 297.4 ± 31.1 mg/dL; *p* = 0.06; *n* = 4 to 7). Compared to the chow diet, the Western diet significantly elevated plasma glucose levels of C3H-*Ldlr*-/- and C3H-*Apoe*-/- mice (*p* ≤ 0.0055).

*Ldlr*-/- and C3H-*Apoe*-/- mice (*p* ≤ 0.0055).

**Figure 3.** Fasting plasma glucose levels of male and female C3H-*Apoe*-/- and C3H-*Ldlr*-/- mice fed a chow or Western diet. Results are means ± SE of 4 to 29 mice per group. \* *p* < 0.05 vs. *Apoe*-/- mice and + *p* < 0.05 vs. chow diet. **Figure 3.** Fasting plasma glucose levels of male and female C3H-*Apoe*-/- and C3H-*Ldlr*-/- mice fed a chow or Western diet. Results are means ± SE of 4 to 29 mice per group. \* *p* < 0.05 vs. *Apoe*-/- mice and + *p* < 0.05 vs. chow diet.

C3H-*Apoe*-/- counterparts (381.7 ± 25.4 vs. 297.4 ± 31.1 mg/dL; *p* = 0.06; *n* = 4 to 7). Compared to the chow diet, the Western diet significantly elevated plasma glucose levels of C3H-

#### *3.3. Glucose Tolerance Test (GTT) and Insulin Tolerance Test (ITT) 3.3. Glucose Tolerance Test (GTT) and Insulin Tolerance Test (ITT)*

Intraperitoneal GTT and ITT were performed on both male and female C3H-*Ldlr*-/ and C3H-*Apoe*-/- mice fed the Western diet. In response to injected glucose, blood glucose levels rose quickly to the peak at the 20th min for male C3H-*Ldlr*-/- mice and the 30th min for male *Apoe*-/- mice (Figure 4). Then, glucose levels fell gradually and returned to preinjection levels by the 120th min. Compared to C3H-*Apoe*-/- counterparts, male C3H-*Ldlr*-/ mice displayed significant glucose tolerance (*p* < 0.001; *n* = 4 to 17), having lower glucose levels at the 10th, 20th, and 30th min. For female mice, blood glucose levels quickly reached the peak at the 10th min and then fell gradually. There was no significant difference in glucose tolerance between female C3H-*Ldlr*-/- and C3H-*Apoe*-/- mice (*p* = 0.25; *n* = 4 to 17). Intraperitoneal GTT and ITT were performed on both male and female C3H-*Ldlr*-/ and C3H-*Apoe*-/- mice fed the Western diet. In response to injected glucose, blood glucose levels rose quickly to the peak at the 20th min for male C3H-*Ldlr*-/- mice and the 30th min for male *Apoe*-/- mice (Figure 4). Then, glucose levels fell gradually and returned to pre-injection levels by the 120th min. Compared to C3H-*Apoe*-/- counterparts, male C3H-*Ldlr*-/- mice displayed significant glucose tolerance (*p* < 0.001; *n* = 4 to 17), having lower glucose levels at the 10th, 20th, and 30th min. For female mice, blood glucose levels quickly reached the peak at the 10th min and then fell gradually. There was no significant difference in glucose tolerance between female C3H-*Ldlr*-/- and C3H-*Apoe*-/- mice (*p* = 0.25; *n* = 4 to 17).

In response to injected insulin, male C3H-*Ldlr*-/- mice showed a gradual fall in blood glucose levels till the 30th min, which remained low till the 45th min and recovered at the 60th min (Figure 5A). In contrast, male C3H-*Apoe*-/- mice showed an increase in blood glucose levels at the 15th min though glucose levels were comparable to the levels of C3H-*Ldlr*-/- mice from the 30th to 60th min (16% reduction from the basal) (Figure 5C). Female C3H-*Ldlr*-/- mice showed a more obvious fall in blood glucose levels than female C3H-*Apoe*-/- mice (47% vs. 18% decrease from the basal level) (Figure 5B,D). In addition, the basal non-fasting blood glucose level (at 0 min) was significantly higher in female C3H-*Ldlr*-/- mice than in the C3H-*Apoe*-/- counterparts (165.4 ± 13.7 vs. 118.5 ± 5.4 mg/dL; *p* = 0.006; *n* = 4 to 20).

#### *3.4. Plasma insulin Concentration*

Fasting plasma insulin levels were measured for female C3H-*Ldlr*-/- and C3H-*Apoe*-/ mice fed the Western diet. C3H-*Ldlr*-/- mice had higher insulin levels than the C3H-*Apoe*-/ mice (0.622 ± 0.121 vs. 0.487 ± 0.074 ng/mL), although the difference was not statistically significant (*p* = 0.33; *n* = 7 to 13) (Figure 6).

**Figure 4.** Glucose tolerance test (GTT) (**A**,**B**) and calculated area under the curve (**C**,**D**) for male and female C3H-*Apoe*-/- and C3H-*Ldlr*-/- mice fed a Western diet. Overnight-fasted mice were intraperitoneally injected with 1 g glucose per kg body weight. Blood glucose concentrations were determined with a glucometer using blood taken from cut tail tips at the indicated time points. Values are means ± SE of 4 to 17 mice per group. \* *p* < 0.05 vs. *Apoe*-/- mice. **Figure 4.** Glucose tolerance test (GTT) (**A**,**B**) and calculated area under the curve (**C**,**D**) for male and female C3H-*Apoe*-/- and C3H-*Ldlr*-/- mice fed a Western diet. Overnight-fasted mice were intraperitoneally injected with 1 g glucose per kg body weight. Blood glucose concentrations were determined with a glucometer using blood taken from cut tail tips at the indicated time points. Values are means ± SE of 4 to 17 mice per group. \* *p* < 0.05 vs. *Apoe*-/- mice. *Biomedicines* **2022**, *10*, x FOR PEER REVIEW 7 of 13

In response to injected insulin, male C3H-*Ldlr*-/- mice showed a gradual fall in blood

**Figure 5.** Insulin tolerance test (ITT) for male and female C3H-*Apoe*-/- and C3H-*Ldlr*-/- mice fed a Western diet. Non-fasted mice were intraperitoneally injected with 0.75 U/kg of insulin. Blood glucose concentrations were determined with a glucometer using blood taken from cut tail tips at the indicated time points (**A**,**B**). Values are means ± SE of 4 to 20 mice per group. \* *p* < 0.05 vs. *Apoe*-/ mice. (**C**,**D**), Glucose concentrations were expressed as % of the basal. **Figure 5.** Insulin tolerance test (ITT) for male and female C3H-*Apoe*-/- and C3H-*Ldlr*-/- mice fed a Western diet. Non-fasted mice were intraperitoneally injected with 0.75 U/kg of insulin. Blood glucose concentrations were determined with a glucometer using blood taken from cut tail tips at the indicated time points (**A**,**B**). Values are means ± SE of 4 to 20 mice per group. \* *p* < 0.05 vs. *Apoe*-/ mice. (**C**,**D**), Glucose concentrations were expressed as % of the basal.

Fasting plasma insulin levels were measured for female C3H-*Ldlr*-/- and C3H-*Apoe*-/ mice fed the Western diet. C3H-*Ldlr*-/- mice had higher insulin levels than the C3H-*Apoe*-/-

*3.4. Plasma insulin Concentration*

significant (*p* = 0.33; *n* = 7 to 13) (Figure 6).

**Figure 5.** Insulin tolerance test (ITT) for male and female C3H-*Apoe*-/- and C3H-*Ldlr*-/- mice fed a Western diet. Non-fasted mice were intraperitoneally injected with 0.75 U/kg of insulin. Blood glucose concentrations were determined with a glucometer using blood taken from cut tail tips at the indicated time points (**A**,**B**). Values are means ± SE of 4 to 20 mice per group. \* *p* < 0.05 vs. *Apoe*-/-

Fasting plasma insulin levels were measured for female C3H-*Ldlr*-/- and C3H-*Apoe*-/ mice fed the Western diet. C3H-*Ldlr*-/- mice had higher insulin levels than the C3H-*Apoe*-/-

mice. (**C**,**D**), Glucose concentrations were expressed as % of the basal.

*3.4. Plasma insulin Concentration*

significant (*p* = 0.33; *n* = 7 to 13) (Figure 6).

**Figure 6.** Plasma insulin levels of female C3H-*Apoe*-/- and C3H-*Ldlr*-/- mice fed a Western diet. Blood samples were collected after mice were fasted overnight. Values are means ± SE of 7 or 13 mice per group. **Figure 6.** Plasma insulin levels of female C3H-*Apoe*-/- and C3H-*Ldlr*-/- mice fed a Western diet. Blood samples were collected after mice were fasted overnight. Values are means ± SE of 7 or 13 mice per group.

#### *3.5. Body Weight 3.5. Body Weight*

At 3 months of age on the chow diet, either male or female C3H-*Ldlr*-/- mice had a body weight similar to that of the C3H-*Apoe*-/- mice (male: 23.1 ± 0.9 vs. 23.2 ± 0.7 g; female: 17.2 ± 0.3 vs. 18.1 ± 0.5 g) (Figure 7). After being fed the Western diet for 12 weeks, C3H-*Ldlr*-/- and C3H-*Apoe*-/- mice also had a similar body weight (male: 33.2 ± 0.8 vs. 35.0 ± 0.8 g; female: 23.1 ± 1.2 vs. 23.8 ± 0.8 g). Males had significantly heavier body weight than females for both the C3H-*Ldlr*-/- and C3H-*Apoe*-/- mice fed either chow or a Western diet (*p* < 0.05; *n* = 4 to 26). At 3 months of age on the chow diet, either male or female C3H-*Ldlr*-/- mice had a body weight similar to that of the C3H-*Apoe*-/- mice (male: 23.1 ± 0.9 vs. 23.2 ± 0.7 g; female: 17.2 ± 0.3 vs. 18.1 ± 0.5 g) (Figure 7). After being fed the Western diet for 12 weeks, C3H-*Ldlr*-/- and C3H-*Apoe*-/- mice also had a similar body weight (male: 33.2 ± 0.8 vs. 35.0 ± 0.8 g; female: 23.1 ± 1.2 vs. 23.8 ± 0.8 g). Males had significantly heavier body weight than females for both the C3H-*Ldlr*-/- and C3H-*Apoe*-/- mice fed either chow or a Western diet (*p* < 0.05; *n* = 4 to 26).

**Figure 7.** Body weight (g) of male and female C3H-*Apoe*-/- and C3H-*Ldlr*-/- mice fed a chow or a Western diet. Chow-fed mice were weighed at 3 months of age, and Western diet-fed mice were weighed after being euthanized. Results are means ± SE of 4 to 26 mice. + *p* < 0.05 vs. female mice. **Figure 7.** Body weight (g) of male and female C3H-*Apoe*-/- and C3H-*Ldlr*-/- mice fed a chow or a Western diet. Chow-fed mice were weighed at 3 months of age, and Western diet-fed mice were weighed after being euthanized. Results are means ± SE of 4 to 26 mice. + *p* < 0.05 vs. female mice.

#### **4. Discussion 4. Discussion**

In this study, we characterized type 2 diabetes-associated phenotypes in C3H-*Ldlr*-/ mice through a comparison with C3H-*Apoe*-/- mice, which develop type 2 diabetes on a Western diet [28]. Our results here indicate that Western diet consumption and ensuing In this study, we characterized type 2 diabetes-associated phenotypes in C3H-*Ldlr*-/ mice through a comparison with C3H-*Apoe*-/- mice, which develop type 2 diabetes on aWestern diet [28]. Our results here indicate that Western diet consumption and ensuing

hyperlipidemia lead to the development of type 2 diabetes in hyperlipidemic mice, irrespective of underlying genetic causes. On a chow diet, plasma total and non-HDL choles-

were the fasting glucose levels. When fed a Western diet, C3H-*Ldlr*-/- mice developed just as severe hyperlipidemia as C3H-*Apoe*-/- mice, and the magnitude of hyperglycemia was similar. Male C3H-*Ldlr*-/- mice were more tolerant to glucose loading and more sensitive to insulin than male C3H-*Apoe*-/- mice, while female C3H-*Ldlr*-/- mice were just as tolerant to glucose and sensitive to insulin as female C3H-*Apoe*-/- mice. Moreover, male but not female C3H-*Ldlr*-/- and C3H-*Apoe*-/- mice developed moderate obesity on the Western diet. A major finding of this study is that C3H-*Ldlr*-/- mice developed type 2 diabetes with fasting plasma glucose levels ranging from 300 to 400 mg/dL after 12 weeks of a Western diet. Diabetes is defined by fasting hyperglycemia. In humans, fasting blood glucose levels of ≥ 126 mg/dL are considered diabetic. For mice, plasma instead of whole blood is often used for glucose measurements. Fasting plasma glucose exceeding 250 mg/dL is considered diabetic for mice [30]. Thus, both male and female C3H-*Ldlr*-/- mice developed diabetes on the Western diet. Compared to the C3H-*Apoe*-/- males, the C3H-*Ldlr*-/- males had lower total and non-HDL cholesterol and higher HDL cholesterol levels, and their plasma glucose levels were also lower. Similarly, C3H-*Ldlr*-/- and C3H-*Apoe*-/- females had hyperlipidemia lead to the development of type 2 diabetes in hyperlipidemic mice, irrespective of underlying genetic causes. On a chow diet, plasma total and non-HDL cholesterol levels were significantly lower in C3H-*Ldlr*-/- mice than in C3H-*Apoe*-/- mice and so were the fasting glucose levels. When fed a Western diet, C3H-*Ldlr*-/- mice developed just as severe hyperlipidemia as C3H-*Apoe*-/- mice, and the magnitude of hyperglycemia was similar. Male C3H-*Ldlr*-/- mice were more tolerant to glucose loading and more sensitive to insulin than male C3H-*Apoe*-/- mice, while female C3H-*Ldlr*-/- mice were just as tolerant to glucose and sensitive to insulin as female C3H-*Apoe*-/- mice. Moreover, male but not female C3H-*Ldlr*-/- and C3H-*Apoe*-/- mice developed moderate obesity on the Western diet.

A major finding of this study is that C3H-*Ldlr*-/- mice developed type 2 diabetes with fasting plasma glucose levels ranging from 300 to 400 mg/dL after 12 weeks of a Western diet. Diabetes is defined by fasting hyperglycemia. In humans, fasting blood glucose levels of ≥126 mg/dL are considered diabetic. For mice, plasma instead of whole blood is often used for glucose measurements. Fasting plasma glucose exceeding 250 mg/dL is considered diabetic for mice [30]. Thus, both male and female C3H-*Ldlr*-/- mice developed diabetes on the Western diet. Compared to the C3H-*Apoe*-/- males, the C3H-*Ldlr*-/- males had lower total and non-HDL cholesterol and higher HDL cholesterol levels, and their plasma glucose levels were also lower. Similarly, C3H-*Ldlr*-/- and C3H-*Apoe*-/- females had comparable total, non-HDL, and HDL cholesterol levels, and their fasting glucose levels were comparable. These results hint at the significance of lipid profiles in determining blood glucose homeostasis.

Schreyer et al. [31] observed a time-dependent increase in fasting glucose levels of B6-*Ldlr*-/- mice but not in B6-*Apoe*-/- mice within 16 weeks on a diabetogenic diet containing 35.5% fat, 36.6% carbohydrates, and no cholesterol, although the increase did not meet the threshold of 250 mg/dL for the diagnosis of diabetes. Phillips et al. [32] found that only the Western diet but not a diabetogenic diet containing 13% fat and 67% carbohydrates raised blood glucose level of B6-*Apoe*-/- mice.

C3H-*Ldlr*-/- mice developed milder hypercholesterolemia on the chow diet than C3H-*Apoe*-/- mice, having lower total and non-HDL cholesterol levels but higher HDL cholesterol levels. These results are in line with what has been found with the two knockouts on the C57BL/6 background [33]. Lipid profiles vary by strains [28], but the distinctions between *Ldlr*-/- and *Apoe*-/- mice in total and HDL and non-HDL cholesterol levels remain on the C3H background. What is more distinct are the fasting glucose levels, which were much lower in the C3H-*Ldlr*-/- mice than in the C3H-*Apoe*-/- mice of both sexes. Differential fasting glucose levels of the two knockouts were in parallel with their differing lipid profiles, with lower non-HDL and higher HDL cholesterol levels being linked with lower fasting glucose levels. Consistently, in multiple F2 cohorts derived from *Apoe*-/- mouse strains, plasma glucose levels are positively correlated with non-HDL cholesterol levels [11–13] and inversely correlated with HDL levels [13].

Although both male and female C3H-*Ldlr*-/- and C3H-*Apoe*-/- mice developed significant hyperglycemia on the Western diet, only C3H-*Apoe*-/- males exhibited noticeable glucose intolerance. Indeed, male C3H-*Apoe*-/- mice showed an increase in blood glucose concentration after intraperitoneal glucose injection. As there was little fall in blood glucose concentration after insulin injection, these mice developed strong insulin resistance on the Western diet. Thus, insulin resistance should be responsible in part for the glucose intolerance and hyperglycemia observed in male *Apoe*-/- mice. Obesity and hyperlipidemia were two obvious factors contributing to the development of insulin resistance. Both C3H-*Ldlr*-/- and C3H-*Apoe*-/- males fed the Western diet developed moderate obesity as evidenced by substantial increases in body weight when compared to age-matched chow-fed mice (33.2 ± 0.8 vs. 24.1 ± 1.0 g for C3H-*Ldlr*-/- mice, 35.0 ± 0.8 vs. 26.2 ± 1.2 g for C3H-*Apoe*-/- mice). However, the finding that the two knockouts had similar body weight but male *Ldlr*-/- mice did not develop obvious insulin resistance suggests that other factors than obesity also contributed to glucose intolerance and insulin resistance of male C3H-*Apoe*-/- mice. The consumption of a high-fat diet leads to hyperlipidemia, which is

a major driver of oxidative stress and systemic chronic inflammation [34,35]. Besides its role in lipid metabolism, ApoE has antioxidant effects and suppresses inflammation in the body [36]. *Apoe*-/- mice show enhanced responses to oxidative stress and inflammatory stimuli compared to *Ldlr*-/- mice [37,38]. The present finding that female C3H-*Apoe*-/- mice also exhibited an increased insulin resistance relative to the *Ldlr*-/- counterparts supports the speculation on enhanced oxidative stress and inflammatory responses in *Apoe*-/- mice.

Unlike their male counterparts and most other mouse models of type 2 diabetes that develop significant obesity [39], female C3H-*Ldlr*-/- and C3H-*Apoe*-/- mice had no overweight or obesity. Despite the absence of obesity, female C3H-*Ldlr*-/- and C3H-*Apoe*-/ mice developed just as severe hyperglycemia as their male counterparts. In humans, although most type 2 diabetic patients are obese or overweight, a fraction of patients have normal body weight [40]. On the Western diet, the two female knockouts had similar lipid profiles, including total, HDL, non-HDL cholesterol, and triglyceride. Interestingly, they also had comparable fasting plasma glucose and insulin levels. This coincidence supports the significance of lipid profiles in the development of type 2 diabetes. There are quantitative but not qualitative differences in plasma insulin levels between male and female hyperlipidemic mice fed a Western diet [41]. For more than 100 inbred strains examined, male mice have higher plasma insulin levels than female mice. Previous studies showed that a high-fat diet had little or no influence on plasma glucose levels of wild-type B6 mice [42,43], probably due to the moderateness in the increase in plasma lipid levels. This speculation appears applicable to wild-type C3H mice in that they only developed modest hyperlipidemia (plasma cholesterol level: 181 ± 14 mg/dL) and showed a modest increase in fasting glucose levels on the Western diet (148 ± 14 vs. 106 ± 11 mg/dL) (Supplementary Materials).

In humans, *APOE* and *LDLR* polymorphisms have been associated with obesity [44]. We previously observed a deficit in growth and weight gain during postnatal development of *Apoe*-/- mice as compared to wild-type mice [45]. Male *Apoe*-/- mice showed resistance to Western diet-induced obesity [46]. *Ldlr*-/- mice also showed a decreased susceptibility to Western diet-induced obesity due to an increased thermogenesis [47]. Here we also observed that male *Ldlr*-/- mice had a smaller body weight in comparison to male *Apoe*-/ mice on the Western diet.

#### **5. Conclusions**

*Ldlr* deficiency results in increased levels of ApoB-100 containing LDL due to both increased hepatic lipoprotein production and impaired clearance [48,49], while ApoE deficiency leads to the accumulation of ApoB48 containing chylomicron or VLDL remnants due to a delayed clearance into hepatocytes by the LDL receptor, LDL receptor-related protein 1, and cell surface heparan sulphate proteoglycans [50]. Despite different lipoprotein profiles, *Ldlr*-/- mice and *Apoe*-/- mice developed diet-induced type 2 diabetes that is independent of obesity. This finding provides direct evidence for the significance of a Western-type diet and its induced hyperlipidemia in the etiology of type 2 diabetes. The current observations are not specific to C3H mice because *Ldlr*-/- mice with the B6;129 genetic background also developed hyperglycemia on the Western diet, which was alleviated after mice were switched to a low-fat diet [51]. We previously observed that the Western diet-induced increases in plasma glucose and triglyceride levels of *Apoe*-/- mice are time-dependent within the 12-week feeding period [52,53], while the current study only examined one time point on the Western diet. Another limitation is that wild-type C3H mice were not included in this study. In addition, sample sizes in some experiments were small despite the fact that the two knockouts were inbred and tended to have small intragroup variation in the phenotypes tested. Nevertheless, there is no doubt that the two mouse models are useful for investigating type 2 diabetes that is related or unrelated to obesity.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/biomedicines10061429/s1. All data reported in the article are included in the Supplementary Materials and also accessible via 10.6084/m9.figshare.20032229.

**Author Contributions:** Conceptualization, W.S. and Z.L.; methodology, W.S., J.L. and M.-H.C.; formal analysis, W.S. and J.L.; investigation, W.S., J.L., K.B., M.-H.C. and Z.L.; resources, W.S.; data curation, W.S., J.L. and K.B.; writing—original draft preparation, W.S. and Z.L.; writing—review and editing, W.S. and Z.L.; supervision, W.S. and Z.L.; project administration, W.S.; funding acquisition, W.S. and Z.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** The work was supported by NIH grants R01 DK116768 and HL112281 and Commonwealth Health Research Board (CHRB) Virginia.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All data reported in the article are included in the Supplementary Materials and also accessible via 10.6084/m9.figshare.20032229.

**Conflicts of Interest:** The authors declared they have no conflict of interest.

#### **References**

## *Article* **Orphan GPR26 Counteracts Early Phases of Hyperglycemia-Mediated Monocyte Activation and Is Suppressed in Diabetic Patients**

**Zahra Abedi Kichi 1,2 , Lucia Natarelli 2 , Saeed Sadeghian 3 , Mohammad ali Boroumand 4 , Mehrdad Behmanesh 1,\* and Christian Weber 2,\***

**Abstract:** Diabetes is the ninth leading cause of death, with an estimated 1.5 million deaths worldwide. Type 2 diabetes (T2D) results from the body's ineffective use of insulin and is largely the result of excess body weight and physical inactivity. T2D increases the risk of cardiovascular diseases, retinopathy, and kidney failure by two-to three-fold. Hyperglycemia, as a hallmark of diabetes, acts as a potent stimulator of inflammatory condition by activating endothelial cells and by dysregulating monocyte activation. G-protein couple receptors (GPCRs) can both exacerbate and promote inflammatory resolution. Genome-wide association studies (GWAS) indicate that GPCRs are differentially regulated in inflammatory and vessel cells from diabetic patients. However, most of these GPCRs are orphan receptors, for which the mechanism of action in diabetes is unknown. Our data indicated that orphan GPCR26 is downregulated in the PBMC isolated from T2D patients. In contrast, GPR26 was initially upregulated in human monocytes and PBMC treated with high glucose (HG) levels and then decreased upon chronic and prolonged HG exposure. GPR26 levels were decreased in T2D patients treated with insulin compared to non-insulin treated patients. Moreover, GPR26 inversely correlated with the BMI and the HbA1c of diabetic compared to non-diabetic patients. Knockdown of GPR26 enhanced monocyte ROS production, MAPK signaling, pro-inflammatory activation, monocyte adhesion to ECs, and enhanced the activity of Caspase 3, a pro-apoptotic molecule. The same mechanisms were activated by HG and exacerbated when GPR26 was knocked down. Hence, our data indicated that GPR26 is initially activated to protect monocytes from HG and is inhibited under chronic hyperglycemic conditions.

**Keywords:** diabetes mellitus; GPCRs; GPR26; monocytes; peripheral blood cells; hyperglycemia

## **1. Introduction**

*Diabetes mellitus* (DM) is a chronic, metabolic disease characterized by elevated levels of blood glucose (hyperglycaemia). Type 2 diabetes (T2D) is the most common type of diabetes, accounting for approximately 90% of all cases of DM [1]. Hyperglycaemia, a hallmark in T2D, affects the metabolic homeostasis of several organs, like kidney, pancreas, and eyes, and induces vascular cell dysfunction and associated vascular diseases, including cardiovascular diseases (CVD), diabetic nephropathy, and neuropathies [2,3].

Inflammation plays a crucial role in the pathogenesis of T2D and its complications. Vascular cells, especially immune cells, such as lymphocytes and monocytes, are involved

**Citation:** Kichi, Z.A.; Natarelli, L.; Sadeghian, S.; Boroumand, M.a.; Behmanesh, M.; Weber, C. Orphan GPR26 Counteracts Early Phases of Hyperglycemia-Mediated Monocyte Activation and Is Suppressed in Diabetic Patients. *Biomedicines* **2022**, *10*, 1736. https://doi.org/10.3390/ biomedicines10071736

Academic Editor: Alfredo Caturano

Received: 9 June 2022 Accepted: 13 July 2022 Published: 19 July 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

in the development of vascular inflammation, and promote endothelial activation and monocyte adhesion to the endothelium, sustaining diabetic vascular complications [2,4,5]. Despite data on the involvement of neutrophils and blood monocytes in diabetic patients still being contradictory, increased glucose uptake by monocytes from diabetic patients induces monocyte activation and enhances abnormal leukocyte–endothelial interaction [6,7]. Moreover, monocytes from diabetic patients are characterized by a moderate decrease in the activity of enzymes involved in the modulation of monocyte metabolism and inflammatory activation [8]. Mechanistically, high glucose (HG) levels, deriving from diabetes-associated chronic hyperglycaemia, induce inflammation via a variety of mechanisms, including ROS accumulation, pro-inflammatory MAPK cascade signalling, and NF-κB activation [9], which promotes the transcription of pro-inflammatory cytokines and adhesion molecules and related monocyte activation [6,10,11]. Moreover, HG can regulate monocyte apoptosis, although the role of this programmed cell death in diabetic patients is still controversial [12,13].

Cellular receptors are fine-tuned regulators of cell response to environmental changes via translating extracellular signals into intracellular activation cascades. The failure of a physiological process, such as the receptor-derived physiological processes in monocytes, dysregulates their function and mediates monocyte pro-inflammatory activation [3]. G-protein-coupled receptors (GPCRs) are the largest family of cell surface receptors in the human genome that play pivotal roles in a wide variety of physiological processes [14]. GPCRs emerged as novel receptors modulating the response of monocytes and other cells to environmental changes, such as hyperglycaemia [15]. Indeed, GPCRs can regulate the expression of cytokines and adhesion molecules to both exacerbate inflammation or promote its resolution [16,17]. Genome-wide association studies (GWAS) on diabetic patients indicate that GPCRs are differentially regulated in T2D patients and can influence the development and progression of T2D and vascular complications [17–20]. However, most of these GPCRs are orphan receptors, for which the mechanism of action in immune cells in diabetic patients is largely unknown [21]. Only a few studies exist on GPR119, free fatty acid receptor 1 (FFAR1/GPR40) and 4 (FFAR4/GPR120), and the bile acid receptor GPBAR1 (TGR5) as a developer of insulin resistance and *β*-cell dysfunction [22], receiving particular attention as targets for therapeutic interventions in diabetic patients [22]. Since orphan GPCRs emerged as the most differentially regulated sub-class of GPCRs in diabetic patients, the scientific community started to focus more on this sub-class of receptors [15,16,22]. Three orphan GPCRs have been reported so far to be involved in the development of diabetes and cardiovascular complications: GPR135 [19], GPR55 [23,24], and GPR39 [25]. However, data are still too preliminary and mainly focused on adipocyte-related diabetic complications [19,23–25]. Given the potential role of monocytes and orphan GPCR receptors in diabetes and associated cardiovascular complications, we investigated the role of GPCRs on monocytes from diabetic patients. Among almost 800 GPCRs existing, we focused on orphan GPCRs as the most important target receptor family for therapeutic purposes in biomedicine [21].

#### **2. Materials and Methods**

#### *2.1. Bioinformatics Analysis of GWAS Available Databases*

GWAS studies on single nucleotides polymorphisms and microarrays data from blood or peripheral blood mononuclear cells (PBMC) samples collected from diabetic patients (T1D and T2D) (GDS3963, GDS3874) were used to identify GPCRs differentially expressed and related to diabetes (Supplement Excel File S1). Among all identified GPCR candidates, we excluded the T1D-related GPCRs and focused on those related to T2D. In the next step, we focused on orphan GPCRs [18,19,21,22]. Finally, GPR26 was selected as a T2Drelated orphan GPCR. To predict functional associations between GPR26 and attributes (e.g., genes, proteins, diseases, etc.), we performed a bioinformatics analysis using the Harmonizome (2022 Database, Oxford) [26], JASPAR (9th release, V4.0, 2022) [27], KEGG

(V 103.0, July 2022, Kanehisa Laboratories) [28], and STRING (V11.5, STRING Consortium 2022) databases [29].

#### *2.2. Specimen Collection and Laboratory Investigations*

Whole blood samples were obtained from 32 diabetic patients and 32 healthy donors referred to the outpatient clinic of Tehran Heart Center, Iran. The diagnosis of T2D was based on WHO criteria, and diabetic patients were selected based on standard criteria (fasting blood glucose (FBS)), N 125 mg/dL (6.9 mmol/L), and HbA1c N 6.5% (47 mmol/mol) (https://apps.who.int/iris/handle/10665/70523, accessed on 1 June 2022). To exclude any intervening condition due to background diseases, all of participants were examined for T1D, liver and kidney dysfunction, auto-immune diseases, current or prior cancers, acute or chronic inflammatory diseases, and any other unrelated disease. In addition, pregnant, smoking individuals and patients who were receiving immunosuppressive or hormone-containing drugs were excluded from the study. The healthy donors showed normal FBS and HbA1C values and did not report a T2D history, autoimmune disease, obesity, or any other chronic disease. This group was matched to diabetic cases based on gender, age, and ethnicity. Human PBMC were obtained from volunteers following an oral and written informed consent. Institutional approval was obtained from the Ethics Committees of Tarbiat Modares University (IR.MODARES.REC.1397.109). The study was conducted in accordance with the ethical guidelines of the Helsinki declaration.

#### *2.3. Biochemical Analysis of Blood Samples from T2D and Healthy Donors*

Whole blood samples were collected from precipitants after an overnight fasting. FBS was determined by glucose hexokinase method (Cobas Integra 400, Roche Diagnostics). HbA1c was quantified using an enzymatic method according to the manufacturer's instructions (Cat no. DZ168A, Diazyme Laboratories, Inc., 12889 Gregg Ct. Poway, CA 92064 USA).

#### *2.4. PBMC Isolation from Iranian Diabetic and Healthy Volunteers*

PBMCs were isolated from fresh whole blood samples by density gradient using Ficoll Histopaque (10771, Sigma-Aldrich, St. Loius, MO, USA). In detail, 2 mL of Ficoll Histopaque per 1 mL of blood were placed in a 50 mL conical centrifuge tube. Fifteen mL of anticoagulated blood was diluted with an equal volume of Phosphate-buffered saline (PBS). The diluted blood was layered over the Ficoll Hypaque solution gently. Gradients were centrifuged at 400× *g* for 30 min at room temperature with no brake. Then, the PBMC layer was collected, washed with cold PBS, and centrifuged 10 min at 400× *g* (4 ◦C). The supernatant was discarded, and a wash step was repeated with PBS. PBMCs were finally collected and used for downstream analyses.

#### *2.5. RNA Extraction and qPCR*

Total RNA was extracted from PBMCs using TRIzol Reagent (15596026, Thermofisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions. One µg of RNA was reverse-transcribed with a high-capacity cDNA reverse transcription kit according to the manufacturer's instruction (4368813, Thermofisher Scientific). Gene expression was evaluated by qPCR using the GoTaq qPCR master mix (A6001, Promega, Madison, WI, USA). All samples were tested at least in duplicate (technical replicates). The relative gene expression analysis of GPR26 was according to the Livak method [30]. Expression levels were normalized to B2M as stable internal control gene and expressed as 2 −∆Ct. Primer efficiencies for the tested genes were comparable to those for reference genes. Primer sequences are reported in Supplement Table S1.

#### *2.6. Cell Culture and Treatments*

PBMCs for cell culture experiments were obtained from additional 12 healthy volunteers after giving oral and written informed consent. Human monocyte cells (THP-1, ATCC,

Rockville, MD, USA) were cultured in a Roswell Park Memorial Institute 1640 medium (RPMI 1640, 11530586, Gibco, Termofisher Scientific) supplemented with 2 mM L-glutamin, 1% penicillin/streptomycin, and 10% fetal bovine serum (Gibco), and incubated at 37 ◦C in a humidified atmosphere containing 5% CO2. CTHP-1 were exposed for 24, 48, and 72 h to media containing either 5 mM D-glucose (normal glucose, NG), or 25 mM D-glucose (high glucose, HG) to simulate a condition closed to the diabetic hyperglycemia. PMBCs were treated similarly for 24 and 48 h. Cell viability in each experiment was >90%, as indicated by trypan blue staining. THP-1 and PBMC total RNA was isolated as described above.

### *2.7. Antisense LNA GapmeRs Cell Transfection*

THP-1 were chemically transfected (Lipofectamine RNAiMAX transfection reagent, 13778075, Life Technologies (Waltham, MA, USA), Termofisher Scientific) for 24, 48, and 72 h with LNA single-stranded antisense oligonucleotides (LNA-GapmeRs, 50 nM) designed for highly effective, strand-specific, knockdown of GPR26 mRNA, or with scrambled controls, alone or in combination with NG or HG. RNA isolation and cDNA synthesis were performed as mentioned before. Efficiency of GPR26 Gapmers was validated by qPCR and western blot. All data belong to at least three biological replicates.

#### *2.8. Western Blot Analysis*

Whole proteins were extracted from THP-1 using a RIPA lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% Igepal, 0.5% sodium deoxycholate, 0.1% SDS) containing phosphatase and proteinase inhibitor cocktails (cOmplete Mini Tablets and PhosSTOP,<PHONE_NUMBER>1, Roche, Basel, Switzerland). The protein content was determined using the Pierce BCA Protein Assay Kit (23227, Thermo Fisher Scientific). Twenty µg of total proteins were loaded on a 4–12% precast polyacrylamide gel (NuPAGE™ 4 bis 12%, Bis-Tris, 1.0–1.5 mm, Mini-Protein-Gel, NP0321PK2, Invitrogen, Waltham, MA, USA) and electrotransferred onto polyvinylidene difluoride (PVDF) 0.45 µm membranes according to the manufacturer's instructions. Proteins were incubated with antibodies indicated in the Supplementary Table S2. The β-actin and α-tubulin proteins were used for protein quantification and internal loading control.

#### *2.9. Reactive Oxygen Species (ROS) Assay*

To examine the possible role of GPR26 in generation of ROS, its amount was measured using the OxiSelect Intracellular ROS Assay Kit (STA-342, Cell Biolabs, San Diego, CA, USA) based on the manufacturer's instructions. Briefly, THP-1 cells were transfected with GPR26 GapmeRs and exposed to NG or HG as indicated before. Then, cells were incubated with 2 0 , 70 -Dichlorodihydrofluorescin diacetate (DCFH-DA) during the last 30 min of treatment. At the end of the incubation, cells were washed three times with PBS to remove any DCFH-DA excess. Fluorescence was analyzed at 485 nm excitation/530 nm emission using a fluorescent plate reader (Infinite® 200 PRO, Tecan, Männedorf, Switzerland). ROS levels were expressed as DCFH-DA relative fluorescence intensity and reported as fold change of the control.

#### *2.10. Caspase-3 Activity Assay*

THP-1 cells were cultured and transfected with GPR26 GapmeRs as mentioned above. To determine the activity of caspase-3, cells were incubated at 37 ◦C with a luminogenic caspase-3 substrate containing the tetrapeptide sequence DEVD (Caspase-Glo® 3/7 Assay Systems, G8091, Promega). Luminescence is proportional to the amount of caspase activity present. Luminescence was measured in a plate luminometer (Infinite® 200 PRO, Tecan). Six technical replicates per condition were performed for each of the 3 biologically independent experiments.

#### *2.11. Monocyte Adhesion Using Calcein-AM*

THP-1 were transfected with GPR26 GapmeRs for 24, 48 and 72 h. At the end of the experiment, cells were labeled with 5 µM/L × 106 cells calcein-AM (Calcein, AM, Zellfarbstoff, C1430, Invitrogen, Life Technologies) for 45 min as previously described [31], with some modifications. Human Aortic Endothelial cells (HAoECs criopreserved, 500.000 cells, C-12271, PromoCell, Heidelberg, Germany) were cultured on gelatin-coated 96-well black plates. Confluent HAoEC monolayers were co-cultured with fluorescently labeled THP-1 cells for 20 min at 37 ◦C. Then, the monolayers were washed three times with PBS to remove non-adherent cells. Fluorescence was acquired at 492 nm (excitation) and 535 nm (emission) on a plate reader (Tecan). Relative fluorescence from treated monocytes adherent to HAoECs was normalized on that from the relative monocytes control groups. Comparisons were made between all four groups, as indicated in the Results. Representative images were acquired with a Thunder Imager Live Cell inverted microscope (Leica microsystem, Leica Mikrosysteme Vertrieb GmbH, Wetzlar, Germany) and equipped with a dual fluorescent and brightfield camera. Images were acquired for each of the 4 technical replicates and for all 3 biological replicates.

#### *2.12. 3D Confocal Immunofluorescence for GPR26 Spatial Localization*

THP-1 monocytes were plated on 8-well chamber slides (Ibidi) and treated as described before. Cells were fixed using the ViewRNA Cell Plus Assay (Fix & PERM™ Zellpermeabilisierungskit, GAS003, Affymetrix, Thermofisher Scientific), and incubated with antibodies against GPR26 (250 ng/mL dilution, NBP2-57693, R87210, Novusbio) and β-catenin (200 ng/mL, ab16051, Abcam). Proteins were detected using appropriate fluorescently labeled secondary antibodies feasible for 3D confocal microscopy, such as STAR-488-conjugated anti-rabbit IgG antibody (Molecular Probes, #A11034), and Cy™3 AffiniPure Donkey Anti-Rabbit IgG (H+L) (711-165-152, Jackson Immuno Research). Cells were embedded in Vectashield Antifade Mounting Medium with DAPI (H-1200-10, Vector Laboratories, Burlingame, CA, USA) to counterstain nuclei.

For detailed GPR26 localization studies, three-dimensional confocal laser scanning microscopy (CLSM) was performed with a Leica SP8 3X microscope equipped with a 63×/1.40 (Leica) oil immersion objective. Optical zoom was used where applicable. A UV laser (405 nm) was used for excitation of DAPI. A tunable white light laser for selective excitation of Star635P and Cy3 fluorochromes was used for the detection of GPR26 and βcatenin, respectively. Time gating of the detected emission signals (0.8–6.0 ns) was used for all STED channels to enhance image resolution. All data were acquired in three dimensions and voxel size was determined according to Nyquist sampling criterion. Image reconstructions were performed using the LAS X software package v.3.0.2 (Leica) and deconvolution was performed combining the Huygens Professional software package v.19.10 (Scientific Volume, Hilversum, The Netherlands) using the unsupervised CLSM algorithms.

GPR26 quantification and cell localization were performed using the Imaris 8.4.2 software equipped with the imaging processing toolbox MATLAB. Spatial distribution of automatically calculated GPR26 voxels (0.950 µm spot detection value) was defined according to the voxel localization to the cell surface, which corresponded to the membrane surface calculated using the spatial distribution of the β-catenin (0.289 µm smoothing, 3.46 µm threshold). A voxel value of 0.5 µm was used as threshold to discriminate membrane versus cytosolic GPR26. Multiple wells per experimental conditions were performed in independent experiments. The analysis of the staining was performed in a blinded manner.

#### *2.13. Statistical Analysis*

Statistical analysis was performed using GraphPad Prism 9.3.1 (GraphPad Software, Inc., San Diego, CA, USA). Data were initially evaluated for normal distribution with the Shapiro-Wilk test. Comparisons between two groups with normally distributed variables were analyzed by Student's unpaired *t* test. Not normally distributed variables were compared by the Mann–Whitney test. Analysis of variance (ANOVA) was used to determine

the significant differences between more than two groups. The correlation between the expression levels of GPR26 and the clinical features of patients were assessed by Spearman or Pearson tests based on the normality test result. In all cases, *p* values < 0.05 were considered significant.

#### *2.14. Data and Resource Availability*

The raw data are available in the Supplementary Files.

#### **3. Results**

#### *3.1. GPCRs SNPs and Correlation with Their Expression in Diabetic Patients*

*Diabetes mellitus* is a chronic, metabolic disease characterized by elevated blood glucose levels, which leads to additional chronic complications, such as cardiovascular, inflammatory, and kidney diseases [32]. Diabetes is among the top 10 leading causes of death worldwide, following a significant percentage increase of 70% since 2000 (WHO, https: //www.who.int/news-room/fact-sheets/detail/the-top-10-causes-of-death, accessed on 1 June 2022). In Iran, diabetes is the 6th leading cause of death, with a death rate of 27.47 per 100.000 of population (WHO 2018 and World Health Rankings, https://www. worldlifeexpectancy.com/iran-diabetes-mellitus, accessed on 1 June 2022). Genome-wide association studies (GWAS) provided evidence that occurrence of certain single nucleotide polymorphisms (SNPs) in GPCRs are associated with an increased risk for diabetes and cardiovascular complications [33]. Hence, we analysed GWAS available datasets to depict all GPCRs with a reported SNP in diabetic patients. We identified 31 potential GPCRs candidates (Figure 1a). GPCRs are divided into six sub-classes based on their sequence and function. Accordingly, 15 out of 31 GPCRs were members of the Class A-rhodopsin-like receptors, 8 were members of the Class B-secretin family, 5 were members of the Class C-metabotropic of glutamate receptors, and 3 were members of the Frizzled or other GPCR subclasses [34] (Figure 1a).

Next, we analyzed the expression of the selected 31 GPCRs using available GEO datasets from blood samples isolated from T2D patients to define whether the presence of SNPs in the GPCR sequences correlated with a different expression of selected GPCRs in diabetic patients compared to healthy subjects. Twenty-nine out 31 GPCRs were differentially regulated in the blood or PBMC samples (Figure 1b,c). Next, we focused on orphan GPCRs as more attractive candidates for further studies. We end up with three orphan GPCRs, such as GPR35, GPR158, and GPR26. However, since GPR158 expression did not significantly change in the blood nor in the PBMC isolated from diabetic patients, and GPR35 is also known to be modulated in T1D, we end up with the orphan GPR26 as a promising candidate for our subsequent experiments (Figure 1b,c). Indeed, GPR26 was the most promising candidate among other orphan GPCRs, for which expression was decreased in the blood of T2D patients compared to healthy donors [33,35] (Figure 1c). GPR26 expression was slightly decreased in PBMC samples, probably due to the origin of the samples from young patients with an early T2D diagnosed disease.

*Biomedicines* **2022**, *10*, x FOR PEER REVIEW 7 of 23

**Figure 1.** GPCRs candidates from GWAS, GEO datasets, and T2D patients. (**a**) GPCRs showing SNPs associated to diabetes. Analysis of available microarray GEO datasets and relative heatmap of 31 GPCRs differentially expressed (**b**) in the blood and (**c**) in the PBMC isolated from diabetic patients. (**d**) Expression analysis of GPR26 in diabetic patients (*n* = 32) in comparison to non-diabetic individuals (*n* = 32) (\*\*\* *p* < 0.001). (**e**) *GPR26* expression in patients treated with insulin (*n* = 11) compared to non-insulin treated patients (*n* = 21) (\* *p* < 0.05). GPR26 mRNA expression was normalized to the corresponding B2M expression and expressed as power ∆Ct. (**e**–**g**) Correlation of GPR26 expression with (**e**) FBS, (**f**) HbA1c (%), and (**g**) BMI in non-diabetic and diabetic patients. Pearson correlation and r values are indicated. **Figure 1.** GPCRs candidates from GWAS, GEO datasets, and T2D patients. (**a**) GPCRs showing SNPs associated to diabetes. Analysis of available microarray GEO datasets and relative heatmap of 31 GPCRs differentially expressed (**b**) in the blood and (**c**) in the PBMC isolated from diabetic patients. (**d**) Expression analysis of GPR26 in diabetic patients (*n* = 32) in comparison to nondiabetic individuals (*n* = 32) (\*\*\* *p* < 0.001). (**e**) *GPR26* expression in patients treated with insulin (*n* = 11) compared to non-insulin treated patients (*n* = 21) (\* *p* < 0.05). GPR26 mRNA expression was normalized to the corresponding B2M expression and expressed as power ∆Ct. (**e**–**g**) Correlation of GPR26 expression with (**e**) FBS, (**f**) HbA1c (%), and (**g**) BMI in non-diabetic and diabetic patients. Pearson correlation and r values are indicated.

To predict functional associations between orphan GPR26 and attributes, like genes and proteins associated with diabetes and related complications, we performed a bioinformatics analysis using the Harmonizome [26], JASPAR [27], KEGG [28], and STRING [29] databases. Harmonizome database results indicated that orphan GPR26 showed 1.763 functional associations with biological entities spanning 8 categories (molecular profile, organism, functional term, phrase or reference, disease, phenotype or trait, chemical, structural feature, cell line, cell type or tissue, gene, protein, or microRNA) extracted from 59 datasets (Supplement Excel File S2). Analysis of functional associations predicted that orphan GPR26 might be involved in the regulation of cAMP catabolic processes (GO:0006198), vesicle formation and transport (GO:0071805), and of ADP-ribosylation factor (ARF) protein signal transduction (GO:0032012), which acts as regulator of phagocytosis and of immunity-related autophagy [36,37]. KEGG pathway analysis indicated that GPR26 might be involved in the cAMP, cGMP-PKG, insulin pancreatic secretion, MAPK, VEGF, oxidative phosphorylation, chemokine, mTOR, autophagy regulation, ABC transporters, T1D, and T2D pathways (Supplement Excel File S3). Similar results were obtained using STRING, where G-protein coupled receptors, cyclic AMP-responsive element-binding proteins, and lysosome-related organelle complexes were predicted as functional partners of GPR26.

#### *3.2. GPR26 Is down Regulated in the PBMC from Diabetic Patients and Negatively Correlates with Their BMI, HbA1c, and Insulin*

GPR26 expression was first investigated in the blood collected from T2D and healthy Iranian donors. Demographic and clinical characteristics of control participants and patients are presented in Table 1. In detail, blood samples were collected from 32 patients (19 male, 13 females) with a diagnosed T2D and from 32 healthy donors (13 males, 19 females). None of the healthy donors were smokers or were hypertensive. Eleven diabetic patients got insulin routinely or had a history of using insulin. The diabetic and non-diabetic subjects did not significantly differ in gender, age, and body mass index (BMI) (Table 1). We did not observe sex-related differences within the diabetic or healthy donors in glycated hemoglobin (HbA1c), an index of long-term blood glucose concentrations, fasting blood glucose (FBS), nor on BMI. FBS and HbA1c were high in diabetic patients compared to healthy donors (Table 2). GPR26 expression was downregulated in diabetic patients compared to healthy donors (Figure 1d). We divided the diabetic patients according to their insulin supplementation (Table 2 and Figure 1d). In detail, 11 out of 32 T2D patients got insulin and mainly metformin + glibenclamide, an antidiabetic tablet medication, to lower the hyperglycemic levels and increase the insulin. The other 21 T2D patients did not get insulin but mainly the antidiabetic Glibenclamide medication (Table 2). Analysis of GPR26 expression in insulin versus non-insulin-treated patients indicated that GPR26 expression was decreased in patients taking insulin routinely or showing a history of using insulin compared to antidiabetic medication tablets treated patients, although the glycemic levels (HbA1c) were not different between the two groups (Table 3).

This data suggested that insulin-mediated alleviation of hyperglycemic adverse effects might counteract monocyte activation and GPR26 expression. Accordingly, we evaluated the association of *GPR26* expression with the glycemic profile, the HbA1c, and the BMI. *GPR26* was not correlated with FBS (Figure 1e) but showed a significant correlation with HbA1c (r = −0.38 *p* = 0.02) in diabetic patients (Figure 1f, Table 3). The association of *GPR26* with the BMI was also significant in both diabetic (r = −0.41 *p* = 0.01) and healthy donors (r = −0.35 *p* = 0.04) (Figure 1g, Table 3). Taken together, these data indicated that GPR26 is downregulated in diabetic patients and that insulin treatment might prevent GPR26 putative protective activation.

**Table 1.** Demographic characteristics of the diabetic patients and healthy control.

a : Body mass index; b : Low density lipoproteins; c : Glycated hemoglobin; Data are presented as mean (SEM) for variables with normal distribution and median (interquartile range) for those without normal distribution. *i* Chi-square or Fisher's exact test is performed to compare variables between diabetic and non-diabetic patients. *ii* Student's *t*-test or Mann–Whitney *U* test is performed to compare variables between diabetic and non-diabetic patients.

**Table 2.** Glucose-lowering medications used to treat diabetic patients.

**Table 3.** Correlation analysis between GPR26 mRNA expression and clinical characteristic of diabetic patients and non-diabetic individuals.

### *3.3. Hyperglycemia Upregulated GPR26 in PBMC and THP-1 Cultured Cells*

We analyzed the expression of GPR26 in human cultured monocytes treated with different doses of glucose to simulate a hyperglycemic condition and to study GPR26 functional role. Accordingly, THP-1 cells were treated with 5 mmol/L (normal glucose, NG) or 25 mmol/L (high glucose, HG) of glucose for 24, 48, and 72 h. Interestingly, treatment of THP-1 with HG increased the expression of GPR26 (Figure 2a). The protein levels of GPR26 decreased over time when THP-1 cells were treated with HG, which increased GPR26 levels only at 24 and slightly at 48, but not at 72 h (Figure 2a). These data suggested that monocytes might response to hyperglycemic conditions by enhancing GPR26 expression, the translation of which into a protein is ineffective when cells are exposed to prolonged and chronic doses of HG.

*Biomedicines* **2022**, *10*, x FOR PEER REVIEW 11 of 23

**localization.** THP-1 and PBMC were treated with D-glucose 5.5 mM (normal glucose, NG) or 25 mM D-glucose (high glucose, HG) for 24, 48, and 72 h to analyze GPR26 in THP-1 (**a**) at mRNA (left) **Figure 2. Effect of HG on GPR26 mRNA and protein levels in human monocytes and cellular localization.** THP-1 and PBMC were treated with D-glucose 5.5 mM (normal glucose, NG) or 25 mM D-glucose (high glucose, HG) for 24, 48, and 72 h to analyze GPR26 in THP-1 (**a**) at mRNA (left) and protein (right) level. A representative Western blot (left) and relative expression of GPR26 (right).

**Figure 2. Effect of HG on GPR26 mRNA and protein levels in human monocytes and cellular**

Densitometry plots report the fold change versus control. GPR26 intensity values were normalized to the corresponding β-tubulin value. GPR26 mRNA expression was normalized to the corresponding B2M expression and expressed as log10 (*n* = 4 independent experiments) (**b**) GPR26 mRNA (left) and protein (right) levels in PBMCs isolated from healthy donors and treated as indicated for THP-1. Densitometry plots reporting the fold change versus control. GPR26 intensity values were normalized to the corresponding β-actin value. GPR26 mRNA expression was normalized to the corresponding B2M expression and expressed as power ∆Ct (*n* = 4 independent experiments) (**c**) Analysis of GPR26 whole levels and cellular localization in THP-1 cells. Confocal 3D scanning images are representative of 3 independent experiments. White arrowhead indicates GPR26 localizing at the membrane surface. GPR26 levels were normalized to the cell surface, and on total number of cells (*n* = 3 ± SEM). Scale bar: 5 µm. \* *p* < 0.05 \*\* *p* < 0.01 versus NG, # *p* < 0.05 48 h versus 24 h, % *p* < 0.05 72 h versus 24 h.

To corroborate the data, human PBMC were isolated from healthy donors and treated with NG or HG for 24 and 48 h. Accordingly, we found that GPR26 expression was increased only at 24 but not at 48 h of HG exposure (Figure 2b). Accordingly, HG increased GPR26 protein levels at 24 but not at 48 h (Figure 2b).

Next, we analyzed GPR26 cellular localization and relative levels to test whether HG affected GPR26 exposure on the monocyte membrane surface. THP-1 were treated with NG or HG for 24, 48, and 72 h and stained for GPR26 and β-catenin as membrane marker. Immunofluorescence analysis 3D z-scan confocal images confirmed that HG increased whole GPR26 levels at 24, 48 but not at 72 h (Figure 2c). The number of voxels relative to GPR26 close to the β-catenin surface were reduced compared to that far from the surface, a value relative to the cytoplasmic GPR26 (Figure 2c). HG increased both cytoplasmic- and membrane-associated GPR26 levels at 24 and 48 h (Figure 2c). However, analysis of GPR26 localization at 72 h, a time point at which the GPR26 protein levels are no longer increased by HG, indicated that HG impaired GPR26 localization on the membrane surface of THP-1 and increased GPR26 cytoplasmic levels (Figure 2c).

Taken together, these data suggested that GPR26 might be activated to counteract the deleterious effects promoted by HG, which in turn impaired GPR26-related protective effect by inhibiting its membrane localization on the monocytes from T2D patients.

#### *3.4. Knockdown of GPR26 Induced ROS Generation in THP1*

To study the functional role of GPR26, THP-1 cells cultured in NG or HG were transfected for 24, 48, and 72 h with 50 nM of LNA single-stranded antisense oligonucleotides to knockdown GPR26, or with scrambled controls. GPR26 knockdown was confirmed by the analysis of GPR26 at mRNA and protein levels at all time points (Figure 3a). Transfection of THP-1 with LNA GapmeRs suppressed HG-mediated increase of GPR26 expression at levels lower than cells cultured with NG and transfected with scrambled controls (Figure 3a). Knockdown of GPR26 was confirmed at protein levels, in NG and HG treated THP-1 cells at 24 and 48 h (Figure 3a). No differences were visible at 72 h on GPR26 protein levels (Figure 3a). Immunofluorescence analysis confirmed that the main effect exerted by knockdown of GPR26 was in its cellular localization. Indeed, HG increased whole GPR26 levels at 24 and 48 h and decreased GPR26 at 72 h (Figure 3b). Knockdown of GPR26 inhibited the HG-mediated membrane localization and enhanced GPR26 cytoplasmic levels (Figure 3b). Taken together, these data suggested that GPR26 might be exposed on monocyte membrane to promote a mechanism of defense in monocytes, which is then inhibited by prolonged and chronic exposure to HG.

**Figure 3. GPR26 levels and localization in HG-treated THP-1 transfected with GPR26 Gapmers**. THP-1 were treated with NG or HG for 24, 48, and 72 h and transfected with GPR26 (+) or scrambled GapmeRs (−). (**a**) GPR26 mRNA expression was normalized to the corresponding B2M expression and expressed as relative expression (log10) (**left**). Densitometry plots reporting the fold change versus control. GPR26 intensity values were normalized to the corresponding β-actin value (**right**). (**b**) Confocal laser scanning microscopy images of THP-1 stained for GPR26 to analyze GPR26 whole **Figure 3. GPR26 levels and localization in HG-treated THP-1 transfected with GPR26 Gapmers**. THP-1 were treated with NG or HG for 24, 48, and 72 h and transfected with GPR26 (+) or scrambled GapmeRs (−). (**a**) GPR26 mRNA expression was normalized to the corresponding B2M expression and expressed as relative expression (log10) (**left**). Densitometry plots reporting the fold change versus control. GPR26 intensity values were normalized to the corresponding β-actin value (**right**).

(**b**) Confocal laser scanning microscopy images of THP-1 stained for GPR26 to analyze GPR26 whole levels, cytoplasmic, and membrane localization levels. White arrowhead indicates GPR26 localizing at the membrane surface. GPR26 levels were normalized to the cell surface, and then on total number of the cells. All values are represented as mean of at least 3 independent experiments (*n* = 3 ± SEM). (**c**) Intracellular ROS production. Data are expressed as percentage (%) of DCFH-DA fluorescence intensity relative to control. All values are represented as mean of at least 3 independent experiments. Scale bar: 5 µm. \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001 versus NG.

Accumulating evidence indicates that high levels of glucose promote dysfunctional monocyte activation, characterized by an increased inflammatory and apoptotic activity, partly by enhancing the production and accumulation of reactive oxygen species (ROS). To study the role of GPR26 on HG-mediated production of ROS, GPR26 was knocked down in THP-1 cells treated with NG or HG for 24, 48, and 72 h. Accordingly, we confirmed that HG increased ROS levels in HG-induced THP-1 cells compared to NG throughout the entire 72 h (Figure 3c). Knockdown of GPR26 exacerbated ROS production in a time dependent manner (Figure 3c), and enhanced HG-mediated ROS production (Figure 3c). Taken together, these data indicated that GPR26 played a protective role against the formation of free radical oxygen species, the production of which is enhanced by HG through the inhibition of GPR26 exposure on monocyte membrane and through GPR26 cytoplasmic localization. *Biomedicines* **2022**, *10*, x FOR PEER REVIEW 14 of 23 levels, cytoplasmic, and membrane localization levels. White arrowhead indicates GPR26 localizing at the membrane surface. GPR26 levels were normalized to the cell surface, and then on total number of the cells. All values are represented as mean of at least 3 independent experiments (*n* = 3 ± SEM). (**c**) Intracellular ROS production. Data are expressed as percentage (%) of DCFH-DA fluorescence intensity relative to control. All values are represented as mean of at least 3 independent experiments. Scale bar: 5 µm. \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001 versus NG.

#### *3.5. Knockdown of GPR26 Increased ERK1/2 and p38 MAPK Activation in THP1 Cells 3.5. Knockdown of GPR26 Increased ERK1/2 and p38 MAPK Activation in THP1 Cells*

High levels of glucose can promote the activation of the MAPK signaling pathway, including p38 and ERK1/2, to promote monocytes-endothelial cell adhesion [38,39]. Moreover, recent findings indicate that ERK1/2 is regulated in response to the activation of various types of GPCRs [40,41], and that GPCRs mediate p38 MAPK activation to control monocyte inflammatory gene expression [42,43]. To explore whether GPR26 affected p38 and ERK1/2 activation, we assessed the levels of phosphorylated ERK1/2 and p38 by western blot, in THP-1 transfected with GPR26 GapmeRs, alone or in combination with NG or HG. Our results indicated that knockdown of GPR26 enhanced ERK1/2 and p38 MAPK activation in NG-cultured THP-1 at all time points (Figure 4a,b). ERK1/2 activation was increased in HG-treated THP-1 cells at 24 and 48 h. However, ERK1/2 activation persisted at 72 h only in HG-induced THP-1 cells where GPR26 was knocked down (Figure 4a). High levels of glucose can promote the activation of the MAPK signaling pathway, including p38 and ERK1/2, to promote monocytes-endothelial cell adhesion [38,39]. Moreover, recent findings indicate that ERK1/2 is regulated in response to the activation of various types of GPCRs [40,41], and that GPCRs mediate p38 MAPK activation to control monocyte inflammatory gene expression [42,43]. To explore whether GPR26 affected p38 and ERK1/2 activation, we assessed the levels of phosphorylated ERK1/2 and p38 by western blot, in THP-1 transfected with GPR26 GapmeRs, alone or in combination with NG or HG. Our results indicated that knockdown of GPR26 enhanced ERK1/2 and p38 MAPK activation in NG-cultured THP-1 at all time points (Figure 4a,b). ERK1/2 activation was increased in HG-treated THP-1 cells at 24 and 48 h. However, ERK1/2 activation persisted at 72 h only in HG-induced THP-1 cells where GPR26 was knocked down (Figure 4a).

ERK1/2. Densitometry plot reporting the fold change versus control. ERK1/2 and p-ERK1/2 intensity values were normalized to the corresponding β-actin value and the ratio of p-ERK1/2 to ERK1/2 are

values were normalized to the corresponding β-actin value and the ratio of p-p38 MAPK to p38 MAPK are presented as P38 MAPK activation. Images report one representative experiment out of at least 3 independent experiments. All values are represented as mean of at least 3 independent

Knockdown of GPR26 increased p38 MAPK activation at all time points (Figure 4b). P38 phosphorylation was promoted by HG only at 48 h, but remarkably induced at all

experiments (*n* = 3 ± SEM). \* *p* < 0.05 versus NG).

GapmeRs (−), as indicated in the figures. (**a**) A representative Western blot of ERK1/2 and p-ERK1/2. Densitometry plot reporting the fold change versus control. ERK1/2 and p-ERK1/2 intensity values were normalized to the corresponding β-actin value and the ratio of p-ERK1/2 to ERK1/2 are presented as ERK1/2 activation. (**b**) A representative Western blot of p38 MAPK and p-p38 MAPK. Densitometry plot reporting the fold change versus control. p38 MAPK and p-p38 MAPK intensity values were normalized to the corresponding β-actin value and the ratio of p-p38 MAPK to p38 MAPK are presented as P38 MAPK activation. Images report one representative experiment out of at least 3 independent experiments. All values are represented as mean of at least 3 independent experiments (*n* = 3 ± SEM). \* *p* < 0.05 versus NG).

Knockdown of GPR26 increased p38 MAPK activation at all time points (Figure 4b). P38 phosphorylation was promoted by HG only at 48 h, but remarkably induced at all time points when GPR26 was knocked down (Figure 4b). Taken together, these data indicate that GPR26 inhibits MAPK signaling pathways piloted by p38 and ERK1/2, independently from HG.

### *3.6. Knockdown of GPR26 Induced NF-κB p65 Activation and Monocyte Adhesion*

Treatment of human monocytes with HG promotes the phosphorylation of p38 MAPK and ERK1/2, leading to NF-κB transactivation and related monocyte inflammatory activation [44]. GPRCs can regulate leukocyte activation and binding to the vessel wall through the endothelial-mediated expression of chemokines [45]. Despite controversy existing on the role of GPCRs as pro- or anti-inflammatory molecules, especially in diabetic patients, emerging evidence suggests that GPCRs and NF-κB might be connected in the regulation of inflammatory diseases [46].

To explore whether GPR26 affected NF-κB (p65) activation, THP-1 were transfected with GPR26 GapmeRs alone or in combination with NG or HG incubation [47]. Our results indicated that knockdown of GPR26 increased p65 expression at 24 and 48 h of NG (Figure 5a). HG and GPR26 knockdown showed similar effects on p65 activation (Figure 5a). However, p65 was enhanced at 72 h only when GPR26 was knocked down in HG-treated cells (Figure 5a). Hence, inhibition of GPR26 in diabetic patients might sustain hyperglycemia-mediated NF-κB activation.

Next, we examined the role of GPR26 on hyperglycemia-mediated monocyte inflammatory activation and adhesion to HAoECs. Accordingly, knockdown of GPR26 increased the adhesion of THP-1 to HAoECs at 48 and 72 h of NG (Figure 5b). HG-induced THP-1 adhesion to HAoECs was increased at all time points (Figure 5b). Knockdown of GPR26 enhanced the adhesion of THP-1 treated with HG compared to their respective HG-treated cells (Figure 5b). Moreover, knockdown of GPR26 enhanced the number of THP-1 cells adhering to HAoECs at all time points, especially of those co-treated with HG compared to NG (Figure 5b). Taken together, these data indicated that hyperglycemia promotes monocyte adhesion to ECs by suppressing GPR260 s anti-inflammatory role.

pendently from HG.

regulation of inflammatory diseases [46].

hyperglycemia-mediated NF-κB activation.

**Figure 5. The effect of GPR26 knockdown and HG on NF-kB and monocyte adhesion.** THP-1 were transfected with GPR26 gapmers (+) or scrambled GapmeRs (−) and treated with NG or HG to analyze NF-κβ (p65) activation and monocyte adhesion at 24, 48, and 72 h indicated in the figures. Panels show (**a**) a representative Western blot of NF-κβ. Densitometry plots reporting the fold change versus control. NF-κβ intensity values were normalized to the corresponding β-actin value. (**b**) Monocyte adhesion is reported as fold of change versus the basal adhesion of monocyte to HAoECs control (cells cultured in NG) that was set to 1. Right panels are fluorescence images of the monocytes, confirming that the small and bright spherical cells visible under microscope are indeed monocytes that were pre-labeled with Calcein AM. Images were taken using a 10X objective. All values are represented as mean of at least 3 independent experiments (*n* = 3 ± SEM). Scale bar: 50 µm. \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001 versus NG. Next, we examined the role of GPR26 on hyperglycemia-mediated monocyte inflam-**Figure 5. The effect of GPR26 knockdown and HG on NF-kB and monocyte adhesion.** THP-1 were transfected with GPR26 gapmers (+) or scrambled GapmeRs (−) and treated with NG or HG to analyze NF-κβ (p65) activation and monocyte adhesion at 24, 48, and 72 h indicated in the figures. Panels show (**a**) a representative Western blot of NF-κβ. Densitometry plots reporting the fold change versus control. NF-κβ intensity values were normalized to the corresponding β-actin value. (**b**) Monocyte adhesion is reported as fold of change versus the basal adhesion of monocyte to HAoECs control (cells cultured in NG) that was set to 1. Right panels are fluorescence images of the monocytes, confirming that the small and bright spherical cells visible under microscope are indeed monocytes that were pre-labeled with Calcein AM. Images were taken using a 10X objective. All values are represented as mean of at least 3 independent experiments (*n* = 3 ± SEM). Scale bar: 50 µm. \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001 versus NG.

time points when GPR26 was knocked down (Figure 4b). Taken together, these data indicate that GPR26 inhibits MAPK signaling pathways piloted by p38 and ERK1/2, inde-

Treatment of human monocytes with HG promotes the phosphorylation of p38 MAPK and ERK1/2, leading to NF-κB transactivation and related monocyte inflammatory activation [44]. GPRCs can regulate leukocyte activation and binding to the vessel wall through the endothelial-mediated expression of chemokines [45]. Despite controversy existing on the role of GPCRs as pro- or anti-inflammatory molecules, especially in diabetic patients, emerging evidence suggests that GPCRs and NF-κB might be connected in the

To explore whether GPR26 affected NF-κB (p65) activation, THP-1 were transfected with GPR26 GapmeRs alone or in combination with NG or HG incubation [47]. Our results indicated that knockdown of GPR26 increased p65 expression at 24 and 48 h of NG (Figure 5a). HG and GPR26 knockdown showed similar effects on p65 activation (Figure 5a). However, p65 was enhanced at 72 h only when GPR26 was knocked down in HGtreated cells (Figure 5a). Hence, inhibition of GPR26 in diabetic patients might sustain

*3.6. Knockdown of GPR26 Induced NF-κB p65 Activation and Monocyte Adhesion*

#### matory activation and adhesion to HAoECs. Accordingly, knockdown of GPR26 in-*3.7. Knockdown of GPR26 Increased Caspase-3 Activity Promoted by HG*

creased the adhesion of THP-1 to HAoECs at 48 and 72 h of NG (Figure 5b). HG-induced THP-1 adhesion to HAoECs was increased at all time points (Figure 5b). Knockdown of Several in vitro studies indicate that HG induces cell apoptosis in monocytes [48,49]. Caspase 3 plays a pivotal role in the apoptotic cell death process and therefore is widely used as a pro-apoptotic marker. Therefore, we investigated the effect of GPR26 knockdown, alone or in combination with HG, on THP-1 apoptosis by evaluating caspase 3 activation (cleavage of caspase 3 and its activity). Our data indicated that knockdown of GPR26 slightly affected Caspase 3 cleavage at 48 h (Figure 6a). Caspase 3 cleavage was increased when GPR26 was knocked down in THP-1 cells incubated with HG for 24 and 48 h, and the effect persisted even at 72 h (Figure 6a). HG alone significantly enhanced the ratio of cleaved caspase3 on total caspase 3 compared to NG at 24 and 48 h (Figure 6a). To confirm the functional activation of cleaved Caspase 3, we measured caspase-3 and -7 activity using an enzymatic assay. Our data indicated that knockdown of GPR26 increased Caspase 3/7 activity at levels comparable to that of HG at all time points (Figure 6b). Caspase 3/7 activity was significantly enhanced when GPR26 was knocked down in HG-treated cells at all time points (Figure 6b). These results confirmed that GPR26 plays an anti-apoptotic role and that GPR26 in monocytes might counteract HG-mediated increase of THP1 apoptosis. Indeed, inhibition of GPR26 increased the pro-apoptotic activity of caspase 3/7 mediated by HG after 72 h.

caspase 3/7 mediated by HG after 72 h.

**Figure 6. The effect of GPR26 knockdown and HG on Caspase 3 activity.** The effect of GPR26 knockdown alone or in combination with HG on caspase activation at 24, 48, and 72 h with GPR26 (+) or scrambled GapmeRs (−), as indicated in the figures. (**a**) A representative Western blot of cleaved caspase-3 and total caspase-3. Densitometry plot reporting the fold change versus control. Cleaved caspase-3 and total caspase-3 intensity values were normalized to the corresponding βactin value and the ratio of cleaved caspase-3 to total caspase-3 were presented as caspase activity. (**b**) Caspase 3,7 activity was measured using an enzymatic assay. Data are expressed as fold of change versus control (cells cultured in NG) that was set to 1. All values are represented as mean of at least 3 independent experiments (*n* = 3 ± SEM). (\* *p* < 0.05 \*\* *p* < 0.01, \*\*\* *p* < 0.001 versus NG). **Figure 6. The effect of GPR26 knockdown and HG on Caspase 3 activity.** The effect of GPR26 knockdown alone or in combination with HG on caspase activation at 24, 48, and 72 h with GPR26 (+) or scrambled GapmeRs (−), as indicated in the figures. (**a**) A representative Western blot of cleaved caspase-3 and total caspase-3. Densitometry plot reporting the fold change versus control. Cleaved caspase-3 and total caspase-3 intensity values were normalized to the corresponding β-actin value and the ratio of cleaved caspase-3 to total caspase-3 were presented as caspase activity. (**b**) Caspase 3,7 activity was measured using an enzymatic assay. Data are expressed as fold of change versus control (cells cultured in NG) that was set to 1. All values are represented as mean of at least 3 independent experiments (*n* = 3 ± SEM). (\* *p* < 0.05 \*\* *p* < 0.01, \*\*\* *p* < 0.001 versus NG).

GPR26 enhanced the adhesion of THP-1 treated with HG compared to their respective HG-treated cells (Figure 5b). Moreover, knockdown of GPR26 enhanced the number of THP-1 cells adhering to HAoECs at all time points, especially of those co-treated with HG compared to NG (Figure 5b). Taken together, these data indicated that hyperglycemia promotes monocyte adhesion to ECs by suppressing GPR26′s anti-inflammatory role.

Several in vitro studies indicate that HG induces cell apoptosis in monocytes [48,49]. Caspase 3 plays a pivotal role in the apoptotic cell death process and therefore is widely used as a pro-apoptotic marker. Therefore, we investigated the effect of GPR26 knockdown, alone or in combination with HG, on THP-1 apoptosis by evaluating caspase 3 activation (cleavage of caspase 3 and its activity). Our data indicated that knockdown of GPR26 slightly affected Caspase 3 cleavage at 48 h (Figure 6a). Caspase 3 cleavage was increased when GPR26 was knocked down in THP-1 cells incubated with HG for 24 and 48 h, and the effect persisted even at 72 h (Figure 6a). HG alone significantly enhanced the ratio of cleaved caspase3 on total caspase 3 compared to NG at 24 and 48 h (Figure 6a). To confirm the functional activation of cleaved Caspase 3, we measured caspase-3 and -7 activity using an enzymatic assay. Our data indicated that knockdown of GPR26 increased Caspase 3/7 activity at levels comparable to that of HG at all time points (Figure 6b). Caspase 3/7 activity was significantly enhanced when GPR26 was knocked down in HGtreated cells at all time points (Figure 6b). These results confirmed that GPR26 plays an anti-apoptotic role and that GPR26 in monocytes might counteract HG-mediated increase of THP1 apoptosis. Indeed, inhibition of GPR26 increased the pro-apoptotic activity of

*3.7. Knockdown of GPR26 Increased Caspase-3 Activity Promoted by HG*

#### *3.8. GPR26 down Regulation Significantly Inhibited Autophagy* Autophagy is an important physiological process activated to maintain cellular ho-*3.8. GPR26 down Regulation Significantly Inhibited Autophagy*

meostasis and to adapt to extracellular cues. Autophagy is closely associated with ROS Autophagy is an important physiological process activated to maintain cellular homeostasis and to adapt to extracellular cues. Autophagy is closely associated with ROS generation and inflammation [50]. Indeed, HG inhibits autophagy to promote ROS-mediated inflammasome activation and cytokine secretion in immune cells [50]. Microtubule-associated protein 1A/1B-light chain 3 (LC3) is a soluble protein that exists in the two forms of LC3-I and LC3-II, the second converted from LC3-I to initiate the formation and lengthening of the autophagosome. Measurement of LC3-II/LC3-I ratio consents the quantification of autophagy activation levels [51]. In addition, measurement of p62, a selective substrate of autophagy, is an additional method to measure autophagy, since it inversely correlates with the autophagy activation [52]. Thus, to explore the role of GPR26 in regulating autophagy in monocytes, we examined LC3-II/LC3-I ratio and p62 levels in THP-1 cells treated with NG or HG and transfected for 24, 48, and 72 h with GPR26 Gapmers. Our results indicated that knockdown of GPR26 decreased LC3-II/LC3-I ratio at 24 and 48 h (Figure 7a). HG decreased LC3-II at all time points compared to NG treated cells (Figure 7a). A significant decrease in LC3-II/LC3-I ratio was detected in THP-1, knocked down for GPR26, and treated with HG at 24, 48, and 72 h, a time point at which only the knockdown of GPR26 in combination with HG showed a significant effect on LC3-II decrease (Figure 7a). Next, we analyzed the levels of p62, the levels of which should inversely correlate with that of LC3-II. Accordingly, knockdown of GPR26 increased p62 levels in THP-1 cultured in NG at 24 h and 48 h (Figure 7b). HG increased p62 levels only at 48 h (Figure 7b). Knockdown of GPR26 enhanced p62 levels in HG-treated THP-1 at 24, 48, and 72 h, a time point at which neither GPR26 knockdown nor HG increased p62 levels (Figure 7b). Taken together, these data indicated that GPR26 promotes autophagy and might counteract HG-mediated autophagy inhibition. Hence, hyperglycemia might impair GPR26 to inhibit autophagy in monocytes.

**Figure 7. The effect of GPR26 knockdown and HG on autophagy.** The effect of GPR26 knockdown alone or in combination with HG on autophagy at 24, 48, and 72 h with GPR26 (+) or scrambled GapmeRs (−), indicated in the figures. Panels show (**a**,**b**) a representative Western blot of LC3II, LC3I, and p62. Densitometry plot reporting the fold change versus control. LC3II, LC3I, and p62 intensity values were normalized to the corresponding β-actin value. The ratio of LC3II, LC3I were presented as the levels of activated autophagy. Images report one representative experiment out of at least 3 independent experiments. All values are represented as mean of at least 3 independent experiments (*n* = 3 ± SEM). \* *p* < 0.05; versus NG. **Figure 7. The effect of GPR26 knockdown and HG on autophagy.** The effect of GPR26 knockdown alone or in combination with HG on autophagy at 24, 48, and 72 h with GPR26 (+) or scrambled GapmeRs (−), indicated in the figures. Panels show (**a**,**b**) a representative Western blot of LC3II, LC3I, and p62. Densitometry plot reporting the fold change versus control. LC3II, LC3I, and p62 intensity values were normalized to the corresponding β-actin value. The ratio of LC3II, LC3I were presented as the levels of activated autophagy. Images report one representative experiment out of at least 3 independent experiments. All values are represented as mean of at least 3 independent experiments (*n* = 3 ± SEM). \* *p* < 0.05; versus NG.

generation and inflammation [50]. Indeed, HG inhibits autophagy to promote ROS-mediated inflammasome activation and cytokine secretion in immune cells [50]. Microtubuleassociated protein 1A/1B-light chain 3 (LC3) is a soluble protein that exists in the two forms of LC3-I and LC3-II, the second converted from LC3-I to initiate the formation and lengthening of the autophagosome. Measurement of LC3-II/LC3-I ratio consents the quantification of autophagy activation levels [51]. In addition, measurement of p62, a selective substrate of autophagy, is an additional method to measure autophagy, since it inversely correlates with the autophagy activation [52]. Thus, to explore the role of GPR26 in regulating autophagy in monocytes, we examined LC3-II/LC3-I ratio and p62 levels in THP-1 cells treated with NG or HG and transfected for 24, 48, and 72 h with GPR26 Gapmers. Our results indicated that knockdown of GPR26 decreased LC3-II/LC3-I ratio at 24 and 48 h (Figure 7a). HG decreased LC3-II at all time points compared to NG treated cells (Figure 7a). A significant decrease in LC3-II/LC3-I ratio was detected in THP-1, knocked down for GPR26, and treated with HG at 24, 48, and 72 h, a time point at which only the knockdown of GPR26 in combination with HG showed a significant effect on LC3-II decrease (Figure 7a). Next, we analyzed the levels of p62, the levels of which should inversely correlate with that of LC3-II. Accordingly, knockdown of GPR26 increased p62 levels in THP-1 cultured in NG at 24 h and 48 h (Figure 7b). HG increased p62 levels only at 48 h (Figure 7b). Knockdown of GPR26 enhanced p62 levels in HG-treated THP-1 at 24, 48, and 72 h, a time point at which neither GPR26 knockdown nor HG increased p62 levels (Figure 7b). Taken together, these data indicated that GPR26 promotes autophagy and might counteract HGmediated autophagy inhibition. Hence, hyperglycemia might impair GPR26 to inhibit au-

#### **4. Discussion 4. Discussion**

tophagy in monocytes.

Diabetes is among the top 10 leading causes of death worldwide (WHO, https://www.who.int/news-room/fact-sheets/detail/the-top-10-causes-of-death, last accessed on 1 June 2022). T2D is one of the major public health diseases diffused in Iran, the Diabetes is among the top 10 leading causes of death worldwide (WHO, https:// www.who.int/news-room/fact-sheets/detail/the-top-10-causes-of-death, last accessed on 1 June 2022). T2D is one of the major public health diseases diffused in Iran, the Middle East's second-largest country, due to its high prevalence rate [53]. Inflammation plays a crucial role in the pathogenesis of T2D and related complications [2,5]. The dysregulation of immune cells is strongly linked to T2D and vascular complications. Vascular inflammation is thought to be regulated by changes in monocyte and macrophage numbers, function, and related imbalance of pro-inflammatory signaling cascade [5]. Hyperglycemia acts as a strong activator of inflammatory response by activating endothelial cells and by dysregulating monocyte activation, leading to a chronic pro-inflammatory phenotype in monocytes and immune cells [2,5].

GPCRs play pivotal roles in a wide variety of immunological processes and are the target of nearly a third of all clinically utilized drugs [17]. Recently, several GPCRs involved in the development of insulin resistance and pancreatic β-cell dysfunction, such as GPR119, GPR146, GPR40, GPR120, and TGR5, have received attention as targets for therapeutic interventions in diabetic patients [54–56]. Although the mechanisms by which GPCRs regulate insulin sensitivity and immunological processes are unknown, the existence of GPCRs receptors as a very large and conserved family suggests that they might potentially modulate the response of immune cells in pathologies such as diabetes, atherosclerosis, and chronic inflammatory disease [17].

Accordingly, emerging studies on GPCRs, especially on orphan GPCRs, indicates their involvement in the regulation of immune cells biology and function [57]. For example, GPCR MRGPRX2 is expressed by monocytes to activate the intracellular pathways for an immunomodulatory action, leading to monocyte interaction with endothelial cells [58–60]. Our data indicated that orphan GPR26 is downregulated in diabetic patients and might be initially activated in monocytes and PBMCs to counteract the pro-inflammatory and apoptotic activation mediated by hyperglycemia. Chronic and prolonged exposure of monocytes to hyperglycemia impaired GPR26 protective effects and lead to its internalization and ineffective activation. Despite GPR26 ligand and mechanism of action needing to be further investigated, it emerged as a promising target to develop therapeutic strategies against inflammatory and diabetic diseases.

Analysis of GWAS studies from diabetic patients indicated that occurrence of certain SNPs in GPCRs are associated with an increased risk for diabetes and cardiovascular complications [33]. In addition, recent studies indicated that certain GPCRs are differentially regulated in diabetic patients [17–20]. However, the majority of these GPCRs are orphan receptors, for which the mechanism of action in diabetes and related complications is still unknown. In this study, we used GWAS studies to identify potential GPCR candidates with diabetes-associated SNPs and differentially expressed in diabetic samples. We selected orphan GPR26 as a promising candidate, presenting certain SNPs and downregulated in the blood samples from T2D patients. GPR26 is a central orphan GPCR that is most closely related to the serotonin receptor 5-HT5A and gastrin releasing hormone BB2 receptor, suggesting a possible role in the regulation of energy homeostasis and cell metabolism [61,62]. So far, the knowledge we have on GPR26 role belong to studies from *Caenorabditis elegans*, where depletion of GPR26 increases fat storage, and from rodents, where GPR26 deficiency in the hypothalamus increases genetic susceptibility to the onset of obesity, including glucose intolerance, hyperinsulinemia, and dyslipidemia (61). However, data regarding the role of GPR26 in diabetes and inflammatory associated complications are missing. We analyzed the expression of orphan GPR26 in the PBMC isolated from T2D patients and confirmed that GPR26 was downregulated. However, GPR26 expression was initially enhanced in human monocytes (THP-1) and human PBMCs treated with HG, a surrogate of hyperglycemia, whereas it was decreased when cells were exposed to prolonged and chronic HG levels. Two hypotheses were generated to explain these results. First, the group of diabetic patients was composed of insulin and non-insulin treated patients, which might have influenced the expression of GPR26. Second, GPR26 might be initially activated to protect monocytes against HG, whereas prolonged and chronic monocyte exposure to HG might impair GPR26 protective role. Our data indicated that both hypotheses might be reliable. Indeed, when diabetic patients were divided according to their insulin treatment, we found that GPR26 expression was further downregulated in insulin patients compared to non-insulin treated patients. This data might be due to insulin-mediated alleviation of hyperglycemic adverse effects on monocyte activation. In line with this, we noticed that the expression of GPR26 negatively correlated with the BMI and HbA1c percentage in diabetic and non-diabetic subjects. In addition, GPR26 exposure on monocyte membranes was decreased by chronic levels of HG, whereas GPR26 accumulated in the cytoplasm. Hence, these data supported the hypothesis that GPR26 might be activated to protect monocytes against hyperglycemia-mediated detrimental effects.

In the diabetic state, chronic or intermittent hyperglycemia promotes a phenotype named "glucose toxicity", which is often associated with an imbalance of the cellular redox state, inflammation, and related apoptosis [63]. Indeed, several signaling pathways are altered and promote the formation of ROS, as well as the secretion of pro-inflammatory cytokines and cellular death (pathological autophagy and/or apoptosis). ROS plays a pivotal roles in triggering all these diabetic complications [63]. Accordingly, we investigated the role of GPR26 against all these HG-related complications. Our data indicated that orphan GPR26 knockdown promoted ROS production, MAPK-related inflammatory activation, monocyte adhesion, and apoptosis. Autophagy, commonly activated in immune cells to regulate inflammation following oxidative injury in diabetes [64], was impaired by GPR26 knockdown. Hence, our data revealed that GPR26 tightly regulates all crucial pathways related to monocyte cell biology and metabolism. Our data indicated that GPR26 protected monocytes from HG-mediated increase of inflammation and ROS production.

Previous studies showed that elevated ROS production leads to p38 MAPK andERK1/2 activation, which in turn promotes NF-κB-mediated transcription of pro-inflammatory genes and monocyte activation [38,44]. Our data indicated that lack of GPR26 protection under chronic hyperglycemic conditions enhanced MAPK and NF-κB activation. Hence, these data supported our hypothesis about GPR260 s protective role against T2D.

ROS induced -p38 MAPK and ERK1/2 activation results in caspase 3 activation and apoptotic cell death [65], despite several in vitro studies indicating that HG induces cell apoptosis in monocytes [48,49], Contradictory data exist regarding the role of apoptosis in monocytes from diabetic patients [12,13]. Indeed, inhibition of monocyte apoptosis impedes wound healing in diabetic mice, but it also promotes the development of diabetic complications in T1D patients [12,13]. Our data indicated that knockdown of GPR26 increased apoptosis and enhanced HG-mediated Caspase 3 activation, suggesting that GPR26 plays an anti-apoptotic role against HG.

Autophagy is fundamental to maintain cell homeostasis, removal of ROS, and inflammation [50]. Our results provided evidence that orphan GPR26 might play a protective role against HG by activating autophagy. Moreover, we identified a new role for GPR26 in promoting autophagy that is independent from HG. Although we did not investigate the mechanism in detail, HG and GPR26 might influence autophagic flux partly through p38 MAPK.

To the best of our knowledge, this is the first work demonstrating the expression and role of orphan GPR26 in monocytes from diabetic patients. Although additional data are needed to prove the protective role of GPR26 against T2D in vivo, we started to depict possible signaling pathways differentially regulated by GPR26 in THP1, independently or not from HG.

Indeed, our study has some limitations. Expression assay of GPR26 in PBMCs of diabetic patients was performed in limited samples, and future studies with a large number of samples are needed to confirm our results. Moreover, analysis of GPR26 expression and function in other cell types related to diabetes and cardiovascular complications, such as endothelial cells that are closely related to immune cell activation in diabetic patients, could provide more information and better clarify the role of GPR26 in T2D. Since the ligands of GPR26 are still unknown, future studies devoted to their identification might offer the basis for modulating the expression and activation of GPR26 in T2D patients.

#### **5. Conclusions**

The results obtained in the present study defined a new role for GPR26 in human monocytes and PBMCs in T2D patients. Our data suggested that THP1 might initially activate a mechanism of defense in response to hyperglycemia, which is partly mediated by a positive feedback loop that involves GPR26 activation. The protective effect might be impaired by chronic and intermittent hyperglycemic conditions. Treatment of T2D patients with insulin might counteract HG-mediated pathological activation of monocytes, a phenomenon partly reflected from a reduction in GPR26 expression. Moreover, our data suggested that GPR26 exerts a protective role independently from HG by inhibiting pro-inflammatory monocyte activation, ROS production, and apoptosis.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/biomedicines10071736/s1, Table S1: Sequence of primers against the human genes analyzed by qPCR; Table S2: List of antibodies for western blot (WB); File S1: GPR26 assotiations-Microarray values blood GPCRs from GWAS SNP; File S2: GPR26 assotiations; File S3: KEGG predicted pathways.

**Author Contributions:** Z.A.K. researched data and wrote the manuscript. L.N. contributed to discussion and edited the manuscript. S.S. contributed to sample collections. M.a.B. contributed to sample collections and reviewed the manuscript. C.W. contributed to reviewed/edited the manuscript. M.B. contributed to reviewed/edited the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** Funding for the present work was provided by Zahra Abedi Kichi, from the Deutsche Forschungsgemeinschaft (DFG), TRR267, and SFB1123 to C.W. and L.N.

**Institutional Review Board Statement:** The study was conducted in accordance with the Declaration of Helsinki, and approved by the Institutional Review Board (or Ethics Committee) of Tarbiat Modares University (protocol code REC.1397.109, date of approval: 17 October 2018).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** C.W. and M.B. had full access to all data in the study and take responsibility for the integrity of data and accuracy of the data analysis.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

## *Article* **Tetrahydrobiopterin (BH4) Supplementation Prevents the Cardiorenal Effects of Diabetes in Mice by Reducing Oxidative Stress, Inflammation and Fibrosis**

**Ulises Novoa 1 , Karen Soto 1 , Cristian Valdés 2 , Jorge Villaseñor 3 , Adriana V. Treuer 4 and Daniel R. González 1,\***

**Abstract:** Background: The effects of diabetes on the cardiovascular system as well as in the kidney are profound, which include hypertrophy and fibrosis. Diabetes also induces oxidative stress, at least in part due to the uncoupling of nitric oxide synthase (NOS); this is a shift in NO production toward superoxide production due to reduced levels of the NOS cofactor tetrahydrobiopterin (BH4 ). With this in mind, we tested the hypothesis that BH4 supplementation may prevent the development of diabetic cardiomyopathy and nephropathy. Methods: Diabetes was induced in Balb/c mice with streptozotocin. Then, diabetic mice were divided into two groups: one group provided with BH4 (sapropterin) in drinking water (daily doses of 15 mg/kg/day, during eight weeks) and the other that received only water. A third group of normoglycemic mice that received only water were used as the control. Results: Cardiac levels of BH4 were increased in mice treated with BH4 (*p* = 0.0019). Diabetes induced cardiac hypertrophy, which was prevented in the group that received BH4 (*p* < 0.05). In addition, hypertrophy was evaluated as cardiomyocyte cross-sectional area. This was reduced in diabetic mice that received BH4 (*p* = 0.0012). Diabetes induced cardiac interstitial fibrosis that was reduced in mice that received BH4 treatment (*p* < 0.05). We also evaluated in the kidney the impact of BH4 treatment on glomerular morphology. Diabetes induced glomerular hypertrophy compared with normoglycemic mice and was prevented by BH4 treatment. In addition, diabetic mice presented glomerular fibrosis, which was prevented in mice that received BH4 . Conclusions: These results suggest that chronic treatment with BH4 in mice ameliorates the cardiorenal effects of diabetes,, probably by restoring the nitroso–redox balance. This offers a possible new alternative to explore a BH4 -based treatment for the organ damage caused by diabetes.

**Keywords:** nitric oxide synthase uncoupling; sapropterin; diabetic nephropathy

#### **1. Introduction**

Diabetes mellitus is one of the most common chronic diseases worldwide, and continues to increase in numbers and significance, with characteristics of an epidemic, as modern lifestyles lead to reduced physical activity and increased obesity [1]. Diabetic cardiomyopathy is the manifestation in the myocardium of the alterations produced by the altered homeostasis of glucose metabolism, independent of coronary artery disease [2]. This cardiomyopathy is initially characterized by diastolic dysfunction and cardiac hypertrophy, with preserved ejection fraction. As diabetes progresses, systolic dysfunction and reduced ejection fraction develop. This process of cardiac deterioration in diabetes

**Citation:** Novoa, U.; Soto, K.; Valdés, C.; Villaseñor, J.; Treuer, A.V.; González, D.R. Tetrahydrobiopterin (BH4) Supplementation Prevents the Cardiorenal Effects of Diabetes in Mice by Reducing Oxidative Stress, Inflammation and Fibrosis. *Biomedicines* **2022**, *10*, 2479. https://doi.org/10.3390/ biomedicines10102479

Academic Editors: Alfredo Caturano and Raffaele Galiero

Received: 27 June 2022 Accepted: 5 September 2022 Published: 4 October 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

includes altered metabolism, inflammation, and oxidative stress, which result in apoptosis and fibrosis that further deteriorate the myocardium [3–6]. In addition, altered calcium handling was characterized in the diabetic cardiac myocytes. The reduced capacity of the sarcoplasmic reticulum Ca2+ pump SERCA2 results in a diminished storage capacity of Ca2+, which impairs cardiac contractility. Importantly, it also alters cardiac relaxation, which is evidenced in the diastolic dysfunction [7].

Diabetic nephropathy is another of the main complications of diabetes. In advanced stages, it is characterized by urinary albumin excretion [8]. It begins with a series of cellular and molecular changes that lead to morphological alterations, first in the glomerulus, then, in more advanced stages, in the tubules and interstitial space [8]. Glomeruli undergo hypertrophy, with a thickening of the basal membrane and basal tubular membrane, with a progressive accumulation of extracellular matrix components [9]. These ultrastructure changes are responsible for the functional alterations observed in diabetic nephropathy, such as proteinuria, hypertension, and, finally, renal failure. After hyperglycemia is chronically established, oxidative stress is one the main biochemical alterations that occur in the kidney [10,11], leading to a proinflammatory state [11].

Current treatments for the cardiorenal complications of diabetes are based on the control of blood glucose levels, mainly with metformin and sulfonylureas in type 2 diabetes mellitus and insulin mainly in type 1 diabetes [12]. More recently, clinical trials evaluating the organ target damage such as cardiac and renal complications with the use of di-peptidyl peptidase-4 (DPP4) inhibitors, glucagon-like peptide (GLP1) receptor agonists, and sodiumglucose co-transporter 2 (SGLT2) inhibitors have shown promising results [13–16]. At the preclinical level, pharmacological strategies are now directly focusing on end-organ damage processes such as fibrosis, inflammation, and oxidative stress [3].

In diabetes, several sources may contribute to the observed oxidative stress, such as xanthine oxidoreductase, nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, mitochondria, and uncoupled nitric oxide synthases (NOS) [17]. A direct consequence of the increased production of reactive oxygen species (ROS) is the uncoupling of nitric oxide synthase [18]. This is due to the oxidation of tetrahydrobiopterin (BH4), an essential cofactor for NOS activity. When NOS is uncoupled, its activity is redirected toward the production of superoxide, instead of NO, further contributing to the oxidative process [19]. Because BH4 oxidation may also occur in oxidative states in the kidney, this leads to endothelial NOS uncoupling, which generates endothelial dysfunction in the kidney vasculature, including the glomerular capillaries, and afferent and efferent arterioles [20].

We tested the hypothesis that in diabetes, tetrahydrobiopterin supplementation leads to the recoupling of nitric oxide synthase 1 (NOS1), preventing cardiac remodeling and the advance of diabetic nephropathy, two of the main complications of chronic diabetes.

These findings in the diabetic heart and kidney represent a potential translational tool with therapeutic value. The proposed investigation may have a translational impact and contribute to the basic knowledge of NOS uncoupling in the setting of diabetic cardiomyopathy.

#### **2. Methods**

#### *2.1. Experimental Model and Protocol*

Diabetes was induced in Balb/c mice (*n* = 30, male, 30–40 g) with the intraperitoneal injection of three doses (100, 100, and 200 mg/kg) of streptozotocin (Sigma, St. Louis, MO, USA) in 10 mM citrate buffer, pH 4.5. The control group received an injection of citrate. Then, diabetic mice were divided into two groups: one group provided with BH4 (sapropterin, Inpheno, InnoPharmax, Inc., Taipei City, Taiwan, Lot # 6P001) in drinking water (daily doses of 15 mg/kg/day, during eight weeks), and the other received only water. A third group of normoglycemic mice that received only water were used as the control. The protocol was approved by the Bioethics Committee of Universidad de Talca (# 2015-087- DG). Mice were kept in the animal facility of the institution, at room temperature (22 ◦C), under a 12 h light/dark cycle.

#### *2.2. Sample Collection and Storage*

At the end of the eight weeks, animals were induced anesthesia with ketamine 90 mg/kg and 10 mg/kg xylazine. Then, a midline incision was made and blood was obtained from the cava vein. After blood withdrawing, the heart and kidney were extracted.

#### *2.3. Plasma Biochemical Measurements*

For plasma biochemical measurements, we used a brain natriuretic peptide (BNP) Kit ELISA mouse (Elabscience Biotech Co., Ltd., Wuhan, China). Plasma glucose was determined using a kit from Valtek (Santiago, Chile). Insulin was determined using an ELISA from EMD Millipore, Billerica, MA, U.S.A.

#### *2.4. Histological Analyses*

Cardiac and renal sections were obtained for pathology analysis. For this, hearts and kidneys were fixed in Bouin solution. Then, pieces of the organs were dissected, dehydrated in alcohol-xylol solutions and included in Paraplast. In a microtome, 5 µm sections were obtained and mounted in 0.1% polylysine-treated slides. After this, sections were rehydrated and prepared for hematoxylin–eosin, Masson's trichrome, and periodic acid Schiff's staining.

Glomerular pathological analysis was performed by a blinded investigator, scoring the degree of fibrosis, glomerular hypertrophy, and mesangial expansion according to previous reports [21–24].

#### *2.5. TUNEL Assay*

Cardiac sections were probed with a Click-iT™ TUNEL Colorimetric IHC Detection Kit (Catalogue N◦ C10625, Thermo Fisher Scientific Inc., Carlsbad, CA, USA), for detection of apoptotic cells, as previously described [25,26].

#### *2.6. Confocal Microscopy*

Cardiac and renal sections were prepared for confocal microscopy, as previously described [27]. Renal sections were stained with anti-α-smooth muscle actin or F4/80 (Santa Cruz Biotechnology, Dallas, Texas, USA), followed by fluorescein isothiocyanate conjugated (FITC) antimouse (Jackson Inmunoresearch, West Grove, PA, USA). Nuclei were counterstained with propidium iodine (100 µM). Cardiac sections were probed for F4/80 to detect macrophages. Images were obtained with an LSM700 confocal microscope (Carl Zeiss, Jena, Germany).

## *2.7. Tetrahydrobiopterin (BH4) Quantification*

Plasma and cardiac BH4 were determined as previously described [25,28] using a differential oxidation of biopterins protocol. Briefly, samples were submitted either to acidic or basic conditions (pH 3 or 9). Then, samples were oxidized with iodine. BH2 and BH4 contents were quantified by HPLC using HPLC (Perkin Elmer series 200, Waltham, MA) with fluorescence detection with excitation at 350 nm and emission at 450 nm.

#### *2.8. Western Blot*

Cardiac proteins were prepared as previously described for Western blot analysis [29]. Cardiac homogenates (30 µg) were mixed with loading buffer and summited to SDS-PAGE in 7.5% gels. Then, proteins were electro-transferred to nitrocellulose membranes. After blocking with Tween-buffered saline solution supplemented with 5% nonfat milk, membranes were incubated overnight at 4 ◦C with specific antibodies antinitrotyrosine (Badrilla, Leeds, U.K.). For NOS, SDS-PAGE was performed in nonreducing conditions of the loading buffer, and electrophoresis was run with the chamber immersed in ice. After electro-blotting, nitrocellulose membranes were incubated with anti-NOS1 antibody (Cell Signaling, Danvers, MA, USA) or NOS3 (BD Biosciences, Franklin Lakes, NJ, USA).

#### *2.9. Statistical Analysis*

Data are presented as means ± SEM, compared using ANOVA (normally distributed data) or Kruskal–Wallis test (nonparametric data) with Tukey's or Dunn's post hoc tests for comparisons between groups. A value of *p* < 0.05 was considered statistically significant.

#### **3. Results**