*Article* **Enhancement of Sphingomyelinase-Induced Endothelial Nitric Oxide Synthase-Mediated Vasorelaxation in a Murine Model of Type 2 Diabetes**

**Éva Ruisanchez 1,2,† , Anna Janovicz 1,2,†, Rita Cecília Panta 1 , Levente Kiss 3 , Adrienn Párkányi 1 , Zsuzsa Straky 1 , Dávid Korda 1 , Károly Liliom 4 , Gábor Tigyi 1,5 and Zoltán Benyó 1,2,\***

**Abstract:** Sphingolipids are important biological mediators both in health and disease. We investigated the vascular effects of enhanced sphingomyelinase (SMase) activity in a mouse model of type 2 diabetes mellitus (T2DM) to gain an understanding of the signaling pathways involved. Myography was used to measure changes in the tone of the thoracic aorta after administration of 0.2 U/mL neutral SMase in the presence or absence of the thromboxane prostanoid (TP) receptor antagonist SQ 29,548 and the nitric oxide synthase (NOS) inhibitor L-NAME. In precontracted aortic segments of non-diabetic mice, SMase induced transient contraction and subsequent weak relaxation, whereas vessels of diabetic (*Leprdb*/*Leprdb*, referred to as db/db) mice showed marked relaxation. In the presence of the TP receptor antagonist, SMase induced enhanced relaxation in both groups, which was 3-fold stronger in the vessels of db/db mice as compared to controls and could not be abolished by ceramidase or sphingosine-kinase inhibitors. Co-administration of the NOS inhibitor L-NAME abolished vasorelaxation in both groups. Our results indicate dual vasoactive effects of SMase: TP-mediated vasoconstriction and NO-mediated vasorelaxation. Surprisingly, in spite of the general endothelial dysfunction in T2DM, the endothelial NOS-mediated vasorelaxant effect of SMase was markedly enhanced.

**Keywords:** sphingolipids; sphingomyelinase; vasorelaxation; endothelial nitric oxide synthase; type 2 diabetes; thromboxane prostanoid receptor

### **1. Introduction**

Sphingolipids, derived from sphingomyelin metabolism, have been implicated as important mediators in the physiology and pathophysiology of the cardiovascular system [1–6]. Sphingomyelinase (SMase) catalyzes the conversion of sphingomyelin to ceramide, which is the precursor of other sphingolipid mediators, including ceramide-1 phosphate (C1P), sphingosine (Sph), and sphingosine-1-phosphate (S1P) [7]. The majority of S1P-induced biological effects are mediated by G-protein-coupled receptors (GPCRs), termed S1P1–5 [8]. Other sphingolipid mediators may exert biological effects by directly interacting with membrane or intracellular protein targets, independently of the activation of S1P receptors [5,9–11].

Based on the optimal pH for their catalytic activity, SMase isoforms can be divided into three groups: alkaline, acidic, and neutral [12]. The expression and known functions of alkaline SMases are mostly restricted to the gastrointestinal system, whereas acidic and

**Citation:** Ruisanchez, É.; Janovicz, A.; Panta, R.C.; Kiss, L.; Párkányi, A.; Straky, Z.; Korda, D.; Liliom, K.; Tigyi, G.; Benyó, Z. Enhancement of Sphingomyelinase-Induced Endothelial Nitric Oxide Synthase-Mediated Vasorelaxation in a Murine Model of Type 2 Diabetes. *Int. J. Mol. Sci.* **2023**, *24*, 8375. https://doi.org/10.3390/ ijms24098375

Academic Editors: Simona Gabriela Bungau and Cosmin Mihai Vesa

Received: 28 February 2023 Revised: 30 April 2023 Accepted: 4 May 2023 Published: 6 May 2023

**Copyright:** © 2023 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/).

neutral SMases are more widely expressed and involved in physiological and pathophysiological reactions in many systems, including the cardiovascular system. In the vasculature, SMases are implicated in the regulation of vascular tone and permeability as well as in causing atherosclerotic lesions and vascular wall remodeling [13]. Interestingly, neutral SMase has been reported to induce a wide range of changes in the vascular tone, depending on the species, vessel type, and experimental conditions (Table 1). Taken into account the large number of biologically active mediators (including ceramides, C1P, Sph, and S1P) that can be generated both extra- and intracellularly upon triggering the sphingolipid biosynthesis by neutral SMase, the diversity of vascular effects is not unexpected.

**Table 1.** Reported vasoactive effects of neutral SMase.

NO, nitric oxide; PKC, protein kinase C; VDCC, voltage-gated calcium channel; MAP, mitogen activated protein; eNOS, endothelial NO synthase; PLA2, phospholipase A2; TXA2, thromboxane A2; COX, cyclooxygenase.

SMase enzymes are reportedly upregulated in certain cardiovascular and metabolic disorders, such as type 2 diabetes mellitus (T2DM) [13,26,27]. Sphingolipids have been implicated as important regulators of inflammatory processes in diabetes [28]. Stress conditions initiate changes in sphingolipid metabolism [29], and sphingolipids have emerged as key mediators of stress responses [30,31]. Extracellular stressors induce sphingolipid synthesis and turnover, thereby 'remodeling' sphingolipid profiles and their topological distribution within cells [32]. Emerging evidence not only demonstrates profound changes in sphingolipid pools and distribution under conditions of overnutrition [33–35], but also implicates sphingolipids in mediating cell-signaling responses that precipitate pathology associated with obesity [36]. In spite of the marked alterations in the metabolism and actions of sphingolipids in diabetes and recent observations indicating that ceramide may contribute to the development of diabetic endothelial dysfunction [37], relatively little is known about the effects of sphingolipids on vascular functions in T2DM. In the present

study, we analyzed the effects of SMase on vascular tone under diabetic conditions in order to elucidate the signaling mechanisms involved. db/db mice as compared to non-diabetic control littermates. Furthermore, the serum phosphorylcholine level was also significantly increased in the diabetic group (Figure 1C),

conditions in order to elucidate the signaling mechanisms involved.

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implicated as important regulators of inflammatory processes in diabetes [28]. Stress conditions initiate changes in sphingolipid metabolism [29], and sphingolipids have emerged as key mediators of stress responses [30,31]. Extracellular stressors induce sphingolipid synthesis and turnover, thereby 'remodeling' sphingolipid profiles and their topological distribution within cells [32]. Emerging evidence not only demonstrates profound changes in sphingolipid pools and distribution under conditions of overnutrition [33–35], but also implicates sphingolipids in mediating cell-signaling responses that precipitate pathology associated with obesity [36]. In spite of the marked alterations in the metabolism and actions of sphingolipids in diabetes and recent observations indicating that ceramide may contribute to the development of diabetic endothelial dysfunction [37], relatively little is known about the effects of sphingolipids on vascular functions in T2DM. In the present study, we analyzed the effects of SMase on vascular tone under diabetic

First, we verified the general metabolic and vascular phenotypes of the T2DM mice

tested in the present study. Db/db mice reportedly develop obesity with elevated blood glucose levels and insulin resistance [38–40]. Accordingly, the body weight increased almost 2-fold (Figure 1A), whereas blood glucose levels increased 3-fold (Figure 1B) in

#### **2. Results** which is consistent with the reported enhancement of SMase activity in type 2 diabetes

**2. Results**

First, we verified the general metabolic and vascular phenotypes of the T2DM mice tested in the present study. Db/db mice reportedly develop obesity with elevated blood glucose levels and insulin resistance [38–40]. Accordingly, the body weight increased almost 2-fold (Figure 1A), whereas blood glucose levels increased 3-fold (Figure 1B) in db/db mice as compared to non-diabetic control littermates. Furthermore, the serum phosphorylcholine level was also significantly increased in the diabetic group (Figure 1C), which is consistent with the reported enhancement of SMase activity in type 2 diabetes [13,26,27]. According to literature data, acetylcholine (ACh)-evoked vasorelaxation of the aorta prepared from db/db mice is completely NOS-dependent [41], thus ACh was used to characterize the endothelial function. The vessels of db/db animals showed marked endothelial dysfunction, as indicated by the impairment of the dose-response relationship of ACh-induced vasorelaxation after precontraction with 10 µmol/L PE (Figure 1D). The Emax value decreased to 50.8 ± 2.0% in diabetic vessels as compared to controls (65.8 ± 3.9%). However, there was no significant difference in the EC50 values (34.7 ± 16.0 nM vs. 55.7 ± 15.7 nM), indicating unchanged potency in spite of the reduced efficacy of endogenous NO upon stimulation of endothelial NOS (eNOS) by ACh. In contrast, reactivity of the vascular smooth muscle to NO remained unaltered, as neither the Emax (105.2 ± 1.8% vs. 103.3 ± 2.2%) nor the EC50 (10.7 ± 1.3 nM vs. 14.1 ± 2.0 nM) values of sodium nitroprusside (SNP)-induced vasorelaxation differed in vessels of db/db animals as compared to controls (Figure 1E). Taken together, these results confirm the T2DM-like metabolic and vascular phenotypes in db/db mice and suggest the in vivo enhancement of SMase activity as well. [13,26,27]. According to literature data, acetylcholine (ACh)-evoked vasorelaxation of the aorta prepared from db/db mice is completely NOS-dependent [41], thus ACh was used to characterize the endothelial function. The vessels of db/db animals showed marked endothelial dysfunction, as indicated by the impairment of the dose-response relationship of ACh-induced vasorelaxation after precontraction with 10 μmol/L PE (Figure 1D). The Emax value decreased to 50.8 ± 2.0% in diabetic vessels as compared to controls (65.8 ± 3.9%). However, there was no significant difference in the EC50 values (34.7 ± 16.0 nM vs. 55.7 ± 15.7 nM), indicating unchanged potency in spite of the reduced efficacy of endogenous NO upon stimulation of endothelial NOS (eNOS) by ACh. In contrast, reactivity of the vascular smooth muscle to NO remained unaltered, as neither the Emax (105.2 ± 1.8% vs. 103.3 ± 2.2%) nor the EC50 (10.7 ± 1.3 nM vs. 14.1 ± 2.0 nM) values of sodium nitroprusside (SNP)-induced vasorelaxation differed in vessels of db/db animals as compared to controls (Figure 1E). Taken together, these results confirm the T2DM-like metabolic and vascular phenotypes in db/db mice and suggest the in vivo enhancement of SMase activity as well.

**Figure 1.** Manifestation of the metabolic and vascular phenotypes of T2DM in db/db mice. Body weight (**A**), as well as non-fasting blood glucose (**B**) and serum phosphorylcholine levels (**C**), increased in db/db mice as compared to controls (\*\* *p* < 0.01, \*\*\* *p* < 0.001 vs. control group; Student's unpaired *t*-test, *n* = 13–22). ACh-induced relaxation diminished (**D**), while the reactivity of the vascular smooth muscle to sodium nitroprusside (SNP) remained unaltered (**E**) in vessels of db/db mice as compared to controls (mean ± SEM, \*\*\* *p* < 0.001 vs. control; dose-response curve fitted to *n* = 12–24).

Next, we determined the effect of nSMase on the active tone of control and db/db vessels (Figure 2A). After 10 µmol/L phenylephrine (PE)-induced precontraction, 0.2 U/mL nSMase elicited additional contraction in control vessels that reached its maximum at 24).

7.2 min before relaxing back to the pre-SMase level by the end of the 20-min observation period. In contrast, nSMase in db/db vessels elicited completely different responses. After a marked initial relaxation elicited by 0.2 U/mL nSMase during the first 5 min, the tone of the db/db vessels remained in a relaxed state below the level of the initial tension. From the shape of the tension curve, it appeared that in addition to the overriding relaxation, there was a delayed and transient constriction response with a time course similar to that observed in control vessels, but it was unable to overcome the robust dilatation. Evaluation of the AUC (Figure 2B) and the maximal changes in the vascular tone (Figure 2C) also supported the conclusion that there is a marked difference in the vascular effects of nSMase between control and db/db mice: contraction dominates in the former, whereas the latter is characterized by reduction of the vascular tone. period. In contrast, nSMase in db/db vessels elicited completely different responses. After a marked initial relaxation elicited by 0.2 U/mL nSMase during the first 5 min, the tone of the db/db vessels remained in a relaxed state below the level of the initial tension. From the shape of the tension curve, it appeared that in addition to the overriding relaxation, there was a delayed and transient constriction response with a time course similar to that observed in control vessels, but it was unable to overcome the robust dilatation. Evaluation of the AUC (Figure 2B) and the maximal changes in the vascular tone (Figure 2C) also supported the conclusion that there is a marked difference in the vascular effects of nSMase between control and db/db mice: contraction dominates in the former, whereas the latter is characterized by reduction of the vascular tone.

**Figure 1.** Manifestation of the metabolic and vascular phenotypes of T2DM in db/db mice. Body weight (**A**), as well as non-fasting blood glucose (**B**) and serum phosphorylcholine levels (**C**), increased in db/db mice as compared to controls (\*\* *p* < 0.01, \*\*\* *p* < 0.001 vs. control group; Student's unpaired *t*-test, *n* = 13–22). ACh-induced relaxation diminished (**D**), while the reactivity of the vascular smooth muscle to sodium nitroprusside (SNP) remained unaltered (**E**) in vessels of db/db mice as compared to controls (mean ± SEM, \*\*\* *p* < 0.001 vs. control; dose-response curve fitted to *n* = 12–

Next, we determined the effect of nSMase on the active tone of control and db/db vessels (Figure 2A). After 10 μmol/L phenylephrine (PE)-induced precontraction, 0.2 U/mL nSMase elicited additional contraction in control vessels that reached its maximum at 7.2 min before relaxing back to the pre-SMase level by the end of the 20-min observation

*Int. J. Mol. Sci.* **2023**, *24*, x FOR PEER REVIEW 4 of 13

**Figure 2.** Effects of nSMase on the vascular tone. Application of 0.2 U/mL nSMase evoked a complex vascular effect with dominant contraction in control vessels and a more pronounced relaxation in vessels of db/db mice. Black and red lines on panel (**A**) represent average changes in tension of PEprecontracted vessels in control and db/db mice, respectively (dotted lines represent SEM). Both area under curve values (**B**) and maximal tension changes (**C**) were significantly different in vessels from db/db animals as compared to controls (mean ± SEM, Student's unpaired *t*-test, \*\*\*\* *p* < 0.0001 **Figure 2.** Effects of nSMase on the vascular tone. Application of 0.2 U/mL nSMase evoked a complex vascular effect with dominant contraction in control vessels and a more pronounced relaxation in vessels of db/db mice. Black and red lines on panel (**A**) represent average changes in tension of PE-precontracted vessels in control and db/db mice, respectively (dotted lines represent SEM). Both area under curve values (**B**) and maximal tension changes (**C**) were significantly different in vessels from db/db animals as compared to controls (mean ± SEM, Student's unpaired *t*-test, \*\*\*\* *p* < 0.0001 vs. control; *n* = 51–49).

vs. control; *n* = 51–49). Our next aim was to differentiate the constrictor and relaxant components of the vascular tension changes in response to nSMase. In porcine coronary arteries [14] and in the carotid arteries of spontaneously hypertensive rats [23–25], prostanoids acting on TP receptors have been implicated in mediating the vasoconstrictor effect of SMase. Therefore, we hypothesized that thromboxane prostanoid (TP) receptors also mediate the nSMaseinduced vasoconstriction in our murine aorta model. To test this hypothesis, the TP receptor antagonist SQ 29,548 was administered to the organ chambers 30 min prior to administration of nSMase. Blockade of TP receptors not only abolished the vasoconstriction but also converted it to a transient vasorelaxation in control vessels (Figure 3A). The maximum relaxation was reached at 5.5 min after the administration of nSMase, and the vascular tone returned to baseline after 10 min. TP receptor inhibition also markedly changed the vascular response to nSMase in the db/db group: the vasorelaxation was enhanced to more than 70% and reached its maximum at 6.5 min. After its peak, the relaxation decreased, Our next aim was to differentiate the constrictor and relaxant components of the vascular tension changes in response to nSMase. In porcine coronary arteries [14] and in the carotid arteries of spontaneously hypertensive rats [23–25], prostanoids acting on TP receptors have been implicated in mediating the vasoconstrictor effect of SMase. Therefore, we hypothesized that thromboxane prostanoid (TP) receptors also mediate the nSMaseinduced vasoconstriction in our murine aorta model. To test this hypothesis, the TP receptor antagonist SQ 29,548 was administered to the organ chambers 30 min prior to administration of nSMase. Blockade of TP receptors not only abolished the vasoconstriction but also converted it to a transient vasorelaxation in control vessels (Figure 3A). The maximum relaxation was reached at 5.5 min after the administration of nSMase, and the vascular tone returned to baseline after 10 min. TP receptor inhibition also markedly changed the vascular response to nSMase in the db/db group: the vasorelaxation was enhanced to more than 70% and reached its maximum at 6.5 min. After its peak, the relaxation decreased, but the vascular tone failed to return to the pre-SMase level even after 20 min. Both the AUC (Figure 3B) and the peak vasorelaxation (Figure 3C) values showed marked differences between the two experimental groups, indicating that the strongly enhanced and prolonged vasorelaxant capacity is responsible for the differences between the vasoactive effects of nSMase in db/db and control vessels. This finding was very surprising in light of the diminished ACh-induced vasorelaxation that we had observed in db/db animals (Figure 1D) and was not consistent with the large body of literature indicating diminished endothelium-dependent vasorelaxation in T2DM.

endothelium-dependent vasorelaxation in T2DM.

*Int. J. Mol. Sci.* **2023**, *24*, x FOR PEER REVIEW 5 of 13

but the vascular tone failed to return to the pre-SMase level even after 20 min. Both the AUC (Figure 3B) and the peak vasorelaxation (Figure 3C) values showed marked differences between the two experimental groups, indicating that the strongly enhanced and prolonged vasorelaxant capacity is responsible for the differences between the vasoactive effects of nSMase in db/db and control vessels. This finding was very surprising in light of the diminished ACh-induced vasorelaxation that we had observed in db/db animals (Figure 1D) and was not consistent with the large body of literature indicating diminished

but the vascular tone failed to return to the pre-SMase level even after 20 min. Both the AUC (Figure 3B) and the peak vasorelaxation (Figure 3C) values showed marked differences between the two experimental groups, indicating that the strongly enhanced and prolonged vasorelaxant capacity is responsible for the differences between the vasoactive effects of nSMase in db/db and control vessels. This finding was very surprising in light of the diminished ACh-induced vasorelaxation that we had observed in db/db animals (Figure 1D) and was not consistent with the large body of literature indicating diminished

**Figure 3.** Effects of TP receptor blockade on nSMase-induced changes in the vascular tone. After inhibition of the TP receptor by 1 μM SQ 29,548, 0.2 U/mL nSMase relaxed both db/db and control vessels, with a significantly higher relaxation in the db/db group (**A**). Black and red lines in panel A represent average tension changes in PE-precontracted vessels of control and db/db mice, respectively, whereas dotted lines represent SEM. Both area under curve values (**B**) and maximal tension changes (**C**) were significantly different in vessels from db/db animals as compared to controls (mean ± SEM, Student's unpaired *t*-test, \*\*\* *p* < 0.001 vs. control; \*\*\*\* *p* < 0.0001 vs. control; *n* = 20). **Figure 3.** Effects of TP receptor blockade on nSMase-induced changes in the vascular tone. After inhibition of the TP receptor by 1 µM SQ 29,548, 0.2 U/mL nSMase relaxed both db/db and control vessels, with a significantly higher relaxation in the db/db group (**A**). Black and red lines in panel A represent average tension changes in PE-precontracted vessels of control and db/db mice, respectively, whereas dotted lines represent SEM. Both area under curve values (**B**) and maximal tension changes (**C**) were significantly different in vessels from db/db animals as compared to controls (mean ± SEM, Student's unpaired *t*-test, \*\*\* *p* < 0.001 vs. control; \*\*\*\* *p* < 0.0001 vs. control; *n* = 20). **Figure 3.** Effects of TP receptor blockade on nSMase-induced changes in the vascular tone. After inhibition of the TP receptor by 1 μM SQ 29,548, 0.2 U/mL nSMase relaxed both db/db and control vessels, with a significantly higher relaxation in the db/db group (**A**). Black and red lines in panel A represent average tension changes in PE-precontracted vessels of control and db/db mice, respectively, whereas dotted lines represent SEM. Both area under curve values (**B**) and maximal tension changes (**C**) were significantly different in vessels from db/db animals as compared to controls (mean ± SEM, Student's unpaired *t*-test, \*\*\* *p* < 0.001 vs. control; \*\*\*\* *p* < 0.0001 vs. control; *n* = 20).

Next, we aimed to analyze the mechanism of the enhanced nSMase-induced vasorelaxation in the vessels of db/db mice. Theoretically, it could be due to the enhancement of eNOS-mediated vasorelaxation or to the onset of an NO-independent mechanism. To clarify this question, the vessels were incubated with the NOS inhibitor L-NAME (100 μM) in addition to the TP receptor blocker SQ 29,548 (1 μM) for 30 min prior to 0.2 U/mL nSMase administration. L-NAME at a concentration of 100 μM abolished the vasorelaxation observed in the presence of 1 μM SQ 29,548 both in control and in db/db vessels (Figure 4A). There were no significant differences between the two groups either in the AUC (Figure 4B) or in the maximal change of tension values (Figure 4C). These results indicate that the same secondary signaling pathways—namely TP receptors and eNOS—mediate the vasoactive effects of nSMase in health and in T2DM. Next, we aimed to analyze the mechanism of the enhanced nSMase-induced vasorelaxation in the vessels of db/db mice. Theoretically, it could be due to the enhancement of eNOS-mediated vasorelaxation or to the onset of an NO-independent mechanism. To clarify this question, the vessels were incubated with the NOS inhibitor L-NAME (100 µM) in addition to the TP receptor blocker SQ 29,548 (1 µM) for 30 min prior to 0.2 U/mL nSMase administration. L-NAME at a concentration of 100 µM abolished the vasorelaxation observed in the presence of 1 µM SQ 29,548 both in control and in db/db vessels (Figure 4A). There were no significant differences between the two groups either in the AUC (Figure 4B) or in the maximal change of tension values (Figure 4C). These results indicate that the same secondary signaling pathways—namely TP receptors and eNOS—mediate the vasoactive effects of nSMase in health and in T2DM. Next, we aimed to analyze the mechanism of the enhanced nSMase-induced vasorelaxation in the vessels of db/db mice. Theoretically, it could be due to the enhancement of eNOS-mediated vasorelaxation or to the onset of an NO-independent mechanism. To clarify this question, the vessels were incubated with the NOS inhibitor L-NAME (100 μM) in addition to the TP receptor blocker SQ 29,548 (1 μM) for 30 min prior to 0.2 U/mL nSMase administration. L-NAME at a concentration of 100 μM abolished the vasorelaxation observed in the presence of 1 μM SQ 29,548 both in control and in db/db vessels (Figure 4A). There were no significant differences between the two groups either in the AUC (Figure 4B) or in the maximal change of tension values (Figure 4C). These results indicate that the same secondary signaling pathways—namely TP receptors and eNOS—mediate the vasoactive effects of nSMase in health and in T2DM.

**Figure 4.** Effects of combined TP receptor and NOS blockade on nSMase-induced changes in vascular tone. After incubation of the vessels with 1 μM SQ 29,548 and 100 μM L-NAME for 30 min, 0.2 U/mL nSMase could no longer evoke a tension change in the thoracic aorta of control or db/db mice (**A**). Black and red lines in panel A represent average tension changes in PE-precontracted vessels **Figure 4.** Effects of combined TP receptor and NOS blockade on nSMase-induced changes in vascular tone. After incubation of the vessels with 1 μM SQ 29,548 and 100 μM L-NAME for 30 min, 0.2 U/mL nSMase could no longer evoke a tension change in the thoracic aorta of control or db/db mice (**A**). Black and red lines in panel A represent average tension changes in PE-precontracted vessels **Figure 4.** Effects of combined TP receptor and NOS blockade on nSMase-induced changes in vascular tone. After incubation of the vessels with 1 µM SQ 29,548 and 100 µM L-NAME for 30 min, 0.2 U/mL nSMase could no longer evoke a tension change in the thoracic aorta of control or db/db mice (**A**). Black and red lines in panel A represent average tension changes in PE-precontracted vessels of control and db/db mice, respectively (dotted lines represent SEM). Area under curve values (**B**) and maximal tension changes (**C**) were not different in vessels from db/db animals as compared to controls (mean ± SEM, *n* = 9–17).

Finally, we aimed to investigate the possible involvement of downstream sphingolipid metabolites in the vasorelaxing effect of nSMase observed in db/db mice. Thus, aortic segments isolated from db/db were treated with either the ceramidase inhibitor D-erythro MAPP (10 µM) or SKI-II (1 µM), a sphingosine kinase inhibitor. In order to examine the vasorelaxant effects exclusively, vessels were pre-treated with 1 µM SQ 29,568. Neither MAPP nor SKI-II could affect the vasorelaxation evoked by 0.2 U/mL nSMase (Figure 5A). In addition, evaluation of AUC (Figure 5B) and maximal vasorelaxing responses (Figure 5C) showed no significant differences in MAPP or SKI-II-treated vessels compared to vehicle

treated ones. These results suggest that the effect of nSMase is not mediated by sphingosine or sphingosine-1-phosphate. compared to vehicle treated ones. These results suggest that the effect of nSMase is not mediated by sphingosine or sphingosine-1-phosphate.

of control and db/db mice, respectively (dotted lines represent SEM). Area under curve values (**B**) and maximal tension changes (**C**) were not different in vessels from db/db animals as compared to

Finally, we aimed to investigate the possible involvement of downstream sphingolipid metabolites in the vasorelaxing effect of nSMase observed in db/db mice. Thus, aortic segments isolated from db/db were treated with either the ceramidase inhibitor Derythro MAPP (10 μM) or SKI-II (1 μM), a sphingosine kinase inhibitor. In order to examine the vasorelaxant effects exclusively, vessels were pre-treated with 1 μM SQ 29,568. Neither MAPP nor SKI-II could affect the vasorelaxation evoked by 0.2 U/mL nSMase (Figure 5A). In addition, evaluation of AUC (Figure 5B) and maximal vasorelaxing responses (Figure 5C) showed no significant differences in MAPP or SKI-II-treated vessels

*Int. J. Mol. Sci.* **2023**, *24*, x FOR PEER REVIEW 6 of 13

controls (mean ± SEM, *n* = 9–17).

**Figure 5.** Effects of ceramidase and sphingosine-kinase inhibitors on nSMase-induced vascular tone changes in db/db mice. After incubating with 1 μM SQ 29,548 for 30 min, db/db vessels were treated with 10 μM D-erythro MAPP or 1 μM SKI-II. Neither MAPP nor SKI-II could affect the vasorelaxation induced by 0.2 U/mL nSMase (**A**). Black, red, and blue lines in panel A represent average tension changes in PE-precontracted vessels of vehicles, MAPP, and SKI-II treated vessels, respectively (dotted lines represent SEM). Area under curve values (**B**) and maximal tension changes (**C**) were not different in MAPP- or SKI-II-treated vessels as compared to vehicle controls (mean ± SEM, *n* = 4–5). **Figure 5.** Effects of ceramidase and sphingosine-kinase inhibitors on nSMase-induced vascular tone changes in db/db mice. After incubating with 1 µM SQ 29,548 for 30 min, db/db vessels were treated with 10 µM D-erythro MAPP or 1 µM SKI-II. Neither MAPP nor SKI-II could affect the vasorelaxation induced by 0.2 U/mL nSMase (**A**). Black, red, and blue lines in panel A represent average tension changes in PE-precontracted vessels of vehicles, MAPP, and SKI-II treated vessels, respectively (dotted lines represent SEM). Area under curve values (**B**) and maximal tension changes (**C**) were not different in MAPP- or SKI-II-treated vessels as compared to vehicle controls (mean ± SEM, *n* = 4–5).

#### **3. Discussion 3. Discussion**

Findings of the present study indicate that nSMase-induced changes in vascular tension involve both vasoconstriction and vasorelaxation in murine vessels. Our results suggest that the former is mediated by the release of prostanoids and activation of TP receptors, whereas the latter is mediated by eNOS. Surprisingly, nSMase-induced eNOS-mediated vasorelaxation is markedly enhanced in the vessels of db/db mice in spite of the endothelial dysfunction indicated by the diminished vasorelaxation evoked by ACh. Therefore, nSMase appears to be able to induce enhanced NO release from endothelial cells in T2DM. Vasoconstriction in response to SMase has been reported in a number of studies, alt-Findings of the present study indicate that nSMase-induced changes in vascular tension involve both vasoconstriction and vasorelaxation in murine vessels. Our results suggest that the former is mediated by the release of prostanoids and activation of TP receptors, whereas the latter is mediated by eNOS. Surprisingly, nSMase-induced eNOSmediated vasorelaxation is markedly enhanced in the vessels of db/db mice in spite of the endothelial dysfunction indicated by the diminished vasorelaxation evoked by ACh. Therefore, nSMase appears to be able to induce enhanced NO release from endothelial cells in T2DM.

hough the mechanisms mediating this effect appear to be highly variable depending on the experimental conditions, including species, vascular region, and integrity of the endothelium (see Table 1). Release of prostanoids and consequent activation of TP receptors have been proposed in porcine coronary arteries [14] as well as in the carotid arteries of spontaneously hypertensive rats [23–25]. In our study, nSMase-induced contraction was found to be TP receptor-dependent in both control and db/db mice, indicating that nSMase stimulates the release of TXA2 from the aortic rings. Vasoconstriction in response to SMase has been reported in a number of studies, although the mechanisms mediating this effect appear to be highly variable depending on the experimental conditions, including species, vascular region, and integrity of the endothelium (see Table 1). Release of prostanoids and consequent activation of TP receptors have been proposed in porcine coronary arteries [14] as well as in the carotid arteries of spontaneously hypertensive rats [23–25]. In our study, nSMase-induced contraction was found to be TP receptor-dependent in both control and db/db mice, indicating that nSMase stimulates the release of TXA2 from the aortic rings.

There might be at least three different sources for the SMase-induced arachidonic acid formation necessary for TXA2 production [42]. One such possibility is that diacylglycerol (DAG) would accumulate while sphingomyelin synthase converted the newly generated ceramide back to sphingomyelin, and DAG lipases would provide arachidonic acid for the production of TXA2 [43]. Another mechanism might relate to the observation that C1P can allosterically activate phospholipase A2 (PLA2) [44], which leads to arachidonic acid formation [45]. It might be important in this context that the gene encoding ceramide kinase (*CERK*) is upregulated in T2DM [46]. Finally, S1P has been reported recently to regulate prostanoid production in a S1P receptor-dependent manner [47]. It should be noted that mechanisms other than prostanoid release might also mediate the vasoconstrictor effect of SMase, i.e., modulation of different ion channels or initiating the Rho signaling pathway [18,19,22,48].

Vasorelaxation in response to nSMase appears to be endothelial NO-dependent, as L-NAME completely abolished the decrease in vascular tone in both control and db/db vessels. Without L-NAME, relaxation was dramatically increased in db/db-derived vascular rings. This is unexpected because endothelial dysfunction with consequential decreased

vasorelaxant capacity is considered to be a hallmark of T2DM-like conditions. A potential explanation may be related to the altered structure of the plasma membrane in T2DM [49]. Normally, sphingomyelin (SM) represents about 10–20% of the lipids in the plasma membrane, mostly residing in the outer leaflet. However, most of these are found in the caveolae, and SMase is thought to be a regulator of lipid microdomains [50,51]. Pilarczyk and colleagues provided evidence that in db/db mice, the endothelial lining of the aorta contains 10-fold larger lipid raft areas enriched in SM as compared to controls [49]. This arrangement might be related to the decreased NO-release in T2DM, as eNOS is inhibited by caveolin-1 [52], which is considered to be an important regulator of eNOS [53–55]. In our experimental setting, nSMase-induced degradation of sphingomyelin could interfere with this caveolar structure and induce the detachment of eNOS from caveolin-1, leading to high amounts of NO released from the endothelium of db/db vascular rings. This hypothesis is supported by the observations of Mogami et al. [21], indicating that SMase causes endothelium-dependent vasorelaxation through Ca2+-independent endothelial NO production in bovine aortic valves and coronary arteries. They also reported SMase-induced translocation of endothelial NOS from plasma membrane caveolae to the intracellular region. Furthermore, protein expression levels of caveolin-1 were reported to be significantly higher in the aorta of db/db mice, and this was thought to be related to the impaired aortic relaxation of C57BL/KsJ mice [56]. In order to find out if downstream sphingolipid mediators such as sphingosine or sphingosine-1-phosphate contribute to the enhanced vasorelaxation seen in db/db aortic segments, we also investigated whether inhibition of ceramidase or sphingosine-kinase could interfere with the vasorelaxation. Neither D-erythro-MAPP nor SKI-II could diminish the nSMase-induced vasorelaxation in db/db vessels; therefore, it is unlikely that the release of these mediators participates in the effect. This finding strengthens our hypothesis that the enhanced relaxation in db/db vessels is mediated by a direct effect of nSMase on the membrane structure. On the other hand, we cannot rule out that the ceramide-related pathway might be involved in the SMase-induced contractions as well [18,23], as we did not address this question in our experiments. Finally, the potentially increased NO-sensitivity of guanylate cyclase (sGC) [57], which could be related to the dysfunctional NO-release observed in T2DM, should also be considered, as this would sensitize sGC to NO and result in enhanced NO-mediated vasorelaxation. However, this mechanism can be excluded in our present experiments, as the SNP dose-response curve remained unchanged in db/db vessels (Figure 1E), indicating that the sensitivity of the vascular smooth muscle to NO was not upregulated.

Sphingolipid metabolism is markedly altered in T2DM and related conditions [58–62], and the observed changes in endothelial lipid rafts [49] might be a consequence of the disrupted plasma membrane lipid metabolism. On the other hand, T2DM has several characteristics that resemble a chronic inflammatory disease [63]. Cytokines that accumulate in chronic inflammation, such as tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1β), can also induce marked changes in sphingolipid metabolism [6,64,65]. Our observation that serum phosphorylcholine levels were increased in the db/db group might be a strong indicator of the altered in vivo sphingolipid metabolism in our animal model and agrees with the literature; however, it cannot be excluded that enzymes other than nSMase also contribute to this increase (e.g., other SMases, choline-kinase, phospholipase C, ENPP, etc.).

As a limitation of our study, it has to be mentioned that the characteristics of the pathophysiological conditions in the db/db mouse model differ from those of human T2DM in some aspects [66]. For example, db/db mice do not necessarily develop hypertension and may have high levels of high-density lipoprotein and a reduced tendency toward atherosclerosis [67]. Therefore, due to the more severe endothelial dysfunction, the enhancement of nSMase-induced eNOS-mediated vasorelaxation may be limited in humans with T2DM. A further limitation of our study is that we tested only one single dose of bacterial nSMase. This 0.2 U/mL dose of exogenously administered bacterial nSMase represents the upper range used in the literature [14–18,20–23], as our aim was to evaluate the

consequences of a robust activation of sphingomyelinase degradation. Further studies may aim to elucidate the exact dose-response relationship for SMase-induced vasorelaxation and vasoconstriction in db/db mice or other T2DM-related conditions, which may also help to clarify the exact molecular mechanisms involved. Though bacterial and different mammalian nSMase orthologues share low overall homology, the 'catalytic core' residues are strongly conserved [68]. Therefore, it is likely that the addition of bacterial nSMase mimics the overactivation of mammalian nSMase and leads to similar cellular effects—in our case, the release of thromboxane and NO [69,70].

#### **4. Materials and Methods**

All procedures were carried out according to the guidelines of the Hungarian Law of Animal Protection (40/2013). The procedures were approved by the National Scientific Ethical Committee of Animal Experimentation (PE/EA/924-7/2021, accepted: 7 September 2021).

#### *4.1. Animals and General Procedures*

The BKS db diabetic mouse strain (JAX stock #000642) was obtained from the Jackson Laboratory (Bar Harbor, ME, USA) and has been maintained in our animal facility by mating repulsion double heterozygotes (*Dock7m* +/+ *Leprdb*). Littermate adult male diabetic (*Leprdb*/*Leprdb*, referred to as db/db) and misty (*Dock7m*/*Dock7m*, referred to as control) mice were selected for experiments. All mice investigated in this research were male and aged between 90 and 180 days. Animals were weighed, and blood samples were collected by cardiac puncture followed by transcardial perfusion with 10 mL of heparinized (10 IU/mL) Krebs solution under deep ether anesthesia, as described previously [71].  Budapest, Hungary). In some experiments, additional blood samples were collected, allowed to clot for 30 min at room temperature, and centrifuged at 2000× *g* for 15 min at 4 ◦C. Serum was snap frozen for a later phosphorylcholine assay, which was based on the method described by Hojjati and Jiang [72] using a commercially available kit (item No. 10009928, Cayman Chemical; Ann Arbor, MI, USA).

#### *4.2. Myography*

The thoracic aorta was removed and cleaned of fat and connective tissue under a dissection microscope (M3Z, Wild Heerbrugg AG; Gais, Switzerland) and immersed in a Krebs solution of the following composition (mmol/L): 119 NaCl, 4.7 KCl, 1.2 KH2PO4, 2.5 CaCl2·2 H2O, 1.2 MgSO4·7 H2O, 20 NaHCO3, 0.03 EDTA, and 10 glucose at room temperature and pH 7.4. Vessels were cut into ~3 mm-long segments and mounted on stainless steel vessel holders (200 µm in diameter) in a conventional myograph setup (610 M multiwire myograph system; Danish Myo Technology A/S; Aarhus, Denmark). Special care was taken to preserve the endothelium.

The wells of the myographs were filled with an 8 mL Krebs solution aerated with carbogen. The vessels were allowed a 30-min resting period, during which the bath solution was warmed to 37 ◦C and the passive tension was adjusted to 15 mN, which was determined to be optimal in a previous study [71]. Subsequently, the tissues were exposed to a 124 mmol/L K+ Krebs solution (made by isomolar replacement of Na+ by K+ ) for 1 min, followed by several washes with normal Krebs solution. Reactivity of the smooth muscle was tested by a contraction evoked by 10 µmol/L PE, and reactivity of the endothelium was tested by following the PE-evoked contraction with administration of 0.1 µmol/L ACh. After repeated washing, during which the vascular tension returned to the resting level, the segments were exposed to a 124 mmol/L K+ Krebs solution for 3 min in order to elicit a reference maximal contraction. Subsequently, after a 30-min washout, increasing concentrations of PE (0.1 nmol/L to 10 µmol/L) and ACh (1 nmol/L to 10 µmol/L) were administered to determine the reactivity of the smooth muscle and the endothelium, respectively. Following a 30-min resting period, the vessels were precontracted to 70–90% of the reference contraction by an appropriate concentration of PE, and after contraction

had stabilized, the effects of 0.2 U/mL nSMase (SMase from *B. cereus*, Sigma-Aldrich; St. Louis, MO, USA) were investigated for 20 min. Bacterial SMase functions at neutral pH and is reportedly a useful tool for mimicking the biological effects of activation of cellular SMase [73,74]. In some experiments, the selective TP receptor antagonist SQ 29,548 (1 µM) with or without the nitric oxide synthase (NOS) inhibitor L-NAME (100 µM) was applied to the baths 30 min prior to administration of nSMase. In order to block ceramidase or sphingosine-kinase enzymes, D-erythro MAPP (10 µM) or SKI-II (1 µM) were used, respectively. Finally, to test the sensitivity of the smooth muscle to NO, SNP (0.1 nmol/L to 10 µmol/L) was administered after a stable precontraction elicited by 1 µmol/L PE.

#### *4.3. Data Analysis*

An MP100 system and AcqKnowledge 3.72 software from Biopac System Inc. (Goleta, CA, USA) were used to record and analyze changes in the vascular tone. All data are presented as mean ± SE, and "*n*" indicates the number of vascular segments tested in myography experiments or the number of animals tested in the case of body weight, blood glucose, and serum phosphorylcholine levels. Maximal changes in the vascular tone were calculated as a percentage of precontraction. To evaluate the temporal pattern of nSMaseinduced vasoactive responses, individual curves were constructed and averaged, showing the changes in vascular tone for 20 min after the application of nSMase. Area under the curve (AUC) values were calculated from individual experiments for quantification of the overall vasoactive effect. The statistical analysis was performed using the GraphPad Prism software v.6.07 from GraphPad Software Inc. (La Jolla, CA, USA). Student's unpaired *t*-test was applied when comparing two variables, and a *p* value of less than 0.05 was considered to be statistically significant. The effects of cumulative doses of PE and ACh were evaluated by dose-response curve fitting for the determination of Emax and EC50 values. All data presented in this study are available in the Supplementary Material File (Figures S1–S5).

#### *4.4. Reagents*

All reagents in this study, including nSMase, were purchased from Sigma-Aldrich (St. Louis, MO, USA), except SQ 29,548, which was from Santa Cruz Biotechnology (Dallas, TX, USA).

#### **5. Conclusions**

Administration of nSMase induces TP receptor-mediated vasoconstriction and eNOSmediated vasorelaxation in murine vessels. In spite of endothelial dysfunction in db/db mice, the vasorelaxant effect of nSMase is markedly augmented. SMase-mediated disruption of SM in endothelial lipid rafts might represent a possible mechanism responsible for enhanced NO generation in T2DM. An intriguing interpretation of our finding is that the retraction of eNOS in sphingomyelin-rich microdomains of the endothelial plasma membrane could contribute significantly to the development of vascular dysfunction in T2DM.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ijms24098375/s1.

**Author Contributions:** Conceptualization, É.R., L.K., G.T. and Z.B.; methodology, É.R. and Z.B.; validation, K.L., G.T. and Z.B.; investigation, É.R., A.J., R.C.P., A.P., Z.S. and D.K.; resources, Z.B.; data curation, É.R., R.C.P., A.J., A.P., Z.S. and D.K.; writing—original draft preparation, É.R., A.J., L.K., K.L., G.T. and Z.B.; visualization, É.R. and A.J.; supervision, L.K., K.L., G.T. and Z.B.; project administration, Z.B.; funding acquisition, É.R. and Z.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Hungarian National Research, Development, and Innovation Office (OTKA K-112964, K-125174, K-139230, and PD-132851) and by the Ministry of Innovation and Technology of Hungary from the NRDI Fund (2020-1.1.6-JÖVO-2021-00010, 2020-1.1.6-JÖV ˝ O- ˝ 2021-00013 and TKP2021-EGA-25).

**Institutional Review Board Statement:** The animal experiments were carried out according to the guidelines of the Hungarian Law of Animal Protection (40/2013) and were approved by the National Scientific Ethical Committee of Animal Experimentation (PE\_EA\_924-7\_2021, Approval date: 7 September 2021).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available in the Supplemental File.

**Acknowledgments:** The authors are grateful to Margit Kerék for expert technical assistance and to Erzsébet Fejes for critically reading the manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study, in the collection, analysis, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results.

#### **References**

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*2.9. Inhibition of Hazelnut Oil Oxidation Assay*

The oil-in-water emulsion was prepared according to a previous study. To obtain oil without any antioxidants, hazelnut crude oil was stripped by silica gel, activated charcoal, and sucrose [28]. The aqueous phase of the emulsion was prepared by dispersing 0.5 wt% Tween 20 in 10 mM phosphate buffer at pH 7.0 followed by stirring at room temperature for 20 min to ensure complete dispersion. Hazelnut oil-in-water emulsions were prepared by homogenizing 10 wt% oil phases with 90 wt% aqueous phases at ambient temperature using a high-speed blender for 2 min, followed by ultrasonic vibration for 20 min. Peptides or TBHQ (tert-Butylhydroquinone, as positive control) were added to this emulsion with a final concentration of 0.02% to test the inhibition ability of peptides against oil. Sodium azide (NaN3, 0.02% (*w*/*w*)) was used as an antibacterial agent.

Incubation temperature for the oil-in-water emulsion system was generally set at 37 ◦C [29] or 40 ◦C [30]. In this work, a longer incubation time was required for analyzing the possible oxidative products of oil-in-water emulsion compared to that of linoleic acid oxidation, so an incubation temperature of 37 ◦C was selected. During incubation at 37 ◦C for 14 days, the levels of lipid hydroperoxides as primary lipid oxidation products were monitored at regular intervals to assess the degree of oxidation of hazelnut oil. In brief, 0.3 mL of emulsions were breakdown by 1.5 mL of isooctane/2-propanol (3:1, *v*/*v*), vortexed, and then centrifuged at 1000× *g* for 2 min. Next, 200 μL of organic solvent phases were collected and mixed with 2.8 mL of methanol/1-butanol (2:1, *v*/*v*), followed by 15 μL ferrous iron solution (prepared by mixing 0.132 M BaCl2 and 0.144 M FeSO4, dissolved in 0.4 M HCl), and 15 μL ammonium thiocyanate solution (3.94 M, dissolved in water). The absorbance was measured at 510 nm after 20 min. Lipid hydroperoxides (μmol/g oil) were calculated using a standard curve prepared by cumene hydroperoxide (0, 20, 40, 80, 160, 300, and 400 μM) [31].

## *2.10. Statistical Analysis*

IBM SPSS 26.0 software (IBM Corporation. Armonk, NY, USA) was used for statistical analysis between groups. The data are expressed as the mean ± standard deviation (*n* = 3).


**1. Introduction**

240 Type 1 Diabetes – Complications, Pathogenesis, and Alternative Treatments

Zhang, W., Feng, D., Li, Y., Iida, K., McGrath, B. & Cavener, D.R. (2006). Perk Eif2ak3

Zinszner, H., Kuroda, M., Wang, X., Batchvarova, N., Lightfoot, R.T., Remotti, H., Stevens,

No.11, (Jun), pp.(3864-3874), 0270-7306

4131

pp.(982-995), 0890-9369

Alpha Kinase Is Required for the Development of the Skeletal System, Postnatal Growth, and the Function and Viability of the Pancreas. *Mol Cell Biol,* Vol. 22,

Control of Pancreatic Beta Cell Differentiation and Proliferation Is Required for Postnatal Glucose Homeostasis. *Cell Metab,* Vol. 4, No.6, (Dec), pp.(491-497), 1550-

J.L. & Ron, D. (1998). Chop Is Implicated in Programmed Cell Death in Response to Impaired Function of the Endoplasmic Reticulum. *Genes Dev,* Vol. 12, No.7, (Apr 1),

> L-Arginine is synthesised *in vivo* from L-glutamine, L-glutamate, or L-proline via the intestinal-renal axis (**Fig. 1A**) in humans and most other mammals (Wu et al., 2009). In humans, plasma L-glutamine is the precursor of 80% of plasma L-citrulline while plasma L-citrulline, in turn, is the precursor of 10% of plasma L-arginine (van de Poll et al., 2007). Although the intestine consumes L-glutamine at a high rates, dependent on L-glutamine supply (and production from the skeletal muscle), approximately 13% of L-glutamine taken up by the intestine is converted to L-citrulline, so that quantitatively, L-glutamine is the major precursor for intestinal release of L-citrulline (van de Poll et al., 2007), which can be further converted to L-arginine. These observations highlight the importance of L-arginine/L-glutamine metabolic coupling, especially as L-arginine is one of the most potent secretagogues of insulin from the pancreatic beta cells (Palmer et al., 1976), whereas L-arginine deficiency is associated with insulinopenia and failure to secrete insulin in response to glucose (Spinas et al., 1999). L-Arginine is essential for metabolism and function of multiple body organs, with decreased plasma and cellular levels of L-arginine reported in type 2 diabetic subjects (Pieper & Dondlinger, 1997).

> Since L-arginine is the precursor of nitric oxide (NO)\*, which serves as a key cell signalling molecule in pancreatic islet -cells, restriction in the availability of L-arginine is likely to

<sup>\*</sup> **Abbreviations used:** CAT, catalase; GSH, glutathione; GSSG, glutathione disulphide; GSPx, glutathione peroxidase; GSRd, glutathione disulphide reductase; HSP70, 70-kDa member of heat shock protein family; eHSP70, extracellular heat shock protein of 70 kDa; IFN-, interferon-; IB, a member of the inhibitors of nuclear factor B; IKK, inhibitor of B kinase; IL-1, interleukin-1; IL1-ra, IL-1 receptor antagonist; iNOS, inducible nitric oxide synthase; NF-B, a member of nuclear transcription factor B; NO, nitric oxide free radical ( N=O); PPAR-, peroxisome proliferator activated receptor-; RNS, reactive nitrogen species; ROS, reactive oxygen species; SNOG, *S-*nitrosoglutathione; SOD,

A Novel L-Arginine/L-Glutamine Coupling Hypothesis: Implications for Type 1 Diabetes 243

mechanical activity, where blood glutamine stores may be challenged. Metabolic acidosis, by increasing L-glutamine utilisation by the kidney, may also favour glutamine depletion unless enteral supplementation or enhanced physical activity takes place. This is also the case of psychological-stress motivated inflammatory reactions that may underlie by the activation of sympathetic-CRH-histamine system (**Fig. 3**), which ultimately leads to a Th1 centered immune response that augments glutamine utilisation. Therefore, L-glutamine imbalance, by virtue of deficiently supplying L-arginine to the pancreas, deviates -cell glutamate metabolism from the synthesis of GSH to that of NO, leading to oxidative stress, impairment of insulin release and insulitis. This ongoing inflammation feeds forward NO metabolism, which enhances L-glutamine consumption thus perpetuating this cyclic condition that leads to type 1 diabetes mellitus (T1DM) **(B).** Physical exercise, on the other hand, may improve L-glutamine supply from the skeletal muscle and

counteract Th1-mediated inflammation due to the production of type 2 cytokines, such as

production and other anti-inflammatory mediators. Arrow widths indicate the intensity of

IL-6. Immunomodulatory action of exercise may also involve heat shock protein

the metabolic flux through each pathway.

Fig. 1. The l-arginine/l-glutamine coupling hypothesis of insulin-secreting -cells. (A) Pancreatic islet -cells utilise l-arginine for the biosynthesis of NO and l-glutamate for GSH generation during secretagogue-stimulated insulin secretion. l-Arginine is provided to the pancreas by the intestinal-renal axis from l-glutamine, while l-glutamate is furnished by the liver mainly from muscle-derived alanine. In the -cell, NH4+ may contribute to l-arginine biosynthesis, through the concerted action of carbamoyl phosphate synthetase I (CPS), ornithine transcarbamoylase (OTC), argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL) that eventually produces l-arginine. Skeletal muscle-derived l-glutamine is also substrate for the maintenance of GSH metabolism in -cells, but rapidlyproliferating cells of the gut as well as immune cells compete with -cell for the utilisation of l-glutamine. Hence, any minimal reduction in the supply of l-arginine to the pancreas may shift l-glutamate metabolism towards the synthesis of NO instead of GSH, thus leading to oxidative stress, inhibition of insulin secretion and eventually -cell death. This is the case of undernourishment, cancer states, trauma, sepsis, major burns and low skeletal muscle

superoxide dismutase; TBARS, thiobarbituric acid-reactive substances; TNF-, tumor necrosis factor-; TNFR, TNF- receptor; UCP, uncoupling protein-2.

Fig. 1. The l-arginine/l-glutamine coupling hypothesis of insulin-secreting -cells. (A) Pancreatic islet -cells utilise l-arginine for the biosynthesis of NO and l-glutamate for GSH generation during secretagogue-stimulated insulin secretion. l-Arginine is provided to the pancreas by the intestinal-renal axis from l-glutamine, while l-glutamate is furnished by the liver mainly from muscle-derived alanine. In the -cell, NH4+ may contribute to l-arginine biosynthesis, through the concerted action of carbamoyl phosphate synthetase I (CPS),

argininosuccinate lyase (ASL) that eventually produces l-arginine. Skeletal muscle-derived l-glutamine is also substrate for the maintenance of GSH metabolism in -cells, but rapidlyproliferating cells of the gut as well as immune cells compete with -cell for the utilisation of l-glutamine. Hence, any minimal reduction in the supply of l-arginine to the pancreas may shift l-glutamate metabolism towards the synthesis of NO instead of GSH, thus leading to oxidative stress, inhibition of insulin secretion and eventually -cell death. This is the case of undernourishment, cancer states, trauma, sepsis, major burns and low skeletal muscle

superoxide dismutase; TBARS, thiobarbituric acid-reactive substances; TNF-, tumor necrosis factor-;

ornithine transcarbamoylase (OTC), argininosuccinate synthetase (ASS) and

TNFR, TNF- receptor; UCP, uncoupling protein-2.

mechanical activity, where blood glutamine stores may be challenged. Metabolic acidosis, by increasing L-glutamine utilisation by the kidney, may also favour glutamine depletion unless enteral supplementation or enhanced physical activity takes place. This is also the case of psychological-stress motivated inflammatory reactions that may underlie by the activation of sympathetic-CRH-histamine system (**Fig. 3**), which ultimately leads to a Th1 centered immune response that augments glutamine utilisation. Therefore, L-glutamine imbalance, by virtue of deficiently supplying L-arginine to the pancreas, deviates -cell glutamate metabolism from the synthesis of GSH to that of NO, leading to oxidative stress, impairment of insulin release and insulitis. This ongoing inflammation feeds forward NO metabolism, which enhances L-glutamine consumption thus perpetuating this cyclic condition that leads to type 1 diabetes mellitus (T1DM) **(B).** Physical exercise, on the other hand, may improve L-glutamine supply from the skeletal muscle and counteract Th1-mediated inflammation due to the production of type 2 cytokines, such as IL-6. Immunomodulatory action of exercise may also involve heat shock protein production and other anti-inflammatory mediators. Arrow widths indicate the intensity of the metabolic flux through each pathway.

A Novel L-Arginine/L-Glutamine Coupling Hypothesis: Implications for Type 1 Diabetes 245

Oxidative stress has long been recognised to play an important role in the development of type 1 diabetes and its subsequent complications (Wierusz-Wysock et al., 1997) which are aggravated due to the low activities of oxygen free radical scavenging enzymes in islet cells, especially mitochondrial manganese-type superoxide dismutase (Mn-SOD; Asayama et al., 1986), glutathione peroxidase (GSPx; Malaisse et al., 1982; Mathews & Leiter, 1999) and glutathione disulphide (GSSG) reductase (GSRd; Mathews & Leiter, 1999). Also, the expression of mRNA encoding for several antioxidant enzymes, such as Mn-SOD, cytoplasmic copper-zinc type SOD (Cu/Zn-SOD), GSPx, and catalase (CAT), has been reported to be lower in islets of Langerhans compared with other mouse tissues (Lenzen et al., 1996). Additionally, the administration of antioxidants (nicotinamide, SOD, -tocopherol, probucol and the 21-aminosteroid lazaroids), as well as oxygen free radical scavengers, have been used in vitro to protect islets from the cytotoxic effects of some proinflammatory cytokines (IL-1, TNF and INF), concurrently providing *in vivo* protection against the development of the autoimmune diabetes process (Nomikos et al., 1986). Conversely, studies on MnSOD and CAT transgenics have shown that protection of islets from oxidative stress does not alter cytokine toxicity (Chen et al., 2005), which indicates that, although related to each other, oxidative stress and cytokine-induced islet toxicity may use

An additional complication to this scenario is the fact that -cells express mitochondrial uncoupling protein 2 (UCP2) which dissipates the coupling between electron transport from ATP formation favouring O2- generation. Since O2- anion is a powerful activator of UCP2, a positive feedback mechanism exists in that O2- generation enhances its own formation. This is particularly critical under prolonged hyperglycaemia, where UCP2 activity may be extremely high thus further depressing insulin secretion by -cells (Newsholme et al., 2007). This situation is probably associated with the development of type 2 diabetes. Furthermore, the high-glucose, high fatty-acid environment created by either insulin-deficiency or insulinresistance favours the expression of NAD(P)H oxidase with consequently enhanced ROS

Type 1 diabetic patients exhibit major defects in antioxidant protection compared with healthy, non-diabetic controls. A significant reduction in total antioxidant status in both plasma and serum samples from these patients is typically observed (Maxwell et al., 1997). Diabetic children show significant reduction in GSH and GSPx in erythrocytes, as well as in plasma -tocopherol and -carotene levels (Dominguez et al., 1998). Incubation of rat (Rabinovitch et al., 1992) and human (Rabinovitch et al., 1996) islet cells with a cytotoxic combination of cytokines (IL-1, TNF and IFN) has been reported as an inducing factor for lipid peroxidation (also known as lipoperoxidation). When individually administered, however, the same cytokines have been shown to inhibit insulin release without any increase in lipid peroxidation or cytodestructive effects in rat islets (Sumoski et al., 1989). Taken together, these findings suggest that cytokine-induced inhibition of insulin release may not be oxygen free radical-mediated, whereas the cytodestructive effects of cytokines on -cells do appear to involve free radical-mediated events that induce the formation of toxic derivatives within the islets of Langerhans (Suarez-Pinz et al., 1996). This strongly suggests that type 1 cytokines interfere in -cell metabolism at some point that is intimately related to insulin secretion. But where does reside this extreme sensitivity of -cells to cytokine signals? The expression of iNOS, necessary for the synthesis of NO during insulin

production and -cell death (Morgan et al., 2007, Newsholme et al., 2009b).

specific and diverse pathways to induce -cell death.

secretion, may provide an explanation.

contribute to derangements in the secretion and action of insulin (Newsholme et al., 2009a). Hypertension associated with diabetes is related with a decrease in levels of L-arginine (Spinas, 1999), as are inflammatory conditions characterised by release of L-arginase by activated macrophages (Murphy & Newsholme, 1998). While excessive NO production can trigger oxidative/nitrosative stress and is undoubtedly a key mechanism that results in cell death (Newsholme et al., 2009a; Spinas, 1999; Michalska et al., 2010), good evidence now suggests that lesser amounts of cellular NO, produced by the NF-B-regulated inducible nitric oxide synthase (iNOS, EC <IP_ADDRESS>), encoded by the *NOS-2* gene, serves as an important coupling factor in insulin secreting cells (Newsholme et al., 2009a; Spinas, 1999; Michalska et al., 2010). Recent data from the authors' laboratories has demonstrated that L-arginine is an important stimulator of -cell glucose consumption and intermediary metabolism (M.S. Krause, N.H. McClenaghan, P.R. Flatt, P.I. Homem de Bittencourt Jr., C. Murphy & P. Newsholme, unpublished results). Such actions lead to increased insulin secretion, enhanced antioxidant and protective responses with greater functional integrity when challenged with pro-inflammatory cytokines. Given that insulin-secreting cells have very low expression levels of antioxidant enzymes, such as catalase (CAT) and glutathione peroxidase (GSPx), -cells are particularly prone to chemical stress in the diabetogenic or inflammatory environment typical of type 1 and possibly type 2 diabetes (Newsholme et al., 2009a; Spinas, 1999). In fact, the pathogenesis of type 2 diabetes is now recognised to involve both innate and adaptive immunity, since type 2 diabetes is associated with low-grade systemic inflammation, infiltration of adipose tissue and pancreatic islets with CD8+ T lymphocytes that precede invasion by inflammatory macrophages and activation of these cells resulting in pro-inflammatory cytokine secretion (Mandrup-Poulsen, 2010).

In this chapter, we discuss how the continued supply of L-arginine, physiologically provided by the metabolism of L-glutamine via the intestinal-renal axis and from active skeletal muscle (which will be enhanced during exercise) is essential for -cell functional integrity and indeed for -cell defence, which will be required to avoid/attenuate islet inflammation associated with the pathogenic mechanisms underlying type 1 and type 2 diabetes (**Fig. 1B**). L-arginine is therefore preserved for essential NO generation and stimulation of glucose metabolism, critical for insulin secretion. Additionally, the role of skeletal muscle (during exercise) on these metabolic processes is discussed.

#### **2. Oxidative metabolism and oxidative stress in -cells and type 1 diabetes**

The intense aerobic metabolism, intrinsic to pancreatic -cells, exposes these cells to the deleterious effects of high-turnover oxygen-based reactions. In fact, during secretagoguestimulated insulin secretion, -cells are associated with accelerated mitochondrial flux of electrons and, consequently, elevated tendency towards reactive oxygen species (ROS) production (Newsholme et al., 2007). However and notably, -cells present a very low level of expression of antioxidant enzymes such as CAT and GSPx compared with other tissues and this reduced antioxidant activity is associated with significant increases in lipid hydroperoxides, conjugated dienes and protein carbonyls, which are markers for oxidative stress (Santini et al., 1997), so that -cells are intrinsically prone to oxidative stress.

Moreover, a growing body of evidence indicates that, in the pre-diabetic condition, antioxidant status may be impaired (Rocie et al., 1997). Hence, the low antioxidant defence in certain individuals (even if transiently) may predispose to an enhanced oxidative stress and the eventual -cell death that categorises the onset of type 1 and type 2 diabetes.

contribute to derangements in the secretion and action of insulin (Newsholme et al., 2009a). Hypertension associated with diabetes is related with a decrease in levels of L-arginine (Spinas, 1999), as are inflammatory conditions characterised by release of L-arginase by activated macrophages (Murphy & Newsholme, 1998). While excessive NO production can trigger oxidative/nitrosative stress and is undoubtedly a key mechanism that results in cell death (Newsholme et al., 2009a; Spinas, 1999; Michalska et al., 2010), good evidence now suggests that lesser amounts of cellular NO, produced by the NF-B-regulated inducible nitric oxide synthase (iNOS, EC <IP_ADDRESS>), encoded by the *NOS-2* gene, serves as an important coupling factor in insulin secreting cells (Newsholme et al., 2009a; Spinas, 1999; Michalska et al., 2010). Recent data from the authors' laboratories has demonstrated that L-arginine is an important stimulator of -cell glucose consumption and intermediary metabolism (M.S. Krause, N.H. McClenaghan, P.R. Flatt, P.I. Homem de Bittencourt Jr., C. Murphy & P. Newsholme, unpublished results). Such actions lead to increased insulin secretion, enhanced antioxidant and protective responses with greater functional integrity when challenged with pro-inflammatory cytokines. Given that insulin-secreting cells have very low expression levels of antioxidant enzymes, such as catalase (CAT) and glutathione peroxidase (GSPx), -cells are particularly prone to chemical stress in the diabetogenic or inflammatory environment typical of type 1 and possibly type 2 diabetes (Newsholme et al., 2009a; Spinas, 1999). In fact, the pathogenesis of type 2 diabetes is now recognised to involve both innate and adaptive immunity, since type 2 diabetes is associated with low-grade systemic inflammation, infiltration of adipose tissue and pancreatic islets with CD8+ T lymphocytes that precede invasion by inflammatory macrophages and activation of these

cells resulting in pro-inflammatory cytokine secretion (Mandrup-Poulsen, 2010).

skeletal muscle (during exercise) on these metabolic processes is discussed.

In this chapter, we discuss how the continued supply of L-arginine, physiologically provided by the metabolism of L-glutamine via the intestinal-renal axis and from active skeletal muscle (which will be enhanced during exercise) is essential for -cell functional integrity and indeed for -cell defence, which will be required to avoid/attenuate islet inflammation associated with the pathogenic mechanisms underlying type 1 and type 2 diabetes (**Fig. 1B**). L-arginine is therefore preserved for essential NO generation and stimulation of glucose metabolism, critical for insulin secretion. Additionally, the role of

**2. Oxidative metabolism and oxidative stress in -cells and type 1 diabetes**

stress (Santini et al., 1997), so that -cells are intrinsically prone to oxidative stress.

and the eventual -cell death that categorises the onset of type 1 and type 2 diabetes.

The intense aerobic metabolism, intrinsic to pancreatic -cells, exposes these cells to the deleterious effects of high-turnover oxygen-based reactions. In fact, during secretagoguestimulated insulin secretion, -cells are associated with accelerated mitochondrial flux of electrons and, consequently, elevated tendency towards reactive oxygen species (ROS) production (Newsholme et al., 2007). However and notably, -cells present a very low level of expression of antioxidant enzymes such as CAT and GSPx compared with other tissues and this reduced antioxidant activity is associated with significant increases in lipid hydroperoxides, conjugated dienes and protein carbonyls, which are markers for oxidative

Moreover, a growing body of evidence indicates that, in the pre-diabetic condition, antioxidant status may be impaired (Rocie et al., 1997). Hence, the low antioxidant defence in certain individuals (even if transiently) may predispose to an enhanced oxidative stress Oxidative stress has long been recognised to play an important role in the development of type 1 diabetes and its subsequent complications (Wierusz-Wysock et al., 1997) which are aggravated due to the low activities of oxygen free radical scavenging enzymes in islet cells, especially mitochondrial manganese-type superoxide dismutase (Mn-SOD; Asayama et al., 1986), glutathione peroxidase (GSPx; Malaisse et al., 1982; Mathews & Leiter, 1999) and glutathione disulphide (GSSG) reductase (GSRd; Mathews & Leiter, 1999). Also, the expression of mRNA encoding for several antioxidant enzymes, such as Mn-SOD, cytoplasmic copper-zinc type SOD (Cu/Zn-SOD), GSPx, and catalase (CAT), has been reported to be lower in islets of Langerhans compared with other mouse tissues (Lenzen et al., 1996). Additionally, the administration of antioxidants (nicotinamide, SOD, -tocopherol, probucol and the 21-aminosteroid lazaroids), as well as oxygen free radical scavengers, have been used in vitro to protect islets from the cytotoxic effects of some proinflammatory cytokines (IL-1, TNF and INF), concurrently providing *in vivo* protection against the development of the autoimmune diabetes process (Nomikos et al., 1986). Conversely, studies on MnSOD and CAT transgenics have shown that protection of islets from oxidative stress does not alter cytokine toxicity (Chen et al., 2005), which indicates that, although related to each other, oxidative stress and cytokine-induced islet toxicity may use specific and diverse pathways to induce -cell death.

An additional complication to this scenario is the fact that -cells express mitochondrial uncoupling protein 2 (UCP2) which dissipates the coupling between electron transport from ATP formation favouring O2- generation. Since O2- anion is a powerful activator of UCP2, a positive feedback mechanism exists in that O2- generation enhances its own formation. This is particularly critical under prolonged hyperglycaemia, where UCP2 activity may be extremely high thus further depressing insulin secretion by -cells (Newsholme et al., 2007). This situation is probably associated with the development of type 2 diabetes. Furthermore, the high-glucose, high fatty-acid environment created by either insulin-deficiency or insulinresistance favours the expression of NAD(P)H oxidase with consequently enhanced ROS production and -cell death (Morgan et al., 2007, Newsholme et al., 2009b).

Type 1 diabetic patients exhibit major defects in antioxidant protection compared with healthy, non-diabetic controls. A significant reduction in total antioxidant status in both plasma and serum samples from these patients is typically observed (Maxwell et al., 1997). Diabetic children show significant reduction in GSH and GSPx in erythrocytes, as well as in plasma -tocopherol and -carotene levels (Dominguez et al., 1998). Incubation of rat (Rabinovitch et al., 1992) and human (Rabinovitch et al., 1996) islet cells with a cytotoxic combination of cytokines (IL-1, TNF and IFN) has been reported as an inducing factor for lipid peroxidation (also known as lipoperoxidation). When individually administered, however, the same cytokines have been shown to inhibit insulin release without any increase in lipid peroxidation or cytodestructive effects in rat islets (Sumoski et al., 1989). Taken together, these findings suggest that cytokine-induced inhibition of insulin release may not be oxygen free radical-mediated, whereas the cytodestructive effects of cytokines on -cells do appear to involve free radical-mediated events that induce the formation of toxic derivatives within the islets of Langerhans (Suarez-Pinz et al., 1996). This strongly suggests that type 1 cytokines interfere in -cell metabolism at some point that is intimately related to insulin secretion. But where does reside this extreme sensitivity of -cells to cytokine signals? The expression of iNOS, necessary for the synthesis of NO during insulin secretion, may provide an explanation.

A Novel L-Arginine/L-Glutamine Coupling Hypothesis: Implications for Type 1 Diabetes 247

enzymic activities by the suppressors of cytokine signalling (SOCS) family. Upregulation of either SOCS-1 or SOCS-3 protects -cells against cytokine-induced cell death in vitro and in vivo (Karlsen et al., 2001; Flodstrom et al., 2003). SOCS-3 also protects insulin-producing cells against IL-1–mediated apoptosis via NF-B inhibition (Karlsen et al., 2004). Evidence indicates that the fate of -cells, after cytokine exposure, depends on the duration and

Besides its activation by cytokines, NF-B is also a potential target for reactive oxygen/nitrogen species (ROS/RNS). It is noteworthy that NF-B was the first redoxsensitive eukaryotic transcription factor shown to respond directly to oxidative stress in many types of cells (Dröge, 2002), while its activation leads to the expression of at least a hundred of inducible proteins directly involved in inflammation, such as cyclooxygenase-2 (COX-2), iNOS, TNF and IL-1 (Moynagh, 2005). Therefore, NF-B is, at the same time, both a target and an inducer of inflammation and inflammation-induced oxidative stress. In resting (unstimulated) cells, NF-B dimeric complexes are predominantly found in the cytosol where they are associated with members of the inhibitory IB family (Moynagh, 2005), so that NF-B gene products are entirely inducible proteins whose activation is dictated by specific stimuli that activate IB kinase (IKK) complexes. These stimuli include high intracellular GSSG levels and oxidative stress *per se* (Dröge, 2002). IKKs, in turn, phosphorylate IB proteins directing them to proteasome-mediated degradation, which sets

NF-B activation is responsible for both initiation and amplification of immune and inflammatory responses in all cells. Actually, NF-B activation is *sine qua non* for the control of immune and inflammatory responses (Baldwin, 1996; Nakamura et al., 1997; Winyard et al., 1997), and since inflammatory factors, such as pro-inflammatory cytokines, chemokines, adhesion molecules, colony-stimulating factors and inflammatory enzymes, are NF-Bdependent gene products, dysregulation or aberrant activation of NF-B could initiate inappropriate autoimmune and inflammatory responses. Conversely, inhibition of NF-B activation has been argued as a potential therapeutic approach in several immune and inflammatory-related diseases (Chen et al., 1999). This is why cyclopentenone prostaglandins (cp-PGs), which are powerful inhibitors of NF-B activation (Rossi et al., 2000), are now considered to be the physiological mediators of the "**resolution of inflammation**" (Piva et al., 2005), whereas cp-PG-based pharmacological approaches, *e.g.* LipoCardium technology, which is a liposome contained cp-PG-based formulation specifically directed towards atherosclerotic lesions in arterial walls (Homem de Bittencourt et al., 2007; Gutierrez et al., 2008) have proved to be powerful anti-atherosclerotic strategies

Finally, considering that all the known forms of inflammation finish with the formation of naturally-occurring anti-inflammatory agents (e.g. cp-PGs, IL-10), an important question remains as to how does -cell not resolve inflammation by triggering such responses? A fault in the expression of the anti-inflammatory heat shock proteins may give a clue to this

Heat shock proteins (HSPs) have been found to play a fundamental role in the recovery from multiple stress conditions and to offer protection from subsequent insults (De Maio,

severity of perturbation of key -cell gene networks.

NF-B dimers free to bind to DNA in the nucleus.

(Piva et al., 2005; Ianaro et al., 2003; Homem de Bittencourt Jr., 2007).

question.

**4. Heat shock protein pathways**

NO has incontestably been shown to be a physiological regulator of insulin secretion in -cells, in an elegant experimental protocol designed by Prof. Anne Marie Salapatek's group in Canada and reported in a seminal paper (Smukler et al., 2002). They have also reported that endogenous NO production can be stimulated by glucose, and that this stimulation can be blocked by NOS inhibition, whereas scavenging of NO specifically blocks insulin release stimulated by physiological intracellular concentrations of NO-donors (2 mM), but has no effect on the release stimulated by elevated K+. It has also been reported that NO donation did not elicit a -cell intracellular Ca2+ ([Ca2+]i) response alone, but was able to potentiate a glucose-induced [Ca2+]i response. Since NO is a strong heme-reactant, it partially inhibits the mitochondrial respiratory chain by binding to cytochrome *c* and/or cytochrome oxidase. As a consequence, the mitochondrial membrane potential decreases and Ca2+ leaves the mitochondria. This is followed by restoration of the mitochondrial membrane potential and Ca2+ reuptake by mitochondria (Spinas, 1999). Therefore, overproduction of NO related to inflammatory stimuli may be related to cellular dysfunction but **not** normal levels of NO. As previously argued (Smukler et al., 2002), the precise level of NO is crucial in determining its resultant effect, with low levels being involved in physiological signalling and higher levels becoming cytotoxic (Moncada et al., 1991; Beck et al., 1999). Hence, the supraphysiological elevation of L-arginine, or the application of exogenous NO donors under the condition of already elevated NO, may result in excessive NO production, yielding cytotoxic effects (Smukler et al., 2002).

#### **3. Nuclear factor B-dependent L-arginine metabolism in -cells**

Pancreatic -cells have to constantly express NF-B-regulated iNOS in order to achieve appropriate amounts of NO produced from L-arginine. However, inflammatory cytokines, such as IL-1 and TNF-, activate NF-B in rodent and human islet cells (Eizirik & Mandrup-Poulsen, 2001). Contrarily, prevention of NF-B activation protects pancreatic -cells against cytokine-induced apoptosis (Giannoukakis et al., 2000; Heimberg et al., 2001). It is impressive that about 70 NF-B–dependent genes have been currently identified in cells, including genes encoding for various inflammatory cytokines and iNOS (Darville & Eizirik, 1998). Remarkably, the expression of *ca.* 50% of the -cell genes that may be modified after cytokine exposure is secondary to iNOS-mediated NO formation (Kutlu et al., 2003). It is of note that treatment of human, as well as rodent -cells with purified IL-1 alone is not sufficient to induce apoptosis, but if IL-1 is combined with interferon- (IFN), -cells undergo apoptosis after few days in culture (Eizirik & Mandrup-Pouls, 2001). This suggests that an intracellular IFN signal must synergise with IL-1 signalling pathways in order to trigger -cell apoptosis. IFN binds to cell surface receptors and activates the Janus tyrosine kinases JAK1 and JAK2. These kinases phosphorylate and activate their downstream transcription factor STAT-1 (for signal transducers and activators of transcription), which dimerises and translocates to the nucleus where binding to -activated sites on target genes occurs (Eizirik & Mandrup-Pouls, 2001). STAT-1 mediates the potentiating effect of IFN on IL-1-induced iNOS expression (Darville & Eizirik, 1998). Because excessive activation of JAK/STAT signalling may lead to cell death, STAT transcriptional activity is regulated by multiple negative feedback mechanisms. These include dephosphorylation of JAK and cytokine receptors by cytoplasmic protein-tyrosine phosphatases SHPs (for Src homology 2 domain phosphatases), and inhibition of JAK

NO has incontestably been shown to be a physiological regulator of insulin secretion in -cells, in an elegant experimental protocol designed by Prof. Anne Marie Salapatek's group in Canada and reported in a seminal paper (Smukler et al., 2002). They have also reported that endogenous NO production can be stimulated by glucose, and that this stimulation can be blocked by NOS inhibition, whereas scavenging of NO specifically blocks insulin release stimulated by physiological intracellular concentrations of NO-donors (2 mM), but has no effect on the release stimulated by elevated K+. It has also been reported that NO donation did not elicit a -cell intracellular Ca2+ ([Ca2+]i) response alone, but was able to potentiate a glucose-induced [Ca2+]i response. Since NO is a strong heme-reactant, it partially inhibits the mitochondrial respiratory chain by binding to cytochrome *c* and/or cytochrome oxidase. As a consequence, the mitochondrial membrane potential decreases and Ca2+ leaves the mitochondria. This is followed by restoration of the mitochondrial membrane potential and Ca2+ reuptake by mitochondria (Spinas, 1999). Therefore, overproduction of NO related to inflammatory stimuli may be related to cellular dysfunction but **not** normal levels of NO. As previously argued (Smukler et al., 2002), the precise level of NO is crucial in determining its resultant effect, with low levels being involved in physiological signalling and higher levels becoming cytotoxic (Moncada et al., 1991; Beck et al., 1999). Hence, the supraphysiological elevation of L-arginine, or the application of exogenous NO donors under the condition of already elevated NO, may result in excessive NO production, yielding cytotoxic effects

**3. Nuclear factor B-dependent L-arginine metabolism in -cells**

Pancreatic -cells have to constantly express NF-B-regulated iNOS in order to achieve appropriate amounts of NO produced from L-arginine. However, inflammatory cytokines, such as IL-1 and TNF-, activate NF-B in rodent and human islet cells (Eizirik & Mandrup-Poulsen, 2001). Contrarily, prevention of NF-B activation protects pancreatic -cells against cytokine-induced apoptosis (Giannoukakis et al., 2000; Heimberg et al., 2001). It is impressive that about 70 NF-B–dependent genes have been currently identified in cells, including genes encoding for various inflammatory cytokines and iNOS (Darville & Eizirik, 1998). Remarkably, the expression of *ca.* 50% of the -cell genes that may be modified after cytokine exposure is secondary to iNOS-mediated NO formation (Kutlu et al., 2003). It is of note that treatment of human, as well as rodent -cells with purified IL-1 alone is not sufficient to induce apoptosis, but if IL-1 is combined with interferon- (IFN), -cells undergo apoptosis after few days in culture (Eizirik & Mandrup-Pouls, 2001). This suggests that an intracellular IFN signal must synergise with IL-1 signalling pathways in order to trigger -cell apoptosis. IFN binds to cell surface receptors and activates the Janus tyrosine kinases JAK1 and JAK2. These kinases phosphorylate and activate their downstream transcription factor STAT-1 (for signal transducers and activators of transcription), which dimerises and translocates to the nucleus where binding to -activated sites on target genes occurs (Eizirik & Mandrup-Pouls, 2001). STAT-1 mediates the potentiating effect of IFN on IL-1-induced iNOS expression (Darville & Eizirik, 1998). Because excessive activation of JAK/STAT signalling may lead to cell death, STAT transcriptional activity is regulated by multiple negative feedback mechanisms. These include dephosphorylation of JAK and cytokine receptors by cytoplasmic protein-tyrosine phosphatases SHPs (for Src homology 2 domain phosphatases), and inhibition of JAK

(Smukler et al., 2002).

enzymic activities by the suppressors of cytokine signalling (SOCS) family. Upregulation of either SOCS-1 or SOCS-3 protects -cells against cytokine-induced cell death in vitro and in vivo (Karlsen et al., 2001; Flodstrom et al., 2003). SOCS-3 also protects insulin-producing cells against IL-1–mediated apoptosis via NF-B inhibition (Karlsen et al., 2004). Evidence indicates that the fate of -cells, after cytokine exposure, depends on the duration and severity of perturbation of key -cell gene networks.

Besides its activation by cytokines, NF-B is also a potential target for reactive oxygen/nitrogen species (ROS/RNS). It is noteworthy that NF-B was the first redoxsensitive eukaryotic transcription factor shown to respond directly to oxidative stress in many types of cells (Dröge, 2002), while its activation leads to the expression of at least a hundred of inducible proteins directly involved in inflammation, such as cyclooxygenase-2 (COX-2), iNOS, TNF and IL-1 (Moynagh, 2005). Therefore, NF-B is, at the same time, both a target and an inducer of inflammation and inflammation-induced oxidative stress. In resting (unstimulated) cells, NF-B dimeric complexes are predominantly found in the cytosol where they are associated with members of the inhibitory IB family (Moynagh, 2005), so that NF-B gene products are entirely inducible proteins whose activation is dictated by specific stimuli that activate IB kinase (IKK) complexes. These stimuli include high intracellular GSSG levels and oxidative stress *per se* (Dröge, 2002). IKKs, in turn, phosphorylate IB proteins directing them to proteasome-mediated degradation, which sets NF-B dimers free to bind to DNA in the nucleus.

NF-B activation is responsible for both initiation and amplification of immune and inflammatory responses in all cells. Actually, NF-B activation is *sine qua non* for the control of immune and inflammatory responses (Baldwin, 1996; Nakamura et al., 1997; Winyard et al., 1997), and since inflammatory factors, such as pro-inflammatory cytokines, chemokines, adhesion molecules, colony-stimulating factors and inflammatory enzymes, are NF-Bdependent gene products, dysregulation or aberrant activation of NF-B could initiate inappropriate autoimmune and inflammatory responses. Conversely, inhibition of NF-B activation has been argued as a potential therapeutic approach in several immune and inflammatory-related diseases (Chen et al., 1999). This is why cyclopentenone prostaglandins (cp-PGs), which are powerful inhibitors of NF-B activation (Rossi et al., 2000), are now considered to be the physiological mediators of the "**resolution of inflammation**" (Piva et al., 2005), whereas cp-PG-based pharmacological approaches, *e.g.* LipoCardium technology, which is a liposome contained cp-PG-based formulation specifically directed towards atherosclerotic lesions in arterial walls (Homem de Bittencourt et al., 2007; Gutierrez et al., 2008) have proved to be powerful anti-atherosclerotic strategies (Piva et al., 2005; Ianaro et al., 2003; Homem de Bittencourt Jr., 2007).

Finally, considering that all the known forms of inflammation finish with the formation of naturally-occurring anti-inflammatory agents (e.g. cp-PGs, IL-10), an important question remains as to how does -cell not resolve inflammation by triggering such responses? A fault in the expression of the anti-inflammatory heat shock proteins may give a clue to this question.


*3.3. Survival Analysis*

Initial cMRI evaluation was performed. During a median follow-up of 17 months (IQR 1 to 29 months), 31 patients (17.4%) experienced MACEs: VA (*n* = 14), HF requiring hospitalization (*n* = 11), and SCD (*n* = 6). The patients with VA were majority males, had increased LVESV (mean 107,533 mL, *p* < 0.001), decreased LVEF (28,429%, *p* < 0.0001), increased LGE mass (24,5 g, *p* < 0.0001) and LVSI (0.46, *p* < 0.001). Of them, ten received ICD therapy and 4 were ablated due to implantable cardioverter defibrillator therapy refusal, the last had a mean LVEF around 30%, two of them had NIDCM post-myocarditis and two were idiopathic NIDCM. The incidence of MACEs was significantly higher in the LGE+ group vs. the LGE− group (*n* = 21, 67.7% vs. *n* = 10, 33.4%). The Kaplan-Meier curves for event-free survival showed a significantly higher rate of MACEs in patients with LGE+ (HR = 4.02; 95%CI (1.91–8.45), *p* < 0.001), high LVSI (HR = 3.23; 95%CI (1.59–6.53), *p* < 0.01) and decreased LAS (HR = 3.94; 95%CI (1.93–8.03), *p* < 0.001) (Figure 3).


Preface

Oral health is an essential part of human health and wellbeing. Oral health issues not only affect basic abilities such as eating, speaking, and smiling, but also self-esteem, school or job performance, and the chance of obtaining employment.

Oral diseases have a high prevalence. According to the World Health Organization, nearly 100% of the adult population has at least one cavity, and 20% suffer from severe gum disease.

Oral health issues, ranging from cavities to oral cancer, cause pain and disability. Edentulism may be considered the ultimate stage of oral suffering. Most oral diseases are preventable and can be treated in the early stages. Preventive methods include proper oral hygiene, regular dental checkups and cleaning, a healthy diet, limited sugar intake, and no smoking. Treatment, depending on the oral health issue, involves using a wide range of materials and techniques. All the materials for dental use are supposed to be biocompatible, their characteristics and properties being strongly dependent on their purpose. The currently available dental materials are in constant development, and techniques such as lasers, microscopy, and 3D-bioprinting are becoming more complex. Nowadays, even edentulism is a condition that can be treated effectively by means of dental implants.

The relationship between oral and general health is obvious, for example, certain medications (painkillers, antihistamines, diuretics, antidepressants, etc.) may reduce saliva flow and lead to oral problems. Alternatively, various diseases and conditions, such as endocarditis, cardiovascular disease, and pneumonia, are linked to oral bacteria and inflammation. Diabetes, AIDS, osteoporosis, eating disorders, and Alzheimer's also have a distinct influence on oral condition.

Lack of appropriate health facilities and limited access to primary oral health services is quite common in certain geographical areas. High costs of dental treatment may represent a major barrier for large categories of people. Targeted health programs and specific approaches are attempted to overcome these issues.

This book, consisting of twenty chapters, focuses on different aspects of oral health issues, including prevention, treatment, and management.

#### **Lavinia Cosmina Ardelean**

Department of Technology of Materials and Devices in Dental Medicine, "Victor Babes" University of Medicine and Pharmacy Timisoara, Timișoara, Romania

> **Laura Cristina Rusu** Department of Oral Pathology, "Victor Babes" University of Medicine and Pharmacy Timisoara, Timișoara, Romania

Section 1


**Abstract**

*Pseudomonas aeruginosa* is an opportunistic Gram-negative bacterium accountable for causing life-threatening infections in humans. According to the World Health Organization, *P. aeruginosa* classified as a critical pathogen. Specifically, *P. aeruginosa* in its colonized or biofilm state presents a major infection threat to immunocompromised (HIV) patients, Cystic fibrosis, burns, wounds and surgery associated infection. It is also a common pathogen responsible for causing hospital acquired/ nosocomial infection and Urinary tract infections. *P. aeruginosa* biofilm is made up of bacterial self-synthesized biomolecules includes extracellular DNA, polysaccharides, proteins, RNA, siderophores and metabolites such as pyocyanin. This chapter will elaborate the manifold functions of *P. aeruginosa* secreted biomolecules in establishing and stabilizing biofilms, triggering virulence and pathogenicity in host, and resisting antibiotics and antibacterial agents.

**Keywords:** *Pseudomonas aeruginosa*, pyocyanin, extracellular DNA, biofilms, alginate, rhamnolipids, pyoverdine


**4.3 Galeazzi sign**

The patient is placed supine with the hips and knees bent and the feet resting in the plane of the examination table, observing the difference in height of the knees (**Figure 2**) [17]. The problem side is lowered compared to the opposite side.


**Abstract**

Although treatment of ischemic stroke focuses on re-establishing blood flow to the brain (e.g., thrombolysis), delayed reperfusion may be associated with oxidative damage to brain capillary endothelial cells, resulting in cerebral bleeding and death (hemorrhagic transformation). The goal of this study was to define cellular mechanisms responsible for reperfusion injury to brain capillaries, and to provide a rationale for more effective treatment of stroke. Mechanisms of oxidative injury to cerebral capillary endothelial cells were measured in the presence and absence of experimental inhibitors to define the roles of transport and metabolic pathways. *In vitro* experiments provided evidence that: (1) intracellular calcium is elevated in brain capillary endothelial cells following simulated transient ischemia and reperfusion, due to reverse movement of Na/Ca exchange; (2) a simultaneous increase of calcium and reactive oxygen species (ROS) during re-oxygenation causes mitochondrial dysfunction, thus initiating apoptosis and loss of brain capillary integrity. *In vivo* studies showed that γ-glutamylcysteine (an antioxidant precursor of glutathione) and the experimental compound KB-R7943 (inhibits reverse movement of Na/Ca exchange) protect brain capillary endothelial cells when co-administered just before reperfusion following transient ischemia. The data indicate that these agents may be useful in preventing oxidative injury associated with thrombolysis for ischemic stroke.

**Keywords:** glutathione, gamma-glutamylcysteine, antioxidant, stroke, thrombolysis, hemorrhagic transformation