Impaired mitochondria-dependent vasodilation in cerebral arteries of Zucker obese rats with insulin resistance

Abstract

Mitochondria affect cerebrovascular tone by activation of mitochondrial ATP-sensitive K+ (K ATP) channels and generation of reactive oxygen species (ROS). Insulin resistance accompanying obesity causes mitochondrial dysfunction, but the consequences on the cerebral circulation have not been fully identified. We evaluated the mitochondrial effects of diazoxide, a putative mitochondrial K ATP channel activator, on cerebral arteries of Zucker obese (ZO) rats with insulin resistance and lean (ZL) controls. Diameter measurements showed diminished diazoxide-induced vasodilation in ZO compared with ZL rats. Maximal relaxation was 38 +/- 3% in ZL vs. 21 +/- 4% in ZO rats (P < 0.05). Iberiotoxin, a Ca2+-activated K+ channel inhibitor, or manganese(III) tetrakis(4-benzoic acid)porphyrin chloride, an SOD mimetic, or endothelial denudation diminished vasodilation to diazoxide, implicating Ca2+-activated K+ channels, ROS, and endothelial factors in vasodilation. Inhibition of nitric oxide synthase (NOS) in ZL rats diminished diazoxide-induced vasodilation in intact arteries, but vasodilation was unaffected in endothelium-denuded arteries. In contrast, NOS inhibition in ZO rats enhanced vasodilation in endothelium-denuded arteries, but intact arteries were unaffected, suggesting that activity of endothelial NOS was abolished, whereas factors derived from nonendothelial NOS promoted vasoconstriction. Fluorescence microscopy showed decreased mitochondrial depolarization, ROS production, and nitric oxide generation in response to diazoxide in ZO arteries. Protein and mRNA measurements revealed increased expression of endothelial NOS and SODs in ZO arteries. Thus, cerebrovascular dilation to mitochondria-derived factors involves integration of endothelial and smooth muscle mechanisms. Furthermore, mitochondria-mediated vasodilation was diminished in ZO rats due to impaired mitochondrial K(ATP) channel activation, diminished mitochondrial ROS generation, increased ROS scavenging, and abnormal NOS activity.

Fig. 1.

Responses of cerebral arteries from Zucker obese (ZO) and Zucker lean (ZL) rats to diazoxide (10, 50 and 100 μmol/l). A: endothelium-intact and -denuded cerebral arteries preconstricted by myogenic tone. B: cerebral arteries from ZO and ZL rats preconstricted by serotonin. Values are means ± SE of 10–18 experiments. *Significantly different from lean endothelium-intact arteries (P < 0.05); in some cases, 2 points overlap (**). †Significantly different from obese endothelium-intact arteries (P < 0.05).

Fig. 2.

A: responses of cerebral arteries from ZL rats to diazoxide (10, 50, and 100 μmol/l) in the presence and absence of iberiotoxin (100 nmol/l), manganese(III) tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP, 100 μmol/l), or iberiotoxin + MnTBAP. B and C: diazoxide-induced vasodilation in endothelium-intact and -denuded arteries in the presence and absence of N^ω-nitro-l-arginine methyl ester (l-NAME, 100 μmol/l) and indomethacin (10 μmol/l). Baseline represents diazoxide response in endothelium-intact arteries in the absence of drugs. Values are means ± SE of 5–18 experiments. *Significantly different from lean endothelium-intact arteries (P < 0.05); in some cases, 3 points overlap (***).

Fig. 3.

A: responses of cerebral arteries from ZO rats to diazoxide (10, 50, and 100 μmol/l) in the presence and absence of iberiotoxin (100 nmol/l), MnTBAP (100 μmol/l), or iberiotoxin + MnTBAP. B and C: diazoxide-induced vasodilation in endothelium-intact and -denuded arteries in the presence and absence of l-NAME (100 μmol/l) and indomethacin (10 μmol/l). Baseline represents diazoxide response in endothelium-intact arteries in the absence of drugs. Values are means ± SE of 5–18 experiments. *Significantly different from baseline in obese endothelium-intact arteries (P < 0.05); in some cases, 2 points overlap (**). †Significantly different from obese endothelium-denuded arteries (P < 0.05); in some cases, 2 points overlap (††).

Fig. 4.

A: responses to 3-nitropropionic acid (3-NPA; 3, 30, and 300 μmol/l) in cerebral arteries from ZL and ZO rats. B: maximal responses in cerebral arteries from ZO and ZL rats to 3-NPA (300 μmol/l), diazoxide (100 μmol/l), and diazoxide + 3-NPA. Values are means ± SE of 9–18 experiments. *Significantly different from lean (P < 0.05).

Fig. 5.

A: responses to diazoxide (10, 50, and 100 μmol/l) in cerebral arteries from ZL and ZO rats in the presence and absence of apocynin (100 μmol/l). Values are means ± SE of 6–18 experiments. B: myogenic tone of isolated cerebral arteries from ZL and ZO arteries in response to increments in intraluminal pressure. Values are means ± SE of 9–12 experiments.

Fig. 6.

Membrane depolarization, reactive oxygen species (ROS) generation, and nitric oxide (NO) production in isolated pressurized cerebral arteries of ZO and ZL rats before and after diazoxide (100 μmol/l) administration.A: fluorescence images of the mitochondrial membrane potential-sensitive dye tetramethylrhodamine ethyl ester (TMRE) in ZL (top) and ZO (bottom) arteries in the absence and presence of diazoxide. B: TMRE fluorescence response to diazoxide. Reduction of diazoxide-induced decrease in TMRE fluorescence in ZO compared with ZL arteries indicates reduced mitochondrial depolarization. C: fluorescence images of the ROS sensitive dye hydroethidine (HEt) in ZL (top) and ZO (bottom) arteries following diazoxide administration. D: HEt fluorescence response to diazoxide compared with vehicle. E: fluorescence images of the NO-sensitive dye 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM) in ZL (top) and ZO (bottom) arteries in the absence and presence of diazoxide. F: DAF-FM fluorescence response to diazoxide compared with vehicle. Reduction of diazoxide-induced increase in HEt and DAF-FM fluorescence in ZO compared with ZL arteries indicates decreased generation of ROS and NO, respectively. *Significantly different from lean (P < 0.05).

Fig. 7.

A: immunoblots showing expression of endothelial NO synthase (eNOS), neuronal NO synthase (nNOS), Cu/Zn-SOD, Mn-SOD, and cytochrome c with matching β-actin blots. Protein samples of homogenates from rat cortex were used as positive control (+). L, lean; O, obese. B: cumulative group data. Cyt C, cytochrome c. C: mRNA expression levels of eNOS, cyclooxygenase-1 (COX1), COX2, Cu/Zn-SOD, and Mn-SOD normalized to GAPDH levels. *Significantly different from lean (P < 0.05).

Fig. 8.

Schematic representation of proposed mitochondrial regulation of vascular tone in cerebral arteries. ×, Potential sites of interruption in the pathways leading to impaired mitochondria-dependent vasodilation in ZO arteries; dashed arrows, pathways that have not been resolved. ER, endoplasmic reticulum; SR, sarcoplasmic reticulum; PGs, prostaglandins; K_ATP channels, ATP-sensitive K⁺ channels; IR, insulin-resistant; SDH, succinate dehydrogenase; [Ca²⁺], Ca²⁺ concentration.