James D. Mintz

Obesity Increases Blood Pressure, Cerebral Vascular Remodeling, and Severity of Stroke in the Zucker Rat

Obesity is a risk factor for stroke, but the mechanisms by which obesity increases stroke risk are unknown. Because microvascular architecture contributes to the outcome of stroke, we hypothesized that middle cerebral arteries (MCAs) from obese Zucker rats (OZRs) undergo inward remodeling and develop increased myogenic tone compared with those in lean Zucker rats (LZRs). We further hypothesized that OZRs have an increased infarct after cerebral ischemia and that changes in vascular structure and function correlate with the development of hypertension in OZRs. Blood pressure was measured by telemetry in LZRs and OZRs from 6 to 17 weeks of age. Vessel structure and function were assessed in isolated MCAs. Stroke damage was assessed after ischemia was induced for 60 minutes followed by 24 hours of reperfusion. Although mean arterial pressure was similar between young rats (6 to 8 weeks old), mean arterial pressure was higher in adult (14 to 17 weeks old) OZRs than in LZRs. MCAs from OZRs had a smaller lumen diameter and increased myogenic vasoconstriction compared with those from LZRs. After ischemia, infarction was 58% larger in OZRs than in LZRs. Before the development of hypertension, MCA myogenic reactivity and lumen diameter, as well as infarct size, were similar between young LZRs and OZRs. Our results indicate that the MCAs of OZRs undergo structural remodeling and that these rats have greater cerebral injury after cerebral ischemia. These cerebrovascular changes correlate with the development of hypertension and suggest that the increased blood pressure may be the major determinant for stroke risk in obese individuals.

Deletion of Protein Tyrosine Phosphatase 1b Improves Peripheral Insulin Resistance and Vascular Function in Obese, Leptin-Resistant Mice via Reduced Oxidant Tone

Rationale: Obesity is a risk factor for cardiovascular dysfunction, yet the underlying factors driving this impaired function remain poorly understood. Insulin resistance is a common pathology in obese patients and has been shown to impair vascular function. Whether insulin resistance or obesity, itself, is causal remains unclear. Objective: The present study tested the hypothesis that insulin resistance is the underlying mediator for impaired NO-mediated dilation in obesity by genetic deletion of the insulin-desensitizing enzyme protein tyrosine phosphatase (PTP)1B in db/db mice. Methods and Results: The db/db mouse is morbidly obese, insulin-resistant, and has tissue-specific elevation in PTP1B expression compared to lean controls. In db/db mice, PTP1B deletion improved glucose clearance, dyslipidemia, and insulin receptor signaling in muscle and fat. Hepatic insulin signaling in db/db mice was not improved by deletion of PTP1B, indicating specific amelioration of peripheral insulin resistance. Additionally, obese mice demonstrate an impaired endothelium dependent and independent vasodilation to acetylcholine and sodium nitroprusside, respectively. This impairment, which correlated with increased superoxide in the db/db mice, was corrected by superoxide scavenging. Increased superoxide production was associated with increased expression of NAD(P)H oxidase 1 and its molecular regulators, Noxo1 and Noxa1. Conclusions: Deletion of PTP1B improved both endothelium dependent and independent NO-mediated dilation and reduced superoxide generation in db/db mice. PTP1B deletion did not affect any vascular function in lean mice. Taken together, these data reveal a role for peripheral insulin resistance as the mediator of vascular dysfunction in obesity.

Impact of Leptin-Mediated Sympatho-Activation on Cardiovascular Function in Obese Mice

Although the anorexic effects of leptin are lost in obesity, leptin-mediated sympatho-activation is preserved. The cardiovascular consequences of leptin-mediated sympatho-activation in obesity are poorly understood. We tested the hypothesis that 32 weeks of high-fat diet (HFD) induces metabolic leptin resistance but preserves leptin-mediated sympatho-activation of the cardiovascular system. HFD in mice significantly increased body weight and plasma leptin concentrations but significantly reduced the anorexic effects of leptin. HFD increased heart rate, stroke volume, cardiac output, and plasma aldosterone levels but not blood pressure. As reflected by the contractile response to phenylephrine measured both in vivo and ex vivo, vascular adrenergic reactivity was reduced by HFD, suggesting that reductions in sympathetic tone to the periphery vasculature may mitigate sympatho-activation of the heart and the renin-angiotensin-aldosterone system. Tachyphylaxis was partially restored by symptho-inhibition and not present in ob/ob and db/db mice, despite obesity, arguing for a sympatho-mediated and leptin-specific mechanism. Although infusion of leptin in HFD mice had no effect on heart rate or blood pressure, it further increased aldosterone levels and further reduced vascular adrenergic tone in the absence of weight loss, indicating persistent leptin-mediated stimulation of the cardiovascular system in obesity. In conclusion, these data indicate that, despite metabolic leptin resistance, leptin-mediated stimulation of the heart, the vasculature, and aldosterone production persists in obesity. Blood pressure effects in response to leptin may be limited by a tachyphylactic response in the circulation, suggesting that failure of adrenergic desensitization may be a requisite step for hypertension in the context of obesity.

Protein tyrosine phosphatase 1B, a major regulator of leptin-mediated control of cardiovascular function.

Background— Obesity causes hypertension and sympathoactivation, a process proposed to be mediated by leptin. Protein tyrosine phosphatase 1B (PTP1B), a major new pharmaceutical target in the treatment of obesity and type II diabetes mellitus, constrains the metabolic actions of leptin, but the extent to which PTP1B regulates its cardiovascular effects is unclear. This study examined the hypothesis that PTP1B is a negative regulator of the cardiovascular effects of leptin. Methods and Results— PTP1B knockout mice had lower body fat but higher mean arterial pressure (116±5 versus 105±5 mm Hg, P<0.05) than controls. Leptin infusion produced a greater anorexic effect in PTP1B knockout mice and a marked increase in mean arterial pressure (135±5 mm Hg) in PTP1B knockout mice only. The decrease in mean arterial pressure in response to ganglionic blockade was higher in PTP1B knockout mice (−38±3% versus −29±3%, P<0.05), which suggests increased sympathetic tone. PTP1B deletion blunted mean arterial pressure responses to phenylephrine injection (55±10% versus 93±7%, P<0.05). Phenylephrine-induced aortic contraction was reduced in PTP1B knockout mice (57.7±9% versus 96.3±12% of KCl, P<0.05), consistent with desensitization to chronically elevated sympathetic tone. Furthermore, PTP1B deletion significantly reduced gene expression of 3 α1-adrenergic receptor subtypes, consistent with blunted constriction to phenylephrine. Conclusions— These data indicate that PTP1B is a key regulator of the cardiovascular effects of leptin and that reduced vascular adrenergic reactivity provides a compensatory limit to the effects of leptin on mean arterial pressure.

Exaggerated Cardiovascular Stress Responses and Impaired β-Adrenergic–Mediated Pressor Recovery in Obese Zucker Rats

Clinical studies have demonstrated that the pressor response to acute stress is larger in obese versus lean individuals. We therefore tested the hypotheses that the pressor response to behavioral stress is greater in obese (OZRs) versus lean Zucker rats (LZRs) and that reduced β-adrenergic–mediated vasodilation contributes to the enhanced pressor response. Animals were restrained and subjected to acute pulsatile air jet stress (3 minutes), followed by a poststress period of 20 minutes; β-adrenergic blockade was achieved with propranolol (5 mg/kg, IV) given 15 minutes before the start of air jet stress. Mean arterial pressure (MAP) was continuously monitored by telemetry. Untreated OZRs responded with a greater integrated pressor response (area under the curve [AUC]) to acute stress (41.2±6.1 versus 21.2±3.3 mm Hg×3 minutes, OZR versus LZR; P<0.05) and significantly reduced poststress recovery of MAP. β-Adrenergic blockade had no effect on stress AUC in either LZRs or OZRs but significantly attenuated the poststress recovery of MAP in LZRs only (poststress AUC: −100.1±48.1 versus 49.0±13.5 mm Hg×20 minutes, untreated versus propranolol; P<0.05). In anesthetized animals, significantly smaller increases in mesenteric vascular conductance contributed to blunted depressor responses to isoproterenol in OZRs versus LZRs, suggesting that β-adrenergic stimulation causes a greater reduction in total peripheral resistance in lean versus obese animals. We conclude that β-adrenergic–mediated vasodilation facilitates blood pressure recovery after stress and that this pathway is compromised in an animal model of morbid obesity, resulting in the impaired ability to regulate blood pressure during stress.

Pin1 Prolyl Isomerase Regulates Endothelial Nitric Oxide Synthase

Objective—The Pin1 prolyl isomerase acts in concert with proline-directed protein kinases to regulate function of protein substrates through isomerization of peptide bonds that link phosphoserine or phosphothreonine to proline. We sought to determine whether Pin1 interacts with endothelial nitric oxide synthase (eNOS) in endothelial cells in a manner that depends on proline-directed phosphorylation of the eNOS enzyme and whether this interaction influences basal or agonist-stimulated eNOS activity. Methods and Results—Inhibitors of the extracellular-regulated kinase (ERK) 1/2 MAP kinases inhibit proline-directed phosphorylation of eNOS at serine 116 (Ser116) in bovine aortic endothelial cells (BAECs). Moreover, eNOS and Pin1 can be coimmunoprecipitated from BAECs only when Ser116 is phosphorylated. In addition, phosphomimetic Ser116Asp eNOS, but not wild-type eNOS, can be coimmunoprecipitated with Pin1 coexpressed in COS-7 cells. Inhibition of Pin1 in BAECs by juglone or by dominant negative Pin1 increases basal and agonist-stimulated NO release from the cells, whereas overexpression of wild-type Pin1 in BAECs suppresses basal and agonist-stimulated NO production. Overexpression of wild-type Pin1 in intact aortae also reduces agonist-induced relaxation of aortic rings. Conclusion—Our results demonstrate a novel form of eNOS regulation in endothelial cells and blood vessels through Ser116 phosphorylation–dependent interaction of eNOS with Pin1.

Impact of Obesity on Coronary Microvascular Function in the Zucker Rat

Objective: To test the hypothesis that vasomotor control is impaired in the coronary circulation of prediabetic obese (OZR) relative to lean Zucker rats (LZR).

Preventing increased blood pressure in the obese Zucker rat improves severity of stroke

Obesity is a risk factor for stroke, but the determinants of increased stroke risk in obesity are unknown. We have previously reported that obese Zucker rats (OZRs) have a worse stroke outcome and display evidence of remodeling of the middle cerebral artery (MCA), in parallel with hypertension, compared with lean controls. This study tested the hypothesis that hypertension is an essential determinant of cerebral vascular remodeling and increased stroke damage in OZRs. Blood pressure was measured by telemetry in lean and obese rats with and without hydrochlorthiazide (HCT; 2 mg.kg(-1).day(-1)) from 8 to 15 wk of age. A separate group of rats was also chronically fed a low-sodium (LS) diet. Vessel structure was assessed in isolated, pressurized MCAs. Cerebral ischemia was induced for 60 min using an intralumenal suture technique, followed by 24 h of reperfusion. HCT treatment effectively prevented the increase in blood pressure in obese rats; however, the LS diet did not lower pressure. Importantly, infarct size was normalized by HCT after ischemia-reperfusion injury. Additionally, HCT improved the changes in MCA structure observed in untreated OZRs. There were no benefits of the LS diet on stroke injury or vessel structure. These results indicate that increased pressure is essential for driving the changes in infarct size in OZRs.

Increasing Muscle Mass Improves Vascular Function in Obese (db/db) Mice

Background A sedentary lifestyle is an independent risk factor for cardiovascular disease and exercise has been shown to ameliorate this risk. Inactivity is associated with a loss of muscle mass, which is also reversed with isometric exercise training. The relationship between muscle mass and vascular function is poorly defined. The aims of the current study were to determine whether increasing muscle mass by genetic deletion of myostatin, a negative regulator of muscle growth, can influence vascular function in mesenteric arteries from obese db/db mice. Methods and Results Myostatin expression was elevated in skeletal muscle of obese mice and associated with reduced muscle mass (30% to 50%). Myostatin deletion increased muscle mass in lean (40% to 60%) and obese (80% to 115%) mice through increased muscle fiber size (P<0.05). Myostatin deletion decreased adipose tissue in lean mice, but not obese mice. Markers of insulin resistance and glucose tolerance were improved in obese myostatin knockout mice. Obese mice demonstrated an impaired endothelial vasodilation, compared to lean mice. This impairment was improved by superoxide dismutase mimic Tempol. Deletion of myostatin improved endothelial vasodilation in mesenteric arteries in obese, but not in lean, mice. This improvement was blunted by nitric oxide (NO) synthase inhibitor l‐NG‐nitroarginine methyl ester (l‐NAME). Prostacyclin (PGI2)‐ and endothelium‐derived hyperpolarizing factor (EDHF)‐mediated vasodilation were preserved in obese mice and unaffected by myostatin deletion. Reactive oxygen species) was elevated in the mesenteric endothelium of obese mice and down‐regulated by deletion of myostatin in obese mice. Impaired vasodilation in obese mice was improved by NADPH oxidase inhibitor (GKT136901). Treatment with sepiapterin, which increases levels of tetrahydrobiopterin, improved vasodilation in obese mice, an improvement blocked by l‐NAME. Conclusions Increasing muscle mass by genetic deletion of myostatin improves NO‐, but not PGI2‐ or EDHF‐mediated vasodilation in obese mice; this vasodilation improvement is mediated by down‐regulation of superoxide.

Increasing peripheral insulin sensitivity by protein tyrosine phosphatase 1B deletion improves control of blood pressure in obesity

Obesity is a major risk factor for hypertension. The copresentation of hypertension and insulin resistance (IR) suggests a role for IR in blood pressure (BP) dysregulation. To test this hypothesis, peripheral IR has been genetically subtracted in a model of obesity by crossing leptin receptor mutant mice (KdbHPTP) with mice lacking protein tyrosine phosphatase 1B (insulin desensitizer, HdbKPTP) to generate obese insulin-sensitive mice (KdbKPTP). BP was recorded in lean (HdbHPTP, HdbKPTP) and obese (KdbHPTP, KdbKPTP) mice via telemetry, and a frequency analysis of the recording was performed to determine BP variability. Correction of IR in obese mice normalized BP values to baseline levels (HdbHPTP: 116±2 mm Hg; KdbHPTP: 129±4 mm Hg; KdbKPTP: 114±5 mm Hg) and restored BP variability by decreasing its standard deviation and the frequency of BP values over the upper autoregulatory limit of the kidneys. However, although IR-induced increases in proteinuria (versus 53±13 &mgr;g/d, HdbHPTP) were corrected in KdbKPTP (112±39 versus 422±159 &mgr;g/d, KdbHPTP), glomerular hypertrophy was not. IR reduced plasma aldosterone levels ruling out a role for mineralocorticoids in the development of hypertension. Taken together, these data indicate that correction of IR prevents hypertension, BP variability, and microalbuminuria in obese mice. Although the mechanism remains to be fully determined, increases in aldosterone or sympathoactivation of the cardiovascular system seem to be less likely contributors.