Published online before print September 24, 2009, doi: 10.1161/CIRCRESAHA.109.206318
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© 2009 American Heart Association, Inc.
Integrative Physiology |
Deletion of Protein Tyrosine Phosphatase 1b Improves Peripheral Insulin Resistance and Vascular Function in Obese, Leptin-Resistant Mice via Reduced Oxidant Tone
From the Vascular Biology Center (M.I.A., P.K., E.J.B.d.C., J.D.M., K.M., C.S., S.M.B., D.J.F., M.B.M., D.W.S.) and Departments Pharmacology (D.J.F., M.B.M.) and Physiology (D.W.S.), Medical College of Georgia, Augusta; and Goodman Cancer Center and Department of Biochemistry (M.L.T.), McGill University, Montreal, Quebec, Canada.
Correspondence to David W. Stepp, PhD, Vascular Biology Center, Medical College of Georgia, 1459 Laney Walker Blvd, Augusta, GA 30912. E-mail dstepp{at}mcg.edu
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Abstract |
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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.
Key Words: obesity • leptin resistance • PTP1B
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Introduction |
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The prevalence of obesity and its cardiovascular complications represents a significant health concern in Western societies,1,2 but the root causes of cardiovascular dysfunction in obese individuals remain unclear. Metabolic dysfunction, notably insulin resistance, is evident in obesity.3,4 It has been speculated that insulin resistance, rather than other aspects of obesity, is the underlying cause of cardiovascular injury in obese patients.5–9 This hypothesis has been difficult to test because insulin-sensitizing drugs have off-target effects4,10 and nonobese models of insulin resistance do not evaluate the relative importance of obesity versus insulin resistance.11–17
The insulin receptor is a classic receptor tyrosine kinase18 and, as such, is deactivated by protein tyrosine phosphatases, notably protein tyrosine phosphatase (PTP)1B.19–21 Deletion of PTP1B improves insulin sensitivity in mouse models of obesity,22 and putative PTP1B antagonists have been used pharmacologically to improve glucose tolerance.23–25 Increases in the activity and/or expression of PTP1B correlate with blunted insulin signaling in a variety of tissue types.26–28 Whether PTP1B deletion and amelioration of insulin resistance improves cardiovascular dysfunction associated with obesity remains unknown.
The present study tested the hypothesis that PTP1B deletion attenuates vascular dysfunction in a model of obesity-induced insulin resistance. Four experimental genotypes were generated through breeding of db/db+/– and PTP1B–/– mice to produce double knockout (KO) PTP1B-null, obese mice. Metabolic profiling, insulin receptor phosphorylation, and PTP1B gene expression were used to assess insulin sensitivity in target tissues. Endothelium-dependent and -independent vascular function were determined in vitro. Molecular techniques examined the mechanism by which deletion of PTP1B improved vascular function. Taken together, these studies critically test the hypothesis that insulin resistance in obesity is the underlying risk factor driving vascular dysfunction in obese individuals.
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Methods |
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Animal Models
All mice used are held in the AAALAC-accredited Medical College of Georgia animal housing facilities. The animals are housed in clean cages with adequate food and water. The Medical College of Georgia IACUC monitors all animal usage on campus. All of our experimental protocols have been institutionally approved.
Two parental strains of mice were used in these studies: leptin receptor mutant db/db mice bred on a C57BL/6 background (The Jackson Laboratories) and PTP1B-null mice bred on a BALB/c background (Michel Tremblay, PhD, Cancer Institute of McGill University). Because db/db mice are sterile, progeny were generated from dual heterozygotes (Hdb, heterozygous for mutant leptin receptor; HPTP, PTP1B gene deletion). Dual heterozygotes were interbred, producing obese, PTP1B gene–null, and dual KO mice at 1:4, 1:4, and 1:16 ratios, respectively. In the F4 generation, dual heterozygotes were bred to heterozygotes for the leptin receptor mutation and PTP1B gene–null mice. This breeding strategy yielded obese and dual KO mice at 1:4 and 1:8 ratios, respectively. Dual heterozygous littermates were used as lean controls, and littermates heterozygous for db and PTP1B gene deletion were used as lean PTP1B-null controls. All experiments were conducted in male progeny. In all cases, mice are designated as H or K, indicating heterozygote or KO. The db gene is designated first and the PTP1B second. Thus, HdbHPTP are heterozygous for both genes, HdbKPTP are lean PTP1B KO mice, KdbHPTP are obese mice with intact PTP1B, and KdbKPTP are deficient in both leptin receptors and PTP1B. Mice were genotyped by PCR of genomic DNA. Metabolic phenotyping was accomplished by assessment of glucose tolerance and plasma chemistry (Online Data Supplement, available at http://circres.ahajournals.org).
Insulin Signaling
To determine the effects of PTP1B deletion on insulin receptor phosphorylation, mice were subjected to an insulin stimulation protocol in vivo.29 Briefly, mice were anesthetized with isoflurane, and either saline or insulin (1 mU/g) was injected into a jugular vein catheter. After 12 minutes, mice were euthanized by isoflurane overdose and samples of liver, skeletal muscle, and adipose tissue were obtained and snap-frozen in liquid nitrogen. The time period between overdose and tissue harvesting was less than 2 minutes total and samples were obtained in differing order to avoid collection bias.
Western Blotting
Tissue homogenates (20 to 50 µg) were separated via SDS-PAGE and transferred to Immobilon-P poly(vinylidene fluoride) membranes. To determine the expression of relevant proteins, immunoblots were probed with antibodies for PTP1B (Upstate), actin (Calbiochem), insulin receptor-β (Santa Cruz Biotechnology), endothelial NO synthase, and phosphorylated endothelial NO synthase 1177/79 (BD Transduction Laboratories).
Insulin Receptor Phosphorylation
An anti–insulin receptor-β antibody was used to immunoprecipitate insulin receptor proteins from 400 to 1000 µg of tissue lysates, and phosphorylation status was assessed with phospho-tyrosine antibody (PY4G10, Upstate) (Online Data Supplement).
In Vitro Microvessel Preparation
Small mesenteric arteries (SMAs) are defined in this study as the second and third distal arcuate artery branches secondary to the conduit superior mesenteric artery ranging from 50 to 150 µm in internal diameter. SMAs were dissected and segments (0.25 to 1 mm in length) were mounted in a vessel bath between two glass micropipettes (25 µm-diameter tip) and secured with 10-0 silk ophthalmic suture. SMAs were then placed in a chilled, oxygenated (21% O2, 5% CO2, and 74% N2) Krebs–Ringer bicarbonate solution composed of (in mmol/L) 118.3 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO44, 1.2 KH2PO4, 25 NaHCO3, and 11.1 D-glucose before analysis to hibernate physiological activity. The lumen of the vessel was filled with Krebs buffer through the micropipette and maintained at a constant pressure of 60 mm Hg. Vessels were monitored under a Nikon inverted light microscope (Melville, NY) connected to a video monitor. Internal diameter was continually measured using video calipers and expressed in micrometers. Buffer temperature was increased to 37°C, and microvessels were allowed to develop spontaneous myogenic tone. After tone was developed, vasodilator responses were measured with sequential doses of acetylcholine (1x10–10 to 1x10–5 mol/L), sodium nitroprusside (SNP) (1x10–9 to 1x10–4 mol/L), or papaverine (1x10–9 to 1x10–4 mol/L). Superoxide dismutase (SOD) was used to scavenge superoxide (100 U/mL). N
-Nitro-L-arginine methyl ester (L-NAME) was used to inhibit NO synthase (100 µmol/L). Dose responses are expressed as a percentage of dilation compared to initial diameter and maximum passive diameter. One dose–response curve was performed per vessel per mouse.
Assessment of Superoxide
The quantitative abundance of superoxide was assessed using electron paramagnetic resonance (EPR) spectroscopy. Qualitative assessment of superoxide localization was made using dihydroethidium staining (Online Data Supplement).
Real-Time RT-PCR
Mesenteric arterial cascades were harvested from euthanized animals, removed of nonvascular tissue, and snap-frozen in liquid nitrogen. Total RNA was extracted using TRIzol Plus RNA (Invitrogen), and cDNA was synthesized using the iScript cDNA Synthesis Kit (Bio-Rad). cDNA was then used to assess relative gene expression using real-time RT-PCR (Bio-Rad iQ SYBR Green). Primer sequences for the selected genes are described in Online Table I.
Statistics
All data are expressed as means±SEM. Differences among all 4 genotypes were compared by 1-way ANOVA or by Student t test with Bonferroni correction test used as the post hoc test. A probability value of less than 0.05 was considered statistically significant.
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Results |
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Weight Gain
Increases in body mass are depicted in Online Figure II. Data are presented as a scatter plot of 5 male mice of each genotype. Summary data at age 12 weeks are shown in the Table. Consistent with the inactivating mutation in the leptin receptor gene, KdbHPTP mice displayed morbid obesity compared to HdbHPTP. Deletion of PTP1B did not affect weight gain in either HdbKPTP or KdbKPTP mice compared to PTP1B intact controls.
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HdbKPTP mice had modest reductions in plasma leptin versus HdbHPTP mice, consistent with sensitization of the leptin receptor, which is also a substrate of PTP1B. In db/db mice, plasma leptin levels were markedly increased along with body weight and were unaffected by deletion of PTP1B (Table).
Food and water intake and urine output are also summarized in the Table. HdbHPTP and HdbKPTP mice exhibited normal and similar food and water intake and urine output. KdbHPTP mice displayed hyperphagia, polydypsia, and polyuria, consistent with obesity. Deletion of PTP1B on the obese background did not affect food and water intake.
In summary, these data indicate that the fundamental defects in leptin signaling that drive obesity in db/db mice are not moderated by the deletion of PTP1B. Thus, the metabolic improvements arising from PTP1B deletion must be attributed to changes in insulin receptor signaling and not modification of obesity.
Glucose Metabolism
Baseline levels of plasma glucose and insulin are shown in the Table. Fasting glucose in HdbHPTP mice was euglycemic and deletion of PTP1B in HdbKPTP mice did not alter fasting glucose. The obesity observed in KdbHPTP mice was associated with moderate hyperglycemia. Deletion of PTP1B in KdbKPTP mice did not reduce fasting blood glucose, suggesting persistent hepatic insulin resistance in the KdbKPTP mice. Consistent with these observations, HdbHPTP mice were euinsulinemic but KdbHPTP and KdbKPTP mice had persistent hyperinsulinemia. Insulin levels in HdbKPTP mice were similar to HdbHPTP mice.
In vivo clearance of a glucose bolus is shown in Online Figure III (S3). HdbHPTP mice displayed rapid glucose disposal, and clearance of glucose in HdbKPTP mice was similar. KdbHPTP mice showed markedly blunted glucose clearance, but KdbKPTP mice showed normalization of glucose clearance despite obesity.
Hemoglobin (Hb)A1c levels, an index of total glycemic load, are shown in the Table. HdbHPTP and HdbKPTP mice showed HbA1C levels lower than 5%, consistent with euglycemic control. In contrast, KdbHPTP mice showed markedly elevated HbA1c levels, consistent with their observed glucose intolerance and fasting hyperglycemia. Although not completely normalized, HbA1c levels were significantly reduced in KdbKPTP mice compared to KdbHPTP mice, despite equivalent food intake.
Lipid Metabolism
Plasma concentration of free fatty acids (FFAs), triglycerides, and cholesterol are shown in the Table. HdbHPTP and HdbKPTP mice show normal levels of all 3 lipid compounds. KdbHPTP mice had elevated fasting FFAs and increased triglyceride levels, consistent with the loss of insulin sensitivity in fat cells. KdbKPTP mice displayed largely normal levels of FFAs and triglycerides, suggesting a normalization of adipocyte insulin resistance by deletion of PTP1B. In contrast, total cholesterol was elevated to a similar extent in both KdbHPTP and KdbKPTP mice.
Expression of PTP1B
To determine the effects of obesity on the tissue expression of PTP1B, Western blotting was performed on extracts from liver, muscle, and fat, which are the 3 major targets of the metabolic actions of insulin. The results are shown in Figure 1A through 1C. PTP1B expression was heterogeneous with marked increases in expression in the skeletal muscle (Figure 1B) and adipose tissue (Figure 1C) of obese mice. Expression of PTP1B in the liver (Figure 1A) was not statistically different between lean HdbHPTP and obese KdbHPTP mice.
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Insulin Signaling
Phosphorylation of the insulin receptor was used as a molecular readout of insulin signaling capacity. In skeletal muscle, adipose, and liver tissue samples, insulin provoked a marked increase in receptor tyrosine phosphorylation that was similar in HdbHPTP and HdbKPTP mice. In contrast, in KdbHPTP mice, the tyrosine phosphorylation of the insulin receptor was markedly reduced, consistent with obesity-induced insulin resistance. In skeletal muscle (Figure 1E) and adipose tissue (Figure 1F), insulin receptor phosphorylation was markedly increased in KdbKPTP mice, suggesting that deletion of PTP1B improved insulin signaling. In contrast, insulin receptor phosphorylation remained depressed in the liver (Figure 1D) of KdbKPTP mice, suggesting persistent hepatic insulin insensitivity in these animals.
Vascular Reactivity
Endothelium-dependent, acetylcholine-mediated vasodilation in the SMAs from all mice is shown in Figure 2A. Smooth muscle reactivity to NO was determined using SNP (Figure 2B). Lean mice that are PTP1B-deficient (HdbKPTP) do not have differences in maximum dilation to acetylcholine (70% versus 68%, P=NS) or SNP (96% versus 94%, P=NS) from HdbHPTP mice, indicating that PTP1B deletion does not affect endothelium-dependent or -independent vasodilation. The maximum vasodilator response to acetylcholine was reduced markedly in KdbHPTP compared to HdbHPTP (50% versus 70%, P<0.05), indicating impairment of endothelial function in KdbHPTP mice. A significant deficiency in reactivity to exogenous NO was also detected in KdbHPTP mice (68% versus 94%, P<0.05). The maximum vasodilator response to acetylcholine was markedly reduced in all mice following treatment with 100 µmol/L L-NAME (Figure 2C), indicating NO as the primary dilator mediating the response to acetylcholine. Furthermore, the NO-independent component of acetylcholine-induced vasodilation was not different among all groups of mice. Taken together, these data indicate that vasodilator reactivity is compromised at the level of NO utilization in obese mice. In contrast to the findings observed in KdbHPTP mice, endothelium-dependent vasodilation in obese KdbKPTP was similar to that observed in lean HdbHPTP mice (Figure 2A). The impaired response to exogenous NO (SNP) was also restored by PTP1B deletion (Figure 2B). Vascular dysfunction was not attributable to loss of endothelial NO synthase expression or phosphorylation because these variables were similar in all strains of mice (Online Figure IV; S4). Responses to the NO-independent vasodilator papaverine were similar across all mice (Online Figure V; S5), suggesting that the vasodilation deficiency in KdbHPTP mice is not attributable to a general deficit in vascular dilation but is confined to NO-mediated dilation.
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To determine whether elevated superoxide production was a mechanism of impaired vasodilation in obesity, vascular function was assessed in the presence of 100 U/mL pegylated (PEG)-SOD. PEG-SOD reversed the impaired dilation to acetylcholine (Figure 2D) and SNP (Figure 2E) in KdbHPTP mice, with no effect on vascular function in the other genotypes. To further determine whether PEG-SOD was indeed restoring NO bioactivity, endothelium-dependent dilation was assessed in the presence of PEG-SOD and both the presence and absence of 100 µmol/L L-NAME (Online Figure VI; S6). All mouse vessels exhibited equivalent degrees of L-NAME–resistant dilation, thus confirming that the main dilation mechanism in these microvessels is NO and further that scavenging of superoxide did not improve NO-independent dilation in KdbHPTP mice.
Passive mechanics were assessed in a zero-Ca2+ Krebs solution and results are shown in Online Table II (ST2). Vascular architectural changes were assessed as previously described. Maximal vessel wall thickness and wall to lumen ratio (at 120 mm Hg of intraluminal pressure) were similar across all genotypes. Vascular compliance, as calculated by the exponential fit of a circumferential stress-strain plot (β-coefficient), also remained similar across all genotypes. Taken together, these data indicate that neither obesity nor deletion of PTP1B produce structural changes that could account for observed deficits in vasodilator function.
Superoxide Production
EPR spectroscopy was used to semiquantitatively measure superoxide. The relative PEG-SOD–inhibitable signal was 4 times higher in mesenteric vessels from KdbHPTP mice versus control HdbHPTP mice (Figure 3A) and reversed to control levels by deletion of PTP1B in obese KdbKPTP. The increased superoxide signal in the KdbHPTP mice was nearly eliminated following acetovanillone (apocynin) incubation (Figure 3B), suggesting that it derives from NAD(P)H oxidases. Taken together, peripheral insulin resistance increases vascular superoxide production that is corrected by PTP1B deletion. As a further measure of vascular reactive oxygen species production, we also performed dihydroethidium staining of blood vessels. These results are in agreement with the EPR studies and demonstrate higher levels of superoxide in the blood vessels of KdbHPTP mice compared to controls (Figure 3C). The deletion of PTP1B in obese animals (KdbKPTP) decreased dihydroethidium staining to control levels, and there was no difference in superoxide production resulting from the deletion of PTP1B in lean animals. Dihydroethidium staining was most intense in the medial layer of mesenteric microvessels in all 4 groups of mice.
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The source of elevated superoxide levels in KdbHPTP mice was addressed using real-time quantitative RT-PCR. As shown in Figure 4, the expression levels for Nox1 and its novel activator and organizer (Noxa1 and Noxo1, respectively) are significantly elevated in KdbHPTP mice as compared to control (2–
Ct±SEM in HdbHPTP versus KdbHPTP; Nox1: 1.45±0.23 versus 3.52±1.6; Noxa1: 1.17±0.11 versus 9.94±1.5; Noxo1: 1.14±0.23 versus 8.81±2.8). Nox2 and Nox4 are expressed similarly in all animals (Figure 4), as are p22phox, p47phox, and p67phox (data not shown). Select antioxidant enzymes were also examined, demonstrating a statistically significant increase in SOD2 and SOD3 in KdbKPTP mice compared to both controls and KdbHPTP mice (2–Ct±SEM in KdbHPTP versus KdbKPTP; SOD2: 1.00±0.5 versus 3.36±0.52; SOD3: 0.68±0.10 versus 4.76±1.6). A summary for all genes studied is shown in Online Table III (ST3).
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Discussion |
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The goal of the present study was to determine whether the metabolic consequences of obesity or the state of obesity itself results in vascular dysfunction. To test this hypothesis, we generated obese mice harboring deletion of PTP1B, a tyrosine phosphatase that antagonizes insulin signaling. The key findings of this study are as follows: (1) deletion of PTP1B in db/db mice does not affect obesity and corrects peripheral but not hepatic insulin resistance; (2) correction of peripheral insulin resistance improves NO-mediated dilation in SMAs despite persistent obesity; and (3) correcting peripheral insulin resistance mediates the improvement in NO dilation by decreasing superoxide levels, primarily through reduced expression of Nox1, Noxa1, and Noxo1.
Metabolic Effects of PTP1B Deletion in Obese Mice
Although PTP1B has attracted considerable attention as a target in the treatment of non–insulin-dependent diabetes, the impact of obesity on the relative distribution of PTP1B expression in tissues central to insulin action is unclear. Moreover, expression of PTP1B in models of obesity and diabetes is complicated because it varies with the stage of diabetes and genetic background.30 As shown in Figure 1A through 1C, obesity in the db/db mice used in these studies caused a differential increase in PTP1B expression, with the most prominent increases in muscle and fat and a statistically undetectable difference in the liver. The increases in PTP1B expression correlate with decreased insulin receptor phosphorylation and are reversed by PTP1B deletion.
Deletion of PTP1B did not affect weight gain in either lean or obese mice. Food and fluid intake, urine output, and plasma leptin levels also remained unchanged. These observations are consistent with those of Cheng et al,31 in which ob/ob mice heterozygous for PTP1B and ob/ob mice with deletion PTP1B showed similarity in weight gain. In contrast, adenoviral delivery of PTP1B antisense RNA produces reductions in body weight and fat mass,32 and when ob/ob mice that lack PTP1B are compared to ob/ob mice with wild-type PTP1B expression, an 
15% weight difference is observed.31 It is important to note that in this study, deletion of PTP1B improves glycemic control in ob/ob mice when wild-type (2 copies) and KO (no copies) are compared. However, one could not determine in this setting whether the improvement in glycemic control reflects the actions of PTP1B on insulin signaling or the weight loss in ob/ob mice. In the present study, we evaluated metabolic control between mice in which body weight is identical and the leptin receptor is completely missing. Thus, differences between KdbHPTP and KdbKPTP, which are equally obese, reflect the effects of PTP1B deletion on improvements in the insulin signaling pathway.
To verify the functional importance of the increase in PTP1B expression in KdbHPTP mice, we used physiological (plasma serum chemistry and glucose tolerance) and molecular (phosphorylation of the insulin receptor) measurements as indices of insulin signaling. Consistent with recent observations from Delibegovic et al,33 the elevated expression of PTP1B in skeletal muscle of obese mice correlated with marked impairment in muscle insulin receptor phosphorylation and significant impairment in glucose tolerance and increased HbA1c percentage. These variables were markedly improved by the deletion of PTP1B. These findings, combined with the marked improvement in insulin receptor tyrosine phosphorylation in muscle indicate that PTP1B is a key determinant of skeletal muscle insulin sensitivity in db/db mice.
Reductions in serum triglycerides and FFAs following deletion of PTP1B, combined with the marked increase in PTP1B expression in visceral adipose tissue and improvement in insulin receptor signaling in visceral adipose tissue, indicate that PTP1B also plays a critical role in the adipose tissue of obese mice. In this study, the lack of a leptin receptor results in similar body weight between KdbHPTP and KdbKPTP mice but marked differences in triglyceride and FFA levels in the plasma, indicating an improvement of insulin signaling in fat tissue. Plasma cholesterol was elevated in KdbHPTP mice compared to HdbHPTP mice (Table) but was not affected by PTP1B deletion in HdbKPTP or KdbKPTP mice. Because both KdbHPTP and KdbKPTP mice retain the hyperphagic phenotype, the lack of a difference in plasma cholesterol likely indicates that elevated cholesterol in these animals reflects dietary intake or persistent hepatic insulin resistance.
Fasting blood glucose, primarily driven by hepatic gluconeogenesis, was similar in KdbHPTP and KdbKPTP mice. Plasma insulin levels are also elevated, consistent with the loss of insulin receptor function in the liver.34,35 PTP1B expression was not significantly increased in the liver and insulin receptor phosphorylation remains depressed in KdbKPTP mice despite deletion of PTP1B. Although previous studies have described a role for PTP1B in hepatic insulin signaling in lean mice36,37 or with nongenomic methods,38 our model does not reflect this outcome, likely because of the background of these mice.30 Nevertheless, the lack of improvement in hepatic insulin signaling and moderate fasting hyperglycemia indicate that hepatic insulin resistance cannot explain observed impairments in cardiovascular function.
Effect of Deletion of PTP1B on Vasodilation
Previous studies in obese rodents have indicated that obesity is a risk factor for vascular dysfunction,11,39,40 but the culpable component of obesity has remained elusive. As described above, the dual KO mice developed for these studies remain obese, but peripheral insulin resistance is improved when PTP1B is deleted. Moreover, the results described in these studies indicate that when PTP1B is deleted in obese mice, vasodilation to NO is improved.
The role of PTP1B in improving NO-mediated dilation could be attributed to (1) a direct effect of PTP1B on endothelial function or (2) an improvement in vasodilation secondary to correction of peripheral insulin resistance. A direct effect of PTP1B deletion is refuted by the lack of vascular outcomes in lean HdbKPTP mice, consistent with previous work in which overexpression of PTP1B in cultured endothelial cells did not influence the function of endothelial NO synthase.41 These observations preclude PTP1B as a direct modulator of vasodilation.
Our observations are more consistent with the hypothesis that insulin resistance is the causal factor in vessel dysfunction in obesity. To date, this hypothesis has been primarily based on studies in nonobese models of insulin resistance11,13,42,43 and studies with pharmacological compounds.4,10,42 Clear interpretation of these studies is confounded by off-target effects of drugs and because they lack the physiological context of obesity. In the present study, we have developed a novel double KO model and characterized in detail the metabolic parameters relevant to insulin resistance. The outcome is that we have described a model in which improvement of peripheral insulin resistance improves vascular function, despite persistent obesity and modest hyperglycemia. This normalization provides strong evidence that obesity has minimal impact on vasodilation mediated by NO in the absence of insulin resistance. Because the deletion of PTP1B in this model improves peripheral but not hepatic insulin resistance, we can refine the metabolic hypothesis of vessel dysfunction beyond whole body insulin resistance to specific compartments. Impairment of vascular function correlates with markers of metabolic dysfunction in muscle and fat but not the liver. To our knowledge, this is the first study to localize vascular dysfunction in obesity to peripheral insulin resistance.
Effect of PTP1B Deletion on Reactive Oxygen Species
The mechanisms by which insulin resistance impairs NO-mediated vasodilation are incompletely understood. In the present study, we present evidence that the primary mechanism is an increase in reactive oxygen species. In obese mice, the level of superoxide is increased versus lean controls (Figure 3A and 3C); vasodilation is restored by oxidant scavenging and components of the NAD(P)H oxidase pathway are increased (Figure 2D and 2E; Figure 4; Online Table III; ST3). Blockade of NAD(P)H oxidases normalizes oxidant load. Correction of insulin resistance by deletion of PTP1B in obese mice corrects the augmented reactive oxygen species levels. Scavenging of superoxide restores sensitivity to acetylcholine as does deletion of PTP1B in KdbKPTP mice. The L-NAME–resistant component of endothelium-dependent dilation remains the same in all mice, ruling out differences in NO-independent endothelial vasodilation. Taken together, these data suggest that insulin resistance corrupts endothelial vasodilation by NAD(P)H oxidase–derived oxidants.
Previous studies have attributed increases in superoxide levels to an increase in NAD(P)H oxidase activity.44,45 Nox2 (gp91phox) was originally identified as the primary source of pathological superoxide production in insulin-resistant states, but more recently roles for Nox1 and Nox4 have been identified.46–48 A key finding in the present study is that the expression levels of Nox1 and its regulatory enzymes Noxa1/Noxo1 in the vasculature correlate with obesity-induced insulin resistance. In obese mice, the expression of Nox1, Noxa1, and Noxo1 are markedly increased and these levels are reversed by the deletion of PTP1B. Coexpression of Nox1 with Noxo1 and Noxa1 results in the constitutive and high-level production of superoxide.49,50 The significance of increased Nox1 activity in insulin-resistant states in not clear. The loss of Nox1 attenuates, whereas an increased expression of Nox1 potentiates, angiotensin-induced hypertension.51,52 In large conduit blood vessels, Nox1 expression is unchanged in db/db mice53; however, our study is the first to measure expression levels of these proteins in microvessels. The expression level of other Nox isoforms was examined in addition to Nox1, and in contrast to other studies,53 there were no changes with obesity.
In summary, these experiments provide new evidence that in the context of obesity, the underlying risk factor that impairs both endothelium-dependent and -independent NO dilation is peripheral insulin resistance. Moderate hyperglycemia or morbid obesity does not cause endothelial dysfunction when the enzyme PTP1B is absent. These data indicate that PTP1B may represent an important therapeutic target for not only the metabolic but also cardiovascular therapy of obesity.
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Acknowledgments |
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We gratefully acknowledge the assistance of Dr Brenda Lilly (Medical College of Georgia) in developing a PCR screening strategy for PTP1B. We also acknowledge Jessica Osmond for helpful review of the manuscript. We thank Dr David M. Stern for initial guidance and encouragement.
Sources of Funding
This work was supported by NIH grants 5R01HL076533 and 1R01HL092446 (to D.W.S.); 5R01HL085827 and 1R01HL092446 (to D.J.F.); AHA Pre-Doctoral Fellowship (to M.I.A.); 5R01HL058139 5R01DK061687 (to M.B.M.); and an American Heart Association Established Investigator Award (to D.J.F.).
Disclosures
None.
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Footnotes |
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*Both authors contributed equally to this work.
Both authors contributed equally to this work as senior authors.
Original received September 16, 2008; resubmission received July 30, 2009; revised resubmission received September 4, 2009; accepted September 10, 2009.
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References |
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1. Ritchie SA, Connell JM. The link between abdominal obesity, metabolic syndrome and cardiovascular disease. Nutr Metab Cardiovasc Dis. 2007; 17: 319–326.[Medline] [Order article via Infotrieve]
2. Morisco C, Lembo G, Trimarco B. Insulin resistance and cardiovascular risk: new insights from molecular and cellular biology. Trends Cardiovasc Med. 2006; 16: 183–188.[CrossRef][Medline] [Order article via Infotrieve]
3. Guilherme A, Virbasius JV, Puri V, Czech MP. Adipocyte dysfunctions linking obesity to insulin resistance and type 2 diabetes. Nat Rev. 2008; 9: 367–377.[CrossRef]
4. Goldstein BJ. Insulin resistance: from benign to type 2 diabetes mellitus. Rev Cardiovasc Med. 2003; 4 (suppl 6): S3–S10.
5. Verma S, Leung YM, Yao L, Battell M, Dumont AS, McNeill JH. Hyperinsulinemia superimposed on insulin resistance does not elevate blood pressure. Am J Hypertens. 2001; 14 (5 pt 1): 429–432.[CrossRef][Medline] [Order article via Infotrieve]
6. Steinberg HO, Chaker H, Leaming R, Johnson A, Brechtel G, Baron AD. Obesity/insulin resistance is associated with endothelial dysfunction. Implications for the syndrome of insulin resistance. J Clin Invest. 1996; 97: 2601–2610.[Medline] [Order article via Infotrieve]
7. Katakam PV, Ujhelyi MR, Hoenig ME, Miller AW. Endothelial dysfunction precedes hypertension in diet-induced insulin resistance. Am J Physiol. 1998; 275 (3 pt 2): R788–R792.[Medline] [Order article via Infotrieve]
8. Dimitropoulou C, Han G, Miller AW, Molero M, Fuchs LC, White RE, Carrier GO. Potassium (BK(Ca)) currents are reduced in microvascular smooth muscle cells from insulin-resistant rats. Am J Physiol. 2002; 282: H908–H917.
9. Baron AD. Insulin resistance and vascular function. J Diabetes Complications. 2002; 16: 92–102.[CrossRef][Medline] [Order article via Infotrieve]
10. Chen ZP, Mitchelhill KI, Michell BJ, Stapleton D, Rodriguez-Crespo I, Witters LA, Power DA, Ortiz de Montellano PR, Kemp BE. AMP-activated protein kinase phosphorylation of endothelial NO synthase. FEBS Lett. 1999; 443: 285–289.[CrossRef][Medline] [Order article via Infotrieve]
11. Romanko OP, Stepp DW. Reduced constrictor reactivity balances impaired vasodilation in the mesenteric circulation of the obese Zucker rat. Am J Physiol. 2005; 289: H2097–H2102.
12. D'Angelo G, Elmarakby AA, Pollock DM, Stepp DW. Fructose feeding increases insulin resistance but not blood pressure in Sprague-Dawley rats. Hypertension. 2005; 46: 806–811.
13. Verma S, Bhanot S, Yao L, McNeill JH. Defective endothelium-dependent relaxation in fructose-hypertensive rats. Am J Hypertens. 1996; 9 (4 pt 1): 370–376.[CrossRef][Medline] [Order article via Infotrieve]
14. Shinozaki K, Ayajiki K, Nishio Y, Sugaya T, Kashiwagi A, Okamura T. Evidence for a causal role of the renin-angiotensin system in vascular dysfunction associated with insulin resistance. Hypertension. 2004; 43: 255–262.
15. Lee DH, Lee JU, Kang DG, Paek YW, Chung DJ, Chung MY. Increased vascular endothelin-1 gene expression with unaltered nitric oxide synthase levels in fructose-induced hypertensive rats. Metabolism. 2001; 50: 74–78.[CrossRef][Medline] [Order article via Infotrieve]
16. Kamata K, Yamashita K. Insulin resistance and impaired endothelium-dependent renal vasodilatation in fructose-fed hypertensive rats. Res Commun Mol Pathol Pharmacol. 1999; 103: 195–210.[Medline] [Order article via Infotrieve]
17. Zavaroni I, Sander S, Scott S, Reaven GM. Effect of fructose feeding on insulin secretion and insulin action in the rat. Metabolism. 1980; 29: 970–973.[CrossRef][Medline] [Order article via Infotrieve]
18. White MF, Kahn CR. The insulin signaling system. J Biol Chem. 1994; 269: 1–4.
19. Seely BL, Staubs PA, Reichart DR, Berhanu P, Milarski KL, Saltiel AR, Kusari J, Olefsky JM. Protein tyrosine phosphatase 1B interacts with the activated insulin receptor. Diabetes. 1996; 45: 1379–1385.[Abstract]
20. Bandyopadhyay D, Kusari A, Kenner KA, Liu F, Chernoff J, Gustafson TA, Kusari J. Protein-tyrosine phosphatase 1B complexes with the insulin receptor in vivo and is tyrosine-phosphorylated in the presence of insulin. J Biol Chem. 1997; 272: 1639–1645.
21. Dadke S, Kusari J, Chernoff J. Down-regulation of insulin signaling by protein-tyrosine phosphatase 1B is mediated by an N-terminal binding region. J Biol Chem. 2000; 275: 23642–23647.
22. Elchebly M, Payette P, Michaliszyn E, Cromlish W, Collins S, Loy AL, Normandin D, Cheng A, Himms-Hagen J, Chan CC, Ramachandran C, Gresser MJ, Tremblay ML, Kennedy BP. Increased insulin sensitivity and obesity resistance in mice lacking the protein tyrosine phosphatase-1B gene. Science. 1999; 283: 1544–1548.
23. Chen H, Cong LN, Li Y, Yao ZJ, Wu L, Zhang ZY, Burke TR Jr, Quon MJ. A phosphotyrosyl mimetic peptide reverses impairment of insulin-stimulated translocation of GLUT4 caused by overexpression of PTP1B in rat adipose cells. Biochemistry. 1999; 38: 384–389.[CrossRef][Medline] [Order article via Infotrieve]
24. Malamas MS, Sredy J, Moxham C, Katz A, Xu W, McDevitt R, Adebayo FO, Sawicki DR, Seestaller L, Sullivan D, Taylor JR. Novel benzofuran and benzothiophene biphenyls as inhibitors of protein tyrosine phosphatase 1B with antihyperglycemic properties. J Med Chem. 2000; 43: 1293–1310.[CrossRef][Medline] [Order article via Infotrieve]
25. Winter CL, Lange JS, Davis MG, Gerwe GS, Downs TR, Peters KG, Kasibhatla B. A nonspecific phosphotyrosine phosphatase inhibitor, bis(maltolato)oxovanadium(IV), improves glucose tolerance and prevents diabetes in Zucker diabetic fatty rats. Exp Biol Med. 2005; 230: 207–216.
26. Ahmad F, Considine RV, Bauer TL, Ohannesian JP, Marco CC, Goldstein BJ. Improved sensitivity to insulin in obese subjects following weight loss is accompanied by reduced protein-tyrosine phosphatases in adipose tissue. Metabolism. 1997; 46: 1140–1145.[CrossRef][Medline] [Order article via Infotrieve]
27. Wu X, Hardy VE, Joseph JI, Jabbour S, Mahadev K, Zhu L, Goldstein BJ. Protein-tyrosine phosphatase activity in human adipocytes is strongly correlated with insulin-stimulated glucose uptake and is a target of insulin-induced oxidative inhibition. Metabolism. 2003; 52: 705–712.[CrossRef][Medline] [Order article via Infotrieve]
28. Venable CL, Frevert EU, Kim YB, Fischer BM, Kamatkar S, Neel BG, Kahn BB. Overexpression of protein-tyrosine phosphatase-1B in adipocytes inhibits insulin-stimulated phosphoinositide 3-kinase activity without altering glucose transport or Akt/Protein kinase B activation. J Biol Chem. 2000; 275: 18318–18326.
29. Dummler B, Tschopp O, Hynx D, Yang ZZ, Dirnhofer S, Hemmings BA. Life with a single isoform of Akt: mice lacking Akt2 and Akt3 are viable but display impaired glucose homeostasis and growth deficiencies. Mol Cell Biol. 2006; 26: 8042–8051.
30. Zabolotny JM, Kim YB, Welsh LA, Kershaw EE, Neel BG, Kahn BB. Protein tyrosine phosphatase 1B (PTP1B) expression is induced by inflammation in vivo. J Biol Chem. 2008; 283: 14230–14241.
31. Cheng A, Uetani N, Simoncic PD, Chaubey VP, Lee-Loy A, McGlade CJ, Kennedy BP, Tremblay ML. Attenuation of leptin action and regulation of obesity by protein tyrosine phosphatase 1B. Dev Cell. 2002; 2: 497–503.[CrossRef][Medline] [Order article via Infotrieve]
32. Rondinone CM, Trevillyan JM, Clampit J, Gum RJ, Berg C, Kroeger P, Frost L, Zinker BA, Reilly R, Ulrich R, Butler M, Monia BP, Jirousek MR, Waring JF. Protein tyrosine phosphatase 1B reduction regulates adiposity and expression of genes involved in lipogenesis. Diabetes. 2002; 51: 2405–2411.
33. Delibegovic M, Bence KK, Mody N, Hong EG, Ko HJ, Kim JK, Kahn BB, Neel BG. Improved glucose homeostasis in mice with muscle-specific deletion of protein-tyrosine phosphatase 1B. Mol Cell Biol. 2007; 27: 7727–7734.
34. Michael MD, Kulkarni RN, Postic C, Previs SF, Shulman GI, Magnuson MA, Kahn CR. Loss of insulin signaling in hepatocytes leads to severe insulin resistance and progressive hepatic dysfunction. Mol Cell. 2000; 6: 87–97.[CrossRef][Medline] [Order article via Infotrieve]
35. Fisher SJ, Kahn CR. Insulin signaling is required for insulin’s direct and indirect action on hepatic glucose production. J Clin Invest. 2003; 111: 463–468.[CrossRef][Medline] [Order article via Infotrieve]
36. Xue B, Kim YB, Lee A, Toschi E, Bonner-Weir S, Kahn CR, Neel BG, Kahn BB. Protein-tyrosine phosphatase 1B deficiency reduces insulin resistance and the diabetic phenotype in mice with polygenic insulin resistance. J Biol Chem. 2007; 282: 23829–23840.
37. Haj FG, Zabolotny JM, Kim YB, Kahn BB, Neel BG. Liver-specific protein-tyrosine phosphatase 1B (PTP1B) re-expression alters glucose homeostasis of PTP1B–/– mice. J Biol Chem. 2005; 280: 15038–15046.
38. Zinker BA, Rondinone CM, Trevillyan JM, Gum RJ, Clampit JE, Waring JF, Xie N, Wilcox D, Jacobson P, Frost L, Kroeger PE, Reilly RM, Koterski S, Opgenorth TJ, Ulrich RG, Crosby S, Butler M, Murray SF, McKay RA, Bhanot S, Monia BP, Jirousek MR. PTP1B antisense oligonucleotide lowers PTP1B protein, normalizes blood glucose, and improves insulin sensitivity in diabetic mice. Proc Natl Acad Sci U S A. 2002; 99: 11357–11362.
39. Oltman CL, Richou LL, Davidson EP, Coppey LJ, Lund DD, Yorek MA. Progression of coronary and mesenteric vascular dysfunction in Zucker obese and Zucker diabetic fatty rats. Am J Physiol. 2006; 291: H1780–H1787.
40. Frisbee JC. Impaired dilation of skeletal muscle microvessels to reduced oxygen tension in diabetic obese Zucker rats. Am J Physiol. 2001; 281: H1568–H1574.
41. Fulton D, Harris MB, Kemp BE, Venema RC, Marrero MB, Stepp DW. Insulin resistance does not diminish eNOS expression, phosphorylation, or binding to HSP-90. Am J Physiol. 2004; 287: H2384–H2393.
42. Katakam PV, Ujhelyi MR, Hoenig M, Miller AW. Metformin improves vascular function in insulin-resistant rats. Hypertension. 2000; 35 (1 pt 1): 108–112.
43. Takagawa Y, Berger ME, Hori MT, Tuck ML, Golub MS. Long-term fructose feeding impairs vascular relaxation in rat mesenteric arteries. Am J Hypertens. 2001; 14 (8 pt 1): 811–817.[CrossRef][Medline] [Order article via Infotrieve]
44. Cai H, Griendling KK, Harrison DG: The vascular NAD(P)H oxidases as therapeutic targets in cardiovascular diseases. Trends Pharmacol Sci. 2003; 24: 471–478.[CrossRef][Medline] [Order article via Infotrieve]
45. Dusting GJ, Selemidis S, Jiang F. Mechanisms for suppressing NAD(P)H oxidase in the vascular wall. Mem Inst Oswaldo Cruz. 2005; 100 (suppl 1): 97–103.[Medline] [Order article via Infotrieve]
46. Duncan ER. Effect of endothelium-specific insulin resistance on endothelial function in vivo. Diabetes. 2008; 57: 3307–3314.
47. Lassegue B, Clempus RE. Vascular NAD(P)H oxidases: Specific features, expression, and regulation. Am J Physiol Reg Integr Comp Physiol. 2003; 285: R277–R297.
48. Ding H, Hashem M, Triggle C. Increased oxidative stress in the streptozotocin-induced diabetic apoE-deficient mouse: changes in expression of NAD(P)H oxidase subunits and eNOS. Eur J Pharmacol. 2007; 561: 121–128.[CrossRef][Medline] [Order article via Infotrieve]
49. Banfi B. Two novel proteins activate superoxide generation by the NAD(P)H oxidase NOX1. J Biol Chem. 2003; 278: 3510–3513.
50. Takeya R. Novel human homologues of p47phox and p67phox participate in activation of superoxide-producing NAD(P)H oxidases. J Biol Chem. 2003; 278: 25234–25246.
51. Dikalova A. Nox1 overexpression potentiates angiotensin II-induced hypertension and vascular smooth muscle hypertrophy in transgenic mice. Circulation. 2005; 112: 2668–2676.
52. Gavazzi G. Decreased blood pressure in NOX1-deficient mice. FEBS Lett. 2006; 580: 497–504.[CrossRef][Medline] [Order article via Infotrieve]
53. San Martin A. Reactive oxygen species-selective regulation of aortic inflammatory gene expression in type 2 diabetes. Am J Physiol Heart Circ Physiol. 2007; 292: H2073–H2082.
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