Deletion of protein tyrosine phosphatase 1b improves peripheral insulin resistance and vascular function in obese, leptin-resistant mice via reduced oxidant tone

Abstract

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.

Figure 1

The PTP1B gene was significantly upregulated in obese, insulin resistant mice compared to control in muscle (1B) and fat (1C) but not hepatic tissues (1A). PTP1B gene deletion markedly improves insulin receptor tyrosine phosphorylation in muscle (1E) and fat (1F) of dual KO animals compared to obese, insulin resistant animals. Dual KO hepatic tissue insulin resistance persisted (1D). Control animals and PTP1B KO lean animals displayed similar in insulin receptor signaling in all tissues. [A-F: * = p < 0.05 vs. H_dbH_PTP, n > 5]

Figure 1

The PTP1B gene was significantly upregulated in obese, insulin resistant mice compared to control in muscle (1B) and fat (1C) but not hepatic tissues (1A). PTP1B gene deletion markedly improves insulin receptor tyrosine phosphorylation in muscle (1E) and fat (1F) of dual KO animals compared to obese, insulin resistant animals. Dual KO hepatic tissue insulin resistance persisted (1D). Control animals and PTP1B KO lean animals displayed similar in insulin receptor signaling in all tissues. [A-F: * = p < 0.05 vs. H_dbH_PTP, n > 5]

Figure 1

The PTP1B gene was significantly upregulated in obese, insulin resistant mice compared to control in muscle (1B) and fat (1C) but not hepatic tissues (1A). PTP1B gene deletion markedly improves insulin receptor tyrosine phosphorylation in muscle (1E) and fat (1F) of dual KO animals compared to obese, insulin resistant animals. Dual KO hepatic tissue insulin resistance persisted (1D). Control animals and PTP1B KO lean animals displayed similar in insulin receptor signaling in all tissues. [A-F: * = p < 0.05 vs. H_dbH_PTP, n > 5]

Figure 1

The PTP1B gene was significantly upregulated in obese, insulin resistant mice compared to control in muscle (1B) and fat (1C) but not hepatic tissues (1A). PTP1B gene deletion markedly improves insulin receptor tyrosine phosphorylation in muscle (1E) and fat (1F) of dual KO animals compared to obese, insulin resistant animals. Dual KO hepatic tissue insulin resistance persisted (1D). Control animals and PTP1B KO lean animals displayed similar in insulin receptor signaling in all tissues. [A-F: * = p < 0.05 vs. H_dbH_PTP, n > 5]

Figure 1

The PTP1B gene was significantly upregulated in obese, insulin resistant mice compared to control in muscle (1B) and fat (1C) but not hepatic tissues (1A). PTP1B gene deletion markedly improves insulin receptor tyrosine phosphorylation in muscle (1E) and fat (1F) of dual KO animals compared to obese, insulin resistant animals. Dual KO hepatic tissue insulin resistance persisted (1D). Control animals and PTP1B KO lean animals displayed similar in insulin receptor signaling in all tissues. [A-F: * = p < 0.05 vs. H_dbH_PTP, n > 5]

Figure 1

The PTP1B gene was significantly upregulated in obese, insulin resistant mice compared to control in muscle (1B) and fat (1C) but not hepatic tissues (1A). PTP1B gene deletion markedly improves insulin receptor tyrosine phosphorylation in muscle (1E) and fat (1F) of dual KO animals compared to obese, insulin resistant animals. Dual KO hepatic tissue insulin resistance persisted (1D). Control animals and PTP1B KO lean animals displayed similar in insulin receptor signaling in all tissues. [A-F: * = p < 0.05 vs. H_dbH_PTP, n > 5]

Figure 2

Obese, insulin resistant mice (K_dbH_PTP) demonstrated a significant decrease in endothelium dependent, acetylcholine-mediated dilation (2A) and endothelium independent, SNP-mediated dilation (2B) that is ameliorated by PTP1B gene deletion. Endothelium dependent dilation was nearly abolished by L-NAME (2C). Superoxide scavenging produced a complete restoration of acetylcholine-mediated dilation in obese, insulin resistant mice (2D). Superoxide dismutase incubation improves endothelium independent nitric oxide-mediated dilation in K_dbH_PTP (2E). [A, B: * = p < 0.05 K_dbH_PTP vs. H_dbH_PTP, n > 5; C: * = p < 0.05 (+) L-NAME vs. (−) L-NAME for each mouse, n > 5; D, E: * = p < 0.05 (+) PEG-SOD vs. (−) PEG- SOD, n > 5]

Figure 2

Obese, insulin resistant mice (K_dbH_PTP) demonstrated a significant decrease in endothelium dependent, acetylcholine-mediated dilation (2A) and endothelium independent, SNP-mediated dilation (2B) that is ameliorated by PTP1B gene deletion. Endothelium dependent dilation was nearly abolished by L-NAME (2C). Superoxide scavenging produced a complete restoration of acetylcholine-mediated dilation in obese, insulin resistant mice (2D). Superoxide dismutase incubation improves endothelium independent nitric oxide-mediated dilation in K_dbH_PTP (2E). [A, B: * = p < 0.05 K_dbH_PTP vs. H_dbH_PTP, n > 5; C: * = p < 0.05 (+) L-NAME vs. (−) L-NAME for each mouse, n > 5; D, E: * = p < 0.05 (+) PEG-SOD vs. (−) PEG- SOD, n > 5]

Figure 2

Obese, insulin resistant mice (K_dbH_PTP) demonstrated a significant decrease in endothelium dependent, acetylcholine-mediated dilation (2A) and endothelium independent, SNP-mediated dilation (2B) that is ameliorated by PTP1B gene deletion. Endothelium dependent dilation was nearly abolished by L-NAME (2C). Superoxide scavenging produced a complete restoration of acetylcholine-mediated dilation in obese, insulin resistant mice (2D). Superoxide dismutase incubation improves endothelium independent nitric oxide-mediated dilation in K_dbH_PTP (2E). [A, B: * = p < 0.05 K_dbH_PTP vs. H_dbH_PTP, n > 5; C: * = p < 0.05 (+) L-NAME vs. (−) L-NAME for each mouse, n > 5; D, E: * = p < 0.05 (+) PEG-SOD vs. (−) PEG- SOD, n > 5]

Figure 2

Obese, insulin resistant mice (K_dbH_PTP) demonstrated a significant decrease in endothelium dependent, acetylcholine-mediated dilation (2A) and endothelium independent, SNP-mediated dilation (2B) that is ameliorated by PTP1B gene deletion. Endothelium dependent dilation was nearly abolished by L-NAME (2C). Superoxide scavenging produced a complete restoration of acetylcholine-mediated dilation in obese, insulin resistant mice (2D). Superoxide dismutase incubation improves endothelium independent nitric oxide-mediated dilation in K_dbH_PTP (2E). [A, B: * = p < 0.05 K_dbH_PTP vs. H_dbH_PTP, n > 5; C: * = p < 0.05 (+) L-NAME vs. (−) L-NAME for each mouse, n > 5; D, E: * = p < 0.05 (+) PEG-SOD vs. (−) PEG- SOD, n > 5]

Figure 2

Obese, insulin resistant mice (K_dbH_PTP) demonstrated a significant decrease in endothelium dependent, acetylcholine-mediated dilation (2A) and endothelium independent, SNP-mediated dilation (2B) that is ameliorated by PTP1B gene deletion. Endothelium dependent dilation was nearly abolished by L-NAME (2C). Superoxide scavenging produced a complete restoration of acetylcholine-mediated dilation in obese, insulin resistant mice (2D). Superoxide dismutase incubation improves endothelium independent nitric oxide-mediated dilation in K_dbH_PTP (2E). [A, B: * = p < 0.05 K_dbH_PTP vs. H_dbH_PTP, n > 5; C: * = p < 0.05 (+) L-NAME vs. (−) L-NAME for each mouse, n > 5; D, E: * = p < 0.05 (+) PEG-SOD vs. (−) PEG- SOD, n > 5]

Figure 3

EPR spectroscopy revealed a 5-fold increase in superoxide signal from obese insulin resistant mice, while PTP1B gene deletion ameliorates this increase (3A). Acetovanillone (apocynin) incubation of K_dbH_PTP samples decreased the superoxide signal to levels seen in controls (3B). Dihydroethidium staining revealed broad localization of superoxide signal throughout the arteries of K_dbH_PTP mice that is absent in all other mice. [A, B: * = p < 0.05 K_dbH_PTP vs. H_dbH_PTP , n > 6; † = p < 0.05 K_dbK_PTP vs. K_dbH_PTP , n > 6]

Figure 3

EPR spectroscopy revealed a 5-fold increase in superoxide signal from obese insulin resistant mice, while PTP1B gene deletion ameliorates this increase (3A). Acetovanillone (apocynin) incubation of K_dbH_PTP samples decreased the superoxide signal to levels seen in controls (3B). Dihydroethidium staining revealed broad localization of superoxide signal throughout the arteries of K_dbH_PTP mice that is absent in all other mice. [A, B: * = p < 0.05 K_dbH_PTP vs. H_dbH_PTP , n > 6; † = p < 0.05 K_dbK_PTP vs. K_dbH_PTP , n > 6]

Figure 3

EPR spectroscopy revealed a 5-fold increase in superoxide signal from obese insulin resistant mice, while PTP1B gene deletion ameliorates this increase (3A). Acetovanillone (apocynin) incubation of K_dbH_PTP samples decreased the superoxide signal to levels seen in controls (3B). Dihydroethidium staining revealed broad localization of superoxide signal throughout the arteries of K_dbH_PTP mice that is absent in all other mice. [A, B: * = p < 0.05 K_dbH_PTP vs. H_dbH_PTP , n > 6; † = p < 0.05 K_dbK_PTP vs. K_dbH_PTP , n > 6]

Figure 4

Gene expression of superoxide-generating and anti-oxidant defense enzymes were assessed using quantitative Real-Time RT-PCR. K_dbH_PTP mice showed major increases in Nox1, Noxa1 and Noxo1 gene expression. [* = p < 0.05 all genotypes vs. H_dbH_PTP , n > 6; † = p < 0.05 K_dbK_PTP vs. K_dbH_PTP , n > 6]