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Neprilysin, obesity and the metabolic syndrome.

Standeven KF, Hess K, Carter AM, Rice GI, Cordell PA, Balmforth AJ, Lu B, Scott DJ, Turner AJ, Hooper NM, Grant PJ - Int J Obes (Lond) (2010)

Bottom Line: In a murine model of diet-induced insulin resistance, plasma NEP levels were significantly higher in high-fat diet (HFD)-fed compared with normal chow diet (NCD)-fed animals (1642 ± 529 and 820 ± 487 pg μl(-1), respectively; P<0.01).Tissue NEP was increased in mesenteric fat in HFD compared with NCD-fed mice (P<0.05).NEP knockout mice did not display any changes in insulin resistance, glucose tolerance, or body and epididymal fat pad weight compared with wild-type mice.

View Article: PubMed Central - PubMed

Affiliation: Division of Cardiovascular and Diabetes Research, Leeds Institute of Genetics, Health and Therapeutics, University of Leeds, Leeds, UK.

ABSTRACT

Objective: Neprilysin (NEP), a zinc metalloendopeptidase, has a role in blood pressure control and lipid metabolism. The present study tested the hypothesis that NEP is associated with insulin resistance and features of the metabolic syndrome (MetS) in a study of 318 healthy human subjects and in murine obesity, and investigated NEP production by adipocytes in-vitro.

Methods and results: In 318 white European males, plasma NEP was elevated in the MetS and increased progressively with increasing MetS components. Plasma NEP activity correlated with insulin, homoeostasis model assessment and body mass index (BMI) in all subjects (P<0.01). Quantitative reverse transcriptase PCR (RT-PCR) and western blotting showed that in human pre-adipocytes NEP expression is upregulated 25- to 30-fold during differentiation into adipocytes. Microarray analysis of mRNA from differentiated human adipocytes confirmed high-NEP expression comparable with adiponectin and plasminogen activator inhibitor-1. In a murine model of diet-induced insulin resistance, plasma NEP levels were significantly higher in high-fat diet (HFD)-fed compared with normal chow diet (NCD)-fed animals (1642 ± 529 and 820 ± 487 pg μl(-1), respectively; P<0.01). Tissue NEP was increased in mesenteric fat in HFD compared with NCD-fed mice (P<0.05). NEP knockout mice did not display any changes in insulin resistance, glucose tolerance, or body and epididymal fat pad weight compared with wild-type mice.

Conclusion: In humans, NEP activity correlated with BMI and measures of insulin resistance with increasing levels in subjects with multiple cardiovascular risk factors. NEP protein production in human adipocytes increased during cell differentiation and plasma and adipose tissue levels of NEP were increased in obese insulin-resistant mice. Our results indicate that NEP associates with cardiometabolic risk in the presence of insulin resistance and increases with obesity.

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NEP expression in the human adipocyte SGBS cells during differentiationTotal RNA and total protein were extracted from differentiating SGBS cells over a 0-14 day period every 2 days, starting at day 0. A: NEP gene expression was measured using semiquantitative real time PCR, with simultaneous amplification of GAPDH as an internal control allowing normalisation of target gene expression. Data represents the mean values observed in two independent experiments, with samples analysed in triplicate. Data are presented as a fold increase in NEP gene expression, relative to the undifferentiated SGBS cells (day 0) which was readily detectable (CT value approximately 25). B: NEP protein expression was measured using a semiquantitative western blot approach. Data are presented as a fold increase in mean ± SEM (n=3) intensity values of NEP, relative to the undifferentiated SGBS cells (day 0). C: Gene expression of NEP, adiponectin, retinol binding protein-4 (RBP4) and PAI-1 in 14 day differentiated SGBS cells determined by the Human Genome U133 Plus 2.0 array. Data from nine arrays are represented as mean ± SEM of the corresponding intensity signals derived by the GeneChip Operating Software. D: Western blot showing the increase in NEP protein concentration over the 14d differentiation period.
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Figure 3: NEP expression in the human adipocyte SGBS cells during differentiationTotal RNA and total protein were extracted from differentiating SGBS cells over a 0-14 day period every 2 days, starting at day 0. A: NEP gene expression was measured using semiquantitative real time PCR, with simultaneous amplification of GAPDH as an internal control allowing normalisation of target gene expression. Data represents the mean values observed in two independent experiments, with samples analysed in triplicate. Data are presented as a fold increase in NEP gene expression, relative to the undifferentiated SGBS cells (day 0) which was readily detectable (CT value approximately 25). B: NEP protein expression was measured using a semiquantitative western blot approach. Data are presented as a fold increase in mean ± SEM (n=3) intensity values of NEP, relative to the undifferentiated SGBS cells (day 0). C: Gene expression of NEP, adiponectin, retinol binding protein-4 (RBP4) and PAI-1 in 14 day differentiated SGBS cells determined by the Human Genome U133 Plus 2.0 array. Data from nine arrays are represented as mean ± SEM of the corresponding intensity signals derived by the GeneChip Operating Software. D: Western blot showing the increase in NEP protein concentration over the 14d differentiation period.

Mentions: As shown in Figure 3A, human SGBS pre-adipocytes expressed NEP mRNA (CT value approximately 25), and this was increased ~25-30-fold, during adipocyte differentiation. At the protein level, NEP was similarly increased when human SGBS preadipocytes differentiated into mature adipocytes (Figure 3B). To examine the expression levels of SGBS adipocyte-derived NEP, Affymetrix human 133 plus 2 gene chip arrays (47,400 transcripts) of differentiating SGBS cells indicated expression of NEP mRNA at levels comparable to that of known adipokines such as adiponectin, retinol binding protein 4 and PAI-1 (Figure 3C) and at least five times higher than the average gene signal.


Neprilysin, obesity and the metabolic syndrome.

Standeven KF, Hess K, Carter AM, Rice GI, Cordell PA, Balmforth AJ, Lu B, Scott DJ, Turner AJ, Hooper NM, Grant PJ - Int J Obes (Lond) (2010)

NEP expression in the human adipocyte SGBS cells during differentiationTotal RNA and total protein were extracted from differentiating SGBS cells over a 0-14 day period every 2 days, starting at day 0. A: NEP gene expression was measured using semiquantitative real time PCR, with simultaneous amplification of GAPDH as an internal control allowing normalisation of target gene expression. Data represents the mean values observed in two independent experiments, with samples analysed in triplicate. Data are presented as a fold increase in NEP gene expression, relative to the undifferentiated SGBS cells (day 0) which was readily detectable (CT value approximately 25). B: NEP protein expression was measured using a semiquantitative western blot approach. Data are presented as a fold increase in mean ± SEM (n=3) intensity values of NEP, relative to the undifferentiated SGBS cells (day 0). C: Gene expression of NEP, adiponectin, retinol binding protein-4 (RBP4) and PAI-1 in 14 day differentiated SGBS cells determined by the Human Genome U133 Plus 2.0 array. Data from nine arrays are represented as mean ± SEM of the corresponding intensity signals derived by the GeneChip Operating Software. D: Western blot showing the increase in NEP protein concentration over the 14d differentiation period.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3040694&req=5

Figure 3: NEP expression in the human adipocyte SGBS cells during differentiationTotal RNA and total protein were extracted from differentiating SGBS cells over a 0-14 day period every 2 days, starting at day 0. A: NEP gene expression was measured using semiquantitative real time PCR, with simultaneous amplification of GAPDH as an internal control allowing normalisation of target gene expression. Data represents the mean values observed in two independent experiments, with samples analysed in triplicate. Data are presented as a fold increase in NEP gene expression, relative to the undifferentiated SGBS cells (day 0) which was readily detectable (CT value approximately 25). B: NEP protein expression was measured using a semiquantitative western blot approach. Data are presented as a fold increase in mean ± SEM (n=3) intensity values of NEP, relative to the undifferentiated SGBS cells (day 0). C: Gene expression of NEP, adiponectin, retinol binding protein-4 (RBP4) and PAI-1 in 14 day differentiated SGBS cells determined by the Human Genome U133 Plus 2.0 array. Data from nine arrays are represented as mean ± SEM of the corresponding intensity signals derived by the GeneChip Operating Software. D: Western blot showing the increase in NEP protein concentration over the 14d differentiation period.
Mentions: As shown in Figure 3A, human SGBS pre-adipocytes expressed NEP mRNA (CT value approximately 25), and this was increased ~25-30-fold, during adipocyte differentiation. At the protein level, NEP was similarly increased when human SGBS preadipocytes differentiated into mature adipocytes (Figure 3B). To examine the expression levels of SGBS adipocyte-derived NEP, Affymetrix human 133 plus 2 gene chip arrays (47,400 transcripts) of differentiating SGBS cells indicated expression of NEP mRNA at levels comparable to that of known adipokines such as adiponectin, retinol binding protein 4 and PAI-1 (Figure 3C) and at least five times higher than the average gene signal.

Bottom Line: In a murine model of diet-induced insulin resistance, plasma NEP levels were significantly higher in high-fat diet (HFD)-fed compared with normal chow diet (NCD)-fed animals (1642 ± 529 and 820 ± 487 pg μl(-1), respectively; P<0.01).Tissue NEP was increased in mesenteric fat in HFD compared with NCD-fed mice (P<0.05).NEP knockout mice did not display any changes in insulin resistance, glucose tolerance, or body and epididymal fat pad weight compared with wild-type mice.

View Article: PubMed Central - PubMed

Affiliation: Division of Cardiovascular and Diabetes Research, Leeds Institute of Genetics, Health and Therapeutics, University of Leeds, Leeds, UK.

ABSTRACT

Objective: Neprilysin (NEP), a zinc metalloendopeptidase, has a role in blood pressure control and lipid metabolism. The present study tested the hypothesis that NEP is associated with insulin resistance and features of the metabolic syndrome (MetS) in a study of 318 healthy human subjects and in murine obesity, and investigated NEP production by adipocytes in-vitro.

Methods and results: In 318 white European males, plasma NEP was elevated in the MetS and increased progressively with increasing MetS components. Plasma NEP activity correlated with insulin, homoeostasis model assessment and body mass index (BMI) in all subjects (P<0.01). Quantitative reverse transcriptase PCR (RT-PCR) and western blotting showed that in human pre-adipocytes NEP expression is upregulated 25- to 30-fold during differentiation into adipocytes. Microarray analysis of mRNA from differentiated human adipocytes confirmed high-NEP expression comparable with adiponectin and plasminogen activator inhibitor-1. In a murine model of diet-induced insulin resistance, plasma NEP levels were significantly higher in high-fat diet (HFD)-fed compared with normal chow diet (NCD)-fed animals (1642 ± 529 and 820 ± 487 pg μl(-1), respectively; P<0.01). Tissue NEP was increased in mesenteric fat in HFD compared with NCD-fed mice (P<0.05). NEP knockout mice did not display any changes in insulin resistance, glucose tolerance, or body and epididymal fat pad weight compared with wild-type mice.

Conclusion: In humans, NEP activity correlated with BMI and measures of insulin resistance with increasing levels in subjects with multiple cardiovascular risk factors. NEP protein production in human adipocytes increased during cell differentiation and plasma and adipose tissue levels of NEP were increased in obese insulin-resistant mice. Our results indicate that NEP associates with cardiometabolic risk in the presence of insulin resistance and increases with obesity.

Show MeSH
Related in: MedlinePlus