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Semicarbazide sensitive amine oxidase overexpression has dual consequences: insulin mimicry and diabetes-like complications ¹

CRAIG M. STOLEN², RAMI MADANAT, LUC MARTI^*, SEPPO KARI, GENNADY G. YEGUTKIN, HANNU SARIOLA, ANTONIO ZORZANO^* and SIRPA JALKANEN

MediCity Research Laboratory, University of Turku and National Public Health Institute, Turku, Finland;
^* Departament de Bioquímica I Biogia Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain; and
Developmental Biology Research Program, Institute of Biotechnology, University of Helsinki, Finland

²Correspondence: MediCity Research Laboratory, University of Turku, Tykistökatu 6A, 20520 Turku, Finland. E-mail: craig.stolen{at}utu.fi

SPECIFIC AIMS

This work investigates the in vivo significance of elevated semicarbazide-sensitive amine oxidation (SSAO). To investigate the insulin mimicking capacity of SSAO and test its ability to cause and/or exacerbate vascular complications, we overexpressed vascular adhesion protein-1 (VAP-1), an endothelial cell surface and soluble molecule possessing SSAO activity, in transgenic mice, then chronically challenged the mice for 15 months with additional SSAO substrate or an atherogenic diet.

PRINCIPAL FINDINGS

1. Chronic human VAP-1 overexpression promotes obesity
Despite a decreased caloric intake the transgenic mice had increased weight, body mass index (BMI), and subcutaneous abdominal and epididymal white adipose tissue (WAT) deposits compared with controls.

2. Blood glucose is regulated by SSAO activity
The combination of the transgene and methylamine enhanced glucose uptake when the mice were fasted and challenged with glucose (Fig. 1 A). This increase could be blocked with a small molecule inhibitor of SSAO, BTT 2042 (Fig. 1B ). The fasting glucose levels were decreased by the transgene and HbA1c levels were decreased by the transgene and/or methylamine supplementation (Fig. 1C ). This amelioration of plasma glucose and glucose tolerance can be explained, at least in part, by improved insulin responsiveness of the skeletal muscle (Fig. 1D ).

View larger version (40K): [in this window] [in a new window]   Figure 1. Human VAP-1 overexpression and methylamine supplementation enhance glucose tolerance. The increases in blood glucose over fasting blood glucose levels at 30, 60, 90, and 120 min after glucose challenge (2.0 g IP glucose/kg body weight) are expressed as mean ± SE. A) Glucose challenge of 26-wk-old nontransgenic (open symbols) and mTIEhVAP-1 transgenic mice (filled symbols) with normal tap water (solid lines) or with methylamine supplemented water (dashed lines). Significant effects on the rate of glucose clearance were identified by comparing the areas under the curve (AUC) of individual mice with an ANOVA (methylamine, P=0.0002 and methylamine/transgene interaction, P=0.046) and by comparing glucose levels at individual time points with unpaired Student’s t tests (*P<0.01 vs. nontransgenic mice with normal water; P<0.02 vs. nontransgenic mice with methylamine). B) Glucose challenge of 10- to 12-wk-old transgenic mice with normal tap water (solid line) followed by 16 days of treatment with methylamine supplemented water (squares and dashed lines), and methylamine supplemented water as well as SSAO inhibitor, BTT 2042, injections (triangles and dashed lines). Significant effects were identified with paired Student’s t tests at individual time points (*P<0.01, P=0.037 vs. transgenic mice with normal water) and for the AUC (normal water vs. methylamine-treated, P=0.006; methylamine-treated vs. methylamine-treated + BTT 2042, P=0.0005; normal water vs. methylamine-treated + BTT 2042, P=0.0008). C) The mean ± SE % of glycosylated hemoglobin (HbA1c) was determined from the blood of fasted, 57- to 59-wk-old nontransgenic (white bars) and transgenic (black bars) mice with (gray strips) or without oral methylamine supplementation. Significant effects on HbA1c levels were found with a mixed model ANOVA (transgene, P=0.03 and methylamine supplementation P<0.0001), and a transgene/methylamine interaction was identified (P=0.01). *Unpaired Student’s t tests, P < 0.01 vs. nontransgenic mice with normal food and drink. D) Glucose transport activity of muscles from nontransgenic and mTIEhVAP-1 transgenic mice. The soleus muscles from nontransgenic (NT, white bars) and transgenic (TG, black bars) mice with (gray strips) or without oral methylamine supplementation for 14 days were used for measurement of basal and insulin-stimulated rates of 2-[1-14C]-deoxy-D-glucose transport. Values are represented as the mean ± SE of the fold increase in transport compared with basal transport in nontransgenic mice with no treatment. *P < 0.05 vs. basal nontransgenic samples. Significant difference compared with nontransgenic mice with insulin alone (P=0.02) or nontransgenic mice with insulin + methylamine (P=0.01).

3. SSAO overexpression increases advanced glycation end product (AGE) formation
AGE-peptide levels were measured by fluorescence spectroscopy in the sera of the 64-wk-old mice. The level of AGE-specific fluorescence (ex 360, em 465) was increased in the presence of the transgene (Fig. 2 ). The fluorescence from tryptophan (ex 280, em 333), an internal standard of protein concentration, did not significantly vary between the groups.

View larger version (22K): [in this window] [in a new window]   Figure 2. A) The SSAO reaction product, formaldehyde, promotes AGE formation in vitro. The AGE-specific fluorescence spectra (ex 360/em 465) of protein samples (20 mg/mL RNase) incubated at 37°C with or without 0.5 M glucose and 0.008% formaldehyde was periodically measured for 11 days. AU, arbitrary units. B) The mTIEhVAP-1 transgene increases AGE-peptide levels in the sera of 64-wk-old mice. The AGE-specific fluorescence spectra (ex 360/em 465) of nontransgenic (NT, open bars) and transgenic (TG, solid bars) mice after chronic feeding with normal food (chow) and water (tap), methylamine supplemented water (Methyl.), or an atherogenic diet (athero.). A significant increase in AGE-specific fluorescence was found with the transgene when analyzing all of the treatments together (ANOVA, transgene effect; P=0.05) and when analyzing the mice with just normal food and water (*unpaired Student’s t test, P=0.05). AU, arbitrary units.

4. Methylamine supplementation promotes hypertension
Systolic arterial blood pressure and heart rate were measured in all of the groups using the tail cuff method. Methylamine supplementation significantly elevated blood pressure but did not affect heart rate. A trend for elevated blood pressure was also found with the transgene alone.

5. Methylamine supplementation and the mTIEhVAP-1 transgene modify the progression of atherosclerosis
The percentage of aorta surface area positive for Oil Red O staining was determined for the mice in each group. Methylamine supplementation increased the percentage of lesion formation compared with untreated controls. The transgene decreased the number of lesions found in each aorta while leaving the total area of lesion formation unchanged suggesting that individual lesions are larger.

6. The mTIEhVAP-1 transgene inhibits renal and glomerular hypertrophy while at the same time promoting glomerulosclerosis
Glomerulosclerosis is the main renal lesion in human and experimental diabetes. Its pathogenesis is controversial and the role of renal and glomerular hypertrophy in the pathogenesis of glomerulosclerosis is unclear. In this study, the two renal pathologies were uncoupled.

Total kidney mass was decreased by the transgene. An atherogenic diet induced increase in kidney mass was also inhibited by the transgene. Morphometric analysis further revealed a significant transgene specific reduction of total glomeruli surface areas and an inhibition of atherogenic diet induced glomerular hypertrophy.

In contrast, histological analysis of kidney sections revealed glomerulosclerosis (decreased capillary space, increased cell number, increased extracellular matrix, and a thicker glomerular stalk) in transgenic mice fed the atherogenic diet. Mild glomerular lesions were also found in nontransgenic mice fed the atherogenic diet but to a lesser extent than in the transgenic mice. In addition, occasional glomerular cysts were present in the transgenic mice fed the atherogenic diet but were not present in the similarly fed nontransgenic mice.

CONCLUSIONS AND SIGNIFICANCE

Earlier work has shown that SSAO activity is elevated in the serum of patients with diabetes, congestive heart failure, and specific inflammatory liver diseases. In this comprehensive study we for the first time show in vivo data indicating that this elevation of SSAO is not just a benign byproduct of the various disease states but that it is a cause of physiological and pathological changes. By performing these experiments in transgenic mice we reveal the consequences of increased SSAO without the additional complications of the disease states in which elevated SSAO is normally found.

We suggest that these changes result from the end products of SSAO substrate metabolism: hydrogen peroxide, ammonia, and aldehyde. On the one hand hydrogen peroxide can produce insulin-like effects that may be important for both obesity (lipogenesis promotion and lipolysis inhibition) and diabetes (increased glucose uptake) and on the other hand hydrogen peroxide is a key reactive oxygen species (ROS) that is implicated in endothelial cell toxicity and cardiovascular pathology. In addition, ammonia and formaldehyde are extremely reactive and cytotoxic chemicals that can promote aberrant, nonenzymatic glycation of proteins and may either directly or subsequently contribute to the late complications of diabetes such as hypertension, atherosclerosis, and nephropathy.

Although these findings appear diametrically opposed they are not mutually exclusive. We propose that VAP-1/SSAO may be increased in diabetes as an attempt to regulate blood glucose levels and that vascular damage is subsequently promoted as a secondary consequence of this chronic SSAO activity (Fig. 3 ). This suggests that while manipulation of VAP-1/SSAO has potential to serve as a therapeutic treatment in insulin-resistant conditions, care must be taken to fully understand its impact on obesity and vascular damage.

View larger version (27K): [in this window] [in a new window]   Figure 3. Schematic diagram. Potential roles of VAP-1/SSAO in diabetes. We propose that SSAO activity is increased in diabetes as an attempt to regulate blood glucose levels and that vascular complications are subsequently promoted as a secondary consequence of chronic SSAO activity. When in vivo levels of VAP-1 are increased, the potential for SSAO activity is increased. The deamination of primary amines, such as methylamine, by VAP-1/SSAO produces the biologically active compounds hydrogen peroxide, ammonia, and aldehyde. These compounds in turn can produce beneficial insulin-like effects while at the same time promoting AGE formation and vascular damage that is typical of diabetes.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0562fje; doi: 10.1096/fj.03-0562fje

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