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Angiotensin-converting enzyme 2 (ACE2) is present in the heart and thought to exert protective functions. We conducted studies in ACE2 deficient mice to determine whether enzyme loss would exacerbate the cardiac and vascular pathological responses to chronic subcutaneous (sc) angiotensin II (Ang II) infusion. Eight-week-old male ACE2 knockout (KO) and wild type (WT) mice were infused with Ang II (1000 ng/kg per min, 4 weeks) using mini-osmotic pumps. Blood pressure (radiotelemetry), cardiac function (echocardiography, echo), cardiac/aortic structure (histology, collagen, and oxidative stress), and vascular inflammation were examined. Before Ang II infusion, ACE2 KO mice showed unaltered cardiac function and blood pressure. After 4 weeks of Ang II infusion, the mean arterial pressure (MAP) increased from 96 ± 2 to 136 ± 17 mm Hg (∼40%) in WT and from 104 ± 5 to 141 ± 13 mm Hg (∼ 35%) in ACE2 KO. While there were no differences in MAP between groups, the ACE2 KO responded differently to the hypertensive stimulus. Echo analysis revealed severe myocardial dysfunction in Ang II-infused ACE2 KO (Ang ACE2 KO). Ejection fraction was lower (39% versus 50%) as was fractional shortening (27% versus 38%) in ACE2 KO versus WT, respectively. Cardiac dysfunction was associated with hypertrophic cardiomyopathy shown by increased left-ventricular wall thickness, average cardiomyocyte cross-sectional area, and heart weight/body weight ratio. Collagen staining in the myocardium and aorta revealed increased collagen in Ang ACE2 KO, suggestive of remodeling. Results also showed enhanced oxidative stress in the myocardium and aorta of Ang ACE2 KO. There was a 3-fold elevation in macrophage inflammatory protein 1α (MIP 1α) in the aorta of ACE2 KO. Studies in the ACE2 KO model reveal the importance of ACE2 in the maladaptive cardiac and aortic responses to Ang II stimulation, seen as enhanced remodeling using physiological, structural, and biochemical markers. Results document a cardio- and vascular-protective role of ACE2 under pathological conditions. [ABSTRACT FROM AUTHOR]

Given the growing interest in the role of zinc in the onset and progression of diseases, there is a crucial demand for reliable methods to modulate zinc homeostasis. Using a dietary approach, we provide validated strategies to alter whole-body zinc in mice, applicable across species. For confirmation of zinc status, animal growth rates as well as plasma and urine zinc levels were evaluated. The accessible and cost-effective methodology outlined will increase scientific rigor, ensuring reproducibility in studies exploring the impact of zinc deficiency and repletion on the onset and progression of diseases. NEW & NOTEWORTHY This methods paper details dietary approaches to alter zinc homeostasis in rodents and qualitative and quantitative methods to ensure the zinc status of experimental animals. The outlined accessible and cost-effective protocol will elevate scientific rigor, ensuring reproducibility in studies exploring the impact of zinc deficiency and repletion on the onset and progression of a multitude of health conditions and diseases.

Aims/hypothesis: Inflammation has a major role in diabetic kidney disease. We thus investigated the role of the IL-8-CXCR1/2 axis in favoring kidney damage in diabetes., Methods: Urinary IL-8 levels were measured in 1247 patients of the Joslin Kidney Study in type 2 diabetes (T2D). The expression of IL-8 and of its membrane receptors CXCR1/CXCR2 was quantified in kidney tissues in patients with T2D and in controls. The effect of CXCR1/2 blockade on diabetic kidney disease was evaluated in db/db mice., Results: IL-8 urinary levels were increased in patients with T2D and diabetic kidney disease, with the highest urinary IL-8 levels found in the patients with the largest decline in glomerular filtration rate, with an increased albumin/creatine ratio and the worst renal outcome. Moreover, glomerular IL-8 renal expression was increased in patients with T2D, as compared to controls. High glucose elicits abundant IL-8 secretion in cultured human immortalized podocytes in vitro. Finally, in diabetic db/db mice and in podocytes in vitro, CXCR1/2 blockade mitigated albuminuria, reduced mesangial expansion, decreased podocyte apoptosis and reduced DNA damage., Conclusions/interpretation: The IL-8- CXCR1/2 axis may have a role in diabetic kidney disease by inducing podocyte damage. Indeed, targeting the IL-8-CXCR1/2 axis may reduce the burden of diabetic kidney disease., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Activation of the renin angiotensin system plays a pivotal role in the regulation of blood pressure, which is mainly attributed to the formation of angiotensin-II (Ang II). The actions of Ang II are mediated through binding to the Ang-II type 1 receptor (AT1R) which leads to increased blood pressure, fluid retention, and aldosterone secretion. In addition, Ang II is also involved in cell injury, vascular remodeling, and inflammation. The actions of Ang II could be antagonized by its conversion to the vasodilator peptide Ang (1-7), partly generated by the action of angiotensin converting enzyme 2 (ACE2) and/or neprilysin (NEP). Previous studies demonstrated increased urinary ACE2 shedding in the db / db mouse model of diabetic kidney disease. The aim of the study was to investigate whether renal and urinary ACE2 and NEP are altered in the 2K1C Goldblatt hypertensive mice. Since AT1R is highly expressed in the kidney, we also researched the effect of global deletion of AT1R on renal and urinary ACE2, NEP, and kidney injury marker (KIM-1). Hypertension and albuminuria were induced in AT1R knock out (AT1RKO) and WT mice by unilateral constriction of the renal artery of one kidney. The 24 h mean arterial blood pressure (MAP) was measured using radio-telemetry. Two weeks after 2K1C surgery, MAP and albuminuria were significantly increased in WT mice compared to AT1RKO mice. Results demonstrated a correlation between MAP and albuminuria. Unlike db / db diabetic mice, ACE2 and NEP expression and activities were significantly decreased in the clipped kidney of WT and AT1RKO compared with the contralateral kidney and sham control ( p < 0.05). There was no detectable urinary ACE2 and NEP expression and activity in 2K1C mice. KIM-1 was significantly increased in the clipped kidney of WT and AT1KO ( p < 0.05). Deletion of AT1R has no effect on the increased urinary KIM-1 excretion detected in 2K1C mice. In conclusion, renal injury in 2K1C Goldblatt mouse model is associated with loss of renal ACE2 and NEP expression and activity. Urinary KIM-1 could serve as an early indicator of acute kidney injury. Deletion of AT1R attenuates albuminuria and hypertension without affecting renal ACE2, NEP, and KIM-1 expression., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Alawi, Dhakal, Emberesh, Sawant, Hosawi, Thanekar, Grobe and Elased.)

Alteration in renin-angiotensin system (RAS) has been implicated in the pathophysiology of diabetic kidney disease (DKD). The deleterious actions of angiotensin II (Ang II) could be antagonized by the formation of Ang-(1-7), generated by the actions of angiotensin-converting enzyme 2 (ACE2) and neprilysin (NEP). NEP degrades several peptides, including natriuretic peptides, bradykinin, amyloid beta, and Ang I. Although combination of Ang II receptor and NEP inhibitor treatment benefits patients with heart failure, the role of NEP in renal pathophysiology is a matter of active research. NEP pathway is a potent enzyme in Ang I to Ang-(1-7) conversion in the kidney of ACE2-deficient mice, suggesting a renoprotective role of NEP. The aim of the study is to test the hypothesis that chronic hyperglycemia downregulates renal NEP protein expression and activity in db/db diabetic mice and treatment with rosiglitazone normalizes hyperglycemia, renal NEP expression, and attenuates albuminuria. Mice received rosiglitazone (20 mg kg -1  day -1 ) for 10 weeks. Western blot analysis, immunohistochemistry, and enzyme activity revealed a significant decrease in renal and urinary NEP expression and activity in 16-wk db/db mice compared with lean control (p < .0001). Rosiglitazone also attenuated albuminuria and increased renal and urinary NEP expressions (p < .0001). In conclusion, data support the hypothesis that diabetes decreases intrarenal NEP, which could have a pivotal role in the pathogenesis of DKD. Urinary NEP may be used as an index of intrarenal NEP status. The renoprotective effects of rosiglitazone could be mediated by upregulation of renal NEP expression and activity in db/db diabetic mice., (© 2020 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Angiotensin converting enzyme 2 (ACE2) and neprilysin (NEP) are metalloproteases that are highly expressed in the renal proximal tubules. ACE2 and NEP generate renoprotective angiotensin (1-7) from angiotensin II and angiotensin I, respectively, and therefore could have a major role in chronic kidney disease (CKD). Recent data demonstrated increased urinary ACE2 in patients with diabetes with CKD and kidney transplants. We tested the hypothesis that urinary ACE2, NEP, and a disintegrin and metalloproteinase 17 (ADAM17) are increased and could be risk predictors of CKD in patients with diabetes. ACE2, NEP, and ADAM17 were investigated in 20 nondiabetics (ND) and 40 patients with diabetes with normoalbuminuria (Dnormo), microalbuminuria (Dmicro), and macroalbuminuria (Dmacro) using ELISA, Western blot, and fluorogenic and mass spectrometric-based enzyme assays. Logistic regression model was applied to predict the risk prediction. Receiver operating characteristic curves were drawn, and prediction accuracies were calculated to explore the effectiveness of ACE2 and NEP in predicting diabetes and CKD. Results demonstrated that there is no evidence of urinary ACE2 and ADAM17 in ND subjects, but both enzymes were increased in patients with diabetes, including Dnormo. Although there was no detectable plasma ACE2 activity, there was evidence of urinary and plasma NEP in all the subjects, and urinary NEP was significantly increased in Dmicro patients. NEP and ACE2 showed significant correlations with metabolic and renal characteristics. In summary, urinary ACE2, NEP, and ADAM17 are increased in patients with diabetes and could be used as early biomarkers to predict the incidence or progression of CKD at early stages among individuals with type 2 diabetes.

Cognitive and mood impairments are common central nervous system complications of type 2 diabetes, although the neuronal mechanism(s) remains elusive. Previous studies focused mainly on neuronal inputs such as altered synaptic plasticity. Axon initial segment (AIS) is a specialized functional domain within neurons that regulates neuronal outputs. Structural changes of AIS have been implicated as a key pathophysiological event in various psychiatric and neurological disorders. Here we evaluated the structural integrity of the AIS in brains of db/db mice, an established animal model of type 2 diabetes associated with cognitive and mood impairments. We assessed the AIS before (5 weeks of age) and after (10 weeks) the development of type 2 diabetes, and after daily exercise treatment of diabetic condition. We found that the development of type 2 diabetes is associated with significant AIS shortening in both medial prefrontal cortex and hippocampus, as evident by immunostaining of the AIS structural protein βIV spectrin. AIS shortening occurs in the absence of altered neuronal and AIS protein levels. We found no change in nodes of Ranvier, another neuronal functional domain sharing a molecular organization similar to the AIS. This is the first study to identify AIS alteration in type 2 diabetes condition. Since AIS shortening is known to lower neuronal excitability, our results may provide a new avenue for understanding and treating cognitive and mood impairments in type 2 diabetes.

Genotyping of genetically-engineered mice is necessary for the effective design of breeding strategies and identification of mutant mice. This process relies on the identification of DNA markers introduced into genomic sequences of mice, a task usually performed using the polymerase chain reaction (PCR). Clearly, the limiting step in genotyping is isolating pure genomic DNA. Isolation of mouse DNA for genotyping typically involves painful procedures such as tail snip, digit removal, or ear punch. Although the harvesting of hair has previously been proposed as a source of genomic DNA, there has been a perceived complication and reluctance to use this non-painful technique because of low DNA yields and fear of contamination. In this study we developed a simple, economic, and efficient strategy using Chelex® resins to purify genomic DNA from hair roots of mice which are suitable for genotyping. Upon comparison with standard DNA purification methods using a commercially available kit, we demonstrate that Chelex® efficiently and consistently purifies high-quality DNA from hair roots, minimizing pain, shortening time and reducing costs associated with the determination of accurate genotypes. Therefore, the use of hair roots combined with Chelex® is a reliable and more humane alternative for DNA genotyping.

The renin angiotensin system (RAS) is a highly complex enzymatic system consisting of multiple peptide hormones, enzymes, and receptors. A thorough characterization of angiotensin peptide metabolism is crucial for understanding pathological states associated with an imbalanced RAS. Here, we describe two matrix-assisted laser desorption/ionization (MALDI) mass spectrometric (MS) approaches for the assessment of in vitro and in situ RAS enzymatic activities in the kidney using the natural angiotensin peptide substrates. These MS techniques demonstrate high specificity and are superior over conventional spectrophotometric or colorimetric assays since multiple proteolytic cleavage sites can be detected, thus unraveling the complexity of the RAS.

Cardiovascular and renal pathologies are frequently associated with an activated renin-angiotensin-system (RAS) and increased levels of its main effector and vasoconstrictor hormone angiotensin II (Ang II). Angiotensin-converting-enzyme-2 (ACE2) has been described as a crucial enzymatic player in shifting the RAS towards its so-called alternative vasodilative and reno-protective axis by enzymatically converting Ang II to angiotensin-(1-7) (Ang-(1-7)). Yet, the relative contribution of ACE2 to Ang-(1-7) formation in vivo has not been elucidated. Mass spectrometry based quantification of angiotensin metabolites in the kidney and plasma of ACE2 KO mice surprisingly revealed an increase in Ang-(1-7), suggesting additional pathways to be responsible for alternative RAS activation in vivo. Following assessment of angiotensin metabolism in kidney homogenates, we identified neprilysin (NEP) to be a major source of renal Ang-(1-7) in mice and humans. These findings were supported by MALDI imaging, showing NEP mediated Ang-(1-7) formation in whole kidney cryo-sections in mice. Finally, pharmacologic inhibition of NEP resulted in strongly decreased Ang-(1-7) levels in murine kidneys. This unexpected new role of NEP may have implications for the combination therapy with NEP-inhibitors and angiotensin-receptor-blockade, which has been shown being a promising therapeutic approach for heart failure therapy.

The renin angiotensin system (RAS) plays a vital role in the regulation of the cardiovascular and renal functions. COS7 is a robust and easily transfectable cell line derived from the kidney of the African green monkey, Cercopithecus aethiops. The aims of this study were to 1) demonstrate the presence of an endogenous and functional RAS in COS7, and 2) investigate the role of a disintegrin and metalloproteinase-17 (ADAM17) in the ectodomain shedding of angiotensin converting enzyme-2 (ACE2). Reverse transcription coupled to gene-specific polymerase chain reaction demonstrated expression of ACE, ACE2, angiotensin II type 1 receptor (AT1R), and renin at the transcript levels in total RNA cell extracts. Western blot and immunohistochemistry identified ACE (60 kDa), ACE2 (75 kDa), AT1R (43 kDa), renin (41 kDa), and ADAM17 (130 kDa) in COS7. At the functional level, a sensitive and selective mass spectrometric approach detected endogenous renin, ACE, and ACE2 activities. ANG-(1-7) formation (m/z 899) from the natural substrate ANG II (m/z 1,046) was detected in lysates and media. COS7 cells stably expressing shRNA constructs directed against endogenous ADAM17 showed reduced ACE2 shedding into the media. This is the first study demonstrating endogenous expression of the RAS and ADAM17 in the widely used COS7 cell line and its utility to study ectodomain shedding of ACE2 mediated by ADAM17 in vitro. The transfectable nature of this cell line makes it an attractive cell model for studying the molecular, functional, and pharmacological properties of the renal RAS., (Copyright © 2015 the American Physiological Society.)

Mood disorder patients that are on long-term atypical antipsychotics treatment frequently experience metabolic dysfunctions. In addition to this, accumulating evidences points to increased risk of structural abnormalities, brain volume changes, altered neuroplasticity and behavioral depression with long-term antipsychotics use. However, there is paucity of preclinical evidences for long-term antipsychotic associated depression-like behavior. The objectives of the present study were: (1) to evaluate influence of long-term antipsychotic (olanzapine) treatment on rat behavior in forced swim test (FST) as a model for depression and; (2) to examine impact of glucagon-like peptide 1 (GLP-1) receptor agonist liraglutide - an antidiabetic medication for type II diabetes, on long-term olanzapine associated metabolic and behavioral changes in rats. Daily olanzapine treatment (0.5 mg/kg; p.o.) for 8-9 weeks significantly increased body weights, food and water intake, plasma cholesterol and triglycerides and immobility time in FST with parallel reduction in plasma HDL cholesterol levels. These results points to development of metabolic abnormalities and depression-like behavior with long-term olanzapine treatment. Acute liraglutide (50 μg/kg; i.p.) and imipramine (10 mg/kg, i. p.) treatment per se significantly decreased duration of immobility in FST compared to vehicle treated rats. Additionally, 3-week liraglutide treatment (50 μg/kg; i.p., daily) partially reversed metabolic abnormalities and depression-like behavior with long-term olanzapine-treatment in rats. None of these treatment regimens affected locomotor behavior of rats. In summary, add-on GLP-1 receptor agonists promise novel alternatives to counteract long-term antipsychotics associated behavioral and metabolic complications.

It is well documented that angiotensin (Ang) II contributes to kidney disease progression. The protease prolyl carboxypeptidase (PRCP) is highly expressed in the kidney and may be renoprotective by degrading Ang II to Ang-(1-7). The aim of the study was to investigate whether renal PRCP protein expression and activity are altered in two-kidney, one-clip (2K1C) Goldblatt hypertensive mice. Left renal artery was constricted by using 0.12 mm silver clips. Blood pressure was measured using telemetry over the eleven weeks of study period and revealed an immediate increase in 2K1C animals during the first week of clip placement which was followed by a gradual decrease to baseline blood pressure. Similarly, urinary albumin excretion was significantly increased one week after 2K1C and returned to baseline levels during the following weeks. At 2 weeks and at the end of the study, renal pathologies were exacerbated in the 2K1C model as revealed by a significant increase in mesangial expansion and renal fibrosis. Renal PRCP expression and activity were significantly reduced in clipped kidneys. Immunofluorescence revealed the loss of renal tubular PRCP but not glomerular PRCP. In contrast, expression of prolyl endopeptidase, another enzyme capable of converting Ang II into Ang-(1-7), was not affected, while angiotensin converting enzyme was elevated in unclipped kidneys and renin was increased in clipped kidneys. Results suggest that PRCP is suppressed in 2K1C and that this downregulation may attenuate renoprotective effects via impaired Ang II degradation by PRCP.

Foot ulcers are a severe complication of diabetic patients resulting from nerve and tendon pathologic alterations. In diabetic patients the tendons are thicker, shorter and have increased stiffness. We examined C57BL/KsJ (BKS.Cg-Dock7(m) +/+ Lepr (db) /J) (db/db) mice tendons to determine whether they are an animal model for human diabetic tendon changes. We hypothesized that the Achilles tendons of db/db diabetic mice would be thicker, stiffer, fail at lower loads and stresses, and have degenerative changes compared to control mice. Biomechanical and histologic analyses of the Achilles tendons of 16 week old db/db and control male mice were performed. There was a significant increase in tendon diameter and significant decreases in maximum load, tensile stress, stiffness and elastic modulus in tendons from diabetic mice compared to controls. Mild degenerative and neutrophil infiltration was observed near the tendon insertions on the calcaneous in 25% of db/db mice. In summary, hyper-glycemia and obesity lead to severe changes in db/db mice will be a useful model to examine mechanisms for tendon alterations.

Angiotensin II (Ang II) is involved in induction and progression of renal damage in diabetes. Angiotensin converting enzyme 2 (ACE2) is highly expressed in the kidney and has been shown to be renoprotective by degrading Ang II to Ang-(1-7). A disintegrin and metalloproteinase 17 (ADAM17)-mediated shedding of renal ACE2 contribute to diabetic nephropathy pathogenesis. Lifestyle modification and metformin are recommended as initial therapies for most patients with type 2 diabetes. The aim of this study was to investigate whether exercise training and/or metformin improve glucose homeostasis and albuminuria and downregulate renal ADAM17 and ACE2 shedding in db/db mice. Seven-week-old normal and db/db mice were subjected either to a sedentary existence or exercise training with and without metformin (150 mg/kg per day) for 10 weeks. Exercise training significantly lowered blood glucose, urinary albumin and ACE2 excretion in db/db mice. ADAM17 and ACE2 proteins were co-localized in cortical tubules of the kidney, indicating a possible interaction. Metformin treatment was effective in lowering hyperglycemia only during the first 2 weeks of treatment. Increased renal ADAM17 in 17-week-old db/db mice was corrected by physical exercise but not metformin. In addition, exercise training reduced plasma triglycerides and enhanced insulin levels of db/db mice. In conclusion, exercise training alone and in combination with metformin prevented shedding of renal ACE2 by decreasing ADAM17 protein. Urinary ACE2 could serve as a prognostic tool for the progression of kidney damage and its attenuation by exercise may partially contribute to its renal protection.

Aminopeptidase-A (APA) is a less well-studied enzyme of the renin-angiotensin system. We propose that it is involved in cardiac angiotensin (ANG) metabolism and its pathologies. ANG-(1-7) can ameliorate remodeling after myocardial injury. The aims of this study are to (1) develop mass spectrometric (MS) approaches for the assessment of ANG processing by APA within the myocardium; and (2) investigate the role of APA in cardiac ANG-(1-7) metabolism after myocardial infarction (MI) using sensitive MS techniques. MI was induced in C57Bl/6 male mice by ligating the left anterior descending (LAD) artery. Frozen mouse heart sections (in situ assay) or myocardial homogenates (in vitro assay) were incubated with the endogenous APA substrate, ANG II. Results showed concentration- and time-dependent cardiac formation of ANG III from ANG II, which was inhibited by the specific APA inhibitor, 4-amino-4-phosphonobutyric acid. Myocardial APA activity was significantly increased 24 h after LAD ligation (0.82 ± 0.02 vs. 0.32 ± 0.02 ρmol·min(-1)·μg(-1), MI vs. sham, P < 0.01). Both MS enzyme assays identified the presence of a new peptide, ANG-(2-7), m/z 784, which accumulated in the MI (146.45 ± 6.4 vs. 72.96 ± 7.0%, MI vs. sham, P < 0.05). Use of recombinant APA enzyme revealed that APA is responsible for ANG-(2-7) formation from ANG-(1-7). APA exhibited similar substrate affinity for ANG-(1-7) compared with ANG II {Km (ANG II) = 14.67 ± 1.6 vs. Km [ANG-(1-7)] = 6.07 ± 1.12 μmol/l, P < 0.05}. Results demonstrate a novel role of APA in ANG-(1-7) metabolism and suggest that the upregulation of APA, which occurs after MI, may deprive the heart of cardioprotective ANG-(1-7). Thus APA may serve as a potentially novel therapeutic target for management of tissue remodeling after MI.

Angiotensin-converting enzyme 2 (ACE2) is located in several tissues and is highly expressed in renal proximal tubules, where it degrades the vasoconstrictor angiotensin II (ANG II) to ANG-(1-7). Accumulating evidence supports protective roles of ACE2 in several disease states, including diabetic nephropathy. A disintegrin and metalloprotease (ADAM) 17 is involved in the shedding of several transmembrane proteins, including ACE2. Our previous studies showed increased renal ACE2, ADAM17 expression, and urinary ACE2 in type 2 diabetic mice (Chodavarapu H, Grobe N, Somineni HK, Salem ES, Madhu M, Elased KM. PLoS One 8: e62833, 2013). The aim of the present study was to determine the effect of insulin on ACE2 shedding and ADAM17 in type 1 diabetic Akita mice. Results demonstrate increased renal ACE2 and ADAM17 expression and increased urinary ACE2 fragments (≈70 kDa) and albumin excretion in diabetic Akita mice. Immunostaining revealed colocalization of ACE2 with ADAM17 in renal tubules. Renal proximal tubular cells treated with ADAM17 inhibitor showed reduced ACE2 shedding into the media, confirming ADAM17-mediated shedding of ACE2. Treatment of Akita mice with insulin implants for 20 wk normalized hyperglycemia and decreased urinary ACE2 and albumin excretion. Insulin also normalized renal ACE2 and ADAM17 but had no effect on tissue inhibitor of metalloproteinase 3 (TIMP3) protein expression. There was a positive linear correlation between urinary ACE2 and albuminuria, blood glucose, plasma creatinine, glucagon, and triglycerides. This is the first report showing an association between hyperglycemia, cardiovascular risk factors, and increased shedding of urinary ACE2 in diabetic Akita mice. Urinary ACE2 could be used as a biomarker for diabetic nephropathy and as an index of intrarenal ACE2 status.

The db/db mouse is a widely used preclinical model in diabetes research. Recent studies have shown that these mice also display aspects of psychosis and depression-like behaviors as seen in some psychiatric disorders. Here, we have performed multiplex immunoassay and liquid chromatography mass spectrometry profiling of the plasma and brain samples from db/db and control mice to identify altered pathways, which could be related to these behavioral abnormalities. This is the first study to carry out profiling of the brain proteome in this model. Plasma from the db/db mice had increased levels of leptin and insulin, decreased levels of peptide YY, glucagon and prolactin and alterations in inflammation-related proteins, compared with control mice. Frontal cortex tissue from the db/db mice showed changes in proteins involved in energy metabolism, cellular structure and neural functioning, and the hippocampus had changes in proteins involved in the same pathways, with additional effects on cellular signalling proteins. The overlap of these findings with effects seen in type 2 diabetes, schizophrenia, major depressive disorder and Alzheimer's disease might contribute to a common endophenotype seen in metabolic and neurological disorders.

Angiotensin-converting enzyme 2 (ACE2) catalyzes conversion of ANG II to ANG-(1-7). The present study uses newly established proteomic approaches and genetic mouse models to examine the contribution of alternative renal peptidases to ACE2-independent formation of ANG-(1-7). In situ and in vitro mass spectrometric characterization showed that substrate concentration and pH control renal ANG II processing. At pH ≥6, ANG-(1-7) formation was significantly reduced in ACE2 knockout (KO) mice. However, at pH <6, formation of ANG-(1-7) in ACE2 KO mice was similar to that in wild-type (WT) mice, suggesting alternative peptidases for renal ANG II processing. Furthermore, the dual prolyl carboxypeptidase (PCP)-prolyl endopeptidase (PEP) inhibitor Z-prolyl-prolinal reduced ANG-(1-7) formation in ACE2 KO mice, while the ACE2 inhibitor MLN-4760 had no effect. Unlike the ACE2 KO mice, ANG-(1-7) formation from ANG II in PEP KO mice was not different from that in WT mice at any tested pH. However, at pH 5, this reaction was significantly reduced in kidneys and urine of PCP-depleted mice. In conclusion, results suggest that ACE2 metabolizes ANG II in the kidney at neutral and basic pH, while PCP catalyzes the same reaction at acidic pH. This is the first report demonstrating that renal ANG-(1-7) formation from ANG II is independent of ACE2. Elucidation of ACE2-independent ANG-(1-7) production pathways may have clinically important implications in patients with metabolic and renal disease.

Alterations within the renal renin angiotensin system play a pivotal role in the development and progression of cardiovascular and renal disease. Angiotensin converting enzyme 2 (ACE2) is highly expressed in renal tubules and has been shown to be renoprotective in diabetes. The protease, a disintegrin and metalloprotease (ADAM) 17, is involved in the ectodomain shedding of several transmembrane proteins including ACE2. Renal ACE2 and ADAM17 were significantly increased in db/db mice compared to controls. We investigated the effect of the insulin sensitizer, rosiglitazone, on albuminuria, renal ADAM17 protein expression and ACE2 shedding in db/db diabetic mice. Rosiglitazone treatment of db/db mice normalized hyperglycemia, attenuated renal injury and decreased urinary ACE2 and renal ADAM17 protein expression. Urinary excreted ACE2 is enzymatically active. Western blot analysis of urinary ACE2 demonstrated two prominent immunoreactive bands at approximately 70 & 90 kDa. The predominant immunoreactive band is approximately 20 kDa shorter than the one demonstrated for kidney lysate, indicating possible ectodomain shedding of active renal ACE2 in the urine. Therefore, it is tempting to speculate that renoprotection of rosiglitazone could be partially mediated via downregulation of renal ADAM17 and ACE2 shedding. In addition, there was a positive correlation between blood glucose, urinary albumin, plasma glucagon, and triglyceride levels with urinary ACE2 excretion. In conclusion, urinary ACE2 could be used as a sensitive biomarker of diabetic nephropathy and for monitoring the effectiveness of renoprotective medication.

Recent studies indicate a high comorbidity between type-2 diabetes mellitus (T2DM) and neurological disorders. Many are associated with abnormalities in dopamine neurotransmission such as schizophrenia. Because most of the antipsychotic drugs aggravate pre-existing insulin resistance in type-2 diabetics, there is a need to search for alternative antipsychotics. Glucagon like peptide-1 (GLP-1) is a gut hormone primarily involved in glucose homeostasis. GLP-1 agonist (liraglutide) and dipeptidyl peptidase-IV (DPP-IV) inhibitor (sitagliptin) are the US-FDA approved medications for the management of T2DM. However, little is known about their role in dopamine mediated neurological disorders like schizophrenia. To address this, we used apomorphine-induced cage climbing behavior as a murine model for psychosis and examined for potential antipsychotic-like effect of liraglutide and sitagliptin. While acute liraglutide treatment (50 μg/kg; i.p.) significantly attenuated apomorphine (3 mg/kg, s.c.) induced cage climbing, sitagliptin (50mg/kg; i.p.) failed to elicit such effect. This is the first preclinical evidence for antipsychotic-like effect of GLP-1 receptor agonist. These results open an opportunity to explore GLP-1 analogs for their potential to modulate spectrum of dopamine-mediated neurological disorders., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The objective of the present study was to examine the effect of long-term management of insulin resistance and hyperglycemia on neurobehavioral deficits in db/db mice. In this study, 5-week-old db/db and lean control mice were fed with rosiglitazone (20 mg/kg/day) mixed or standard chow for a duration of 5 weeks. Mice were monitored weekly for blood glucose concentration. Five weeks after the onset of treatment, they were subjected to the forced swim test (FST), pre-pulse inhibition (PPI), open field test (OFT) and fear-potentiated startle (FPS) test to examine for depression, psychosis-like behavior, locomotor activity and emotional learning, respectively. Rosiglitazone normalized hyperglycemia and improved glucose tolerance. Rosiglitazone significantly reduced immobility time in the FST in db/db mice, suggesting an antidepressant-like effect. However, rosiglitazone failed to reverse disruption of PPI in db/db mice, indicating its ineffectiveness against psychosis-like behavior. In the OFT, rosiglitazone did not affect the activity of db/db mice, suggesting its antidepressant-like effect was independent of changes in locomotor activity. In the FPS test, db/db mice showed impaired emotional learning and rosiglitazone failed to correct it. In conclusion, long-term blood glucose management in type-2 diabetics may help to limit the co-occurrence of depression but not the psychotic symptoms and ability to cope with stress.

To better understand the tissue distribution and activity of enzymes involved in angiotensin II (Ang II) processing, we developed a novel molecular imaging method using matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. Mouse kidney sections (12 μm) were incubated with 10-1,000 μmol/l Ang II for 5-15 min at 37°C. The formed peptides Ang III and Ang-(1-7) were identified by MALDI-TOF/TOF. A third metabolite, Ang-(1-4), was generated from further degradation of Ang-(1-7). Enzymatic processing of Ang II was dose and time dependent and absent in heat-treated kidney sections. Distinct spatial distribution patterns (pseudocolor images) were observed for the peptides. Ang III was localized in renal medulla, whereas Ang-(1-7)/Ang-(1-4) was present in cortex. Regional specific peptide formation was confirmed using microdissected cortical and medullary biopsies. In vitro studies with recombinant enzymes confirmed activity of peptidases known to generate Ang III or Ang-(1-7) from Ang II: aminopeptidase A (APA), Ang-converting enzyme 2 (ACE2), prolyl carboxypeptidase (PCP), and prolyl endopeptidase (PEP). Renal medullary Ang III generation was blocked by APA inhibitor glutamate phosphonate. The ACE2 inhibitor MLN-4760 and PCP/PEP inhibitor Z-pro-prolinal reduced cortical Ang-(1-7) formation. Our results establish the power of MALDI imaging as a highly specific and information-rich analytical technique that will further aid our understanding of the role and site of Ang II processing in cardiovascular and renal pathologies.

High-fructose diet is known to produce cardiovascular and metabolic pathologies. The objective was to determine whether the timing of high fructose (10% liquid solution) intake affect the metabolic and cardiovascular outcomes. Male C57BL mice with radiotelemetric probes were divided into four groups: (1) 24 h water (control); (2) 24 h fructose (F24); (3) 12 h fructose during the light phase (F12L); (4) 12 h fructose during the dark phase (F12D). All fructose groups had higher fluid intake. Body weight was increased in mice on restricted access with no difference in total caloric intake. Fasting glycemia was higher in groups with restricted access. F24 mice showed a fructose-induced blood pressure increase during the dark period. Blood pressure circadian rhythms were absent in F12L mice. Results suggest that the timing of fructose intake is an important variable in the etiology of cardiovascular and metabolic pathologies produced by high fructose consumption.

Hypotension in aryl hydrocarbon receptor knockout mice (ahr(-/-)) is mediated, in part, by a reduced contribution of angiotensin (Ang) II to basal blood pressure (BP). Since AHR is highly expressed in endothelial cells (EC), we hypothesized that EC-specific ahr(-/-) (ECahr(-/-)) mice would exhibit a similar phenotype. We generated ECahr(-/-) mice by crossing AHR floxed mice (ahr(fx/fx)) to mice expressing Cre recombinase driven by an EC-specific promoter. BP was assessed by radiotelemetry prior to and following an acute injection of Ang II or chronic treatment with an angiotensin converting enzyme inhibitor (ACEi). ECahr(-/-) mice were hypotensive (ECahr(+/+): 116.1±1.4; ECahr(-/-): 107.4±2.0 mmHg, n=11, p<0.05) and exhibited significantly different responses to Ang II and ACEi. While Ang II increased BP in both genotypes, the increase was sustained in ECahr(+/+), whereas the increase in ECahr(-/-) mice steadily declined. Area under the curve analysis showed that Ang II-induced increase in diastolic BP (DBP) over 30 min was significantly lower in ECahr(-/-) mice (ECahr(+/+) 1297±223 mmHg/30 min; ECahr(-/-)(AUC): 504±138 mmHg/30 min, p<0.05). In contrast, while ACEi decreased BP in both genotypes, the subsequent rise in DBP after treatment was significantly delayed in the ECahr(-/-) mice. ECahr(-/-) mice also exhibited reduced vascular and adipose Ang II type 1 receptor (AT1R) expression, and reduced aortic Ang II-dependent vasoconstriction in the presence of vascular adipose. Taken together these data suggest that hypotension in ECahr(-/-) mice results from reduced vascular responsiveness to Ang II that is influenced by AT1R expression and adipose., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Recent clinical studies indicate neurobehavioral disturbances in type-2 diabetics. However, there is paucity of preclinical research to support this concept. The validity of db/db mouse as an animal model to study type-2 diabetes and related complications is known. The present study was designed to investigate comprehensively the db/db mouse behavior as preclinical evidence of type-2 diabetes related major neurobehavioral complications. We tested juvenile (5-6weeks) and adult (10-11weeks) db/db mice for behavioral depression in forced swim test (FST), psychosis-like symptoms using pre-pulse inhibition (PPI) test, anxiety behavior employing elevated plus maze (EPM) test, locomotor behavior and thigmotaxis using open field test and working memory deficits in Y-maze test. Both juvenile and adult group db/db mice displayed behavioral despair with increased immobility time in FST. There was an age-dependent progression of psychosis-like symptoms with disrupted PPI in adult db/db mice. In the EPM test, db/db mice were less anxious as observed by increased percent open arms time and entries. They were also hypo-locomotive as evident by a decrease in their basic and fine movements. There was no impairment of working memory in the Y-maze test in db/db mice. This is the first report of depression, psychosis-like symptoms and anxiolytic behavior of db/db mouse strain. It is tempting to speculate that this mouse strain can serve as useful preclinical model to study type-2 diabetes related neurobehavioral complications., (Published by Elsevier Inc.)

National Health and Nutrition Examination Survey data show an association between hypertension and exposure to dioxin-like halogenated aromatic hydrocarbons (HAHs). Furthermore, chronic exposure of mice to the prototypical HAH, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), induces reactive oxygen species (ROS), endothelial dysfunction, and hypertension. Because TCDD induces cytochrome P4501A1 (CYP1A1) and CYP1A1 can increase ROS, we tested the hypothesis that TCDD-induced endothelial dysfunction and hypertension are mediated by CYP1A1. CYP1A1 wild-type (WT) and knockout (KO) mice were fed one control or TCDD-containing pill (180 ng TCDD/kg, 5 days/week) for 35 days (n = 10-14/genotype/treatment). Blood pressure was monitored by radiotelemetry, and liver TCDD concentration, CYP1A1 induction, ROS, and aortic reactivity were measured at 35 days. TCDD accumulated to similar levels in livers of both genotypes. TCDD induced CYP1A1 in endothelium of aorta and mesentery without detectable expression in the vessel wall. TCDD also induced superoxide anion production, measured by NADPH-dependent lucigenin luminescence, in aorta, heart, and kidney of CYP1A1 WT mice but not KO mice. In contrast, TCDD induced hydrogen peroxide, measured by amplex red assay, to similar levels in aorta of CYP1A1 WT and KO mice but not in heart or kidney. TCDD reduced acetylcholine-dependent vasorelaxation in aortic rings of CYP1A1 WT mice but not in KO mice. Finally, TCDD steadily increased blood pressure after 15 days, which plateaued after 25 days (+20 mmHg) in CYP1A1 WT mice but failed to alter blood pressure in KO mice. These results demonstrate that CYP1A1 is required for TCDD-induced cardiovascular superoxide anion production, endothelial dysfunction, and hypertension.

It has been postulated that fetal vascular abnormalities in aryl hydrocarbon receptor null (ahr(-/-)) mice may alter cardiovascular homeostasis in adulthood. We tested the hypothesis that blood pressure regulation in adult heterozygous mice (ahr(+/-)) would be normal, compared to ahr(-/-) mice, since no vascular abnormalities have been reported in the heterozygote animals. Mean arterial blood pressure (MAP) was measured using radiotelemetry prior to and during treatment with inhibitors of the autonomic nervous system, nitric oxide synthase (NOS), angiotensin converting enzyme (ACE), or endothelin-1 A receptor (ET(A)). Also, indices of renin-angiotensin system (RAS) activation were measured. ahr(+/-) and ahr(-/-) mice were normotensive and hypotensive, respectively, compared to wild-type (ahr(+/+)) littermates. Responses of all genotypes to autonomic nervous system inhibition were normal. ahr(+/-) mice responded normally to NOS inhibition, while the responses of ahr(-/-) mice were significantly blunted. In contrast, ahr(+/-) mice were significantly more responsive to inhibition of ACE, an ET(A) antagonist, or both, while ahr(-/-) mice were significantly less responsive to ACE inhibition and more responsive to an ET(A) antagonist. ahr(+/-) mice also exhibited significant increases in plasma renin and ACE activity, plasma sodium, and urine osmolality, indicative of RAS activation. Thus, normotension in ahr(+/-) mice appears to be maintained by increased RAS and ET-1 signaling, while hypotension in ahr(-/-) mice may result from decreased RAS signaling. In conclusion, despite the lack of overt fetal vascular abnormalities in ahr(+/-) mice, the loss of a single ahr allele has a significant effect on blood pressure regulation., (2010 Elsevier Inc. All rights reserved.)

Aim: To determine whether pharmacological blockade of angiotensin (Ang) AT1 receptors alters the cardiovascular, metabolic, and angiotensin-converting enzyme (ACE and ACE2) responses to a fructose diet in mice., Methods: C57BL male mice were fed with a 60% fructose diet for 8 weeks in combination with losartan treatment on week 9 (30 mg/kg per day). Blood pressure (BP), heart rate (HR), and autonomic balance were monitored using radiotelemetry with spectral analysis. Renal ACE and ACE2 activity and protein levels as well as Ang II and Ang 1-7 were measured., Results: Fructose impaired glucose tolerance and increased plasma cholesterol and insulin. These effects were not corrected by losartan treatment. Fructose increased BP and HR but only during the dark period. Short-term losartan treatment decreased BP by 16% in the fructose group but had no effect in controls. This was accompanied by a decrease in BP variance and its low-frequency component. Fructose increased Ang II (plasma and kidney) and ACE 2 (renal activity and protein expression). Losartan alone increased plasma Ang II in plasma and ACE2 in kidney. There were no changes in renal Ang 1-7 levels., Conclusions: Losartan reversed the pressor effect of a high fructose diet, demonstrating that there are prominent interactions between a dietary regimen that produces glucose intolerance and an antihypertensive drug that antagonizes Ang signaling. The mechanism of change may be via renal Ang II rather than the ACE2/Ang 1-7 pathway because the fructose losartan combination resulted in lowered renal Ang II without changes in Ang 1-7.

The db/db mice serve as a good model for type 2 diabetes characterized by hyperinsulinaemia and progressive hyperglycaemia. There are limited and conflicting data on the cardiovascular changes in this model. The aim of the present study was to characterize the cardiovascular and autonomic phenotype of male db/db mice and evaluate the role of angiotensin II AT(1) receptors. Radiotelemetry was used to monitor 24 h blood pressure (BP) in mice for 8 weeks. Parameters measured were mean arterial pressure (MAP), heart rate (HR) and their variabilities. In 8-week-old db/db mice, the MAP and BP circadian rhythms were not different from age-matched control mice, while HR and locomotor activity were decreased. With ageing, MAP gradually increased in db/db mice, and the 12 h light values did not dip significantly from the 12 h dark periods. In 14-week-old mice, MAP was increased during light (101 +/- 1 versus 117 +/- 2 mmHg, P < 0.01; control versus db/db mice) and dark phases (110 +/- 1.7 versus 121 +/- 3.1 mmHg, P < 0.01; control versus db/db mice). This increase in MAP was associated with a significant increase in plasma angiotensin-converting enzyme activity and angiotensin II levels. Chronic treatment with losartan (10 mg kg(-1) day(-1)) blocked the increase in MAP in db/db mice, with no effect in control animals. Spectral analysis was used to monitor autonomic cardiovascular function. The circadian rhythm observed in systolic arterial pressure variance and its low-frequency component in control mice was absent in db/db mice. There were no changes in HR variability and spontaneous baroreflex sensitivity between control and db/db mice. The results document an age-related increase in MAP in db/db mice, which can be reduced by antagonism of angiotensin II AT(1) receptors, and alterations in autonomic balance and components of the renin-angiotensin system.

Angiotensin (Ang)-converting enzyme 2 (ACE2) metabolizes Ang II to the vasodilatory peptide Ang(1-7), while neprilysin (NEP) generates Ang(1-7) from Ang I. Experiments used novel Surface Enhanced Laser Desorption Ionization-Time of Flight (SELDI-TOF) mass spectroscopic (MS) assays to study Ang processing. Mass spectroscopy was used to measure proteolytic conversion of Ang peptide substrates to their specific peptide products. We compared ACE/ACE2 activity in plasma, brain and kidney from C57BL/6 and NEP(-/-) mice. Plasma or tissue extracts were incubated with Ang I or Ang II (1296 or 1045, m/z, respectively), and generated peptides were monitored with MS. Angiotensin-converting enzyme 2 activity was detected in kidney and brain, but not in plasma. Brain ACE2 activity was highest in hypothalamus. Angiotensin-converting enzyme 2 activity was inhibited by the specific ACE2 inhibitor, DX600 (10 microm, 99% inhibition), but not by the ACE inhibitor, captopril (10 microm). Both MS and colorimetric assays showed high ACE activity in plasma and kidney with low levels in brain. To extend these findings, ACE measurements were made in ACE overexpressing mice. Angiotensin-converting enzyme four-copy mice showed higher ACE activity in kidney and plasma with low levels in hypothalamus. In hypothalamus from NEP-/- mice, generation of Ang(1-7) from Ang I was decreased, suggesting a role for NEP in Ang metabolism. With Ang II as substrate, there was no difference between NEP-/- and wild-type control mice, indicating that other enzymes may contribute to generation of Ang(1-7). The data suggest a predominant role of hypothalamic ACE2 in the processing of Ang II, in contrast to ACE, which is most active in plasma.

The renin-angiotensin system (RAS) has been implicated in the cardiovascular complications of diabetes. We showed that a high-fructose diet increases blood pressure and plasma angiotensin and impairs glucose tolerance. We investigated the role of angiotensin AT(1a) receptors in the development of fructose-induced cardiovascular and metabolic dysfunction. Male angiotensin AT(1a) knockout (AT1aKO) and wild-type (AT1aWT) mice with arterial telemetric catheters were fed a standard diet or one containing 60% fructose. Fructose increased mean arterial pressure (MAP) in AT1aWT but only during the dark phase (8% increase). In AT1aKO mice, fructose unexpectedly decreased MAP, during both light and dark periods (24 and 13% decrease, respectively). Analytical methods were used to measure systolic arterial pressure (SAP) and pulse interval (PI) variability in time and frequency domains. In fructose-fed AT1aWT mice, there was an increase in SAP variance and its low-frequency (LF) domain (11 +/- 3 vs. 23 +/- 4 mmHg(2), variance, and 7 +/- 2 vs. 17 +/- 3 mmHg(2), LF, control vs. fructose, P < 0.004). There were no changes in SAP variance in AT1aKO mice. Depressor responses to alpha(1)-adrenergic blockade were augmented in fructose-fed AT1a WT compared with AT1aKO mice. Fructose inhibited glucose tolerance with a greater effect in AT1aWT mice. Fructose increased plasma cholesterol in both groups (P < 0.01) and reduced ANG II in AT1aKO mice. Results document prominent interactions between genetics and diet with data showing that in the absence of angiotensin AT(1a) receptors, a fructose diet decreased blood pressure.

Unlabelled: Nutrition is an important variable which may affect the risk for renal disease. We previously showed that a high fructose diet in mice produced hypertension and sympathetic activation [8]. The purpose of this study was to determine if a fructose diet altered renal function. A high fructose diet for 12 weeks impaired glucose tolerance, but caused no change in body weight, blood glucose or plasma insulin. Impairment in renal function was documented by the almost two fold increase in urinary protein excretion (, Control: 6.6+/-0.6 vs. Fructose: 15.0+/-0.7 mmol protein/mmol creatinine; p<0.05) which was also accompanied by increases in urinary volume. The diet produced little change in renal histology, kidney weight or kidney weight/body weight ratio. Urinary excretion of angiotensin II/creatinine (, Control: 78.9+/-16.6 vs. Fructose: 80.5+/-14.2 pg/mmol) and renal angiotensin converting enzyme activity (, Control: 9.2+/-1.6 vs. Fructose: 7.6+/-1.0 ACE units) were not different between groups. There was a positive correlation between mean arterial pressure (r=0.7, p=0.01), blood pressure variability (BPV) (r=0.7, p=0.02), low frequency BPV component (r=0.677, p=0.03) and urinary protein excretion. Results show that consumption of a high fructose diet in mice had deleterious effects on renal function, which were correlated with cardiovascular changes.

Objective: To investigate the effect of fructose consumption on the light/dark pattern of blood pressure, heart rate and autonomic neural function in mice., Background: Insulin resistant diabetes is associated with hypertension and autonomic dysfunction. There is evidence that the increasing incidence of diabetes may be related to dietary changes, including consumption of high levels of fructose., Design/methods: C57/BL mice, instrumented with radiotelemetric arterial catheters, were fed a control or high fructose diet (60%). Cardiovascular parameters measured were light/dark pattern of mean arterial pressure (MAP), heart rate (HR) and variability (time and frequency domain). We also measured plasma insulin, glucose, lipids and angiotensin II (Ang II) as well as glucose tolerance. In situ hybridization was used to measure brainstem expression of tyrosine hydroxylase (TH) and Ang AT1a mRNA., Results: Fructose diet (8 weeks) produced an increase in MAP, variance and low frequency domain (14+/-3 vs. 33+/-4 mm Hg(2), variance and 10+/-2 vs. 26+/-4 mm Hg(2), LF, control vs. fructose, P<0.01). The changes occurred only at night, a period of activity for mice. Glucose tolerance was attenuated in the fructose group. Fructose also increased plasma cholesterol (80+/-1 vs. 126+/-2 mg/dl, control vs. fructose, P<0.05) and plasma Ang II (18+/-5 vs.65+/-12 pg/ml, control vs. fructose, P<0.05). Depressor responses to alpha(1)-adrenergic blockade with prasozin were augmented in fructose-fed mice. Using quantitative in situ hybridization, we found that Ang AT1a receptor and TH mRNA expression were significantly increased in the brainstem locus coeruleus., Conclusion: A high fructose diet in mice produced nocturnal hypertension and autonomic imbalance which may be related to activation of sympathetic and angiotensin systems.

A novel assay was developed for evaluation of mouse angiotensin-converting enzyme (ACE) 2 and recombinant human ACE2 (rACE2) activity. Using surface-enhanced laser desorption/ionization time of flight mass spectrometry (MS) with ProteinChip Array technology, ACE1 and ACE2 activity could be measured using natural peptide substrates. Plasma from C57BL/6 mice, kidney from wild-type and ACE2 knockout mice, and rACE2 were used for assay validation. Plasma or tissue extracts were incubated with angiotensin I (Ang I; 1296 m/z) or angiotensin II (Ang II; 1045 m/z). Reaction mixtures were spotted onto the ProteinChips WCX2 and peptides detected using surface-enhanced laser desorption/ionization time of flight MS. MS peaks for the substrates, Ang I and Ang II, and the generated peptides, Ang (1-7) and Ang (1-9), were monitored. The ACE2 inhibitor MLN 4760 (0.01 to 100 micromol/L) significantly inhibited rACE2 activity (IC50=3 nmol/L). Ang II was preferably cleaved by rACE2 (km=5 mumol/L), whereas Ang I was not a good substrate for rACE2. There was no detectable ACE2 activity in plasma. Assay specificity was validated in a model of ACE2 gene deletion. In kidney extract from ACE2-deficient mice, there was no generation of Ang (1-7) from Ang II. However, Ang (1-7) was produced when Ang I was used as a substrate. In conclusion, we developed a specific and sensitive assay for ACE2 activity, which used the natural endogenous peptide substrate Ang II. This approach allows for the rapid screening for ACE2, which has applications in drug testing, high-throughput enzymatic assays, and identification of novel substrates/inhibitors of the renin-angiotensin system.

This article demonstrates the applicability of quantitative proteomics to assays of proteolytic enzyme activity. A novel assay was developed for measurement of renin and angiotensin-converting enzyme (ACE) activity in plasma. The method was validated in animal models associated with alterations of the renin angiotensin system (RAS). Using surface-enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS) with a ProteinChip Array technology, plasma renin and ACE1 could be measured in <0.5 microL of plasma. Plasma is incubated with peptide substrates for renin and ACE, tetradecapeptide (TDP), and angiotensin I (Ang I), respectively. The reactions mixtures are spotted onto the ProteinChip WCX2 and detected using SELDI-TOF-MS. Peak height or area under curve for TDP, Ang I, and angiotensin II (Ang II) peaks are measured. There was a linear relationship between disappearance of substrate and appearance of products for both renin and ACE (R2=0.95 to 0.98). ACE1 activity was blocked with chelating agents (EDTA and 1,10 phenanthrolene), indicating action of a metalloprotease. The ACE1 inhibitor, captopril, selectively blocked ACE1. Renin activity was specifically blocked with renin inhibitor and was not affected by phenanthrolene or captopril. Animal models tested were Ang AT1a receptor-deficient and streptozotocin (STZ) diabetic mice. Plasma renin activity was increased >2-fold in AT1a(-/-) as compared with AT1a(+/+). In STZ diabetic mice, ACE1 was increased 2-fold as compared with controls. The advantage of the method is that it is tagless, does not require additional purification steps, and is extremely sensitive. The approach can be multiplexed and used for identification of novel substrates/inhibitors of the RAS.

We have previously reported that infection with Plasmodium yoelii, Plasmodium chabaudi, or injection of extracts from malaria-parasitized red blood cells induces hypoglycemia in normal mice and normalizes the hyperglycemia in streptozotocin (STZ)-diabetic mice. P yoelii glycosylphosphatidylinositols (GPIs) were extracted in chloroform:methanol:water (CMW) (10:10:3), purified by high-performance thin layer chromatography (HPTLC) and tested for their insulin-mimetic activities. The effects of P yoelii GPIs on blood glucose were investigated in insulin-resistant C57BL/ks-db/db diabetic mice. A single intravenous injection of GPIs (9 and 30 nmol/mouse) induced a significant dose-related decrease in blood glucose (P < .001), but insignificantly increased plasma insulin concentrations. A single oral dose of 2.7 micromol GPIs per db/db mouse significantly lowered blood glucose (P < .01). P yoelii GPIs in vitro (0.062 to 1 micromol/L) significantly stimulated lipogenesis in rat adipocytes in a dose-dependent manner both in the presence and absence of 10(-8) mol/L insulin (P < .01). P yoelii GPIs stimulated pyruvate dehydrogenase phosphatase (PDH-Pase) and inhibited both cyclic adenosine monophosphate (cAMP)-dependent protein kinase A and glucose-6-phosphatase (G6Pase). P yoelii GPIs had no effect on the activity of the gluconeogenic enzymes fructose-1,6-bisphosphatase (FBPase) and phosphoenolpyruvate carboxykinase (PEPCK). This is the first report of the hypoglycemic effect of P yoelii GPIs in murine models of type 2 diabetes. In conclusion, P yoelii GPIs demonstrated acute antidiabetic effects in db/db mice and in vitro. We suggest that P yoelii GPIs, when fully characterized, may provide structural information for the synthesis of new drugs for the management of diabetes., (Copyright 2004 Elsevier Inc.)

We have previously shown that infection with Plasmodium yoelii malaria or injection of extracts from malaria-parasitized red cells induces hypoglycemia in normal mice and normalizes the hyperglycemia in mice made moderately diabetic with streptozotocin. Inositol phosphoglycans (IPGs) are released outside cells by hydrolysis of membrane-bound glycosylphosphatidylinositols (GPIs), and act as second messengers mediating insulin action. The C57BL/Ks-db/db and C57BL/6J-ob/ob mice offer good models for studies on human obesity and Type 2 diabetes. In the present study, we show that a single iv injection of IPG-A or IPG-P extracted from P. yoelii significantly (P < 0.02) lowers the blood glucose in STZ-diabetic, db/db, and in ob/ob mice for at least 4--6 h. Using rat white adipocytes, IPG-P increased lipogenesis by 20--30% in the presence and absence of maximal concentrations of insulin (10(-8) M) (P < 0.01) and stimulated pyruvate dehydrogenase (PDH) phosphatase in a dose-related manner. Both IPG-A and IPG-P inhibited c-AMP-dependent protein kinase (PKA) in a dose-related manner. Compositional analysis of IPGs after 24 h hydrolysis revealed the presence of myo-inositol, phosphorus, galactosamine, glucosamine, and glucose in both IPG-A and IPG-P. However, hydrolysis of IPGs for 4 h highlighted differences between IPG-A and IPG-P. There are some functional similarities between P. yoelii IPGs and those previously described for mammalian liver. However, this is the first report of the hypoglycemic effect of IPGs in murine models of Type 2 diabetes. We suggest that IPGs isolated from P. yoelii, when fully characterized, may provide structural information for the synthesis of new drugs for the management of diabetes mellitus., (Copyright 2001 Academic Press.)

C57BL/KsJ-db/db and C57BL/KsJ-ob/ob mice are good models for studies on human obesity and type 2 diabetes. We have previously shown that infection with blood-stage malaria or injection of extracts from malaria-parasitized red blood cells induces hypoglycemia in normal mice and normalizes hyperglycemia in mice made moderately diabetic by streptozotocin. In the present study, we show that a single intravenous (IV) injection of Formalin-fixed Plasmodium yoelii YM (FFYM) preparation decreases blood glucose in db/db mice from an initial value of 19 mmol/L to a normal value of 7 mmol/L (P < .0001) for at least 24 hours and reduces food intake. Plasma insulin concentrations in db/db mice were not altered. FFYM was also active in normal and ob/ob mice, an effect associated with an increase in plasma insulin. Although the rate of weight gain in lean ob/+ and lean db/+ was not altered by this treatment, there was a significant reduction in weight gain in db/db and ob/ob mice (P < .001). We suggest that malaria-derived molecules, when fully characterized, may provide structural information for the development of new agents for the management of type 2 diabetes.

Hypoglycaemia in falciparum malaria is associated with a poor prognosis and is correlated with mortality. High levels of serum TNF are also correlated with disease severity and mortality, and it has been suggested that TNF may cause the hypoglycaemia. However hypoglycaemia in mice infected with Plasmodium chabaudi or the lethal strain of P. yoelii YM is related to hyperinsulinaemia. Its development was not prevented by treatments which diminished TNF activity or production without affecting levels of plasma insulin. Conversely, it was inhibited by diazoxide, which inhibited insulin secretion but did not affect TNF production. Furthermore, in mice exhibiting neurological symptoms during infection with P. berghei, blood glucose concentrations were significantly raised when TNF levels were high, and TNF levels in the spleen were highest of all in non-lethal P. yoelii infections in which hypoglycaemia does not occur. Administration of human rTNF to normal animals caused an increase rather than a drop in blood glucose levels. Mice transgenic for human TNF did not develop hypoglycaemia when infected with P. yoelii YM, but showed signs of insulin resistance. In line with current views on the role of TNF in obesity and the control of glucose homeostasis, we conclude that the hypoglycaemia of malaria is not caused by increased levels of TNF, which may in fact be beneficial, but is secondary to a hyperinsulinaemia that is probably stimulated directly by products of the parasite.

We have investigated the metabolic disturbances in 2 murine models of blood-stage malaria, Plasmodium chabaudi and Plasmodium yoelii. Blood glucose, plasma insulin and parasitaemia were measured in normal and infected mice before and after treatment with diazoxide, adrenaline, Sandostatin and quinine. Severe hypoglycaemia and marked hyperinsulinaemia developed during both infections. A single injection of diazoxide (25 mg/kg i.p.) or adrenaline (0.03 mg s.c.) lowered insulin concentrations in normal mice, reversed the hypoglycaemia in both infections and significantly reduced the hyperinsulinaemia in P. chabaudi-infected mice (P < 0.0001). Higher doses of Sandostatin (500 micrograms/kg s.c.) were required to reverse hypoglycaemia. Quinine (25 mg/kg i.p.) significantly increased blood glucose in normal and infected mice (P < 0.0010 and no hypoglycaemia was observed in mice with normal blood glucose for more than 3 h. This study shows that the major cause of hypoglycaemia in murine malaria is hyperinsulinaemia rather than high consumption of glucose by host and parasites or chemotherapy with quinine, and that hypoglycaemia can be reversed by correcting the hyperinsulinaemia.

The present study was undertaken to determine if acute stress induced by exposure to ether resulted in the presence of beta-cell-tropic factors in rat plasma and if this insulinotropic activity was increased by pertussis toxin. Rats pretreated with pertussis toxin (5 micrograms/kg, 5 days previously) showed marked hyperinsulinemia, but only after exposure to ether before blood sampling. This hyperinsulinemia was not modified by adrenal demedullation. Effects on insulin secretion were assessed by incubation of plasma (diluted with Krebs buffer) with collagenase-isolated rat pancreatic islets. When blood was collected by decapitation from normal rats, the subsequently prepared plasma (12.5% to 50%) profoundly inhibited insulin release from rat isolated islets. This inhibition was probably mediated by catecholamines, since it was not seen with plasma from adrenal-demedullated rats and was prevented by alpha 2-adrenoceptor-blocking drugs. Plasma from adrenal-demedullated, pertussis toxin-treated rats stimulated insulin secretion (by 60%) when the donor rats had been exposed to ether before blood sampling. It is suggested that stress may result in the presence of circulating beta-cell-tropic factors, which may contribute to the acute stress-induced hyperinsulinemia seen in pertussis toxin-treated animals.