Mammalian target for rapamycin complex 1 (mTORC1) is a common target for the action of immunosuppressant macrolide rapamycin and glucose-lowering metformin. Inhibition of mTORC1 can exert both ...beneficial and detrimental effects in different pathologies. Here, we investigated the differential effects of rapamycin (1.2 mg/kg per day delivered subcutaneously for 6 weeks) and metformin (300 mg/kg per day delivered orally for 11 weeks) treatments on male Zucker diabetic fatty (ZDF) rats that mimic the cardiorenal pathology of type 2 diabetic patients and progress to insulin insufficiency. Rapamycin and metformin improved proteinuria, and rapamycin also reduced urinary gamma glutamyl transferase (GGT) indicating improvement of tubular health. Metformin reduced food and water intake, and urinary sodium and potassium, whereas rapamycin increased urinary sodium. Metformin reduced plasma alkaline phosphatase, but induced transaminitis as evidenced by significant increases in plasma AST and ALT. Metformin also induced hyperinsulinemia, but did not suppress fasting plasma glucose after ZDF rats reached 17 weeks of age, and worsened lipid profile. Rapamycin also induced mild transaminitis. Additionally, both rapamycin and metformin increased plasma uric acid and creatinine, biomarkers for cardiovascular and renal disease. These observations define how rapamycin and metformin differentially modulate metabolic profiles that regulate cardiorenal pathology in conditions of severe type 2 diabetes.
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While chemotherapeutic agents (anthracyclins, kinase‐inhibitors) have improved 5‐year breast cancer survival, their cardiovascular toxicity cause alarming increase in life‐threatening heart disease ...for millions of cancer survivors. Therefore, developing new cardio‐oncology drugs to protect heart during and after chemotherapy is an immediate critical need. Although literature indicate that Angiotensin II type 2 receptor AT2R attenuates cardiac damage, to date there are no AT2R agonists that are used in clinic to treat heart disease. NP‐6A4 is an AT2R peptide agonist that has recently received orphan drug designation for pediatric cardiomyopathy from the FDA. Our previous studies indicated that NP‐6A4 treatment (1.8mg/kg/day) could improve cardiac function and attenuate cardiomyopathy in diabetic rats and protect human cardiovascular cells from acute nutrient stress. We hypothesized that since AT2R is a regulator of cell growth, NP‐ 6A4‐AT2R signaling will attenuate breast cancer cell growth. Here we report NP‐6A4 treatment of triple negative breast cancer cells (MDA‐MB‐231) and estrogen positive breast cancer cells (MCF‐7) resulted in significant (p<0.05) suppression of cell index (as determined by real time cell analyzer), and survival (as determined by MTS assay), albeit the underlying mechanisms were different. NP‐6A4 treatment of MCF‐7 cells augmented Tamoxifen‐induced inhibition of cell growth (p<0.05), and suppressed proliferation marker Ki67 (20%) and phosphokinases CREB (32%), Jun N‐terminal kinase (JNK) (51%) and Erk1/2 (41%). In MDA‐MB‐231 cells, NP‐6A4 treatment caused increase in apoptotic marker Caspase 3 (2.3‐fold increase), but suppressed its inhibitor phosphokinase p38 (37%) (p<0.05), and also suppressed phosphokinases CREB (30%), JNK (51%) and Erk1/2 (33%). However, doxorubicin (anthracycline)‐induced cell death was not reversed by NP‐6A4. NP‐6A4 treatment increased different tumor suppressors: ATIP 1 mRNA in MCF‐7 and ATIP‐3 mRNA in MDA‐MB‐231 (p<0.05). These data suggest that NP‐6A4 differentially regulates human breast cancer cell growth and has high potential as a synergistic cardio‐oncology drug for breast cancer treatment.
Support or Funding Information
This work was supported by NIH grants: 1R01HL118376‐01 and 1R01HL138988‐01A1
This is from the Experimental Biology 2018 Meeting. There is no full text article associated with this published in The FASEB Journal.
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Abstract only Introduction: Obesity affects 42% of the US population and exacerbates heart disease. Currently there are no effective treatments for obesity-induced cardiac pathology. We reported that ...NP-6A4, a peptide agonist of the Angiotensin receptor AT2R mitigates cardiac dysfunction and damage in male rats with untreated obesity. Since cardiac pathology of male and female rats with untreated obesity differ, we hypothesized that there can be differences in their cardiac response to NP-6A4. Methods and Results: Echocardiographic analysis was performed in 15-week old male and female healthy Wistar (N=8) or Zucker lean (N=7) rats treated with saline, and obese and pre-diabetic Zucker rats treated with saline or NP-6A4 (1.8-10mg/kg/day in saline) for four weeks (N=8/group for females, N=7/group for males; subcutaneous delivery). Compared to healthy males, obese males showed significant (P<0.05) reduction in heart rate and increase in anterior wall thickness and left ventricular (LV) filling pressure (E/E’) suggesting concentric hypertrophy. Compared to healthy females, obese females showed significant (P<0.05) reduction in relative wall thickness, heart rate, and ejection fraction (although preserved) and increase in LV internal diameter at both diastole and systole indicating eccentric hypertrophy. NP-6A4 treatment suppressed the pathologic changes in both obese males and females (P<0.05). In another cohort of obese female rats NP-6A4 treatment from 13-to 17-weeks significantly (P<0.05) reduced abnormal E/A ratio and isovolumic contraction and relaxation time, and improved myocardial radial strain. Treatment with AT2R antagonist PD123319 (N=8: 5mg/kg/day delivered subcutaneously) abolished NP-6A4 effects. Cardiac proteome analysis of 18 tissue samples from this cohort using data independent acquisition (DIA) resulted in a total proteome of 4520 proteins and 296 differentially expressed proteins that were significant (P<0.01). Ingenuity pathway analysis predicted that inhibition of NP-6A4-mediated signaling by PD123319 may increase infection of cells (P=<0.001). Conclusion: NP-6A4 attenuated cardiac pathology induced by untreated obesity by altering different cardiac parameters in male and female rats.
Clinical and experimental studies show that angiotensin II (AngII) promotes vascular pathology via activation of AngII type 1 receptors (AT1Rs). We recently reported that NP‐6A4, a selective peptide ...agonist for AngII type 2 receptor (AT2R), exerts protective effects on human vascular cells subjected to serum starvation or doxorubicin exposure. In this study, we investigated whether NP‐6A4–induced AT2R activation could mitigate AngII‐induced abdominal aortic aneurism (AAA) using AngII‐treated Apoe−/− mice. Male Apoe−/− mice were infused with AngII (1 µg/kg/min) by implanting osmotic pumps subcutaneously for 28 days. A subset of mice was pre‐treated subcutaneously with NP‐6A4 (2.5 mg/kg/day) or vehicle for 14 days prior to AngII, and treatments were continued for 28 days. NP‐6A4 significantly reduced aortic stiffness of the abdominal aorta induced by AngII as determined by ultrasound functional analyses and histochemical analyses. NP‐6A4 also increased nitric oxide bioavailability in aortic tissues and suppressed AngII‐induced increases in monocyte chemotactic protein‐1, osteopontin and proteolytic activity of the aorta. However, NP‐6A4 did not affect maximal intraluminal aortic diameter or AAA incidences significantly. These data suggest that the effects of AT2R agonist on vascular pathologies are selective, affecting the aortic stiffness and proteolytic activity without affecting the size of AAA.
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Over-nutrition and metabolic cardiomyopathy Mandavia, Chirag H; Pulakat, Lakshmi; DeMarco, Vincent ...
Metabolism, clinical and experimental,
09/2012, Volume:
61, Issue:
9
Journal Article
Peer reviewed
Open access
Abstract Cardiovascular disease, which accounts for the highest morbidity and mortality in the United States, has several major risk factors, including aging and diabetes. Overweight and obesity, ...especially abdominal obesity, have been increasingly implicated as independent risk factors in the development of cardiovascular disease. Metabolic and/or diabetic cardiomyopathy has been especially associated with excess body weight caused by chronic over-nutrition and high-fat feeding. In the initial stages, obesity is now understood to cause significant dysregulation of cardiac fatty acid and glucose metabolism. These abnormalities are due, in part, to increased oxidative stress, which in turn can cause deleterious effects on intracellular signaling pathways that control cellular growth and proliferation. This increase in oxidative stress is coupled with reduced anti-oxidant species and dysregulation of metabolic signaling pathways. The cardiomyopathy seen with obesity is associated with increased interstitial fibrosis and diastolic dysfunction. Over time, evolving abnormalities include hypertrophy and systolic dysfunction, eventually leading to heart failure.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Mammalian target for rapamycin complex 1 (mTORC1) is a common target for the action of immunosuppressant macrolide rapamycin and glucose-lowering metformin. Inhibition of mTORC1 can exert both ...beneficial and detrimental effects in different pathologies. Here, we investigated the differential effects of rapamycin (1.2 mg/kg per day delivered subcutaneously for 6 weeks) and metformin (300 mg/kg per day delivered orally for 11 weeks) treatments on male Zucker diabetic fatty (ZDF) rats that mimic the cardiorenal pathology of type 2 diabetic patients and progress to insulin insufficiency. Rapamycin and metformin improved proteinuria, and rapamycin also reduced urinary gamma glutamyl transferase (GGT) indicating improvement of tubular health. Metformin reduced food and water intake, and urinary sodium and potassium, whereas rapamycin increased urinary sodium. Metformin reduced plasma alkaline phosphatase, but induced transaminitis as evidenced by significant increases in plasma AST and ALT. Metformin also induced hyperinsulinemia, but did not suppress fasting plasma glucose after ZDF rats reached 17 weeks of age, and worsened lipid profile. Rapamycin also induced mild transaminitis. Additionally, both rapamycin and metformin increased plasma uric acid and creatinine, biomarkers for cardiovascular and renal disease. These observations define how rapamycin and metformin differentially modulate metabolic profiles that regulate cardiorenal pathology in conditions of severe type 2 diabetes.
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DOBA, FSPLJ, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract only Introduction & Hypothesis: Untreated obesity induces insulin resistance and heart disease affecting 42% of the US population. Autophagy, a conserved homeostatic process for cellular ...quality control, plays a critical role in maintaining health. Heart tissues of obese humans and rodent models show impaired autophagy. Drugs that can regulate autophagy are thus of high clinical significance. We reported recently that NP-6A4, a new peptide agonist of the cardiovascular protective angiotensin receptor AT2R, mitigated obesity-induced cardiac dysfunction and damage in rats with untreated obesity and pre-diabetes. We hypothesized that activation of autophagy by NP-6A4 is involved in NP-6A4-AT2R-induced cardioprotection in untreated obesity. Methods & Results: 7-week old female ZDF rats fed high fat diet (D12468, Research Diet) for 6 weeks were randomized to receive either NP-6A4 (10mg/kg/day), NP6A4+AT2R antagonist PD123319 (PD, 5mg/kg/day) delivered subcutaneously (N=6/group), or vehicle (N=5) for 4 weeks. Echocardiography showed that NP-6A4 reduced abnormal E/A ratio from 2.1 to 1.45 (P<0.006), and isovolumic contraction (P<0.004) and relaxation times (P<0.02), and increased myocardial radial strain (P<0.019) and radial strain rate (P<0.033). PD reversed these effects. Immunohistochemistry (IHC) and Western blot analysis revealed a significant 5.9-fold increase (p<0.0006) in LC3 puncta (Fig. A-B) and 2.2-fold increase (p<0.0008) in lipidated autophagy marker LC3-II (Fig. C) compared to vehicle controls. The autophagy activation by NP-6A4 was not seen with the co-administration of PD (Fig. A-C). Cardiac proteome analysis showed that Sec16a, an important partner of autophagy initiators ULK1/2 is increased in ZDF rat heart by NP-6A4 (2.75 fold; P<0.020), but PD suppressed this effect. Conclusions: We identified NP-6A4’s dual targeting of AT2R and novel autophagy regulation to mitigate cardiac dysfunction induced by untreated obesity in female rats
Abstract only
Background
Aged women have greater vascular stiffness and vasomotor dysfunction vs. aged men, leading to a greater incidence of cardiovascular disease (CVD). However, the underlying ...mechanisms that mediate these differences are unclear. The hormone angiotensin II promotes vascular aging via the AngII type 1 receptor (AT1R). However, AngII also acts through the AngII type 2 receptor (AT2R), which is vasoprotective. Previous studies report sex and age‐related differences in AT2R function in the kidney. However, the role for the AT2R in male and female vascular aging is unknown.
Methods/Results
All studies were conducted in accordance with the FASEB Statement of Principles for the use of Animals in Research and Education. We measured aortic pulse wave velocity, an index of vascular stiffness, in 3,12, and 18 month old male and female C57/Bl6 mice. Our results show that males develop increases in vascular stiffness from 3 to 12 months compared to 3 to 18 months in females (p<0.05). To determine the potential role of the AT2R in the vascular protection observed in 12 month old female mice, we treated 12 and 18 month old male and female mice with AT2R antagonist PD‐123319 (3mg/kg/day) or vehicle via osmotic minipumps for 4 weeks. Chronic inhibition of the AT2R increases vascular stiffness in 12 month old females (p<0.05), but not 12 month old males or 18 month old males and females, suggesting that the AT2R contributes to the delay in aging‐associated vascular stiffness in female mice. Next, we measured AT1R‐mediated vasoconstriction and AT2R‐mediated vasodilation in second and third order mesenteric resistance vessels from 3,12, and 18 month old male and female mice. AT1R‐mediated vasoconstriction increases with age in both sexes, however, this increase occurs at 12 months in the males compared to 18 months in the females (p<0.05). Additionally, 18 month old female mice have enhanced AT1R‐mediated vasoconstriction compared to 18 month old males (p<0.05). AT2R‐mediated dilation was measured in response to sequential doses of AT2R agonist Compound‐21. For these studies, vessels were pretreated with AT1R blocker Losartan, and then preconstricted with phenylephrine. Our results show that AT2R‐mediated dilation is enhanced in 3 month females vs. 3 month males (p<0.05) and that this response is reduced with age in females (p<0.05), not males. Together, the resistance vessel studies suggest that aging‐associated alterations in angiotensin II receptor function are sexually dimorphic and may contribute to the differential manifestations of cardiovascular dysfunction observed in aging men and women.
Conclusion
These data indicate sex differences in the time course of the development of aging‐associated vascular stiffness and enhanced vasoconstriction with aging in addition to sex‐specific alterations in vascular angiotensin II receptor function with aging. Specifically, these data suggest that reductions in AT2R function contribute specifically to female vascular aging only and that AT2R restoration and/or activation may represent a novel sex‐specific therapeutic strategy in the treatment of CVD in the aging female population.
Support or Funding Information
1K12 HD092535‐01
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We have previously reported that the DPP-4 inhibitor linagliptin (LINA) prevents diastolic dysfunction and myocardial fibrosis in a mouse model of diet-induced obesity, a pre-clinical model of ...prediabetes. LINA suppressed WD-induced pro-inflammatory mediators, such as TRAF3IP2 and downstream signaling intermediates, NF-κB, AP-1 and p38-MAPK in the heart. Herein, we extend that study by testing whether LINA slows progression of diastolic dysfunction in diabetic db/db mice, and improves the underlying maladaptive immune/inflammatory response. To accomplish this, we fed db/db mice normal mouse chow with or without linagliptin (38 mg/kg chow). After performing echocardiography on mice treated or not for 10 weeks, we analyzed expression of 200 cytokines in myocardial extracts and performed Ingenuity Pathway Analysis (IPA) in an effort to determine what signaling pathways are predicted to be differentially activated in the heart. Mice treated with linagliptin had improved diastolic function indicated by lower LV filling pressure compared to untreated mice (E/E’; 35 vs. 50; p<0.05). IPA predicted that LINA activates pathways contributing to cardiovascular development of the vasculature (32 molecules, Activiation z-score = 3.7), angiogenesis (31 molecules, Activation z-score = 3.7) and vasculogenesis (27 molecules, Activation z-score = 3.7). Cardiomyocyte size (i.e., cross-sectional area) was reduced by 23% (533±30 vs. 411±18 μm2; p<0.05) and myocardial capillary density was two-fold higher with LINA treatment compared to untreated mice (p<0.05). In addition, the proteome analysis showed a significant decrease in TNF-α related apoptosis inducing ligand, a cytokine that induces endothelial nitric oxide, in untreated mice, however, this deficiency was prevented by LINA. These data suggest that linagliptin exerts a beneficial immune/inflammatory response in the diabetic heart that confers myocardial vasculoprotection.
Disclosure
R. Toedebusch: None. A.R. Aroor: None. J. Habibi: None. T. Klein: Employee; Self; Boehringer Ingelheim GmbH. L. Pulakat: None. V. DeMarco: Research Support; Self; Boehringer Ingelheim Pharmaceuticals, Inc..
Diabetic marker miRNA‐29 (miR‐29) is up‐regulated by pro‐inflammatory cytokines and hyperglycemia. miR‐29 induced suppression of MCL‐1, a protein essential for mitochondrial function and cell ...survival, occurs in pancreatic β‐cell death. We hypothesized that conditions that increase miR‐29 levels in heart would promote suppression of MCL‐1 and increase cardiomyocyte cell death. Since Rapamycin (Rap) is known to promote diabetes, we further posited that Rap treatment (750 µg/kg/day; 6 weeks) would increase miR‐29 and suppress MCL‐1 in heart tissues of Zucker Diabetic Fatty (ZDF) rat, a rodent model for the progression of Type 2 Diabetes. Rap treatment attenuated plasma insulin (INS) levels implying development of insulinopenia; however, hyperglycemia persisted. q‐PCR showed a significant increase in miR‐29a, b, and c and suppression of MCL‐1 (p<0.01) in ZDF heart. Rap treatment also resulted in significant loss of cardiomyocytes as visualized by histopathological analysis by H & E and Masson's trichrome staining. Moreover, Rap treatment increased miR‐29 levels and suppressed MCL‐1 mRNA and protein levels in mouse atrial cardiomyocytes (10nM; 12hr) and in human coronary artery smooth muscle cells (20nM; 12hr). These data suggest that MCL‐1 regulation via miR‐29 family in INS‐responsive tissues is conserved across species and contributes to cardiomyocyte loss that can promote heart failure in diabetes.
Grant Funding Source: NIH: 1R01HL118376‐01
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