Chen, Horng

Background: The cardiac hormone atrial natriuretic peptide is the endogenous ligand for the guanylyl cyclase A (GC-A) receptor. The ANP/GC-A/cGMP pathway is a key regulator of cardiorenal and metabolic homeostasis. Beyond its classical natriuretic and vasodilatory roles, emerging evidence reveals that ANP via GC-A stimulates lipolysis in human adipocytes and modulates insulin secretion. Notably, the ANP gene variant rs5068, which enhances ANP production, is associated with reduced risk of obesity and metabolic syndrome in humans. We recently engineered CRRL191, a novel best-in-class GC-A agonist, with superior receptor activation and greater resistance to degradation by neprilysin and insulin degrading enzyme resulting in greater blood pressure lowering and natriuretic effects than ANP. Given the metabolic properties of GC-A signaling, we investigated CRRL191’s metabolic effects on lipolysis and insulin secretion in pancreatic, including its interaction with GLP-1. Hypothesis: CRRL191 exerts potent lipolytic and insulinotropic effects and synergizes with GLP-1 to enhance insulin secretion. Methods: Human visceral adipocytes were treated with CRRL191 (10 -8 -10 -6 M) for 6 hrs. Lipolysis was assessed by measuring glycerol and non-esterified fatty acids (NEFA) in supernatants. Rat pancreatic beta cells (INS-1) were stimulated treated with CRRL191 (10 -10 -10 -6 M), GLP-1 (10 -10 -10 -6 M) alone or their combination for 1 hr. Insulin secretion was quantified by ELISA. Results: CRRL191 stimulated lipolysis in human visceral adipocytes (10 -8 -10 -6 M), increasing glycerol and NEFA release. CRRL191 induced greater glycerol release than GLP-1 across all tested doses, with low dose CRRL191 (10 -10 M) notably enhancing NEFA production. In INS-1 cells, CRRL191 dose dependently increased insulin secretion under hyperglycemic conditions, demonstrating superior potency at lower concentrations (10 -10 and 10 -8 M) compared to GLP-1. Remarkably, co-administration of CRRL191 with GLP-1 amplified insulin release beyond levels achieved by either peptide alone. Conclusion: CRRL191 is a potent GC-A agonist that robustly stimulates lipolysis in visceral adipocytes and enhances insulin secretion in pancreatic beta cells. Its synergistic interaction with GLP-1 highlights a novel therapeutic avenue targeting the GC-A/cGMP pathway for integrated treatment of cardiometabolic diseases. These findings support further evaluation of CRRL191 as a promising candidate for cardiometabolic drug development.

Background: Diabetic cardiomyopathy is a common complication of diabetes, which can progress to symptomatic heart failure. Current conventional medical therapy does not reverse the deleterious features of diabetic cardiomyopathy. The natriuretic peptide-cGMP system has been proven to provide a cardioprotective role in diabetic rats. Therefore, we developed CRRL094, a n bispecific peptide that simultaneously activates the natriuretic peptide system and glucagon-like peptide-1 (GLP-1) receptor. In this study, we aimed to characterize the cardioprotective role of CRRL094 and elucidate its downstream molecular mechanisms. Methods: In vitro, human cardiomyocytes (HCMs) were pretreated with PBS or CRRL094 (5 µM) for 6 hours, then exposed to high glucose treatment (HGT, 25 mM) for 48 hours. HCM hypertrophy was determined by assessing mRNA levels of hypertrophic gene markers (NPPA and NPPB) using real-time PCR. Lipid droplet staining was performed using BODIPY 493/503 dye and fluorescence images were analyzed. The protein levels of CIDEC (lipid droplet-related gene) and phosphorylation-AMPK and total AMPK (a key regulator of lipid metabolism) were analyzed by Western blot. In vivo, a type 2 diabetes (DM2) rat model was established by low-dose intraperitoneal Streptozotocin (STZ) injection and a high-fat diet in Wistar rats (male, 4- to 6-week-old). Diabetes was confirmed by fasting blood glucose > 350 mg/dL. Rats were then allocated to two groups: saline-treated controls (n=6) or CRRL094-treated (30 pmol/kg/min; n=6), with treatment delivered via osmotic pumps for 28 days. Heart tissues were collected and analyzed. Results: HGT in HCMs significantly increased the mRNA levels of NPPA and NPPB, whereas CRRL094 significantly attenuated markers of hypertrophy. Further, HGT increased lipid accumulation in HCMs, which was decreased with CRRL094 treatment. Moreover, CRRL094 increased p-AMPK protein levels and decreased CIDEC protein levels in HCMs under HGT. In a DM2 rat model, chronic CRRL094 therapy was associated with suppression of NPPA and NPPB, downregulation of CIDEC, and upregulation of p-AMPK expression in the heart tissue. Conclusion: CRRL094 protects against HGT-induced hypertrophy and inhibits lipid accumulation through regulating the AMPK/CIDEC pathway in vitro and in vivo . These findings support CRRL094 as a novel therapeutic candidate to prevent diabetic cardiomyopathy.

BACKGROUND: Atrial natriuretic peptide (ANP) and glucagon-like peptide-1 (GLP-1) are endogenous hormones known for roles in cardiovascular regulation and metabolic homeostasis. ANP mediates its actions via the GC-A receptor, while GLP-1 via the GLP-1 receptor (GLP-1R). Both peptides may stimulate insulin secretion from pancreatic beta cells and augment lipid metabolism in adipocytes, yet potential synergistic effects remain unexplored. To optimize ANP activation of GC-A, we developed a novel 15 amino acid miniaturized GC-A activating peptide CRRL555. CRRL555 possesses greater GC-A binding and activating properties than ANP. AIMS: We investigated the metabolic properties of CRRL555/GC-A and its interaction with GLP-1/GLP-1R in vitro. METHODS: Rat pancreatic beta cells (INS-1) were stimulated with high glucose (20mM) and treated with GLP-1 or CRRL555 (10 -10 -10 -6 M) and together for 1 hr. Supernatant was collected and insulin was measured by ELISA. Human visceral adipocytes were differentiated for 10 days and then treated with GLP-1 or CRRL555 (10 -10 -10 -6 M) or together for 6 hrs. Supernatant was collected and glycerol and free fatty acid (NEFA), indicators of lipolysis, were measured. RESULTS: CRRL555 (p<0.05) and GLP-1 (p<0.05), dose dependently, stimulated insulin secretion in INS-1 cells in the presence of high glucose. Importantly, CRRL555 combined with GLP-1, resulted in a greater enhancement (p<0.05) of insulin secretion compared to either peptide alone. While high dose CRRL555 and GLP-1 (10 -6 M) stimulated lipolysis in visceral adipocytes with increases in glycerol and NEFA, only CRRL555 at low dose (10 -10 M), and not low dose GLP-1 (10 -10 M), stimulated glycerol and NEFA production (p<0.05). When low dose GLP-1 (10 -10 M) was added to low dose CRRL555(10 -10 M), lipolysis was like CRRL555 alone. CONCLUSION: We report a novel synergy in insulin release from beta cells with combined stimulation of the potent and miniaturized GC-A designer peptide CRRL555 with GLP-1. CRRL555 possesses greater lipolytic properties compared to GLP-1 in visceral adipocytes at low doses advancing novel metabolic actions of CRRL555 alone or combined with GLP-1R activation. These in vitro studies of dual receptor activation of GC-A and GLP-1R highlight the therapeutic potential of combining CRRL555 with GLP-1-based therapies for the treatment of cardiometabolic diseases, thus supporting studies of dual GC-A/GLP-1R activation in experimental cardiometabolic disease states.

Background Sustained Acting Atrial Natriuretic Peptide (SA-ANP) is a 40 AA peptide consisting of native ANP (1–28) with a 12 AA C-terminus extension which we synthesized based upon a familial ANP gene frameshift mutation. SA-ANP possesses markedly greater and more sustained mean arterial pressure (MAP) lowering, diuretic/natriuretic, angiotensin-aldosterone suppressing, and cGMP activating properties compared to ANP in normal dogs consistent with a super agonist of the ANP receptor (NPR-A) ( JACC in press ). Here we evaluated SA-ANP in a model of acute hypertension with the hypothesis that SA-ANP would have potent MAP lowering and renal enhancing properties. Methods Integrated physiological studies were performed in a canine model of ANG induced hypertension (20 ng/kg/min continuous infusion) before, during, and up to 3 hours following a 45 minute infusion of SA-ANP (30 pmol/kg/min) (n=6) or vehicle (n=6). Results MAP is shown in Figure 1 . SVR significantly decreased and despite a significant reduction in MAP both renal blood flow (RBF) and GFR were significantly increased following SA-ANP infusion. There were significant reductions in proximal and distal tubule sodium reabsorption resulting in a significant increase (>50 fold) in diuresis and natriuresis with SA-ANP. These renal actions were associated with significant increases in plasma and urine cGMP and a significant inhibition of aldosterone activation. Discussion We report for the first time in an acute model of hypertension that SA-ANP reduced MAP while enhancing RBF, GFR, and sodium/water excretion and thus may serve as a potential therapeutic agent in the treatment of hypertension in the setting of volume overload.

Introduction: The prevalence of diabetes mellitus (DM) is increasing, and it is associated with cardiorenal dysfunction. Natriuretic peptides (NPs) have cardiorenal protective actions and enhance insulin sensitivity. We designed a dual-action bispecific peptide (CRRL 094) that simultaneously activates the natriuretic peptide system and induces insulin secretion. Methods: To test the in vivo effects of the peptide, Wistar rats (male, 6-8-week-old) were made diabetic by intraperitoneal Streptozotocin (STZ) injection (65 mg/kg). One week after STZ injection, diabetes was confirmed (fasting blood glucose > 350 mg/dL). Rats were then divided into 2 groups: controls (Ctrl group, n=6) and treatment group (Tx group, n= 9). The peptide (1 pmol/kg/min) was infused in the Tx group for 150 min. Results: After 150 minutes of peptide infusion, plasma insulin (mean± standard deviation) increased more than 3.7x among the Tx group compared to the Ctrl group (0.294± 0.199 ng/mL vs 0.078± 0.021 ng/mL, p=0.01). Additionally, plasma glucose decreased among Tx group (405.3±96.4 mg/dL) compared to Ctrl group (545.6± 52.8 mg/dL, p=0.003). Furthermore, the rate of natriuresis increased significantly among the Tx group compared to controls (1.582±0.7 vs 0.639±0.21, p= 0.009), as did the rate of kaliuresis (3.1± 1.1 vs 2.0±0.6, p=0.045). Importantly, the glomerular filtration rate increased by 1.5x among the Tx group compared to the controls (1.6±0.5 vs 1.0±0.3, p= 0.03). There were no significant differences in mean arterial pressure or heart rate. Conclusions: The dual-action insulin-secreting natriuretic peptide (CRRL 094) was effective in increasing endogenous insulin secretion, enhancing natriuresis, and improving renal clearance. Further studies are warranted to assess its long-term safety and efficacy in diabetes.

Introduction: Atrial natriuretic peptide (ANP) is the most potent endogenous activator of the guanylyl cyclase A receptor (GC-A) mediating cardiorenal protective actions by increasing cGMP production. To test the hypothesis that acute decompensated heart failure (ADHF) is associated with increased circulating ANP but with reduced GC-A potency, we developed an ex-vivo precision medicine assay to quantify the potency of circulating ANP in plasma from healthy and ADHF subjects. Further, we assessed the ex-vivo potency of MANP, a novel ANP analog with greater cGMP activation than ANP, also using plasma of healthy and ADHF subjects. Methods: For the potency assay, we engineered HEK293 cells to overexpress human GC-A. Plasma from individual healthy (n=4) and ADHF (n=4) subjects was incubated in the assay and cGMP was assessed. The endogenous ANP derived cGMP was evaluated against the cGMP response of equimolar synthetic ANP (S ANP ) to assess potency of ANP from each cohort. MANP mediated cGMP generation was also assessed in the assay using healthy and ADHF plasma. Results: Healthy plasma, in which plasma ANP was 26±5 pg/mL, generated 10.3±0.7 pmol/mL of cGMP. S ANP added to the assay to mimic endogenous ANP levels in healthy subjects produced 7.8±0.5 pmol/mL of cGMP. ADHF plasma with markedly elevated ANP (350±57pg/mL) generated 23.5±3.1 pmol/mL of cGMP. S ANP mimicking plasma ANP in ADHF produced 59.7±13.8 pmol/mL of cGMP, which was greater than cGMP generated from ADHF patient-derived plasma (p<0.05), consistent with reduced potency of endogenous ANP in ADHF. Low treatment dose of MANP (LD MANP ; 10 -8 M) in healthy plasma increased cGMP to 45±5.5 pmol/ml while high treatment dose (HD MANP ; 10 -6 M) increased cGMP to 167.1±14.1 pmol/mL. In markedly elevated ANP ADHF plasma, LD MANP increased cGMP to 60.4±9.7 pmol/mL while HD MANP increased cGMP to 200.8±5.4 pmol/mL (p<0.03 for both) demonstrating similar cGMP production by MANP in healthy and ADHF plasma. Conclusions: Employing a novel ex-vivo precision medicine ANP/GC-A/cGMP assay, we conclude that ADHF is characterized by elevated ANP yet with impaired potency in GC-A activation and cGMP generation. MANP overcomes this impairment and serves as a potential efficacious GC-A activating therapeutic in heart failure.

Introduction: The prevalence of metabolic syndrome (MS), and diabetes mellitus (DM) is increasing, and the use of biomarkers to identify subjects at risk of developing MS or DM may be useful for disease prevention. Natriuretic peptides (NPs) are well known for their role as biomarkers in heart failure (HF), but are increasingly being recognized for their favorable metabolic actions and potential role as risk modifiers for the development of DM and MS. To date, however, this risk relationship has not been well defined in the general population. Hypothesis: Adults in the general population with lower NP levels are at higher risk for developing DM or MS. Methods: A retrospective review of the Olmsted County Heart Function Study cohort, a population-based random sample of 2042 Olmsted County, MN residents aged ≥ 45 years. A subgroup of community-dwelling subjects without MS or DM at Visit 1 was identified and subjects returned for followup after 4 years (Visit 2). We compared the characteristics of subjects that developed MS or DM after 4 years to those that did not. Results: Of the 911 subjects who did not have MS or DM at visit 1, 93 (10.2%) developed either MS or DM at visit 2. Subjects who developed MS or DM at visit 2 were more likely to have higher baseline BMI (p <0.001), lower HDL (p<0.001), higher triglycerides (p < 0.001), higher insulin (p< 0.001), higher aldosterone (p < 0.001) levels, lower NT-proANP (p<0.001) and lower NT-proBNP levels (p < 0.01) compared to those that did not develop MS or DM. Those that developed MS or DM were also at higher risk for development of HF when controlling for age and sex (HR 2.73 CI 1.25-5.96). On univariate analysis, higher baseline levels of NT-proANP and NT pro-BNP had a significant negative association with developing new MS or DM (ORs per 1 SD increase: 0.64, CI 0.51-0.79 p < 0.0001 and 0.76, CI 0.62-0.95 p=0.016, respectively). Conclusions: Among adults aged ≥ 45 years in the community, low levels of NT- ANP and NT-proBNP are associated with increased risk of development of DM and MS. Further studies are warranted to determine if potentiating the natriuretic peptide system would delay the development of DM or MS.

Introduction: The atrial natriuretic peptide (ANP) regulates blood pressure (BP) and cardiorenal homeostasis via activation of guanylyl cyclase A (GC-A) receptor and second messenger cGMP. We recently demonstrated the safety, tolerability, and BP-lowering properties of MANP, an ANP analog and best-in-class GC-A activator, in subjects with essential hypertension (HTN). However, the effects of MANP on human metabolome during HTN remains undefined. Hypothesis: A single subcutaneous (SQ) administration of MANP can trigger metabolomic changes in humans. Methods: Twenty-two patients with HTN and metabolic syndrome (MetS) were randomized to receive placebo (N=5) or MANP (N=17, 2.5 μg/kg). Untargeted liquid chromatography mass spectrometry-based metabolomics and informatics were conducted on plasma of all subjects at baseline, 1 h post injection (hpi), and 6 hpi. Top circulating metabolites associated with MANP treatment were identified via a comparison of abundance between before and after MANP administration. Results: Compared to placebo, MANP resulted in the most prominent cGMP increase at 1 hpi and BP lowering effect at 6 hpi. With untargeted approach, we identified totally 1384 circulating metabolites. Principal component analysis supported distinct metabolic signatures at baseline, 1 hpi and 6 hpi in subjects receiving MANP. In MANP group, we found 31 metabolites at 1 hpi and 148 metabolites at 6 hpi showing significant change in abundance (adjusted P value < 0.05) compared to baseline. By contrast, <10 metabolites with difference in circulating abundance at either 1 hpi or 6 hpi compared to baseline were found in placebo group. Specifically, 8 annotated metabolites were identified with significant and progressive changes at both 1 and 6 hpi after treatment of MANP but not placebo, which included spisulosine, paecilaminol, quinine ethyl carbonate, thyrotropin releasing hormone, 3-epideoxycholic acid, hellicoside, pentanorvitamin D3, and tetracosanal. Conclusion: SQ administration of MANP triggers changes in a wide range of circulating metabolites beyond cGMP activation and BP reduction in humans. The biological outcome and clinical impact of these MANP-associated metabolites may help guide a precision medicine approach in HTN with MetS.

Introduction: Cardiorenal dysfunction with impaired cyclic guanosine monophosphate (cGMP) response to volume load is a hallmark of heart failure. Phosphodiesterase V (PDEV) is known to be upregulated and may explain the dysfunction of renal response. Hypothesis: We tested the hypothesis that PDEV inhibition in combination with low dose intravenous (IV) B-type natriuretic peptide (BNP) would improve renal function and potentiate urinary sodium and cGMP excretion in patients with acute heart failure. Methods: Randomized open label study in 67 patients admitted to the hospital with acute heart failure. These patients were randomized to standard care, low dose IV BNP (0.005μg/kg/min), or combination low dose BNP/PDEV inhibition with sildenafil (25 mg q12 hrs) for 48 hours. Plasma and urine studies were obtained at baseline, 24 hours, and 48 hours after study drug initiation to assess renal function. The primary endpoint was the percent change in estimated glomerular filtration rate (eGFR) and blood urea nitrogen (BUN) from baseline to 48 hours. Changes from baseline were summarized with median and quartiles and groups were compared using two-sample t-test. [ClinicalTrials.gov Identifier: NCT00972569] Results: Baseline characteristics were similar between groups. Median age was 78 years and median ejection fraction 39%. Treatment with BNP and BNP/PDEV inhibitor significantly increased plasma cGMP at 24 hr (% increase of 25.6 (8.9, 13.1) and 60.8 (32.3, 103.8) for BNP and BNP/PDEV vs % decrease of 13.5 (-29.1, 14.2) for placebo, p=0.001). BNP levels were significantly higher in both groups at 48 hr compared with placebo. However, there was no significant change in eGFR, BUN, or urinary sodium/cGMP excretion between groups. Hypotension was more common in the BNP/PDEV inhibitor group. Conclusions: Low dose IV BNP and combination BNP/PDEV inhibition increased plasma cGMP in patients with acute heart failure but did not improve renal function or urinary sodium/cGMP excretion. Our study does not support the use of low dose IV BNP with or without PDEV inhibition to enhance renal function in patients admitted with acute heart failure.

Introduction: Atrial natriuretic peptide (ANP) plays an important role in blood pressure (BP) regulation via the second messenger cGMP. ANP also induces lipolysis by increasing plasma levels of non-esterified fatty acids (NEFA). MANP is a new Mayo Clinic designed ANP analog consisting of the native ANP with a novel 12-amino-acid C-terminus extension. In experimental models, MANP is more potent than ANP in reducing BP and inducing natriuresis. These actions are mediated by the activation of the pGC-A receptor and generation of cGMP. To date, the BP pressure lowering, cGMP generating and lipolytic actions of MANP remain unexplored along with its safety and tolerability in subjects with hypertension (HTN) associated with metabolic disease. Hypothesis: In subjects with HTN, obesity and metabolic syndrome (MS), MANP administration is safe, well tolerated, activates cGMP and exerts BP lowering and lipolytic properties. Methods: Double-blind, placebo-control clinical trial in 22 subjects (17 receiving MANP) with HTN, obesity and MS involving a single subcutaneous (SQ) injection of MANP (2.5 μg/Kg) or placebo. Subjects were monitored and cardiovascular, cGMP and metabolic parameters were collected during 24 hours after the injection. Results: In all subjects, MANP was safe and well-tolerated with no serious adverse side effects. MANP increased mean cGMP plasma levels after 30 minutes (delta: 4.8 ± 2.0 pmol/mL, p=0.02) and 1 hour (delta: 2.9 ±1.3 pmol/mL, p=0.03) when compared to baseline. Six hours after MANP injection systolic BP decreased by 5.7 ± 2.9 mmHg (p=0.06) whereas subjects receiving placebo had no decrease in blood pressure values compared to baseline. During fasting period, one hour after the administration of MANP, NEFA plasma levels increased by 108.2 ± 37.4 μM (p=0.01). Conclusions: In subjects with HTN, obesity and MS, SQ MANP is safe, well tolerated and activates the pGC-A receptor as measured by a significant increase in the second messenger cGMP. Subjects receiving MANP showed a borderline significant decrease in systolic BP and a significant increase in NEFA circulating levels supporting anti-hypertensive and lipolytic properties. Our findings support MANP as a potential novel therapy for HTN associated with metabolic disease.