A novel dual PPAR-γ agonist/sEH inhibitor treats diabetic complications in a rat model of type 2 diabetes
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The metabolic syndrome is a cluster of risk correlates that can progress to type 2 diabetes. The present study aims to evaluate a novel molecule with a dual action against the metabolic syndrome and type 2 diabetes.
We developed and tested a novel dual modulator, RB394, which acts as a soluble epoxide hydrolase (sEH) inhibitor and a peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist in rat models of the metabolic syndrome—the obese spontaneously hypertensive (SHROB) rat and the obese diabetic Zucker fatty/spontaneously hypertensive heart failure F1 hybrid (ZSF1) rat. In SHROB rats we studied the ability of RB394 to prevent metabolic syndrome phenotypes, while in ZSF1 obese diabetic rats we compared RB394 with the ACE inhibitor enalapril in the treatment of type 2 diabetes and associated comorbid conditions. RB394 (10 mg/kg daily) and enalapril (10 mg/kg daily) were administered orally for 8 weeks.
RB394 blunted the development of hypertension, insulin resistance, hyperlipidaemia and kidney injury in SHROB rats and reduced fasting blood glucose and HbA1c, improved glucose tolerance, reduced blood pressure and improved lipid profiles in obese ZSF1 rats. A reduction in liver fibrosis and hepatosteatosis was evident in RB394-treated obese ZSF1 rats. Unlike RB394, enalapril did not demonstrate any positive effects in relation to diabetes, hyperlipidaemia or liver dysfunction in obese ZSF1 rats. RB394 ameliorated diabetic nephropathy by reducing renal interstitial fibrosis and renal tubular and glomerular injury in obese diabetic ZSF1 rats. Intriguingly, enalapril demonstrated a weaker action against diabetic nephropathy in obese ZSF1 rats.
These findings demonstrate that a novel sHE inhibitor/PPAR-γ agonist molecule targets multiple risk factors of the metabolic syndrome and is a glucose-lowering agent with a strong ability to treat diabetic complications.
KeywordsDiabetic nephropathy Drug development Liver steatosis Metabolic syndrome Soluble epoxide hydrolase
Epithelial sodium channel
Monocyte chemoattractant protein-1
Non-alcoholic fatty liver disease
Peroxisome proliferator-activated receptor-γ
Systolic blood pressure
Soluble epoxide hydrolase
Obese spontaneously hypertensive
Unilateral ureteral obstruction
Zucker diabetic fatty/spontaneously hypertensive heart failure F1 hybrid
Metabolic complications due to obesity, hypertension, hyperlipidaemia and insulin resistance are considered a major health concern and are collectively known as the metabolic syndrome . The metabolic syndrome has been recognised as a major risk factor for diseases in recent years as its prevalence has steadily increased. Indeed, the metabolic syndrome per se has a great impact on cardiovascular morbidity and mortality. Epidemiological studies have revealed an increased risk of developing type 2 diabetes as well as diabetic and cardiovascular complications in individuals with the metabolic syndrome [1, 2].
Developing adequate therapeutic and preventive measures for this multifactorial syndrome has been challenging. The complex pathophysiology of the metabolic syndrome and type 2 diabetes mean that treatment needs to involve multiple therapies aimed at regulation of lipid and glucose homeostasis as well as blood pressure. However, a multitherapy approach to treatment can result in decreased efficacy and enhanced toxicity of each drug and result in drug interactions and compromised patient compliance . Drugs that can simultaneously target various metabolic syndrome components could avoid these limitations.
We developed a novel multi-target molecule that modulates soluble epoxide hydrolase (sEH) and peroxisome proliferator-activated receptor-γ (PPAR-γ). Small-molecule sEH inhibitors are antihypertensive, organ protective and have beneficial actions on glucose metabolism [4, 5, 6, 7]. Our novel molecule combined sEH inhibitor activity with PPAR-γ agonism, the latter being widely known to regulate glucose and lipid metabolism. The efficacy of this novel dual modulator in treating the metabolic syndrome-related pathologies was determined using two different pre-clinical rat models, obese spontaneously hypertensive (SHROB) rats and obese diabetic Zucker diabetic fatty/spontaneously hypertensive heart failure F1 hybrid (ZSF1) rats [8, 9, 10, 11]. We hypothesised that the novel small dual modulator molecule RB394 would treat the metabolic syndrome, type 2 diabetes and associated complications.
The chemistry and synthesis processes for the dual PPAR-γ agonist/sEH inhibitor RB394 (see electronic supplementary material [ESM] Fig. 1) are as described previously . Enalapril was purchased from Lab Express International (Fairfield, NJ, USA). All other chemicals used were purchased from Sigma Aldrich (St Louis, MO, USA) unless otherwise mentioned.
Animals and experimental design
The Medical College of Wisconsin Institutional Animal Care and Use Committee, which abides by the National Institutes of Health Guidelines, approved animal studies. Rat strains were purchased from Charles River Laboratories (Wilmington, MA, USA) and were housed at the Medical College of Wisconsin with a 12 h light–dark cycle and free access to water and rat chow. Mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA) and were housed at the Medical College of Wisconsin under conditions similar to rats.
Rat experiments were carried out using two different protocols (Protocols 1 and 2). In Protocol 1, SHROB (SHROB/KolGmiCrl-Leprcp/Crl) rats were used as a pre-clinical model of the metabolic syndrome and Wistar–Kyoto (WKY) rats were used as control. Rats were treated with vehicle or RB394 (10 mg/kg daily) orally for 8 weeks (56 days) (ESM Fig. 2). Throughout the 8 week treatment protocol, glucose tolerance tests and tail-cuff blood pressure measurements were performed. Urine samples were collected and the rats were euthanised for blood and kidney tissue collection at the end of the 8 week treatment. See ESM Methods for details of Protocol 1. In Protocol 2, 16-week-old male obese ZSF1 (ZSF1-LeprfaLeprcp/Crl, Obese Strain code 378) rats were used as a model of type 2 diabetes and diabetic nephropathy. At this age ZSF1 obese rats develop hyperglycaemia, hypertension and proteinuria (ESM Fig. 3). These rats, along with control ZSF1 lean (ZSF1-LeprfaLeprcp/Crl, Lean Strain Code 379) rats, were treated with vehicle, RB394 (10 mg/kg daily) or enalapril (10 mg/kg daily) in drinking water for 8 weeks. During the (56 days) 8 week treatment period, glucose tolerance tests, blood pressure measurements and urine collections were performed (ESM Fig. 2). After the final urine collection, rats were euthanised for plasma and tissue collection. See ESM Methods for Protocol 2 details.
Mouse experiments were carried out in Protocol 3 wherein we used a renal fibrosis unilateral ureteric obstruction (UUO) model in C57Bl/6J mice. Mice with UUO or sham surgery were treated with vehicle or RB394 (10 mg/kg daily) for 10 days followed by kidney tissue collection. See ESM Methods for further details of Protocols 1, 2 and 3.
Glucose tolerance test
An intraperitoneal glucose tolerance test was carried out in rats at different time points, depending on the experimental protocol. See ESM Methods for details.
Biochemical assays were carried out to assess glycaemic status, lipid profile, liver function, and renal injury in rats. See ESM Methods for details.
Real-time PCR analysis
Formalin-fixed and paraffin-embedded kidney (rat and mouse) and liver (rat) were cut into 4 μm sections for histological analysis. Frozen liver sections (10 μm) were stained with Oil Red O dye to assess liver steatosis in ZSF1 rats. See ESM Methods for details.
Immune cell infiltration in the kidney for the different experimental groups rat was determined using immunohistopathological analysis. Kidney sections were immunostained with anti-CD68 (1:100; Serotec, Raleigh, NC, USA) to determine macrophage/monocyte infiltration. Biotinylated rat anti-mouse secondary antibody (1:200) was used for development with avidin-biotinylated horseradish peroxidase complex (Vectastain ABC Elite kit; Vector Laboratories, Burlingame, CA, USA) followed by counterstaining with haematoxylin. See ESM Methods for details.
Formalin-fixed and paraffin-embedded ZSF1 rat kidney sections were immunostained with anti-nephrin (1:100; Santa Cruz Biotechnology, Dallas, TX, USA) to determine nephrin expression. Donkey anti-rabbit IgG H&L (Alexa Fluor 488) secondary antibody (1:200; Abcam, Cambridge, MA, USA) was used for development with fluorescence quenching liquid (Vector Laboratories). See ESM Methods for details.
Western immunoblotting was done to determine the renal expression of TGF-β1 and fibronectin. Primary antibodies against TGF-β1 (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), fibronectin (1:2000; Cell Signaling Technology, Danvers, MA, USA) and β-tubulin (1:1000; Cell Signaling Technology) were used. Antibodies were checked against a species-specific positive control tissue and species-specific recombinant protein and tested by quenching with a competing peptide. See ESM Methods for details.
Data are reported as mean ± SEM. Statistical significance between groups was determined by a two-tailed unpaired Student’s t test (and among more than two groups determined by repeated measure one-way ANOVA followed by Tukey’s post hoc test) using GraphPad Prism Version 4.0 software (GraphPad Software, La Jolla, CA, USA). Difference between two groups was considered significant when p ≤ 0.05.
Metabolic abnormalities are ameliorated in RB394-treated SHROB rats
RB394 treatment decreases kidney injury in SHROB rats
RB394 effectively reduces diabetes and hypertension in ZSF1 obese rats
Hyperlipidaemia is reduced by RB394 in diabetic ZSF1 obese rats
RB394 ameliorates liver complications and steatosis in diabetic ZSF1 obese rats
RB394 reduces kidney injury in diabetic ZSF1 obese rats
ZSF1 obese rats exhibited renal inflammation associated with higher urinary MCP-1 excretion and a greater number of CD68-positive cells in the kidney (Fig. 8i–k). RB394 reduced renal inflammation in ZSF1 obese rats. Interestingly, enalapril had a smaller effect on renal inflammation than RB394 in obese ZSF1 rats: enalapril reduced urinary MCP-1 excretion and renal infiltration of CD68 cells by only 13–20% (p > 0.05).
RB394 reduces kidney injury in UUO in mice
The ability of RB394 to reduce kidney injury independent of concomitant changes in the metabolic syndrome variables was tested in a mouse model of UUO renal injury. RB394 treatment reduced kidney fibrosis and kidney fibronectin levels (ESM Fig. 4). These data demonstrate that RB394 can decrease kidney injury independent of changes in metabolic variables.
We developed a novel dual-acting molecule, RB394, which simultaneously acts as an inhibitor of sEH and as a PPAR-γ agonist. Using two distinct approaches, we examined the efficacy of RB394 in pre-clinical models of the metabolic syndrome and type 2 diabetes. In one model (SHROB rats, which exhibit features of the metabolic syndrome) we investigated the ability of RB394 to prevent pathophysiological features of the metabolic syndrome and renal injury . In another model (obese ZSF1 rat model of type 2 diabetes), we compared RB394 with an ACE inhibitor, enalapril, in treating diabetic complications since ACE inhibitors are widely used for blood pressure control and are particularly beneficial for treating diabetic nephropathy in hypertensive individuals with type 2 diabetes [14, 15, 16, 17].
In SHROB rats, we demonstrated that RB394 blunts the development of hypertension, hyperlipidaemia and insulin resistance. Interestingly, in the diabetic obese ZSF1 rats, we demonstrated that RB394 but not enalapril effectively reduces hyperglycaemia, hyperlipidaemia and insulin resistance. We suggest that this ability of RB394 to provide multifaceted actions in the metabolic syndrome and type 2 diabetes can be attributed to its biological actions associated with sEH inhibition and PPAR-γ agonism. Indeed, it has been reported that sEH inhibitors reduce hypertension  as well as improve lipid profiles [6, 13] and insulin sensitivity  in the metabolic syndrome. Likewise, in the metabolic syndrome PPAR-γ agonists exert similar effects to reduce hypertension, hyperlipidaemia and insulin resistance [18, 19].
RB394 treatment did not alter body weight in SHROB and obese ZSF1 rats. This is an interesting finding as according to current views obesity and insulin resistance are closely and reciprocally interrelated . It is, however, reported that sEH inhibitors do not affect body weight gain while improving insulin sensitivity in the metabolic syndrome . PPAR-γ agonists do not reduce body weight and can even increase body weight in animals and humans . It is important to note that the increase in body weight caused by PPAR-γ agonists can be a result of oedema, a serious side effect observed in certain populations . It is reported that PPAR-γ agonists stimulate kidney epithelial sodium channels (ENaCs), which play a critical role in body water and electrolyte balance . Interestingly, sEH inhibition can block ENaC activity by elevating endogenous epoxyeicosatrienoic acids, which are potent ENaC inhibitors [24, 25, 26]. These findings support the notion that RB394 can oppose the PPAR-γ agonistic activation of ENaC and subsequent oedema.
Our findings collectively demonstrate that unlike the current option of treatment with an ACE inhibitor, the novel dual-acting molecule can act on multiple cardinal metabolic events in the metabolic syndrome. We demonstrate that RB394 as a single entity prevents the development of the metabolic syndrome and reduces the severity of type 2 diabetes and associated comorbid conditions. This is an important finding as type 2 diabetes is associated with high morbidity and mortality, largely due to the inability of currently available drugs to control comorbid conditions, such as hypertension and hyperlipidaemia, and to prevent the development of diabetic complications . We suggest that RB394 would be beneficial in reducing diabetic complications including diabetic nephropathy.
Indeed, the increasing population with type 2 diabetes has created a major impact on the prevalence of diabetic nephropathy, which occurs in 20–40% of individuals with type 2 diabetes and is the leading cause of end-stage renal disease [28, 29]. Few new drugs aimed at reducing kidney complications have been successfully developed over the past 16 years . This could be due to the complexity of aetiological factors associated with diabetic nephropathy. The prevention and treatment of diabetic nephropathy requires a multiple risk factor approach, the goal being to achieve glycaemic control along with reduction of hypertension and dyslipidaemia . This medical need is, as yet, unmet. In the current study SHROB and ZSF1 rats exhibited marked renal fibrosis and glomerular injury. We demonstrated that RB394 prevented nephropathy development in SHROB rats and, more importantly, it effectively ameliorated diabetic nephropathy and was comparable or better than enalapril in obese diabetic ZSF1 rats (model of type 2 diabetes).
The marked beneficial renal actions of RB394 can be attributed to its ability to improve insulin sensitivity, lower blood pressure and decrease plasma lipids in the metabolic syndrome. Impaired glucose homeostasis, hypertension and hyperlipidaemia are known to contribute to the progression of diabetic nephropathy . One prominent metabolic abnormality in the metabolic syndrome is insulin resistance, which leads to hyperinsulinaemia followed by glomerular hyperfiltration, endothelial dysfunction and eventual albuminuria . Impaired insulin sensitivity is also associated with altered renal cellular metabolism, mesangial hyperplasia, increased endothelial cell proliferation and lipid and hyaluronate deposition, which contributes to kidney injury . In SHROB and ZSF1 rats we demonstrated marked insulin resistance and in the obese ZSF1 rats insulin resistance was accompanied by hyperinsulinaemia. RB394 prevented and reduced insulin resistance and hyperinsulinaemia in SHROB and ZSF1 rats. These actions of RB394 could be related to the biological actions of the two pharmacophores that comprise RB394. Indeed, genetic deletion or pharmacological inhibition of sEH improves insulin sensitivity and glucose tolerance in mice [5, 6] and PPAR-γ agonists improve insulin sensitivity and glucose homeostasis .
In the present study enalapril had no effect on insulin sensitivity in obese ZSF1 rats. It should be noted that ACE inhibitors have been shown to improve insulin sensitivity by increasing nitric oxide levels . However, this action of ACE inhibitors on insulin sensitivity is controversial and there are multiple studies demonstrating no such action. Indeed, no increases in insulin sensitivity were found in healthy individuals, hypertensive individuals with essentially normal insulin sensitivity or hypertensive insulin-resistant individuals with type 2 diabetes following treatment with trandolapril  or enalapril . Animal studies have shown similar results—enalapril did not cause positive effects on glycaemic control in a rat model of type 2 diabetes/obesity —and support our current findings.
The renal actions of RB394 demonstrated in the current study are also related to its antihypertensive and lipid-lowering effects. In clinical studies, blood pressure reduction has been identified as a potent cardiovascular disease risk reducer in the metabolic syndrome and type 2 diabetes. Better blood pressure control in type 2 diabetes has been shown to decrease the onset or degree of albuminuria and vascular complications . In the current study, RB394 prevented and reduced hypertension in rat models of the metabolic syndrome and type 2 diabetes. It should be noted that sEH inhibition and PPAR-γ agonism are known to produce an antihypertensive effect [13, 40]. As expected, the ACE inhibitor enalapril reduced hypertension in the obese ZSF1 rats and, interestingly, the antihypertensive actions of RB394 and enalapril were equipotent.
Akin to hypertension, there is increasing evidence for an association between dyslipidaemia and renal disease progression in individuals with type 2 diabetes . Lipid-lowering agents demonstrate beneficial renal outcomes besides their beneficial effects on cardiovascular disease in type 2 diabetes . In the present study, the lipid-lowering effect of RB394 can be attributed to the concerted action of its two pharmacophores [43, 44]. Unlike RB394, enalapril did not improve the lipid profile in obese ZSF1 rats. It is important to note that an earlier study suggested that enalapril may prevent the development of elevated cholesterol levels in obese ZSF1 rats after 12 and 24 weeks of treatment . However, in this study enalapril was used in a preventive manner and it is not known whether enalapril or any other ACE inhibitor can reduce established hyperlipidaemia in a pre-clinical diabetic nephropathy model.
Apart from metabolic abnormalities per se, inflammation associated with metabolic abnormalities plays a crucial role in metabolic syndrome kidney injury. Chronic inflammation is a hallmark of the metabolic syndrome and its severity depends on the presence of different metabolic syndrome pathophysiologies . It is proposed that metabolic syndrome-induced disruption in physiological regulatory systems due to excessive energy intake provokes stressor stimuli that trigger inflammation . The substantial inflammatory macrophage infiltration and cytokines in the abdominal and peri-renal fat seen in animal models of the metabolic syndrome could serve as a channel for cytokines to access the kidney . We earlier demonstrated renal inflammation and injury associated with increased MCP-1 excretion and elevated renal immune cell infiltration in animal models of the metabolic syndrome [8, 13]. The present study demonstrates that SHROB and obese diabetic ZSF1 rats exhibit renal inflammation and injury. Interestingly, we demonstrate that RB394 reduces renal inflammation by reducing MCP-1 excretion, renal immune cell infiltration and TGF-β expression. Conversely, the ACE inhibitor enalapril did not reduce renal inflammation in diabetic obese ZSF1 rats.
The anti-inflammatory and renal actions of RB394 can be attributed to sEH inhibition and/or PPAR-γ agonism. Anti-inflammatory activity has been demonstrated for sEH inhibitors in the metabolic syndrome, type 2 diabetes and hypertension [7, 13]. Similarly, PPAR-γ agonists demonstrate renal anti-inflammatory activity in SHROB and ZSF1 obese rats [13, 38]. It is possible that the anti-inflammatory and renal actions of sEH inhibitors and PPAR-γ agonist in the metabolic syndrome are due to their ability to reduce metabolic abnormalities and that they do not have any true and independent anti-inflammatory or renal protective actions. Contrary to this notion, anti-inflammatory and renal protective actions for sEH inhibitors and PPAR-γ agonists have been demonstrated in pathological conditions that do not depend on antihypertensive or glucose-lowering effects [47, 48, 49, 50]. These findings support the notion that RB394 possesses direct renal protective activity. Experiments conducted in obstructive nephropathy demonstrated that RB394 possesses kidney protective effects independent of its actions on metabolic pathologies. Overall, we demonstrate that RB394 reduces diabetic nephropathy by exerting a multifaceted action in the metabolic syndrome and in other renal pathologies not associated with the metabolic syndrome, hypertension or diabetes.
Individuals with type 2 diabetes have an increased risk of developing chronic liver disease, especially non-alcoholic fatty liver disease (NAFLD) . The prevalence of NAFLD in diabetic individuals is higher than in those without diabetes, possibly as high as 75%, and the NAFLD can progress to liver fibrosis [52, 53, 54]. We examined whether RB394 or enalapril could treat liver dysfunction, fibrosis and steatosis in obese ZSF1 rats. Enalapril had no effect on liver dysfunction or hepatosteatosis, supporting earlier findings that enalapril did not reduce hepatosteatosis in obese ZSF1 rats . Indeed, the hepatic action of ACE inhibitors is controversial and there are reports of ACE-inhibitor-associated hepatotoxicity in individuals with diabetes . Ramipril, a well-acclaimed ACE inhibitor used in diabetic hypertensive patients, has been reported to cause liver dysfunction [56, 57]. In contrast, we demonstrated striking effects for RB394 in ameliorating liver dysfunction, fibrosis and steatosis in obese ZSF1 rats. This is an interesting finding as sEH and PPAR-γ differ in their actions in fatty liver and steatohepatitis. The role of PPAR-γ in hepatic steatosis is complex. Mouse models of the metabolic syndrome and diabetes, including ob/ob and KKAy mice, demonstrate the development of fatty livers in which expression of PPAR-γ is enhanced. These results suggest that PPAR-γ might be implicated in fatty liver pathophysiology in mice . Nevertheless, PPAR-γ agonists have beneficial actions in non-alcoholic steatohepatitis as they reduce liver collagen levels . sEH inhibitors reduced hepatic steatosis in a high-fat-diet-induced model of the metabolic syndrome . Moreover, sEH inhibitors demonstrate strong antifibrotic activity in carbon tetrachloride-induced cirrhotic hepatitis . Although there is an apparent difference in the actions of the two pharmacophores of RB394, we suggest that the common biological actions of sEH and PPAR-γ on hyperglycaemia and hyperlipidaemia are associated with the liver protection. Indeed, there are roles for hyperglycaemia and hyperlipidaemia in NAFLD pathogenesis and consequent liver dysfunction and fibrosis . In addition, hyperglycaemia is suggested to contribute to the pathophysiology of liver fibrosis in the metabolic syndrome and type 2 diabetes [62, 63]. Overall, we demonstrated that apart from its ability to treat diabetic nephropathy, RB394 can treat fatty liver and steatohepatitis in type 2 diabetes.
In summary, we demonstrate the therapeutic potential for a dual modulator, RB394, which acts as an sEH inhibitor and PPAR-γ agonist in rat models of the metabolic syndrome and type 2 diabetes. This novel molecule prevented the development of metabolic abnormalities and kidney injury in a model of the metabolic syndrome. Most importantly, we demonstrate the ability of RB394 to ameliorate type 2 diabetes and its comorbid conditions, such as insulin resistance, hypertension and hyperlipidaemia, and to reduce multiple diabetic complications such as diabetic nephropathy and liver injury. Overall, we demonstrate a promising dual-ligand small molecule, RB394, which can be developed as a therapeutic agent for the metabolic syndrome, type 2 diabetes and associated complications.
MAHK, JDI and EP conceived the study and wrote the manuscript. LK and MS carried out all biochemical, protein expression, gene expression, histopathological and immunohistopathological analyses and the final presentation of the data. EP, RB and MH designed and synthesised RB394. LK, MS, RB and MH contributed to drafting and revision of the manuscript. JDI and EP take full responsibility for the work and the conduct of the study, had access to the data and controlled the decision to publish.
A National Institute of Health (NIH) grant (DK103616) and Dr. Ralph and Marian Falk Medical Research Trust Bank of America, N.A., Trustee grant to JDI supported this study. Research was supported by the Deutsche Forschungsgemeinschaft (Sachbeihilfe PR 1405/2-2, Heisenberg-Professur PR 1405/4-1 and SFB 1039 Teilprojekt A07) to EP. RB thanks Else-Kröner-Fresenius Foundation graduate college for Translational Research Innovation–Pharma (TRIP) for a PhD scholarship. MH thanks German Cancer Consortium (DKTK) for a PhD scholarship. The funding sources were not involved in the design of the study, the collection, analysis and interpretation of data, writing the manuscript or the decision to submit the manuscript for publication.
Duality of interest
JDI, EP, MAHK and RB have a patent application that covers the composition of matter for RB394. All other authors declare that there is no duality of interest associated with their contribution to this article.
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