Oleanolic, Ursolic, and Betulinic Acids as Food Supplements or

Review pubs.acs.org/JAFC

Oleanolic, Ursolic, and Betulinic Acids as Food Supplements or Pharmaceutical Agents for Type 2 Diabetes: Promise or Illusion? Filomena S. G. Silva,† Paulo J. Oliveira,§ and Maria F. Duarte*,† †

J. Agric. Food Chem. 2016.64:2991-3008. Downloaded from pubs.acs.org by OPEN UNIV OF HONG KONG on 01/23/19. For personal use only.

Centro de Biotecnologia Agrı ́cola e Agro-Alimentar do Alentejo (CEBAL)/Instituto Politécnico de Beja (IPBeja), Apartado 6158, 7801-908 Beja, Portugal § CNC, Center for Neuroscience and Cellular Biology, UC-Biotech Building, Biocant Park, University of Coimbra, 3060-107 Cantanhede, Portugal ABSTRACT: Oleanolic (OA), ursolic (UA), and betulinic (BA) acids are three triterpenic acids (TAs) with potential effects for treatment of type 2 diabetes (T2DM). Mechanistic studies showed that these TAs act as hypoglycemic and antiobesity agents mainly through (i) reducing the absorption of glucose; (ii) decreasing endogenous glucose production; (iii) increasing insulin sensitivity; (iv) improving lipid homeostasis; and (v) promoting body weight regulation. Besides these promising beneficial effects, it is believed that OA, UA, and BA protect against diabetes-related comorbidities due to their antiatherogenic, antiinflammatory, and antioxidant properties. We also highlight the protective effect of OA, UA, and BA against oxidative damage, which may be very relevant for the treatment and/or prevention of T2DM. In the present review, we provide an integrative description of the antidiabetic properties of OA, UA, and BA, evaluating the potential use of these TAs as food supplements or pharmaceutical agents to prevent and/or treat T2DM. KEYWORDS: oleanolic acid, ursolic acid, betulinic acid, antidiabetic, hepatic disease, cardiovascular disease, antiobesity

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INTRODUCTION Diabetes is a chronic metabolic disorder that is currently assuming epidemic proportions. According to statistics, it was estimated that about 9% of the adult worldwide population had diabetes in 2014, with 90% among those having type 2 diabetes mellitus (T2DM).1 Type 2 diabetes is characterized by hyperglycemia resulting from insulin resistance, as well as insufficient insulin secretion by pancreatic β-cells. Accumulating evidence demonstrates that T2DM is associated with obesity,2 as well as with the development of several comorbidities, including hepatic, cardiac, and renal disorders.3 Management of T2DM requires an integrated approach, which includes hyperglycemia and obesity treatments, as well as prevention of diabetic comorbidities. However, the currently available antidiabetic drugs have limited efficacy and/or safety concerns and only temporarily improve blood glucose levels, failing in the treatment of obesity, as well as in the prevention of diabetes complications. Therefore, the identification of new therapeutic agents, which may have the potential to prevent or treat diabetes and its related comorbidities, is an unmet need. Consumption of different natural compounds is known to result in antidiabetic effects, offering exciting possibilities for the future development of successful therapies. Terpenoids are a diverse group of phytochemical agents derived from squalene or related acyclic and cyclic 30-carbon precursors, which are currently receiving attention because of their wide range of biological activity.4 An increasing number of publications have focused on pentacyclic triterpenoids, lupane-, oleanane-, and ursane-type structures, particularly oleanolic (3β-hydroxyolean12-en-28-oic, OA), ursolic (3β-hydroxyurs-12-en-28-oic, UA), and betulinic (3β-hydroxy-lup-20-en-28-oic, BA) acids (Figure 1) due to their antidiabetic properties (reviewed in refs 5−7). © 2016 American Chemical Society

Oleanolic, ursolic, and betulinic acids have several important biological activities, including antitumoral, anti-inflammatory, and antioxidant effects,4 as well as hepato-,8−11 cardio-,12−14 and nephroprotective8,15 roles. The three triterpenic acids (TAs) described above also have a potential application for T2DM management and its associated comorbidities. The biological effects mainly result from their anti-hyperglycemic,16−19 anti-hyperlipidemic,20−23 antiatherogenic,24−26 antioxidant,27−29 and anti-inflammatory27,30,31 actions. Published data indicate that OA, UA, and BA, or plants extracts rich in those TAs, reduce the absorption and uptake of glucose, lower endogenous glucose production, increase insulin biosynthesis, secretion, and sensitivity, and protect against diabetic complications.6,7 These protective effects occur without noticeable hepatotoxicity, at the dose used in the experimental studies, as indicated by the decreased values of aspartate aminotransferase and alanine aminotransferase measured.21,22,32 This relevant fact is contrary to most currently used antidiabetic drugs, including sulfonylureas, α-glucosidase inhibitors, biguanides, and thiazolidinediones, which can cause liver toxicity.33,34 The low toxicity profile of these compounds has also been evidenced in several in vivo studies, where OA (10−1700 mg/ kg),35−39 UA (25−2500 mg/kg),10,40,41 and BA (25−50 mg/ kg)42 protected the liver from oxidative damage at concentrations equivalent to or higher than those used in diabetic studies, when administered to rodents pretreated with different hepatotoxicants. The protective effects of OA, UA, and BA Received: Revised: Accepted: Published: 2991

December March 25, March 25, March 25,

21, 2015 2016 2016 2016 DOI: 10.1021/acs.jafc.5b06021 J. Agric. Food Chem. 2016, 64, 2991−3008

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Journal of Agricultural and Food Chemistry

Figure 1. Chemical structures of some bioactive triterpenic acids: oleanolic, ursolic, and betulinic acids. Adapted from ref 4.

against oxidative damage may be critical for their use in the treatment and prevention of diabetes and its comorbidities. In this context, in the present unique review, we make an integrative description of the hypoglycemic and antiobesity properties of OA, UA, and BA, as well as their effects on diabetic comorbidities in the management of T2DM. We here emphasize the main targets of action and limitations of these compounds, exploring the current knowledge and open questions. The present work focuses on the main issues that research in this field should take into account, emphasizing what should be clarified in the future and evaluating the potential use of these TAs as food supplements or pharmaceutical agents to prevent and/or treat T2DM.

reduces the synthesis of glycogen by increasing gluconeogenesis and glycogenolysis and by reducing glycogenesis.56 Oleanolic, ursolic, and betulinic acids (IC50 = 14.9, 9, and 43 μM, respectively) inhibit glycogen phosphorylase (GP),57 the rate-limiting step of glycogenolysis, which decreases glycogenolysis and also stimulates glycogen synthesis.58 Moreover, UA treatment (5 mg/kg/day or 0.01−0.05%, w/w) increases glycogen content by increasing hepatic glucokinase activity in vivo mice models,17,20,59 a key regulatory step in liver glycogen formation,60 and also decreases blood glucose levels by inhibiting hepatic glucose-6-phosphatase (G6-Pase) activity,17,20,59 the ultimate step in glucose production.61 In line with increased hepatic glycogen, UA (2 mg/kg/day) also increases glycogen deposition in healthy rats,62 most likely by promoting the phosphorylation and inactivation of glycogen synthase kinase-3 (GSK-3), an enzyme that inhibits the glycogen synthesis pathway.63 Additionally, OA, UA, and BA (10 μM) stimulate glucose uptake and glycogen synthesis in human HepG2 hepatoma cells via the 5′ AMP-activated protein kinase (AMPK)-GSK-3β pathway.64 Once activated, AMPK, an energy sensor that regulates cellular metabolism, leads to the inhibition of glucose production by increasing GSK-3β phosphorylation. Activation of AMPK decreased gluconeogenesis by regulating gluconeogenic targets, such as phosphoenolpyruvate carboxykinase (PEPCK) and G6-Pase.65 In vivo treatment with OA (20 mg/kg/day) decreased G6-Pase and PEPCK protein contents,18 which may result in lower gluconeogenesis rates. A similar in vitro study, but using BA instead (10 and 20 μM), demonstrated an effect on hepatic gluconeogenesis by decreasing PEPCK and G6-Pase mRNA, resulting from AMPK activation.19 Another study also evidenced that once activated by BA, the AMPK pathway promoted glucose utilization and glucose transport across the cell membrane, by induction of glycolysis and by up-regulation of GLUT-1 and -2 protein expression in mouse embryonic fibroblasts. This study also demonstrated that glycolysis stimulation by BA was accompanied by a reduction of glucose oxidation66 (Table 1). According to the results, OA, UA, and BA reduced glucose availability in the body by limiting endogenous glucose production, also promoting glycogen synthesis in T2DM, contributing to the hypoglycemic effect of these compounds. However, future studies are warranted to confirm these results using more in vivo assays, including in humans. Insulin Resistance. T2DM mainly results from insulin resistance and β-cell dysfunction. The improvement of insulin sensitivity represents a good strategy to prevent T2DM development.67 Numerous studies have supported the notion that OA, UA, and BA improve insulin responses by acting as insulin

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MECHANISMS BEHIND HYPOGLYCEMIC EFFECTS Numerous research studies have described the hypoglycemic effects for OA, UA, and BA using in vitro and in vivo models. The description of hypoglycemic effects of these three TAs is summarized in Table 1. Absorption and Uptake of Glucose. Inhibition of carbohydrate hydrolysis and down-regulation of glucose transporters in the gastrointestinal tract decrease postprandial hyperglycemia,43,44 suppressing, at least in part, the development of T2DM.45 Oleanolic, ursolic, and betulinic acids inhibit pancreatic αamylase activity in vitro ([TAs] = 5−50 μg/mL ≈ 10−100 μM),46,47 and in vivo (5−20 mg/kg/day during 15 weeks) in high-fat diet (HFD)-induced obese mice.32,48,49 Additionally, other authors also showed that these three TAs have also the capacity to decrease α-glucosidase (IC50 = 0.1−231 μM)47,50−54 activity in vitro. Also, OA inhibits human pancreatic α-amylase activity (IC50 = 0.1 mg/mL ≈ 200 μM),55 and decreases postprandial hyperglycemia in diabetic rats by inhibiting the activities of α-amylase (IC50 = 3.60 μM), and α-glucosidase (IC50 = 12.40 μM), and simultaneously down-regulating the expression of sodium-dependent glucose cotransporter (SGLT1) and glucose transporter-2 (GLUT-2) in the small intestine16 (Table 1). Taken together, these results suggest that inhibition of α-amylase and α-glucosidase, as well as down-regulation of glucose transporters by the above-mentioned TAs, can delay glucose absorption in the small intestine in diabetes, consequently preventing postprandial plasma glucose increase. However, the reduced numbers of animal tests and the lack of human studies raise questions about the true effectiveness of these compounds in vivo, highlighting the need for more studies in living systems. Endogenous Glucose Production and Glycogen Synthesis. T2DM increases endogenous glucose production and 2992

DOI: 10.1021/acs.jafc.5b06021 J. Agric. Food Chem. 2016, 64, 2991−3008

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Journal of Agricultural and Food Chemistry Table 1. Summary of Studies Reporting the Hypoglycemic Effects of OA, UA, and BAa

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Journal of Agricultural and Food Chemistry Table 1. continued

a

Abbreviations used: Adipo-IR, adipose tissue insulin resistance; AMPK, 5′ AMP-activated protein kinase; Akt, phosphoinositide-dependent serine/ threoninekinase; BA, betulinic acid; CAMKK, Ca2+-calmodulin-dependent protein kinase; cAMP, cyclic adenosine monophosphate; CAMs, cell adhesion molecules; CAT, catalase; CCL2/MCP-1, monocyte chemotactic protein-1; CHO/IR, Chinese hamster ovary cells expressing human insulin receptor; CREB, cAMP response element-bindinprotein; DM, diabetes mellitus; ERK, extracellular-signal regulated kinase; Gclc, glutamate− cysteine ligase catalytic subunit; Gclm, glutamate−cysteine ligase modified subunit; GLP-1, glucagon-like peptide-1; GK, glucokinase; G6-Pase, glucose-6-phosphatase; GSK-3, glycogen synthase kinase-3; GLUT-2 and -4, glucose transporter-2 and -4; GP, glycogen phosphorylase; GPx, glutathione peroxidase; HbA1C, hemoglobin A1c; HFD, high-fat diet; Ho-1, heme oxygenase-1; HOMA-IR, homeostasis model assessment of ́ insulin resistance index; HUVEC, human umbilical vein endothelial cells; ICAM, intracellular adhesion molecule-1; IECs, intestinal epithelial cells; IFN-γ, interferon-γ; IR, insulin receptor; IRS, insulin receptor substrate; LPS, lipopolysaccharides; MDA, malondialdehyde; MEF, mouse embryonic fibroblasts; NA, nicotinamide; NF-κB, nuclear factor kappa B; Nqo1, NAD(P)H:quinone oxidoreductase; OA, oleanolic acid; PDK, phosphoinositide-dependent kinase; PEPCK, phosphoenolpyruvate carboxykinase; PGC-1α, peroxisome proliferator-activated receptor-γ coactivator-1α; PI3KB, phosphatidylinositol 3-kinase-dependent; PKC, protein kinase C; PTP-1B, protein tyrosine phosphatase-1B; PYY, peptide YY; ROS, reactive oxygen species; SHP-2, src homology phosphatase-2; SGLT-1, sodium-dependent glucose cotransporter; SOD, superoxide dismutase; STZ, streptozotocin; TCPTP, T-cell protein tyrosine phosphatase; TNF-α1, tumor necrosis facto- α1; UA, ursolic acid; UCP-1 and -2, 2994


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uncoupling protein-1 and -2; VCAM, vascular cell adhesion molecule-1; VEGF, vascular endothelial growth factor; Δψm, mitochondrial membrane potential; ↑, enhances/up-regulates; ↑↑, activates; ↑↑↑, improves; ↓, reduces/down-regulates; ↓↓, inhibits; ↓↓↓, blocks; →, preserves or prolongs; → →, modulates; dependents; ∼, no effects.

like peptide-1 (GLP-1) release.85 GLP-1 is a potent glucosedependent insulin-stimulating hormone95 that stimulates β-cell proliferation and pro-insulin gene expression96 and which inhibits glucagon expression.97 Oleanolic acid (10−50 μM) also activated TGR5 in pancreatic β-cells, leading to TGR5mediated glucose-stimulated insulin release86 (Table 1). Contradictorily, a human phase I clinical trial showed that the hypoglycemic effect of intraduodenal OA perfusions (1 mM) was not accompanied by the release of GLP-1 in healthy male volunteers (University Hospital, Basel, Switzerland, coded NCT01674946). Improvement of β-Cell Survival. Current evidence shows that decreased function of β-cells always precedes the development of T2DM and induces profound metabolic dysfunction. Therefore, β-cell function preservation or recovery can be an effective therapeutic strategy to prevent or treat T2DM.98 It is known that UA (0.05%, w/w) preserved the integrity of β-cells68 and islet architecture69 in the pancreas, contributing to the prevention of the metabolic dysfunction of β-cells and consequently the development of T2DM. Oleanolic acid (0.5 mg/day during 2 days) prolonged the survival of transplanted islets due to its anti-inflammatory, immuneregulatory, and antioxidant properties. Beneficial effects were suggested to be mediated by decreased γ-interferon-inducible protein 10 and IL-4 in serum, the number of T cells secreting γinterferon, IL-4, IL-7, and IL-2, and the infiltration of CD4 and CD8 cells, as well as decreased reactive oxygen species (ROS) production,87 supporting the notion that the anti-inflammatory and antioxidant effects of these compounds can also contribute to preserve the integrity of β-cells and consequently the development of T2DM. Anti-inflammatory and Antioxidant Properties. Different studies demonstrated that OA,99 UA,30 and BA31 downregulate the nuclear factor kappa B (NF-κB) signaling pathway in different cell models, which may decrease several inflammatory cytokines and, consequently, improve insulin response in diabetic patients,100 supporting the notion that the anti-inflammatory effects of these compounds may in turn prevent T2DM. Similarly, it was also described that the reduction of inflammatory cytokines tumor necrosis factor-α (TNF-α) and IL-6, mediated by reduction of NF-κB promoted by OA, may in turn down-regulate the expression of IRS-1 and GLUT-4 in the HepG2 cell line,27 evidencing the crucial role between the reduction of oxidative stress and the suppression of inflammatory response in the prevention of T2DM. Another study also suggested that OA (20 mg/kg/day during 2 weeks) improves hepatic insulin resistance by acting as an antiinflammatory, down-regulating IL-1β, IL-6, and TNF-α1, and as antioxidant by increasing superoxide dismutase (SOD), catalase (CAT) protein contents and mitochondrial reduced glutathione pool in diabetic mice. In the same study, OA also increased nuclear factor (erythroid-derived 2)-like 2 (Nrf2) protein expression,18 which promoted the transcriptional induction of several antioxidant genes and suppression of inflammatory responses,101 contributing thus to the improvement of insulin resistance.102 The authors suggested that this increase of Nrf2 may in turn be responsible for reducing

secretagogue and insulinomimetic agents in different contexts.68−73 According to different studies, the three TAs interfere with insulin biosynthesis, secretion, and sensitivity involving multitarget mechanisms, mainly by inhibiting different protein tyrosine phosphatases,74−79 by stimulating the phosphatidylinositol 3-kinase-dependent (PI3KB)/protein kinase Akt pathway,72,73,80−82 by acting as TGR-5 receptors,83−86 by improving the survival of β-cells,68,69,87 and/or by acting as anti-inflammatory and antioxidant agents.27,28,31 Protein Tyrosine Phosphatases and the PI3KB/Akt Pathway. Several in vitro studies provided evidence that OA, UA, and BA directly inhibited protein tyrosine phosphatase 1B (PTP-1B),74−79 thus improving insulin resistance in T2DM and obesity.88−91 PTP-1B is a molecule that negatively regulates insulin signaling. Insulin regulates glucose homeostasis through (i) binding to its receptor to initiate a signaling cascade, (ii) activating and promoting phosphorylation of insulin receptor substrate proteins, and (iii) mediating the PI3K/Akt pathway.92,93 Akt pathway activation facilitated glucose uptake into adipose tissue, cardiac muscle, and skeletal muscle, by mediating the translocation of glucose transporter-4 (GLUT4) to the plasma membrane and by inhibiting gluconeogenesis.5 However, PTP-1B inhibited the PI3K/Akt signaling pathway resulting in insulin resistance. The mechanism is thought to result from the inhibition of GLUT-4 translocation to the plasma membrane, decreasing glucose re-uptake and gluconeogenesis. Therefore, the direct inhibition of PTP-1B by OA, UA, and BA may improve insulin sensitivity. Nevertheless, it was described that UA acted directly in the insulin receptor (IR).80 In vitro studies performed by Jung et al. in cultured adipocytes and Chinese hamster ovary cells expressing human IR80 showed that UA is an insulin sensitizer, leading to an increase in IRβ autophosphorylation and to a subsequent activation of downstream PI3K signaling pathway. The activation of this pathway would result in phosphorylation and inactivation of GSK-3, leading to an increase of glycogen synthesis,63 as described before. Also, OA increased IR and insulin receptor substrate-1 (IRS-1) mRNA and up-regulated total IRS-1 protein expression in adipose tissue insulin resistance rats, suggesting that this compound improves insulin resistance via the IRS-1/PI3k/Akt pathway.81 With regard to UA (10−20 μM), this molecule was described to increase insulin sensitivity, by promoting GLUT-4 translocation in 3T3-L1 adipocytes,80,82 probably by activating the PI3K pathway.82 In a similar way, in vivo studies showed that treatment with UA or BA (10 mg/kg) increases glycogen content and glucose uptake in muscle by acting as insulin secretagogue and insulinomimetic agents via PI3K and MAPK, stimulating GLUT-4 synthesis and translocation in muscle.72,73 All of these effects are summarized in Table 1. TGR5 Receptors. Accumulating evidence suggests that OA,83 UA, and BA84 act in vitro as selective TGR5 agonists (EC50 = 1.04−2.25 μM). TGR5 is a G-protein-coupled receptor identified as the first cell surface receptor involved in energy homeostasis that is activated by bile acids. Its activity ameliorates insulin resistance.94 Indeed, OA (10−30 μM) activated TGR5 in enteroendocrine cells, leading to glucagon2995


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Journal of Agricultural and Food Chemistry Table 2. Summary of Studies Reporting the Antiobesity Effects of OA, UA, and BAa

a

Abbreviations used: ACC, acetyl-coA carboxylase; ACO, acyl-CoA oxidase; ACS, acyl-CoA synthetase; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; AMPK, 5′ AMP-activated protein kinase; ATGL, adipose triglyceride lipase; BA, betulinic acid; cAMP, cyclic adenosine monophosphate; C/EBP-α and -β, CCAAT element binding protein -α and -β; CPT-1α, carnitine palmitoyltransferase-1α; DGAT, diacylglycerol acyltransferase; DM, Diabetes mellitus; FABP-4, fatty-acid-binding protein 4; FATP-4, fatty acid transport protein-4; FAS, fatty acid synthase; FFA, free fatty acid; GK, glucokinase; Gpam, glycerol-3-phosphate acyltransferase; G6-Pase, glucose-6-phosphatase; GPx, glutathione peroxidase; hACAT-2, cholesterol acyltransferase 2; HbA1c, hemoglobin A1c; HDLc, high-density lipoprotein cholesterol; HFD, high-fat diet; HSL, hormone-sensitive lipase; LC3-II, microtubule-associated protein 1A/1B-light chain 3-II; LDLc, low-density lipoprotein cholesterol; LKB1, liver kinase B1; MDA, malondialdehyde; OA, oleanolic acid; PPAR-α and -γ, peroxisome proliferator activated receptor-α and -γ; PPRE, peroxisome proliferator response element; RER, respiratory exchange rate; SCD-1 and -2, stearoyl-CoA desaturase; Sirt1, silent mating type information regulation 2 homologue 1; SOCS-3, suppressor of cytokine signaling-3; SOD, superoxide dismutase; SREBP-1c, sterol-regulatory2996


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element-binding protein-1c; Srebf-1, sterol regulatory element binding factor-1; STAT-1 and -3, signal transducer and activator of transcription factor; STZ, streptozotocin; TG, triglycerides; UA, ursolic acid; UCP-3, uncoupling protein-3; VLDLc, very-low-density lipoprotein cholesterol; ↑, enhances/up-regulates; ↑↑, activates; ↑↑↑, improves; ↓, reduces/down-regulates; ↓↓, inhibits; →, preserves/prolongs/promotes; ∼, no effects.

true hypolipidemic effects of these compounds, clearly highlighting the need for future human studies. Lipid Absorption. Postprandial hyperlipidemia in diabetes is a risk factor for the development of hyperlipidemia complications. Hence, the absorption of dietary lipids by the small intestine plays an important role in the regulation of postprandial hyperlipidemia. Ursolic107 and betulinic acids107,108 exert hyperlipidemic effects, at least in part, by inhibiting pancreatic lipase (IC50 values between 15.83 and 21.10 μM), which prevent the absorption of lipids from the small intestine. Other studies showed that OA and BA have inhibitory activities on human cholesterol acyltransferase-2 (hACAT-2),115 an isoenzyme responsible for cholesterol absorption in the intestine.116 When OA and BA were compared, the latter exhibited more potent hACAT-2 inhibitory activity, with an IC50 value of 28.8 μM.115 Taken together, these results suggest that OA and BA also decrease the lipid absorption process in intestinal mucosal cells (Table 2). Lipogenesis and Fatty Acid β-Oxidation. The inhibition of free fatty acids (FFA) synthesis (lipogenesis)117 and increasing fatty acid β-oxidation118 are two potential pathways that contribute to the reduction of hepatic fat accumulation in T2DM patients. Compounds that inhibit the transcription of sterol regulatory element-binding protein-1c (SREBP-1c), and consequently mediate the down-regulation of hepatic genes involved in lipogenesis, such as acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS),119 result in a reduction of FFA synthesis. Additionally, agents that stimulate the up-regulation of carnitine palmitoyltransferase-1 (CPT-1),120 acyl-CoA oxidase (ACO),121 and peroxisome proliferator activated receptor-α (PPAR-α),122 increase fatty acid β-oxidation. An in vivo study showed that OA (25−50 mg/kg/day) reduced the expression of lipogenic genes including ACC,109,110 glycerol-3phosphate acyltransferase,110 and FAS,109 thus inhibiting lipid synthesis and, consequently, the retention of lipids in hepatocytes. Treatment with UA (5 mg/kg/day) downregulated SREBP-1c, FAS, and ACC transcripts, while promoting the inhibition of lipogenesis in liver, and increased fatty acid oxidation (by up-regulating CPT-1 and PPAR-α).22 Other studies described the hypolipidemic effects of UA in vitro and in vivo as primarily achieved by the activation of hepatic PPAR-α, which acts as an antagonist. Once activated, PARP-α promotes fatty acid uptake and fatty acid β-oxidation and decreases lipogenesis. Ursolic acid also increased the expression of fatty acid transport protein 4, enhanced hepatic fatty acid uptake, and induced acyl-CoA synthetase-1, CPT-1, and ACO1, improving hepatic fatty acid β-oxidation.23,111 Besides that, UA decreased FAS and SREBP-1c transcripts while inhibiting lipogenesis.23 Interestingly, Jia et al. showed that UA induces hepatic autophagy, which could also contribute to the degradation of lipid droplets in hepatocytes and, consequently, regulate hypolipidemic effects of UA.23 It was also proposed that UA reduces intracellular fat storage in skeletal muscle cells by increasing FFA uptake and β-oxidation via activation of AMPK pathway and up-regulation of uncoupling protein (UCP)-3,123 an inner mitochondrial membrane protein

mitochondrial redox balance, also improving mitochondrial dysfunction and also promoting the activation of AMPK signaling in diabetic mice.18 Although this study suggested that Nrf2-mediated protection may be a key mechanism in improving insulin resistance induced by OA, no more studies showed this effect in T2DM. However, the induction of Nrf2dependent genes was detected when OA (22.5−90 mg/kg/day for 3 or 4 days)103−105 and UA (25 and 50 mg/kg/daily for 1 week)41 were administered as hepatoprotective agents against different hepatotoxicants in vivo studies. This supported the notion the two TAs suppressed inflammatory and oxidative response through the Nrf2 pathway and perhaps contributed to reducing insulin resistance through that same mechanism. Another study also reported that UA (0.125, 0.25, and 0.5% during 6 weeks) promoted anti-inflammatory effects by decreasing TNF-α, monocyte chemotactic protein-1 (CCL2/ MCP-1), IL-1β, IL-2, IL-6, and IL-8, and antioxidant effects, mediated by increasing SOD, CAT, and glutathione peroxidase (GPx) activities, and by decreasing malondialdehyde (MDA), which also indirectly contributed to improve insulin resistance.28 This study supported the notion that UA contributed to improving insulin resistance due to its anti-inflammatory28,30 and antioxidant properties.28,40,41 With regard to BA, although its anti-inflammatory31 and antioxidant properties29,31 are extensively described, there are no studies that suggest the direct contribution of those effects in the improvement of insulin resistance. Instead, a recent study indicated that BA was not able to activate the Nrf2 pathway in Chinese hamster ovary cells.66 Altogether, the data support the notion that the antiinflammatory and antioxidant properties of these compounds contribute, at least in part, to prevent the development of T2DM, although doubts still exist on the role of Nrf2 regulation.

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ANTIOBESITY EFFECTS AND THEIR MECHANISMS OF ACTION In association with improved glucose tolerance and insulin sensitivity, OA, UA, and BA are also antiobesity promoters (Table 2). Several studies demonstrated the improvement of glucose tolerance being often accompanied by alterations in the lipid profile, by decreasing total cholesterol, triglycerides (TG), and LDLc contents, and by increasing levels of HDLc in streptozotocin-106 and alloxan-21 -diabetic rats treated with OA (60−200 mg/kg/day, during 40 days). The use of UA and BA (10 mg/kg/day during 15 days) was associated with decreased glucose, total cholesterol, and TG contents in HFDinduced obese mice.32,48,49 Additionally, treatment with OA, BA (5−20 mg/kg/day, during 15 weeks), and UA (5−10 mg/ kg/day or 0.05% w/w during 5−15 weeks) also reduced body weight, visceral adiposity,32,48,49 free fatty acids,22 and hepatic lipid accumulation,20,69 restoring the normal adipocyte size20 in obese diabetic rodents. Accumulating evidence suggests that the three TAs act as antiobesity agents by reducing lipid absorption,107,108 improving lipid homeostasis,109−114 and promoting food intake and body weight regulation.32,48,49 However, the lack of studies in humans places into question the 2997


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Table 3. Summary of Studies Reporting the Protective Effects of OA, UA, and BA against Some Diabetic Comorbiditiesa

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a

Abbreviations used: ACC, acetyl-coA carboxylase; ACh, acetylcholine; AGEs, advanced glycation end-products, Akt, phosphoinositide-dependent serine/threoninekinase; ALT, alanine aminotransferase; AMPK, AMP-activated protein kinase; AP-1, activator protein-1; AR, aldose reductase; AST, aspartate aminotransferase; BA, betulinic acid; CAMKK, activate Ca2+-calmodulin dependent protein kinase kinase; CAT, catalase; CCL2/MCP-1, monocyte chemotactic protein-1; CK, creatine kinase; CML, carboxymethyllysine; CPT-1, carnitine palmitoyltransferase-1; DGAT, diacylglycerol acyltransferase; DM, diabetes mellitus; EDR, endothelium-dependent relaxation; eNOS, endothelial NOS; ERK 1/2, extracellularly regulated kinase1 and -2; E-selectin, endothelial-selectin; FAS, fatty acid synthase; FAT/CD36, fatty acid translocase; FFA, free fatty acid; FOXO1, forkhead box O1; GLI, glyoxalase I; G6-Pase, glucose-6-phosphatase; GPx, glutathione peroxidase; GR, glutathione reductase; Grx1, glutaredoxin 1; GSH, glutathione; GSSG, glutathione (oxidized form); GSK-3β, glycogen synthase kinase-3β; GST, glutathione S-transferase; ICAM-1, intercellular adhesion molecule1; IR, homeostasis model assessment of insulin resistance index; HbA1c, hemoglobin A1c; HFD, high-fat diet; HOMA- HUVEC, human umbilical vein endothelial cells; IFN-γ, interferon-γ; iNOS, inducible nitric oxide synthase; LDH, lactate dehydrogenase; LDLc, low-density lipoprotein cholesterol; MAPK, mitogen-activated protein kinase; MCP-1, monocyte chemotactic protein 1; MDA, malondialdehyde; NF-AT, nuclear factor of activated T cells; NF-κB, nuclear factor kappa B; Nox4, NADPH oxidase 4; Nrf2, nuclear factor erythroid 2-related factor 2; OA, oleanolic acid; PPAR-α, peroxisome proliferator activated receptor-α; PRX1, peroxiredoxin-1; RAGE, receptor for AGE; ROS, reactive oxygen species; SCD-1, stearoyl-CoA desaturase-1; SDH, sorbitol dehydrogenase; SOD, superoxide dismutase; SREBP-1c, sterol-regulatory-element-binding protein-1c; STAT-3, signal transducer and activator of transcription factor-3; STZ, streptozotocin; tBHP, tert-butyl hydroperoxide; TBARs, thiobarbituric acid reactive substances; TC, total cholesterol; TG, triglycerides; TGF-β1, transforming growth factor β1; TNF-α, tumor necrosis factor-α; α-TOC, αtocopherol; UA, ursolic acid; VCAM-1, vascular cell adhesion molecule-1; ↑, enhances/up-regulates; ↑↑, stimulates/activates; ↑↑↑, improves; ↓, reduces/down-regulates/reverses/attenuates; ↓↓, inhibits; ↓↓↓, blocks/abolishes; ∼, no effects.

adipogenesis by stimulating fatty acid oxidation and promoting also the inhibition of fatty acids synthesis.113 Other studies reported that OA (31.7 μM)114 and BA (IC50 = 9.6 μM)129 decrease adiposity by inhibiting the activity of diacylglycerol acetyltransferase, involved in TG deposition in adipocytes. However, OA and UA (25−100 μM) also decreased adiposity by increasing lipolysis in fat cells, by promoting hormonesensitive lipase translocation,126,127 the rate-limiting enzyme in adipose tissue lipolysis that hydrolyzes stored triglycerides into monoglycerides and FFAs.130 In vitro studies demonstrated that UA (1−1000 μM) and BA (2.5−10 μM) also promote lipolysis by inhibiting phosphodiesterase activity,108,128 thus preventing or reversing obesity and insulin resistance in T2DM. Regulation of Food Intake and Body Weight. Adipocytes and the gastrointestinal tract release a wide range of hormones, such as adiponectin, resistin, leptin, and ghrelin, which regulate appetite and energy metabolism. Altered production or secretion of these hormones caused by excess adipose tissue and body weight contribute to obesity and insulin resistance.131 Therefore, the regulation of adiponectin, resistin, leptin, and ghrelin contribute to avoid obesity and to improve insulin sensitivity in T2DM. Oleanolic, ursolic, and betulinic acids (5−20 mg/kg/day during 15 days) increased leptin and also decreased the amount of ghrelin synthesized on HFD-induced obesity in mice,32,48,49 thus regulating appetite in

involved in mitochondrial uncoupling and fatty acid metabolism,124 contributing thus to explaining also the antiobesity effects induced by this compound. Interestingly, BA (20−40 μM) decreased lipogenesis and lipid accumulation, suppressing SREBP-1, FAS, and SCD-1 mRNA expression and SREBP-1 nuclear translocation through the AMPK pathway.125 This strengthens the notion that the activation of AMPK may be involved in the hypoglycemic and hypolipidemic effects of these compounds. Hypolypidemic effects of the compounds are described in Table 2. Adipogenesis and Lipolysis. Several studies demonstrated that OA,112,114,126 UA,113,127,128 and BA108,129 regulate adipogenesis by inhibiting the expression and/or activation of different adipogenic transcriptional factors, which may in turn reduce obesity and ultimately prevent the insulin resistance in T2DM. Oleanolic acid (1−25 μM) attenuated adipogenesis by reducing protein and mRNA expression of peroxisome proliferator receptor-γ (PPAR-γ) and CCAAT element binding protein α (C/EBP-α).112 With regard to UA (2.5−10 μM), this molecule was shown to decrease protein expression of C/EBPα and -β, PPAR-γ, and SREBP-1c, promoting the increase of ACC phosphorylation, as well as CPT-1 protein expression, and to decrease protein contents of FAS and fatty-acid-binding protein-4 through the liver kinase B1/AMPK pathway. Therefore, these results also suggested that UA reduces 2999


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by decreasing hydroxyl and superoxide radicals and by increasing the expression/activity of antioxidant enzymes, including SOD, CAT, glutathione reductase, GPx, and thioredoxin peroxidase (TPx), overall resulting in lipid peroxidation inhibition. Although the mechanisms of antioxidant effects are still not clarified, it has been proposed that antioxidant and anti-inflammatory effects of OA and UA are mediated, to a great extent, through the activation of Nrf2 transcription, as described above. Nevertheless, the known role of Nrf2 on the regulation of hepatic lipogenesis,123 associated with the expression of anti-inflammatory genes, lead us to propose that the activation of Nrf2 pathway by OA and UA may in turn prevent the development of NAFLD by promoting anti-inflammatory and antioxidant pathways reducing, at the same time, hepatic lipid accumulation. Although BA is unable to activate the Nrf2 pathway, it has been shown that its antioxidant mechanisms responsible for the hepatoprotective activities may also be related with an improvement of the tissue redox system, thus decreasing lipid peroxidation.29 Cardiovascular Diseases (CVD). Diabetes is associated with increased atherosclerosis and the development of CVD. Several studies suggested that OA, UA, and BA are potential therapeutic agents for diabetic-related CVD, mainly to prevent the development of atherosclerotic lesions and myocardial dysfunction. Besides their hypolipidemic effects, these TAs have also antiatherogenic properties. Oleanolic and ursolic acids protected against low-density lipoprotein oxidation140 and reduced pro-inflammatory cytokine production, including NFκB, TNF-α, and IL-1β.24,141 In accordance with this, Jiang et al. showed that the activation of the Nrf2 signaling pathway by OA promoted the reduction of low-density lipoprotein oxidation, contributing thus to the prevention of atherosclerosis.123 Other studies suggested that UA exerts at least some of its antiatherogenic effects by preventing the accumulation of inflammatory monocytes in the blood, reducing in vivo monocyte chemotactic activity, thus decreasing plaque size, and macrophage content in atherosclerotic lesions in diabetic mice.25,26 It was previously shown that BA vascular protection occurs by inhibition of ROS and NF-κB, which consequently reduces the expression of cell adhesion molecules such as vascular cell adhesion molecule-1, intracellular adhesion molecule-1, and endothelial selectin, contributing to suppression inflammatory responses,31 suggesting that the antioxidant and anti-inflammatory effects of these compounds are crucial to preventing the development of atherosclerosis. Relevant data on antiatherogenic effects are summarized in Table 3. Another potential mechanism that may delay the development of atherosclerosis may involve a decrease in blood pressure level. The anti-hypertensive effects of OA and UA may be mainly attributed to their potent anti-hyperlipidemic and antioxidant effects, combined with diuretic, natriuretic, and saluretic effects, mostly due to the inhibition of Na+ and K+ reabsorption in the early portion of the distal tubule.142 However, it was also reported that the anti-hypertensive and antiatherogenic properties of these compounds may also result from their effects on endothelium-dependent vasorelaxation. Impaired endothelial function, due to decreased bioavailability of NO, is a hallmark of atherosclerosis. In contrast, an increase in NO induces vasorelaxation, preventing the development of atherosclerosis.143 In this context, UA and BA (0.1−100 μM) displayed vasorelaxing properties, mediated by an increase of NO release and relaxation by activation of endothelial nitric

this animal model. In contrast, Jia et al. showed that UA (50 and 200 mg/kg) decreases leptin levels on HFD-induced obese mice as a consequence of the reduction in adipocyte tissue observed in this study. In the same work, it was also reported that UA increases adiponectin,23 regulating lipid and glucose metabolism. Oleanolic acid (1−25 μM) suppressed the production of resistin during adipogenic differentiation of 3T3-L1 adipocytes,132 suggesting that this is mechanism contributes to the improvement of obesity and insulin resistance in T2DM (Table 2).

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COMORBIDITIES T2DM leads to the progression of macrovascular and microvascular comorbidities, including atherosclerosis and cardiovascular and hepatic diseases, as well as nephropathy, which are the main risk factors for death among diabetic patients.3 Therefore, the prevention of these related complications may reduce the patients’ mortality risk. Hepatic Diseases. End-stage liver disease is one of the main causes of death in T2DM patients. Among liver diseases in diabetes, nonalcoholic fatty liver disease (NAFLD) is an initial stage of a continuing spectrum of liver alterations, which ultimately can be fatal. 133−135 Because OA 109,110 and UA22,23,111 decrease lipogenesis and stimulate fatty acid βoxidation in hepatocytes (Table 2), it is possible that both OA and UA provide effective treatment options for NAFLD, thus avoiding progression of the disease. In agreement with this, Zhou et al. reported that OA administration (100 mg/kg day during 4 weeks) activates AMPK,136 an energy sensor that regulates fatty acid oxidation,137 and reduces lipogenesis and lipid accumulation in the liver.138 Once activated by OA, AMPK induces the inhibition of SREBP and also leads to decreases in hepatic ACC, FAS, and stearoyl-CoA desaturase-1 (SCD-1) in T2DM mice (Table 3), thus normalizing hepatic lipid metabolism.136 Betulinic acid activates AMPK, which in turn leads to suppression of mRNA expression and nuclear translocation of SREBP-1, repression of FAS and SCD-1 gene expression, and increases of PPAR-α and CD36 gene levels in human HepG2 hepatoma cells, primary hepatocytes, and liver tissue from HFD-fed mice. These results suggest that BA can also effectively improve intracellular lipid accumulation in liver cells, suppressing de novo lipogenesis and increasing lipolysis through AMPK pathway activation.125 However, because UA promotes FFA β-oxidation in muscle by activation of the AMPK pathway,123 a similar effect is expected in the liver. Nonetheless, it is known that UA improves HFD-induced hepatic steatosis by regulating lipid metabolism through the PPAR-α pathway, decreasing mRNA/protein content for FAT/ CD36, SREBP-1c, ACC, and FAS and increasing mRNA/ protein content of CPT-1. It was suggested that UA improves insulin resistance by decreasing insulin level and improving the homeostasis model assessment of insulin resistance index (HOMA-IR), by decreasing inflammatory signaling by downregulating TNF-α, CCL2/MCP-1, IL-1β, IL-2, IL-6, and IL-8, and by stimulating the oxidative stress network, increasing SOD, CAT, and GPx activities, resulting in decreased MDA content,28 as described in Table 3. Taken all together, those effects may contribute to the improvement of hepatic steatosis and reduction of metabolic dysfunctions on NAFLD. In addition to hypolipidemic effects, the antioxidant activity of TAs may also prevent hepatic dysfunction in diabetes. It has been proposed that the hepatoprotective effects of OA139 and UA10 are also mediated by a direct ROS scavenging role, that is, 3000


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Figure 2. Mechanisms of oleanolic (OA), ursolic (UA), and betulinic (BA) acids on diabetes type 2 and related comorbidities. The different hypoglycemic (orange continuous arrow) and antiobesity (red dash arrows) properties of these TAs are schematized. These TAs promote the reduction of glucose absorption (1) by inhibition of pancreatic α-amylase and α-glucosidase or by down-regulation of sodium-dependent glucose cotransporter (SGLT-1) and glucose transporter 2 (GLUT2) as well as reducing the endogenous glucose production (EGP) and increasing glycogen synthesis; (2) as consequence of the reduction of gluconeogenesis and glycogenolysis and stimulation of glycogenesis. The improvement of insulin signaling (3) by OA, UA, and BA is highlighted and mediated (i) by inhibition of protein tyrosine phosphatase (PTP), (ii) by improvement of β-cell survival and direct stimulation of phosphatidylinositol 3-kinase-dependent (PI3KB)/protein kinase Akt pathway, (iii) by translocation of GLUT-4, (iv) by activation TGR5 and glucagon-like peptide-1 (GLP-1) release, and also (v) by inhibition of ROS production and suppression of inflammatory processes. Antiobesity effects occur due to the reduction of lipid absorption (4) by inhibition of pancreatic α-lipase or human cholesterol acyltransferase-2 (hACAT-2); regulation of lipid metabolism (5) by reduction of lipogenesis and stimulation of fatty acid β-oxidation; and also by promoting reduction adipogenesis, stimulating the lipolysis in adipose tissue. The regulation of appetite (6) is another effect of these compounds, which occurs as a consequence of the reduction of ghrelin and resistin and the increase of adiponectin and leptin levels, although the effect of UA on leptin remains controversial. The protective properties of OA, UA, and BA against diabetes-related comorbidities (blue dash arrows) may act on preventing the development of nonalcoholic fatty liver disease (NAFLD) (7), cardiovascular disease (CVD) (8), or diabetic nephropathy (9), as a consequence of their hypolipidemic, antiatherogenic, hypotensive, anti-inflammatory, and antioxidant properties and also by preserving kidney structure and promoting the inhibition of polyol pathway. ↔, regulates; ↑, enhances/stimulates/promotes; (+), up-regulates/activates; ↑↑↑= improves; ↓, reduces; (−), down-regulates; ⦶, inhibits.

oxide synthase (eNOS).144−146 It was previously described that BA increased the production of NO and the activation of eNOS mediated by the AMPK signaling pathway,147 suggesting that this compound used the same pathway to regulate also hepatic glucose production19 (Table 1) and intracellular lipid accumulation125 (Table 2). Nevertheless, it was also supposed that UA and BA (1−10 μM) up-regulate eNOS expression and, simultaneously, reduce nicotinamide adenine dinucleotide phosphate (NADPH) oxidase expression in human endothelial cells.148,149 As NADPH oxidase represents a major source of ROS in CVD,150 this decreased expression may limit vascular oxidative stress and increase bioactive NO, which contributes to preventing the development of CVD. In contrast, Messner et al. showed that UA (3−50 μM) induced pro-atherogenic effects, promoting DNA damage, p53-mediated mitochondrial- and caspase-dependent human endothelial cell apoptosis, and accelerating atherosclerotic plaque formation in vivo (Table 3). These authors supposed that proatherogenic activities

observed in this study are exerted at least in part by UA metabolite effects, instead of directly by UA.11 However, further studies are needed to identify their metabolites and clarify these effects. Other studies also showed that OA12 and UA151 exhibit cardioprotective effects against hyperglycemia-induced myocardial damage, at least in part due to their antioxidant and antiapoptotic properties. Although the precise mechanisms for these TA-mediated antioxidant effects in the heart are unclear, earlier data described that OA (0.6−1.2 mmol/kg/day during 3 days ≈ 274−550 mg/kg/day)152 and UA (35 mg/kg/day during 8 weeks)151 increase the expression of several enzymatic and nonenzymatic antioxidants, including α-tocopherol, GPx, CAT, TPx, and SOD in the heart tissue. Oleanolic and ursolic acids are also believed to act as direct free radical scavengers and to reduce lipid peroxidation.153 Thus, it is possible that TA antioxidant effects may prevent cardiometabolic complications in diabetes. In agreement, Mapanga et al. proposed that OA12 3001


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Besides these promising beneficial effects, OA, UA, and BA may also contribute to protect against diabetes comorbidities, such as kidney injury and cardiometabolic and hepatic diseases (Figure 2). It is interesting to note that many of their multiple antidiabetic effects occur by mediation of common signaling pathways, which may in turn result in different outcomes. Among multiple effects, it should be noted that AMPK and Nrf2 pathways are involved in many mechanisms of action of these compounds, AMPK signaling being responsible by hypoglycemic, hypolipidemic, and antiatherosclerotic mechanisms of these TAs, whereas Nrf2 may modulate many of the antioxidant, anti-inflammatory, and hypolipidemic effects of OA and UA, making them important action targets of these compounds. Although the high potential of these three TAs in the treatment and prevention of T2DM is very promising, clinical trials with OA, UA, and BA are practically nonexistent, with the exception of a 2005 Phase II clinical trial with UA at the University of Guadalajara, Mexico (NCT02337933). The trial was performed to evaluate its effects on insulin sensitivity and metabolic syndrome, as characterized by overweight/obesity, insulin resistance, hyperglycemia, dyslipidemia, and hypertension and by an inflammatory state, which together increase the risk for CVD or T2DM (University of Guadalajara, Mexico, coded NCT02337933).The results of that clinical trial are still undisclosed. The very limited number of clinical studies puts into question the true effect of these compounds in humans, highlighting the need to clarify the true efficacy, safety, and tolerability of these compounds and the doses required to achieve the antidiabetic effects of these TAs before they can be used in T2DM management. Another major limitation in the use of these compounds is their low bioavailability, supported by the fact that the peak plasma concentration after administration of OA,163 UA,164 and BA165 (10−100 mg/kg) never exceed 1% of the oral dose administered in rats, possibly due to their poor gastrointestinal absorption, limiting their human applications. To improve the bioavailability and efficacy of these compounds, some strategies have been used, either by developing new systems for drug delivery to increase the bioavailability of these compounds or through the development also of new derivative structures. Numerous efforts have been made to improve the bioavailability of these compounds by developing noncovalent complexes with hydrophilic cyclodextrins, as well as the use of nanosuspensions.166 Ursolic acid liposomes (UALs) were until recently the only system approved for use in a phase I clinical trial (2009L00634, China) to improve bioavailability in patients with advanced solid tumors.167 This strategy may increase the therapeutic use of these compounds, although its use with OA and BA, as well as their effects in diabetic patients, remains unstudied. Another strategy has been the development of OA,168 UA,169 and BA84 derivatives to improve the anti-hypoglicemic, antiinflammatory, and antioxidant effects of these natural compounds. However, the only derivative so far studied in clinical trials was the semisynthetic OA derivative, bardoxolone methyl (C-28 methyl ester of 2-cyano-3,12-dioxoolean-1,9dien-28-oic acid), also designated CDDO-Me or RTA 402. Bardoxolone methyl successfully completed phase II clinical trials (coded NCT01574365) for the treatment of patients with type 2 diabetes-associated chronic kidney disease.170,171 However, phase III trials (NCT01351675) with patients in end-stage chronic kidney disease were halted due to a higher

antioxidant effects attenuate myocardial apoptosis and thereby lead to improvement in contractile functional recovery following ischemia−reperfusion of hyperglycemic rat hearts. In vitro, OA is a potent cardioprotective agent against highglucose-induced injury, acting as an antioxidant, anti-apoptotic, and anti-inflammatory agent.13 Although there are no studies concerning UA and BA cardioprotective effects in diabetes, it is known that BA14 and UA154 prevent cardiomyocyte apoptosis. Nephropathy. Diabetic renal injury, or the so-called diabetic nephropathy, is an important diabetic complication, which exacerbates the severity and mortality of T2DM. Nonenzymatic glycation with the formation of Maillard reaction products, also known as advanced glycation endproducts (AGEs), including glycated hemoglobin, N ε (carboxymethyl)lysine (CML) and glycated albumin have been implicated in the pathogenesis of diabetic nephropathy.155,156 Particularly, it is suggested that the accumulation of CML and glycated albumin contributes to the deterioration of diabetic nephropathy.155 Oleanolic acid treatment (100 mg/kg/ day during 2 weeks) on HFD- and streptozotocin-induced diabetes in mice decreases the loss of glucose in urine and improves kidney structure.157 Other studies also indicated that OA and UA (0.01−0.2% for 10−12 weeks) improved kidney function in diabetic mice,158,159 by preserving kidney structure and by inhibiting the polyol pathway. The inhibition of this pathway probably occurred by decreasing renal sorbitol and fructose concentrations159 and by decreasing renal aldose reductase and renal sorbitol dehydrogenase activities,17,59,159 thereby inhibiting AGE formation and preventing kidney and liver injury. Besides that, these two TAs also decrease plasma HbA1c, pentosidine, and CML, as well as urinary glycated albumin in streptozotocin-diabetic mice.159 These results were different from those observed in vitro, where OA inhibited the formation of pentosidine and CML, but UA suppressed only CML, without affecting pentosidine160 (Table 3). Considering also that the oxidative and inflammatory reactions contribute to glycative processes,161 the anti-inflammatory and antioxidant properties of these three TAs can also contribute to reduce AGE production, although there are no studies evidencing these effects. Despite that, UA was found to decrease the receptor for AGE (RAGE) expression in the brain of aging mice,162 which subsequently interact with AGEs in the circulation and tissues to form an AGE−RAGE complex. As it was described that the AGE−RAGE complex generates proinflammatory, pro-thrombotic molecules and ROS (reviewed in ref 161), it is expected that the reduction of the AGE−RAGE complex contributes to reduce the glycative stress and delays the development of CVD and diabetic renal injury. It is clear that human studies are needed to further confirm the antiglycative effect of these TAs in the progression of development of diabetic comorbidities.

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PRESENT AND FUTURE PRESPECTIVES Oleanolic, ursolic, and betulinic acids are three TAs that display a wide range of promising antidiabetic properties, without demonstrating apparent hepatotoxic effects at the dosages administered, contrarily to most antidiabetic agents currently used. A large number of studies have shown that OA, UA, and BA act as hypoglycemic and antiobesity agents mainly through (i) reducing the absorption of glucose, (ii) decreasing endogenous glucose production and increasing glycogen synthesis, (iii) increasing insulin sensitivity, (iv) improving lipid homeostasis, and (v) promoting body weight regulation. 3002


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Journal of Agricultural and Food Chemistry incidence of cardiovascular events.172 Despite this, development of new TA derivatives may eventually represent an excellent strategy to treat diabetes in the near future. Apart from these strategies, a very recent study reported also the ability of OA (100 mg/kg day, during 4 weeks) to synergistically potentiate antidiabetic effects of metformin (250 mg/kg day, during 4 weeks) by improving glucose and insulin homeostasis in diabetic mice.173 It is reasonable to speculate that these compounds may be used to increase the antidiabetic effects and possibly to counteract hepatotoxicity derived from commonly used therapeutics. However, OA’s possible hepatotoxic effect should be confirmed, because it has been reported that OA, at higher concentrations (≥90 mg/kg/day), caused cholestatic liver injury in mice.174,175 With regard to UA and BA, there are no studies reporting their synergistic effects when combined with antidiabetic agents or their potential hepatotoxicity. In this regard, the highly promising effect of these compounds should push clinical assays to confirm the efficacy, safety, and tolerability of OA, UA, and BA individually or in combination with antidiabetic agents. In summary, this review provides a unique and holistic view on the antidiabetic properties of OA, UA, and BA in the management of T2DM, highlighting the strengths and weaknesses to the future application these compounds in humans. A strong point of the three compounds is their global antidiabetic effects mediated by specific mechanisms of action, a peculiar characteristic of these compounds that makes them a true asset for the treatment and prevention of T2DM. Potential weaknesses involve their low bioavailability and the very limited number of clinical trials. More strategies should be designed to increase the bioavailability of these natural compounds, testing also their effects in clinical trials with T2DM patients. From this perspective, urgent needs are also the clarification of the signaling pathways involved in antidiabetic effects and the search for new therapeutic targets that are involved in Nrf2 and AMPK signaling pathways so that new derivatives with selective antidiabetic effects can explore those signaling pathways. Therefore, by merging all of the information described above, we expect the development of new strategies to improve the bioavailability and efficacy of these TAs in managing T2DM. An optimal future scenario would be that these compounds could be used as pharmaceutical agents and/or food supplements for the prevention and/or management of T2DM.

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protein kinase; BA, betulinic acid; CAT, catalase; C/EBP-α and -β, CCAAT element binding protein α and β; CCL2/MCP-1, monocyte chemotactic protein-1; CML, Nε-(carboxymethyl)lysine; CPT-1, carnitine palmitoyltransferase-1; CVD, cardiovascular disease; eNOS, endothelial nitric oxide synthase; FAS, fatty acid synthase; FFA, free fatty acid; GLP-1, glucagon-like peptide-1; GLUT-2 and -4, glucose transporter-2 and -4; GP, glycogen phosphorylase; G6-Pase, glucose-6-phosphatase; GPx, glutathione peroxidase; GSK-3, glycogen synthase kinase-3; hACAT-2, human cholesterol acyltransferase-2; HFD, high-fat diet; HOMA-IR, homeostasis model assessment of insulin resistance index; IR, insulin receptor; IRS-1, insulin receptor substrate-1; MDA, malondialdehyde; NADPH, nicotinamide adenine dinucleotide phosphate; NAFLD, nonalcoholic fatty liver disease; NF-κB, factor nuclear kappa B; Nrf2, nuclear factor erythroid 2-related factor 2; OA, oleanolic acid; PEPCK, phosphoenolpyruvate carboxykinase; PI3KB, phosphatidylinositol 3-kinase-dependent; PPAR-α and -γ, peroxisome proliferator activated receptor-α and -γ; PTP-1B, protein tyrosine phosphatase 1B; RAGE, receptor for AGE; ROS, reactive oxygen species; SCD-1, stearoyl-CoA desaturase-1; SGLT-1, sodium-dependent glucose cotransporter; SOD, superoxide dismutase; SREBP-1c, sterol-regulatory-element-binding protein-1c; TAs, triterpenic acids; T2DM, type 2 diabetes mellitus; TG, triglycerides; TNF-α1, tumor necrosis factor-α1; TPx, thioredoxin peroxidase; UA, ursolic acid; UCP-3, uncoupling protein-3

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REFERENCES

(1) WHO, Diabetes Fact Sheet 312, 2015. (2) Biggs, M. L.; Mukamal, K. J.; Luchsinger, J. A.; Ix, J. H.; Carnethon, M. R.; Newman, A. B.; de Boer, I. H.; Strotmeyer, E. S.; Mozaffarian, D.; Siscovick, D. S. Association between adiposity in midlife and older age and risk of diabetes in older adults. JAMA, J. Am. Med. Assoc. 2010, 303, 2504−2512. (3) Forouhi, N. G.; Wareham, N. J. Epidemiology of diabetes. Medicine 2014, 42, 698−702. (4) Domingues, R. M. A.; Guerra, A. R.; Duarte, M.; Freire, C. S. R.; Neto, C. P.; Silva, C. M. S.; Silvestre, A. J. D. Bioactive triterpenic acids: from agroforestry biomass residues, to promising therapeutic tools. Mini-Rev. Org. Chem. 2014, 11, 382−399. (5) Camer, D.; Yu, Y.; Szabo, A.; Huang, X. F. The molecular mechanisms underpinning the therapeutic properties of oleanolic acid, its isomer and derivatives for type 2 diabetes and associated complications. Mol. Nutr. Food Res. 2014, 58, 1750−1759. (6) Castellano, J. M.; Guinda, A.; Delgado, T.; Rada, M.; Cayuela, J. A. Biochemical basis of the antidiabetic activity of oleanolic acid and related pentacyclic triterpenes. Diabetes 2013, 62, 1791−1799. (7) Alqahtani, A.; Hamid, K.; Kam, A.; Wong, K. H.; Abdelhak, Z.; Razmovski-Naumovski, V.; Chan, K.; Li, K. M.; Groundwater, P. W.; Li, G. Q. The pentacyclic triterpenoids in herbal medicines and their pharmacological activities in diabetes and diabetic complications. Curr. Med. Chem. 2013, 20, 908−931. (8) Abdel-Zaher, A. O.; Abdel-Rahman, M. M.; Hafez, M. M.; Omran, F. M. Role of nitric oxide and reduced glutathione in the protective effects of aminoguanidine, gadolinium chloride and oleanolic acid against acetaminophen-induced hepatic and renal damage. Toxicology 2007, 234, 124−134. (9) Gao, J.; Tang, X.; Dou, H.; Fan, Y.; Zhao, X.; Xu, Q. Hepatoprotective activity of Terminalia catappa L. leaves and its two triterpenoids. J. Pharm. Pharmacol. 2004, 56, 1449−1455. (10) Martin-Aragon, S.; de las Heras, B.; Sanchez-Reus, M. I.; Benedi. Pharmacological modification of endogenous antioxidant enzymes by ursolic acid on tetrachloride-induced liver damage in rats and primary cultures of rat hepatocytes. Exp. Toxicol. Pathol. 2001, 53, 199−206.

AUTHOR INFORMATION

Corresponding Author

*(M.F.D.) Phone: + 351 284 314 399. E-mail: fatima.duarte@ cebal.pt. Funding

This work is funded by FEDER funds through the Operational Programme Competitiveness Factors − COMPETE and national funds by FCT − Foundation for Science and Technology under Project NEucBark − New valorization strategies for Eucalyptus spp. Bark Extracts (PTDC/AGR-FOR/ 3187/2012) and Strategic Project UID/NEU/04539/2013 (CNC). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED ACC, acetyl-coA carboxylase; ACO, acyl-CoA oxidase; AGEs, advanced glycation end-products; AMPK, 5′ AMP-activated 3003


Review


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