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The molecular basis of the anti-diabetic properties of camel milk
diabetes research and clinical practice
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Contents available at ScienceDirect
Diabetes Research and Clinical Practice journal homepage: www.elsevier.com/locat e/dia bre s
Review
The molecular basis of the anti-diabetic properties of camel milk Mohammed Akli Ayoub *, Abdul Rasheed Palakkott, Arshida Ashraf, Rabah Iratni Biology Department, College of Science, United Arab Emirates University, Al Ain, United Arab Emirates
A R T I C L E I N F O
A B S T R A C T
Article history:
Over the years, strong evidence have been accumulated in favor of the beneficial effects of
Received 11 July 2018
camel milk on glucose homeostasis with significant anti-diabetic properties in both human
Received in revised form
and animal diabetic models. However, the cellular and molecular mechanisms involved in
18 September 2018
such effects remain not understood. In this review, we speculated about the potential
Accepted 5 November 2018
mechanisms and summarized few mechanistic-based studies that investigated the biolog-
Available online 16 November 2018
ical activity of camel milk and its protein components on the different aspects that may be involved in the anti-diabetic effects. A special emphasis is given to the molecular events
Keywords: Diabetes Camel milk Insulin Glucose
engaged by camel milk proteins/peptides on two key aspects: insulin secretion and insulin receptor activity. Thus, the review gives a molecular rationale to the anti-diabetic effects of camel milk. This will help to identify the anti-diabetic agent(s) contained in camel milk and to understand better its mechanism of action in order to use it for the management of diabetes mellitus. Ó 2018 Elsevier B.V. All rights reserved.
Contents 1. 2. 3. 4. 5. 6. 7.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mechanisms of the anti-diabetic properties of camel milk. . . . . . . . . . . . . . . . Camel milk proteins/peptides acting as insulin-like proteins . . . . . . . . . . . . . . Effects of camel milk proteins/peptides on insulin secretion . . . . . . . . . . . . . . Glucagon receptor and GLUT4, the other candidates for camel milk proteins . Effects of camel milk proteins/peptides on the diabetic wound healing . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Funding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Appendix A. Supplementary material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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* Corresponding author at: Biology Department, College of Science, United Arab Emirates University, P.O. Box 15551, Al Ain, United Arab Emirates. E-mail address: [email protected] (M.A. Ayoub). https://doi.org/10.1016/j.diabres.2018.11.006 0168-8227/Ó 2018 Elsevier B.V. All rights reserved.
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Introduction
Many studies reported interesting nutritional and therapeutic characteristics of camel milk due to its chemical composition and its biochemical properties [1–8]. Indeed, camel milk has been shown to have potential benefits in many health issues and diseases with significant anti-bacterial, anti-carcinogenic, anti-oxidant, anti-hypertensive, and anti-diabetic properties, demonstrated in many in vitro and in vivo studies [1,2,4,7,9,10]. Undoubtedly, the hypoglycemic effects of camel milk demonstrated in many in vitro and in vivo studies and its potential applications for the management of diabetes mellitus and its related complications remain the most exciting and studied aspects of camel milk. In fact, in vitro and in vivo studies using either type 1- or type 2-diabetic animals models (rats and rabbits) and also patients with diabetes reported the beneficial effects of camel milk by reducing blood sugar, increasing insulin secretion, decreasing insulin resistance, and improving lipid profiles [2,11–24]. Interestingly, in one populationbased study carried out in India it has been reported that the incidence risk of diabetes was lower in camel breeders and rearing tribal, the Raikas, who regularly consume camel milk, compared to other communities who do not [25,26]. Thus, camel milk has been proposed as a promising adjuvant in the anti-diabetic therapy. Moreover, camel milk has been shown to improve other pathophysiological aspects related to diabetes as a chronic disease such as insulin resistance, obesity, inflammation, wound healing, and oxidative damage [2,9,13,27–30]. However, the secret behind these beneficial effects of camel milk is still unknown since the exact anti-diabetic component(s) contained in camel milk and the molecular and cellular mechanisms involved is/are still not completely understood. This constitutes the challenge for the present and the future studies and investigations in order to identify the anti-diabetic agent in camel milk and to understand its mechanism(s) of action. In this review, we focused on the key studies that aimed at investigating the impact of camel milk components on the different molecular and cellular events leading to the beneficial effects observed in the context of diabetes and its related complications such as wound healing. We attempted to provide a molecular basis for the anti-diabetic properties of camel milk by highlighting recent data and new perspectives with regard to the potential action of camel milk and its components on the two key tissues involved in diabetes, the pancreas and the insulin-sensitive tissues such as liver. The emphasis is given to the effects of camel milk on insulin secretion by the pancreatic b-cells and on insulin function through its receptor in the insulin-sensitive tissues. We also described few studies that attempted to identify the antidiabetic agent(s) in camel milk with an emphasis on protein and peptide components and their plausible mechanism of action investigated at the molecular and cellular levels using various in vitro assays.
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2. Mechanisms of the anti-diabetic properties of camel milk The anti-diabetic properties of camel milk may be very complex involving many molecular and cellular mechanisms and aspects of glucose metabolism and transport as well as insulin synthesis and secretion [2,10,24,31,32]. Obviously, insulin synthesis and secretion, and insulin receptor function constitute the two key aspects that may be involved in the beneficial effects of camel milk on diabetes. Thus, from the molecular and cellular points of view, camel milk may act at different levels including; (i) direct effects on insulin receptor function and glucose transport in the insulin-sensitive tissues, (ii) direct and/or indirect effects on insulin secretion by the pancreatic b-cells, and (iii) effects on the survival, growth, and the overall activity of the pancreatic cells (Fig. 1). The link between the anti-diabetic properties of camel milk and its putative effects on insulin receptor activity appears obvious with the pivotal role of insulin receptor in glucose homeostasis (Fig. 1) [33]. This is also based on the assumption that camel milk contains insulin and/or insulin-like proteins that act directly on the insulinsensitive tissues and that may have specific properties in terms of stability, proteolysis, and absorption in the stomach, and their entry into the circulation before reaching their target tissues [10,24,32]. Even though the insulin content of camel milk is not as clear as many reviews stated, a high concentration of insulin (52 units/liter) has been detected in camel milk [34], and this seems to vary over the lactation period [35] as well as the storage condition [36]. In addition, a comparative study on insulin-like protein content in milk revealed that camel milk contains 3 times more insulinlike proteins than cow milk [37]. The existence of such insulin-like proteins was supported by the fact that amino acid sequence of some of camel milk proteins is found to be rich in half cystine like some insulin family peptides [38]. With this line, a recent study by Abdulrahman et al. brought the first evidence of camel milk proteins directly acting on the insulin receptor and thereby potentiating it activity and signaling in vitro (Fig. 2)[31]. The other hypothesis consists of the direct or indirect action of camel milk on insulin synthesis and secretion by the pancreatic b-cells (Fig. 1). This is mainly supported by in vitro studies suggesting the existence of specific proteins and bioactive peptides that act directly or indirectly on specific cellular pathways controlling insulin synthesis and secretion. Indeed, many hormones and their receptors play a key role in the control of insulin synthesis and secretion by the pancreas. This includes glucagon and glucose-dependent insulinotropic polypeptides, such as gastric inhibitory polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), as well as enzymes like the endoprotease dipeptidyl peptidase IV (DPP-IV) and its proteolytic effects on GIP and GLP-1 [39–41]. Finally, the indirect effects of camel milk may be characterized by the antiinflammatory, the anti-apoptotic, and the anti-oxidative
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Fig. 1 – Speculative representation of the different cellular and molecular targets of camel milk and its mechanisms of action leading to anti-diabetic effects.
Fig. 2 – The positive allosteric effect of camel milk on insulin receptor activity and signaling as demonstrated in vitro by Abdulrahman et al. [31].
effects that restore the secretory activity and the overall functioning of the pancreatic b-cells as demonstrated in many studies [42–44]. In addition, the absence of coagulation of camel milk in the stomach [11] and the protective effects of small size immunoglobulins contained in camel milk on the pancreatic b-cells [12] have also been proposed to explain the hypoglycaemic effects of camel milk.
3. Camel milk proteins/peptides acting as insulin-like proteins The hypoglycemic action of camel milk observed in many in vivo studies on type 1- and type 2-diabetes using animal models as well as patients with diabetes obviously suggested the existence of insulin and/or insulin-like proteins in camel
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milk [2,11–24]. However, as mentioned earlier the insulin content of camel milk is not clear yet and only few quantitative studies were reported [34–36]. The presence of insulin or insulin-like proteins in camel milk argues for the possible direct effect of camel milk on insulin receptor and its function in the major insulin-sensitive tissues [38], but evidence for this assumption still to be brought. Camel milk proteins are represented by two major components, caseins (75%) and whey proteins (25%) [1–3,5]. Camel milk whey mainly contains serum albumin, a-lactalbumin, lactoferrin, lysozyme, immunoglobulins and peptidoglycan recognition protein [1–3,5]. Therefore, the biological activity of camel milk is thought to be mediated by these proteins and/or their peptides upon hydrolysis by the gastrointestinal system [6]. Among the whey proteins, the iron-binding glycoprotein, lactoferrin, constitutes an interesting candidate for a possible activity on insulin and insulin receptor function and signaling. Indeed, many studies linked lactoferrin with insulin resistance and other related disorders such as obesity and inflammation [45–49]. For instance, circulating lactoferrin was negatively associated with hyperglycemia and positively with insulin sensitivity and anti-inflammatory responses [48]. Moreover, lactoferrin has been shown to increase insulin-mediated Akt phosphorylation in human hepatocarcinoma (HepG2) and adipocyte 3T3-L1 cell lines [49] suggesting a direct effect on insulin receptor. The activation of PI3kinase/Akt axis by insulin is the key signaling pathway involved in the transport and the utilization of glucose by the insulin-sensitive tissues through the membrane glucose transporter 4 (GLUT4) [33]. However, it is not clear whether camel milk lactoferrin can really bind to the insulin receptor and modulate its function and signaling which may explain its anti-diabetic effects. This constitutes in fact a legitimate question and an exciting hypothesis to be tested on cells expressing the insulin receptor as well as GLUT4 since lactoferrin has a strong potential of binding to cell membrane components. Recently, an elegant in vitro study reported the first evidence for a direct effect of camel milk on insulin receptor activity and signaling in the transfected human embryonic kidney (HEK293) cell line [31]. In this study, treatment of cells with camel milk weakly affected insulin receptor activity, however this significantly potentiated the agonistic action of insulin indicating a putative allosteric mechanism of action (Fig. 2) [31]. Such a potentiation was observed on insulinmediated Grb2 protein translocation to the activated receptor assessed, in real-time and live cells, using bioluminescence resonance energy transfer (BRET) technology. Interestingly, the fractionation experiments clearly supported the protein/ peptide nature of such a potentiating agent contained in camel milk and this is yet to be identified. Moreover, camel milk has been shown to activate the extracellular signalregulated kinases 1 & 2 (ERK1/2), but not Akt, signaling pathway and strongly potentiated the insulin-mediated ERK1/2 response which is consistent with BRET data [31]. These observations suggest that camel milk acts on the mitogenic (Grb2/Ras/mitogenic protein kinases), but not on the metabolic (PI3-kinase/Akt) pathway, which is somehow difficult to reconcile with the anti-diabetic effects of camel milk observed in vivo. Indeed, the metabolic pathway characterized
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by insulin activating PI3-kinase/Akt axis controls the expression and the translocation of GLUT4 to the plasma membrane and this constitutes the critical step in the transport and the utilization of glucose by the insulin-sensitive tissues through its receptor [33]. By contrast, the study did not show whether the potentiation of the insulin receptor by camel milk was associated with any increase in glucose uptake by the cells. As camel milk candidates and as described later in this review, some bioactive peptides obtained from the tryptic hydrolysis of camel milk whey proteins have been reported to have beneficial effects in the context of diabetes [21,22,50,51]. Therefore, it will be interesting to test these peptides for their direct pharmacological effects on the insulin receptor activity and signaling using similar approaches reported by Abdulrahman et al. [31].
4. Effects of camel milk proteins/peptides on insulin secretion The anti-diabetic properties of camel milk demonstrated in many in vivo studies also suggest potential effects on the pancreas and on insulin secretion by the pancreatic b-cells. In fact, feeding patients and animals (rats and rabbits) with diabetes with camel milk has been reported to restore the production and the release of insulin compared to control patients and animals [2,14,17,18,29,52]. Of course, the exact molecular mechanisms involved in these insulinotropic effects are not understood. This may imply different levels of action on the pancreatic b-cells function including the stimulation of insulin synthesis and release and/or the protection of the pancreatic b-cells from damage and apoptosis. Indeed, camel milk proteins may restore and increase insulin secretion by either (i) stimulating glucose-mediated insulin secretion, (ii) inhibiting the secretion of glucagon by the pancreatic a-cells and its function in liver, and/or (iii) inhibiting the key enzymes that indirectly control the secretion of insulin such as the dipeptidyl peptidase IV (DPP-IV) (Fig. 1). DPP-IV is the metabolic enzyme that cleaves and inactivates incretins; GIP, GLP-1, and polypeptide YY (PYY). These incretins control the proper function of the pancreas by decreasing glucagon release from the pancreatic a-cells and stimulating the synthesis and the secretion of insulin from the pancreatic b-cells [53]. Regarding the effects of camel milk on DPP-IV, recent studies using camel milk whey proteins hydrolysates identified various bioactive peptides that inhibited to different extent the activity of DPP-IV assessed in vitro [21,22,50,51]. Some of these peptides have even been sequenced and characterized as competitive inhibitors of DPP-IV [21,22]. However, the positive effects of the peptides on insulin secretion need to be proven at the level of the enzymatic activity measured in more integrated systems including cell lines and in vivo experiments. Moreover, the plausible positive allosteric effects of camel milk proteins on GIP and GLP-1 receptors expressed in the pancreatic a-cells cannot be excluded (Fig. 1). The identified bioactive peptides as well as other camel milk whey proteins constitute the potential candidates for their binding to and pharmacological modulation of GIP and GLP-1 receptors in a similar way as for the insulin receptor. This aspect consti-
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tutes another interesting mechanistic-based research perspective on camel milk. On the other hand, it is evident that defects in both redox and the immune systems are linked to the damage and the destruction of the pancreatic b-cells resulting in diabetes mellitus [27,29,42–44,54–56]. Therefore, many studies aimed to investigate the beneficial effects of camel milk and its proteins on the redox and the immune systems with a positive impact on the function of the pancreatic b-cells and insulin secretion. These many studies suggested the implication of the immunomodulatory, the antiinflammatory, and the anti-oxidative actions of camel milk components that restore the function of the immune cells as well as the secretory function of the pancreatic b-cells. Indeed, the treatment of type 1-diabetic rats with camel milk whey proteins has been shown to reduce the proinflammatory (IL-1b, IL-6, and tumor necrosis factor (TNFa)) and to increase the anti-inflammatory (IL-2 and IL-4) cytokines [27,55]. From the molecular point of view, camel milk whey proteins decreased the apoptosis in the lymphocytes through the canonical insulin receptor signaling pathway, PI3-kinase/Akt, and NFjB/IjB pathway which increased the phosphorylation of Akt and IjB [27]. Other studies also reported the beneficial effects of camel milk whey proteins on B and T cell proliferation and chemotaxis [55,57,58]. However, it is not clear whether camel milk whey proteins act directly on the chemokines, CCL-21 and CXCL-12, and/ or on their respective receptors on the surface of the lymphocytes. Moreover, the anti-apoptotic effects of camel milk whey proteins have been demonstrated in lymphoid tissues of type 1-diabetic rats through down-regulation of the proapoptotic Bax protein and stimulation of the expression of the anti-apoptotic Bcl-XL protein [30]. Interestingly, some of these studies showed that the effects of camel milk whey proteins were linked to a decrease in the pathological and histological alterations of the pancreas with an improvement in insulin secretion and glycaemia [29,30]. These effects along with the inhibitory effects on DPP-IV and the positive pharmacological effects on GIP and GLP-1 receptors may constitute the molecular and the cellular rationales of the beneficial effects of camel milk in the management of diabetes mellitus.
5. Glucagon receptor and GLUT4, the other candidates for camel milk proteins In addition to insulin receptor in liver and GIP/GLP-1 receptors in the pancreas, the glucagon receptor (GCGR) in liver constitutes another interesting target for the biological and the pharmacological effects of camel milk proteins. In fact, camel milk may also have a hypoglycemic action by exerting a negative allosteric modulation on the glucagon receptor reducing or inhibiting glucose release from liver (Fig. 1). Such effects may be either conjointly or not with the pharmacological action of camel milk on glucagon secretion by the pancreas and/or that on insulin receptor activity in liver. Moreover, the potential targeting of the insulin-sensitive tissues and their modulation by camel milk components may also be possible through the direct positive modulation of GLUT4 activity
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and thereby enhancing glucose uptake (Fig. 2). This may be exerted in insulin receptor-dependent or -independent manners. Thus, investigating and profiling the pharmacological effects of camel milk on the glucagon receptor and GLUT4 in vitro using cell lines expressing these membrane proteins should help to test such a hypothesis and will further give a molecular rationale for the anti-diabetic properties of camel milk.
6. Effects of camel milk proteins/peptides on the diabetic wound healing The other interesting beneficial effects of camel milk in the context of diabetes are those reported on wound healing [9,59–63,24]. Indeed, many recent in vitro and in vivo studies using either camel milk whey proteins or their derived peptide fractions showed their improved wound healing potential in diabetic animals (mice and rats) [59–61]. Such effects were attributed to the anti-oxidative activity of camel milk whey proteins that enhances the proliferation of immune cells and accelerates the wound healing process happened during diabetes. This implies the attenuation of the oxidative and the inflammatory processes through the reduction of the levels of pro-inflammatory cytokines such as TNF-a and IL-6 and the restoration of the anti-inflammatory IL10 levels [59,60,62,64]. Interestingly and as suggested above for the effect of camel milk on insulin receptor, lactoferrin also constitutes a good candidate since it has been reported to regulate the release of TNF-a, IL-1 and IL-6 in vivo [65]. This has been shown to be beneficial for the inflammation and the mortality of animals [65]. Whey supplementation improves wound healing by increasing GSH synthesis and improving the function of the cellular antioxidant defense system [61]. Moreover, the supplementation of diabetic mice with camel milk whey proteins significantly modulated the expression of many chemokines (MIP-1a, MIP-2, KC, CX3CL1) as well as TGF-b in wound tissue compared with untreated diabetic mice [59–61]. This suggests the importance of the chemotaxis of the immune cells such as neutrophils and macrophages, and their function in the wound healing process. Finally, another study correlated the beneficial effects of camel milk proteins in wound healing and the expression of b-defensins 2 and 3 [60]. These skin-derived defensins are known to play a role in promoting wound healing due to their anti-microbial properties as well as their stimulatory effects on wound repair and immune cells [60]. Together these studies indicate that camel milk proteins may be promising candidates for the immune-modulation in chronic diabetic wounds. However, it is not clear whether camel milk proteins/peptides act intracellularly on specific signaling pathways or via binding to specific cell surface receptors leading to the modulation of the different factors involved in wound healing. Many key cell surface receptors such as chemokine and toll-like receptors may be the targets for camel milk proteins/peptides. Further investigations in this direction would be of great importance to understand better the molecular mechanisms of the positive effects of camel milk in diabetes complications.
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Concluding remarks
The anti-diabetic properties continue to be one of the fascinating hallmarks of camel milk. However, a big gap still exists between all the convincing in vivo data using patients with diabetes and animal models and the cellular and the molecular mechanisms of action. As described in this review, these mechanisms are complexes with the implication of different cell types (pancreatic a- and b-cells, liver, immune cells) and molecular components such as DDP-IV enzyme, various receptors (insulin, glucagon, GIP, GLP-1, and cytokines/ chemokines), GLUT4, and other signaling proteins and pathways. The few mechanistic-based studies support the direct biological and pharmacological action of camel milk proteins on the key enzymes and receptors, which really gives a molecular basis for the beneficial effects of camel milk on diabetes. However, the data are still weak to understand the exact mode(s) of action of camel milk and to identify the active anti-diabetic agent(s) contained in camel milk. The future studies should be oriented more toward dissecting the cellular and the molecular mechanisms and pathways that would help better understanding the hypoglycemic effects of camel milk. The cell surface receptors for insulin, glucagon, incretins, cytokines, and chemokines constitute the exciting targets and candidates to be investigated with pharmacological and biochemical emphases. For the identification of camel milk agent(s), the strategy using bioactive peptides from camel milk whey proteins can be considered as a promising one. All these studies constitute the future perspectives in the aim to shed more light on the antidiabetic potentials of camel milk.
Funding This work was supported by the United Arab Emirates University through its 2018–2020 Research Start-up competition grant No G00002595.
Conflict of interest The authors declare no conflict of interest.
Appendix A. Supplementary material Supplementary data to this article can be found online at https://doi.org/10.1016/j.diabres.2018.11.006.
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[25] Agrawal RP, Budania S, Sharma P, et al. Zero prevalence of diabetes in camel milk consuming Raica community of north-west Rajasthan, India. Diabet Res Clin Pract 2007;76:290–6. [26] Bhat DK, Kanga U, Kumar N, et al. The Raikas - a unique combination of high prevalence of type 1 diabetes susceptibility genes and near zero incidence of the disease. Hum Immunol 2014;75:1252–8. [27] Badr G, Sayed LH, Omar HE-DM, et al. Camel whey protein protects B and T cells from apoptosis by suppressing activating transcription factor-3 (ATF-3)-mediated oxidative stress and enhancing phosphorylation of AKT and IjB-a in type I diabetic mice. Cell Physiol Biochem 2017;41:41–54. [28] Korish AA, Abdel Gader AG, Korashy HM, et al. Camel milk attenuates the biochemical and morphological features of diabetic nephropathy: inhibition of Smad1 and collagen type IV synthesis. Chem Biol Interact 2015;229:100–8. [29] Meena S, Rajput YS, Pandey AK, et al. Camel milk ameliorates hyperglycaemia and oxidative damage in type-1 diabetic experimental rats. J Dairy Res 2016;83:412–9. [30] Sayed LH, Badr G, Omar HM, et al. Camel whey protein improves oxidative stress and histopathological alterations in lymphoid organs through Bcl-XL/Bax expression in a streptozotocin-induced type 1 diabetic mouse model. Biomed Pharmacother 2017;88:542–52. [31] Abdulrahman AO, Ismael MA, Al-Hosaini K, et al. Differential effects of camel milk on insulin receptor signaling - toward understanding the insulin-like properties of camel milk. Front Endocrinol 2016;7:4. [32] Malik A, Al-Senaidy A, Skrzypczak-Jankun E, et al. A study of the anti-diabetic agents of camel milk. Int J Mol Med 2012;30:585–92. [33] Haeusler RA, McGraw TE, Accili D. Biochemical and cellular properties of insulin receptor signalling. Nat Rev Mol Cell Biol 2018;19:31–44. [34] Singh R. Annual Report of National Research Centre on Camel. [35] Wernery U, Johnson B, Tawfiq Ishmail W. Insulin content in raw dromedary milk and serum measured over one lactation period. J Camel Pract Res 2006;13:89–90. [36] Wernery U, Nagy P, Bhai I, et al. The effect of heat treatment, pasteurization and different storage temperatures on insulin concentrations in camel milk. Milchwissenschaft 2006;61:25–8. [37] Mehaia MA, Hablas MA, Abdel-Rahman KM, et al. Milk composition of Majaheim, Wadah and Hamra camels in Saudi Arabia. Food Chem 1995;52:115–22. [38] Beg OU, von Bahr-Lindstro¨m H, Zaidi ZH, et al. A camel milk whey protein rich in half-cystine. Primary structure, assessment of variations, internal repeat patterns, and relationships with neurophysin and other active polypeptides. Eur J Biochem 1986;159:195–201. [39] McIntosh CHS, Widenmaier S, Kim S-J. Glucose-dependent insulinotropic polypeptide (gastric inhibitory polypeptide; GIP). Vitam Horm 2009;80:409–71. [40] Seino Y, Yabe D. Glucose-dependent insulinotropic polypeptide and glucagon-like peptide-1: incretin actions beyond the pancreas. J Diabet Investig 2013;4:108–30. [41] Seino Y, Fukushima M, Yabe D. GIP and GLP-1, the two incretin hormones: similarities and differences. J Diabet Investig 2010;1:8–23. [42] Geerlings SE, Hoepelman AIM. Immune dysfunction in patients with diabetes. FEMS Immunol Med Microbiol 1999;26:259–65. [43] Graves DT, Kayal RA. Diabetic complications and dysregulated innate immunity. Front Biosci 2008;13:1227–39. [44] King GL. The role of inflammatory cytokines in diabetes and its complications. J Periodontol 2008;79:1527–34.
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Contents available at ScienceDirect
Diabetes Research and Clinical Practice journal homepage: www.elsevier.com/locat e/dia bre s
Review
The molecular basis of the anti-diabetic properties of camel milk Mohammed Akli Ayoub *, Abdul Rasheed Palakkott, Arshida Ashraf, Rabah Iratni Biology Department, College of Science, United Arab Emirates University, Al Ain, United Arab Emirates
A R T I C L E I N F O
A B S T R A C T
Article history:
Over the years, strong evidence have been accumulated in favor of the beneficial effects of
Received 11 July 2018
camel milk on glucose homeostasis with significant anti-diabetic properties in both human
Received in revised form
and animal diabetic models. However, the cellular and molecular mechanisms involved in
18 September 2018
such effects remain not understood. In this review, we speculated about the potential
Accepted 5 November 2018
mechanisms and summarized few mechanistic-based studies that investigated the biolog-
Available online 16 November 2018
ical activity of camel milk and its protein components on the different aspects that may be involved in the anti-diabetic effects. A special emphasis is given to the molecular events
Keywords: Diabetes Camel milk Insulin Glucose
engaged by camel milk proteins/peptides on two key aspects: insulin secretion and insulin receptor activity. Thus, the review gives a molecular rationale to the anti-diabetic effects of camel milk. This will help to identify the anti-diabetic agent(s) contained in camel milk and to understand better its mechanism of action in order to use it for the management of diabetes mellitus. Ó 2018 Elsevier B.V. All rights reserved.
Contents 1. 2. 3. 4. 5. 6. 7.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mechanisms of the anti-diabetic properties of camel milk. . . . . . . . . . . . . . . . Camel milk proteins/peptides acting as insulin-like proteins . . . . . . . . . . . . . . Effects of camel milk proteins/peptides on insulin secretion . . . . . . . . . . . . . . Glucagon receptor and GLUT4, the other candidates for camel milk proteins . Effects of camel milk proteins/peptides on the diabetic wound healing . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Funding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Appendix A. Supplementary material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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* Corresponding author at: Biology Department, College of Science, United Arab Emirates University, P.O. Box 15551, Al Ain, United Arab Emirates. E-mail address: [email protected] (M.A. Ayoub). https://doi.org/10.1016/j.diabres.2018.11.006 0168-8227/Ó 2018 Elsevier B.V. All rights reserved.
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1.
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Introduction
Many studies reported interesting nutritional and therapeutic characteristics of camel milk due to its chemical composition and its biochemical properties [1–8]. Indeed, camel milk has been shown to have potential benefits in many health issues and diseases with significant anti-bacterial, anti-carcinogenic, anti-oxidant, anti-hypertensive, and anti-diabetic properties, demonstrated in many in vitro and in vivo studies [1,2,4,7,9,10]. Undoubtedly, the hypoglycemic effects of camel milk demonstrated in many in vitro and in vivo studies and its potential applications for the management of diabetes mellitus and its related complications remain the most exciting and studied aspects of camel milk. In fact, in vitro and in vivo studies using either type 1- or type 2-diabetic animals models (rats and rabbits) and also patients with diabetes reported the beneficial effects of camel milk by reducing blood sugar, increasing insulin secretion, decreasing insulin resistance, and improving lipid profiles [2,11–24]. Interestingly, in one populationbased study carried out in India it has been reported that the incidence risk of diabetes was lower in camel breeders and rearing tribal, the Raikas, who regularly consume camel milk, compared to other communities who do not [25,26]. Thus, camel milk has been proposed as a promising adjuvant in the anti-diabetic therapy. Moreover, camel milk has been shown to improve other pathophysiological aspects related to diabetes as a chronic disease such as insulin resistance, obesity, inflammation, wound healing, and oxidative damage [2,9,13,27–30]. However, the secret behind these beneficial effects of camel milk is still unknown since the exact anti-diabetic component(s) contained in camel milk and the molecular and cellular mechanisms involved is/are still not completely understood. This constitutes the challenge for the present and the future studies and investigations in order to identify the anti-diabetic agent in camel milk and to understand its mechanism(s) of action. In this review, we focused on the key studies that aimed at investigating the impact of camel milk components on the different molecular and cellular events leading to the beneficial effects observed in the context of diabetes and its related complications such as wound healing. We attempted to provide a molecular basis for the anti-diabetic properties of camel milk by highlighting recent data and new perspectives with regard to the potential action of camel milk and its components on the two key tissues involved in diabetes, the pancreas and the insulin-sensitive tissues such as liver. The emphasis is given to the effects of camel milk on insulin secretion by the pancreatic b-cells and on insulin function through its receptor in the insulin-sensitive tissues. We also described few studies that attempted to identify the antidiabetic agent(s) in camel milk with an emphasis on protein and peptide components and their plausible mechanism of action investigated at the molecular and cellular levels using various in vitro assays.
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2. Mechanisms of the anti-diabetic properties of camel milk The anti-diabetic properties of camel milk may be very complex involving many molecular and cellular mechanisms and aspects of glucose metabolism and transport as well as insulin synthesis and secretion [2,10,24,31,32]. Obviously, insulin synthesis and secretion, and insulin receptor function constitute the two key aspects that may be involved in the beneficial effects of camel milk on diabetes. Thus, from the molecular and cellular points of view, camel milk may act at different levels including; (i) direct effects on insulin receptor function and glucose transport in the insulin-sensitive tissues, (ii) direct and/or indirect effects on insulin secretion by the pancreatic b-cells, and (iii) effects on the survival, growth, and the overall activity of the pancreatic cells (Fig. 1). The link between the anti-diabetic properties of camel milk and its putative effects on insulin receptor activity appears obvious with the pivotal role of insulin receptor in glucose homeostasis (Fig. 1) [33]. This is also based on the assumption that camel milk contains insulin and/or insulin-like proteins that act directly on the insulinsensitive tissues and that may have specific properties in terms of stability, proteolysis, and absorption in the stomach, and their entry into the circulation before reaching their target tissues [10,24,32]. Even though the insulin content of camel milk is not as clear as many reviews stated, a high concentration of insulin (52 units/liter) has been detected in camel milk [34], and this seems to vary over the lactation period [35] as well as the storage condition [36]. In addition, a comparative study on insulin-like protein content in milk revealed that camel milk contains 3 times more insulinlike proteins than cow milk [37]. The existence of such insulin-like proteins was supported by the fact that amino acid sequence of some of camel milk proteins is found to be rich in half cystine like some insulin family peptides [38]. With this line, a recent study by Abdulrahman et al. brought the first evidence of camel milk proteins directly acting on the insulin receptor and thereby potentiating it activity and signaling in vitro (Fig. 2)[31]. The other hypothesis consists of the direct or indirect action of camel milk on insulin synthesis and secretion by the pancreatic b-cells (Fig. 1). This is mainly supported by in vitro studies suggesting the existence of specific proteins and bioactive peptides that act directly or indirectly on specific cellular pathways controlling insulin synthesis and secretion. Indeed, many hormones and their receptors play a key role in the control of insulin synthesis and secretion by the pancreas. This includes glucagon and glucose-dependent insulinotropic polypeptides, such as gastric inhibitory polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), as well as enzymes like the endoprotease dipeptidyl peptidase IV (DPP-IV) and its proteolytic effects on GIP and GLP-1 [39–41]. Finally, the indirect effects of camel milk may be characterized by the antiinflammatory, the anti-apoptotic, and the anti-oxidative
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Fig. 1 – Speculative representation of the different cellular and molecular targets of camel milk and its mechanisms of action leading to anti-diabetic effects.
Fig. 2 – The positive allosteric effect of camel milk on insulin receptor activity and signaling as demonstrated in vitro by Abdulrahman et al. [31].
effects that restore the secretory activity and the overall functioning of the pancreatic b-cells as demonstrated in many studies [42–44]. In addition, the absence of coagulation of camel milk in the stomach [11] and the protective effects of small size immunoglobulins contained in camel milk on the pancreatic b-cells [12] have also been proposed to explain the hypoglycaemic effects of camel milk.
3. Camel milk proteins/peptides acting as insulin-like proteins The hypoglycemic action of camel milk observed in many in vivo studies on type 1- and type 2-diabetes using animal models as well as patients with diabetes obviously suggested the existence of insulin and/or insulin-like proteins in camel
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milk [2,11–24]. However, as mentioned earlier the insulin content of camel milk is not clear yet and only few quantitative studies were reported [34–36]. The presence of insulin or insulin-like proteins in camel milk argues for the possible direct effect of camel milk on insulin receptor and its function in the major insulin-sensitive tissues [38], but evidence for this assumption still to be brought. Camel milk proteins are represented by two major components, caseins (75%) and whey proteins (25%) [1–3,5]. Camel milk whey mainly contains serum albumin, a-lactalbumin, lactoferrin, lysozyme, immunoglobulins and peptidoglycan recognition protein [1–3,5]. Therefore, the biological activity of camel milk is thought to be mediated by these proteins and/or their peptides upon hydrolysis by the gastrointestinal system [6]. Among the whey proteins, the iron-binding glycoprotein, lactoferrin, constitutes an interesting candidate for a possible activity on insulin and insulin receptor function and signaling. Indeed, many studies linked lactoferrin with insulin resistance and other related disorders such as obesity and inflammation [45–49]. For instance, circulating lactoferrin was negatively associated with hyperglycemia and positively with insulin sensitivity and anti-inflammatory responses [48]. Moreover, lactoferrin has been shown to increase insulin-mediated Akt phosphorylation in human hepatocarcinoma (HepG2) and adipocyte 3T3-L1 cell lines [49] suggesting a direct effect on insulin receptor. The activation of PI3kinase/Akt axis by insulin is the key signaling pathway involved in the transport and the utilization of glucose by the insulin-sensitive tissues through the membrane glucose transporter 4 (GLUT4) [33]. However, it is not clear whether camel milk lactoferrin can really bind to the insulin receptor and modulate its function and signaling which may explain its anti-diabetic effects. This constitutes in fact a legitimate question and an exciting hypothesis to be tested on cells expressing the insulin receptor as well as GLUT4 since lactoferrin has a strong potential of binding to cell membrane components. Recently, an elegant in vitro study reported the first evidence for a direct effect of camel milk on insulin receptor activity and signaling in the transfected human embryonic kidney (HEK293) cell line [31]. In this study, treatment of cells with camel milk weakly affected insulin receptor activity, however this significantly potentiated the agonistic action of insulin indicating a putative allosteric mechanism of action (Fig. 2) [31]. Such a potentiation was observed on insulinmediated Grb2 protein translocation to the activated receptor assessed, in real-time and live cells, using bioluminescence resonance energy transfer (BRET) technology. Interestingly, the fractionation experiments clearly supported the protein/ peptide nature of such a potentiating agent contained in camel milk and this is yet to be identified. Moreover, camel milk has been shown to activate the extracellular signalregulated kinases 1 & 2 (ERK1/2), but not Akt, signaling pathway and strongly potentiated the insulin-mediated ERK1/2 response which is consistent with BRET data [31]. These observations suggest that camel milk acts on the mitogenic (Grb2/Ras/mitogenic protein kinases), but not on the metabolic (PI3-kinase/Akt) pathway, which is somehow difficult to reconcile with the anti-diabetic effects of camel milk observed in vivo. Indeed, the metabolic pathway characterized
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by insulin activating PI3-kinase/Akt axis controls the expression and the translocation of GLUT4 to the plasma membrane and this constitutes the critical step in the transport and the utilization of glucose by the insulin-sensitive tissues through its receptor [33]. By contrast, the study did not show whether the potentiation of the insulin receptor by camel milk was associated with any increase in glucose uptake by the cells. As camel milk candidates and as described later in this review, some bioactive peptides obtained from the tryptic hydrolysis of camel milk whey proteins have been reported to have beneficial effects in the context of diabetes [21,22,50,51]. Therefore, it will be interesting to test these peptides for their direct pharmacological effects on the insulin receptor activity and signaling using similar approaches reported by Abdulrahman et al. [31].
4. Effects of camel milk proteins/peptides on insulin secretion The anti-diabetic properties of camel milk demonstrated in many in vivo studies also suggest potential effects on the pancreas and on insulin secretion by the pancreatic b-cells. In fact, feeding patients and animals (rats and rabbits) with diabetes with camel milk has been reported to restore the production and the release of insulin compared to control patients and animals [2,14,17,18,29,52]. Of course, the exact molecular mechanisms involved in these insulinotropic effects are not understood. This may imply different levels of action on the pancreatic b-cells function including the stimulation of insulin synthesis and release and/or the protection of the pancreatic b-cells from damage and apoptosis. Indeed, camel milk proteins may restore and increase insulin secretion by either (i) stimulating glucose-mediated insulin secretion, (ii) inhibiting the secretion of glucagon by the pancreatic a-cells and its function in liver, and/or (iii) inhibiting the key enzymes that indirectly control the secretion of insulin such as the dipeptidyl peptidase IV (DPP-IV) (Fig. 1). DPP-IV is the metabolic enzyme that cleaves and inactivates incretins; GIP, GLP-1, and polypeptide YY (PYY). These incretins control the proper function of the pancreas by decreasing glucagon release from the pancreatic a-cells and stimulating the synthesis and the secretion of insulin from the pancreatic b-cells [53]. Regarding the effects of camel milk on DPP-IV, recent studies using camel milk whey proteins hydrolysates identified various bioactive peptides that inhibited to different extent the activity of DPP-IV assessed in vitro [21,22,50,51]. Some of these peptides have even been sequenced and characterized as competitive inhibitors of DPP-IV [21,22]. However, the positive effects of the peptides on insulin secretion need to be proven at the level of the enzymatic activity measured in more integrated systems including cell lines and in vivo experiments. Moreover, the plausible positive allosteric effects of camel milk proteins on GIP and GLP-1 receptors expressed in the pancreatic a-cells cannot be excluded (Fig. 1). The identified bioactive peptides as well as other camel milk whey proteins constitute the potential candidates for their binding to and pharmacological modulation of GIP and GLP-1 receptors in a similar way as for the insulin receptor. This aspect consti-
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tutes another interesting mechanistic-based research perspective on camel milk. On the other hand, it is evident that defects in both redox and the immune systems are linked to the damage and the destruction of the pancreatic b-cells resulting in diabetes mellitus [27,29,42–44,54–56]. Therefore, many studies aimed to investigate the beneficial effects of camel milk and its proteins on the redox and the immune systems with a positive impact on the function of the pancreatic b-cells and insulin secretion. These many studies suggested the implication of the immunomodulatory, the antiinflammatory, and the anti-oxidative actions of camel milk components that restore the function of the immune cells as well as the secretory function of the pancreatic b-cells. Indeed, the treatment of type 1-diabetic rats with camel milk whey proteins has been shown to reduce the proinflammatory (IL-1b, IL-6, and tumor necrosis factor (TNFa)) and to increase the anti-inflammatory (IL-2 and IL-4) cytokines [27,55]. From the molecular point of view, camel milk whey proteins decreased the apoptosis in the lymphocytes through the canonical insulin receptor signaling pathway, PI3-kinase/Akt, and NFjB/IjB pathway which increased the phosphorylation of Akt and IjB [27]. Other studies also reported the beneficial effects of camel milk whey proteins on B and T cell proliferation and chemotaxis [55,57,58]. However, it is not clear whether camel milk whey proteins act directly on the chemokines, CCL-21 and CXCL-12, and/ or on their respective receptors on the surface of the lymphocytes. Moreover, the anti-apoptotic effects of camel milk whey proteins have been demonstrated in lymphoid tissues of type 1-diabetic rats through down-regulation of the proapoptotic Bax protein and stimulation of the expression of the anti-apoptotic Bcl-XL protein [30]. Interestingly, some of these studies showed that the effects of camel milk whey proteins were linked to a decrease in the pathological and histological alterations of the pancreas with an improvement in insulin secretion and glycaemia [29,30]. These effects along with the inhibitory effects on DPP-IV and the positive pharmacological effects on GIP and GLP-1 receptors may constitute the molecular and the cellular rationales of the beneficial effects of camel milk in the management of diabetes mellitus.
5. Glucagon receptor and GLUT4, the other candidates for camel milk proteins In addition to insulin receptor in liver and GIP/GLP-1 receptors in the pancreas, the glucagon receptor (GCGR) in liver constitutes another interesting target for the biological and the pharmacological effects of camel milk proteins. In fact, camel milk may also have a hypoglycemic action by exerting a negative allosteric modulation on the glucagon receptor reducing or inhibiting glucose release from liver (Fig. 1). Such effects may be either conjointly or not with the pharmacological action of camel milk on glucagon secretion by the pancreas and/or that on insulin receptor activity in liver. Moreover, the potential targeting of the insulin-sensitive tissues and their modulation by camel milk components may also be possible through the direct positive modulation of GLUT4 activity
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and thereby enhancing glucose uptake (Fig. 2). This may be exerted in insulin receptor-dependent or -independent manners. Thus, investigating and profiling the pharmacological effects of camel milk on the glucagon receptor and GLUT4 in vitro using cell lines expressing these membrane proteins should help to test such a hypothesis and will further give a molecular rationale for the anti-diabetic properties of camel milk.
6. Effects of camel milk proteins/peptides on the diabetic wound healing The other interesting beneficial effects of camel milk in the context of diabetes are those reported on wound healing [9,59–63,24]. Indeed, many recent in vitro and in vivo studies using either camel milk whey proteins or their derived peptide fractions showed their improved wound healing potential in diabetic animals (mice and rats) [59–61]. Such effects were attributed to the anti-oxidative activity of camel milk whey proteins that enhances the proliferation of immune cells and accelerates the wound healing process happened during diabetes. This implies the attenuation of the oxidative and the inflammatory processes through the reduction of the levels of pro-inflammatory cytokines such as TNF-a and IL-6 and the restoration of the anti-inflammatory IL10 levels [59,60,62,64]. Interestingly and as suggested above for the effect of camel milk on insulin receptor, lactoferrin also constitutes a good candidate since it has been reported to regulate the release of TNF-a, IL-1 and IL-6 in vivo [65]. This has been shown to be beneficial for the inflammation and the mortality of animals [65]. Whey supplementation improves wound healing by increasing GSH synthesis and improving the function of the cellular antioxidant defense system [61]. Moreover, the supplementation of diabetic mice with camel milk whey proteins significantly modulated the expression of many chemokines (MIP-1a, MIP-2, KC, CX3CL1) as well as TGF-b in wound tissue compared with untreated diabetic mice [59–61]. This suggests the importance of the chemotaxis of the immune cells such as neutrophils and macrophages, and their function in the wound healing process. Finally, another study correlated the beneficial effects of camel milk proteins in wound healing and the expression of b-defensins 2 and 3 [60]. These skin-derived defensins are known to play a role in promoting wound healing due to their anti-microbial properties as well as their stimulatory effects on wound repair and immune cells [60]. Together these studies indicate that camel milk proteins may be promising candidates for the immune-modulation in chronic diabetic wounds. However, it is not clear whether camel milk proteins/peptides act intracellularly on specific signaling pathways or via binding to specific cell surface receptors leading to the modulation of the different factors involved in wound healing. Many key cell surface receptors such as chemokine and toll-like receptors may be the targets for camel milk proteins/peptides. Further investigations in this direction would be of great importance to understand better the molecular mechanisms of the positive effects of camel milk in diabetes complications.
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Concluding remarks
The anti-diabetic properties continue to be one of the fascinating hallmarks of camel milk. However, a big gap still exists between all the convincing in vivo data using patients with diabetes and animal models and the cellular and the molecular mechanisms of action. As described in this review, these mechanisms are complexes with the implication of different cell types (pancreatic a- and b-cells, liver, immune cells) and molecular components such as DDP-IV enzyme, various receptors (insulin, glucagon, GIP, GLP-1, and cytokines/ chemokines), GLUT4, and other signaling proteins and pathways. The few mechanistic-based studies support the direct biological and pharmacological action of camel milk proteins on the key enzymes and receptors, which really gives a molecular basis for the beneficial effects of camel milk on diabetes. However, the data are still weak to understand the exact mode(s) of action of camel milk and to identify the active anti-diabetic agent(s) contained in camel milk. The future studies should be oriented more toward dissecting the cellular and the molecular mechanisms and pathways that would help better understanding the hypoglycemic effects of camel milk. The cell surface receptors for insulin, glucagon, incretins, cytokines, and chemokines constitute the exciting targets and candidates to be investigated with pharmacological and biochemical emphases. For the identification of camel milk agent(s), the strategy using bioactive peptides from camel milk whey proteins can be considered as a promising one. All these studies constitute the future perspectives in the aim to shed more light on the antidiabetic potentials of camel milk.
Funding This work was supported by the United Arab Emirates University through its 2018–2020 Research Start-up competition grant No G00002595.
Conflict of interest The authors declare no conflict of interest.
Appendix A. Supplementary material Supplementary data to this article can be found online at https://doi.org/10.1016/j.diabres.2018.11.006.
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