Camel and bovine milk lactoferrins activate insulin receptor and its related AKT and ERK1/2 pathways.

Lactoferrin (LF) is a milk protein that may be an interesting candidate for the antidiabetic properties of milk due to its well-documented bioactivity and implication in diabetes. Here, we investigated the functional action of LF purified from camel and bovine milk (cLF, bLF) on insulin receptors (IR) and their pharmacology and signaling in hepatocarcinoma (HepG2) and human embryonic kidney (HEK293) cells. For this, we examined IR activation by bioluminescence resonance energy transfer (BRET) technology and the phosphorylation of its key downstream signaling kinases by western blot. The purified cLF and bLF induced phosphorylation of IR, AKT, and ERK1/2 in HepG2 and HEK293 cells. The BRET assays in HEK293 cells confirm the pharmacological action of cLF and bLF on IR, with a possible allosteric mode of action. This reveals for the first time the bioactivity of LF toward IR function, indicating it as a potential bioactive protein behind the antidiabetic properties of camel milk.

Anti-Insulin Receptor Antibodies in the Pathology and Therapy of Diabetes Mellitus.

Diabetes mellitus (DM) is recognized as the most common and the world's fastest-growing chronic disease with severe complications leading to increased mortality. Many strategies exist for the management of DM and its control, including treatment with insulin and insulin analogs, oral hypoglycemic therapy such as insulin secretion stimulators and insulin sensitizers, and diet and physical training. Over the years, many types of drugs and molecules with an interesting pharmacological diversity have been developed and proposed for their anti-diabetic potential. Such molecules target diverse key receptors, enzymes, and regulatory/signaling proteins known to be directly or indirectly involved in the pathophysiology of DM. Among them, insulin receptor (IR) is undoubtedly the target of choice for its central role in insulin-mediated glucose homeostasis and its utilization by the major insulin-sensitive tissues such as skeletal muscles, adipose tissue, and the liver. In this review, we focus on the implication of antibodies targeting IR in the pathology of DM as well as the recent advances in the development of IR antibodies as promising anti-diabetic drugs. The challenge still entails development of more powerful, highly selective, and safer anti-diabetic drugs.

Molecular basis of the anti-diabetic properties of camel milk through profiling of its bioactive peptides on dipeptidyl peptidase IV (DPP-IV) and insulin receptor activity.

The molecular basis of the anti-diabetic properties of camel milk reported in many studies and the exact active agent are still elusive. Recent studies have reported effects of camel whey proteins (CWP) and their hydrolysates (CWPH) on the activities of dipeptidyl peptidase IV (DPP-IV) and the human insulin receptor (hIR). In this study, CWPH were generated, screened for DPP-IV binding in silico and inhibitory activity in vitro, and processed for peptide identification. Furthermore, pharmacological action of intact CWP and their selected hydrolysates on hIR activity and signaling and on glucose uptake were investigated in cell lines. Results showed inhibition of DPP-IV by CWP and CWPH and their positive action on hIR activation and glucose uptake. Interestingly, the combination of CWP or CWPH with insulin revealed a positive allosteric modulation of hIR that was drastically reduced by the competitive hIR antagonist. Our data reveal for the first time the profiling and pharmacological actions of CWP and their derived peptides fractions on hIR and their pathways involved in glucose homeostasis. This sheds more light on the anti-diabetic properties of camel milk by providing the molecular basis for the potential use of camel milk in the management of diabetes.

Interplay Between Angiotensin II Type 1 Receptor and Thrombin Receptor Revealed by Bioluminescence Resonance Energy Transfer Assay.

The key hormone of the renin-angiotensin system (RAS), angiotensin II (AngII), and thrombin are known to play major roles in the vascular system and its related disorders. Previous studies reported connections between AngII and thrombin in both physiological and pathophysiological models. However, the molecular mechanisms controlling such interplay at the level of their receptors belonging to the family of G protein-coupled receptors (GPCRs) are not fully understood. In this study, we investigated the functional interaction between the AngII type 1 receptor (AT1R) and the thrombin receptor [or protease-activated receptor 1 (PAR1)] in human embryonic kidney 293 (HEK293) cells. For this, we used various bioluminescence resonance energy transfer (BRET) proximity-based assays to profile the coupling to the heterotrimeric Gαq protein, ß-arrestin recruitment, and receptor internalization and trafficking in intact cells. The overall dose-response and real-time kinetic BRET data demonstrated the specific molecular proximity between AT1R and PAR1 resulting in their functional interaction. This was characterized by thrombin inducing BRET increase within AT1R/Gαq and AT1R/ß-arrestin pairs and synergistic effects observed upon the concomitant activation of both receptors suggesting a positive allosteric interaction. The BRET data corroborated with the data on the downstream Gαq/inositol phosphate pathway. Moreover, the selective pharmacological blockade of the receptors revealed the implication of both AT1R and PAR1 protomers in such a synergistic interaction and the possible transactivation of AT1R by PAR1. Interestingly, the positive action of PAR1 on AT1R activation was contrasted with its apparent inhibition of AT1R internalization and its endosomal trafficking. Finally, BRET saturation and co-immunoprecipitation assays supported the physical AT1-PAR1 interaction in HEK293 cells. Our study reveals for the first time the functional interaction between AT1R and PAR1 in vitro characterized by a transactivation and positive allosteric modulation of AT1R and inhibition of its desensitization and internalization. This finding may constitute the molecular basis of the well-known interplay between RAS and thrombin. Thus, our data should lead to revising some findings on the implication of RAS and thrombin in vascular physiology and pathophysiology revealing the importance to consider the functional and pharmacological interaction between AT1R and thrombin receptors.

Positive Modulation of Angiotensin II Type 1 Receptor-Mediated Signaling by LVV-Hemorphin-7.

Hemorphins are hemoglobin ß-chain-derived peptides initially known for their analgesic effects via binding to the opioid receptors belonging to the family of G protein-coupled receptor (GPCR), as well as their physiological action on blood pressure. However, their molecular mechanisms in the regulation of blood pressure are not fully understood. Studies have reported an antihypertensive action via the inhibition of the angiotensin-converting enzyme, a key enzyme in the renin-angiotensin system. In this study, we hypothesized that hemorphins may also target angiotensin II (AngII) type 1 receptor (AT1R) as a key GPCR in the renin-angiotensin system. To investigate this, we examined the effects of LVV-hemorphin-7 on AT1R transiently expressed in human embryonic kidney (HEK293) cells using bioluminescence resonance energy transfer (BRET) technology for the assessment of AT1R/Gαq coupling and ß-arrestin 2 recruitment. Interestingly, while LVV-hemorphin-7 alone had no significant effect on BRET signals between AT1R and Gαq or ß-arrestin 2, it nicely potentiated AngII-induced BRET signals and significantly increased AngII potency. The BRET data were also correlated with AT1R downstream signaling with LVV-hemorphin-7 potentiating the canonical AngII-mediated Gq-dependent inositol phosphate pathway as well as the activation of the extracellular signal-regulated kinases (ERK1/2). Both AngII and LVV-hemorphin-7-mediated responses were fully abolished by AT1R antagonist demonstrating the targeting of the active conformation of AT1R. Our data report for the first time the targeting and the positive modulation of AT1R signaling by hemorphins, which may explain their role in the physiology and pathophysiology of both vascular and renal systems. This finding further consolidates the pharmacological targeting of GPCRs by hemorphins as previously shown for the opioid receptors in analgesia opening a new era for investigating the role of hemorphins in physiology and pathophysiology via the targeting of GPCR pharmacology and signaling.

The molecular basis of the anti-diabetic properties of camel milk.

Over the years, strong evidence have been accumulated in favor of the beneficial effects of camel milk on glucose homeostasis with significant anti-diabetic properties in both human and animal diabetic models. However, the cellular and molecular mechanisms involved in such effects remain not understood. In this review, we speculated about the potential mechanisms and summarized few mechanistic-based studies that investigated the biological 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 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.