**4. Diabetes-associated disturbances in bile acids and gut microflora**

Disturbances in bile acids composition may result in tissue necrosis due to higher than normal concentrations of potent bile acids such as lithocholic acid compared with less potent bile acids such as chenodeoxycholic acid. Secondary bile acids are solely produced by the action of gut microflora on primary bile acids, and thus, microfloral composition is directly linked to secondary bile acid production and bile acid composition. This interaction between bile acid composition and the composition of gut microflora represents the base of the hypothesized link between bile acid, gut microflora and energy balance. However, even though the compositions of bile acids and gut microflora are reported to be different in diabetic patients (Duan et al. 2008; Gebel 2011; Morris 1989; Ogura et al. 1986; Slivka et al. 1979a; Thomson 1983), it is still not clear how these changes directly affect the development and progression of diabetes or its complications. These complications include cardiovascular, tissue necrosis and ulcerations, and metabolic disturbances.

The amino acid taurine, which is used by hepatocytes in bile acid conjugation and bile salts formation, has many other physiological functions including the regulation of intracellular osmolarity, cardiomyocytes functions, and as an antioxidant. Accordingly, a clear link between bile compositions, taurine concentrations and diabetes complications can be discussed. A hypoglycemic effect of taurine, directly or through synergizing the effect of insulin, has also been reported (Kulakowski & Maturo 1984). Conjugated bile acids includes glycine and taurine conjugates, both existing in constant ratio. Glycine conjugated bile acids are less soluble and are harder to excrete compared with taurine conjugated bile acids. This result in bile accumulation noticed in T1D subjects (Bennion & Grundy 1977). In T1D patients, who have increased lipid metabolism, the percentage of taurocholic acid in bile is decreased indicating an altered biosynthesis of taurine (Meinders et al. 1981c). In one study, diabetic patients showed altered taurine metabolism causing consequent cellular dysfunctions that resulted in worsening diabetic neuropathy, cardiomyopathy, platelet

Potentials and Limitations of Bile Acids and Probiotics in Diabetes Mellitus 371

information about a drug's interaction with living tissue, and are more cost-effective compared with *in vivo* animal models (Qin et al. 2010). *In situ* methods can better predict drug absorption compared with *ex vivo* models but *in vivo* models can provide more comprehensive pharmacokinetic profiles and give a better understanding of drug-tissue interactions (Zanchi et al. 1998). I*n vivo* studies are usually carried out where drug therapeutic formulations are administered to animals in order to investigate short and long term safety, to explore various clinical effects and to study different physicochemical parameters before confirming suitability of the formulation to a disease condition(s).

Although there is a surplus of animal models (spontaneous and induced) to study T1D, there is no ideal or standard model for studying the effect of bile acids and probiotics on T1D. Rats lack gall bladder which means bile is not stored before secretion but rather is secreted immediately after food intake. However, this does not seem to stop researches from using rats as an animal model of T1D (Al-Salami et al. 2008e). Rats, mice and hamsters have been used to study bile acids and probiotics applications in T1D, however, future research is needed, to compare the effect of bile acids and probiotics on T1D, using

An ideal animal model should represent a specific medical condition in terms of disease

If we are to create a better model of human T1D, we should carefully consider the disease

The current therapeutics for T1D are inadequate, which necessitate further drug development and *in vivo* studies. Clinical translation of T1D pathophysiology and clinical manifestations, from animal to human, has been limited and rather difficult. This is because very little is known about T1D; the extent of heterogeneity, polymorphism, genetic distance, the exact site of initial immune response (gut or pancreas), and diabetogenic antigens. Creating a suitable animal model for T1D requires the ability to accurately translate the findings to human. These findings include therapeutic efficacy (prevention/treatment), safety and PK/PD profiles. There are various animal models for T1D, with the nonobese diabetic (NOD) mouse being the 'standard' one. Other models are induction models of rats, mice and hamsters using alloxan or streptozotocin to destroy pancreatic beta cells and induce T1D. The NOD mouse represents the best spontaneous model for a human autoimmune disease, in particular, T1D. NOD mouse model allows the investigation of various immunointerventions that can be used in human T1D. Similar to T1D in human,

development, pathophysiology, biological disturbances and short & long term

2. The relevant speed and stages of disease development and progression. 3. Disease complications, their progression and the relevant clinical end point(s).

5. Feasibility of sample collections in terms of tissue site and sample volume.

NOD mice have higher levels of macrophages, dendritic cells, CD4+ and B cells.

The induction of T1D in NOD mouse can be achieved through environmental conditions, mimicking the development of T1D in human. However, the development of T1D in NOD mouse takes place quickly and can produce a significant inflammatory condition that may over-respond to immunomanipulation and exaggerate the effect of a treatment. Also, the

1. Relevant end points including primary, secondary and tertiary.

4. Symptomatic/nonsymptomatic signs of the disease.

6. The incidence in males vs. females.

Various animal models are used to represent various diseases.

different animal models.

effect on the following:

complications.

aggregation and endothelial dysfunction (Hansen 2001). In T1D rats, taurine concentrations were found different in various organs (Goodman & Shihabi 1990; Hansen 2001; Reibel et al. 1979). Taurine concentrations in kidney and liver were low, while they were higher in heart and skeletal muscle. One important diabetic complication, platelet hyperaggregation, has been normalized by the alteration of bile acids composition through the addition of taurine (Franconi et al. 1995). Another complication is T1D retinopathy which have shown significantly less taurine levels in the retina, compared with that in healthy rats (Vilchis & Salceda 1996). Diabetic nephropathy are other major complication of T1D. Taurine consumption has shown to reduce chronic diabetic nephropathy in T1D rats (Trachtman & Sturman 1996). Other diabetic complications can also be reduced or even prevented by the addition of taurine. These include high glucose induced apoptosis in human vascular endothelial cells (Di Wu et al. 1999) and impaired endothelium-dependent vasodilatation in diabetic mice.

Even though the composition of gut microflora has been reported to be different in T1D patients, it may be difficult to quantify or qualify such a difference. Gut microflora interacts closely with the body immune system and has shown to control the immune response to various inflammatory stimuli. The mechanism of action of probiotics could be one or more of the following. Firstly, by competitive exclusion, where gut microfloral bacteria resist colonization of other 'foreign' bacteria. Secondly, by barrier formation where the microflora forms a physical barrier reducing bacterial translocation by forming a wall surrounding the outside part of the gut enterocytes. Thirdly, gut bacteria can produce bacteriocins and change the pH to create a harsher environment for other invading bacteria to settle in the gut. Fourthly, gut microflora can influence the immune system through its effect on gut enterocytes (quorum sensing) and the innate and adaptive immune system (Gareau et al. 2010; Walker 2008a).

It is a common conception that the efficiency of the immune system is compromised in diabetic patients resulting in prolonged healing of infections and diabetic ulcers (Steed et al. 1996). This is also brought about by the higher rates of bacterial infections reported in diabetes and higher rate of antibiotic use (Goldberg & Krause 2009; Paccagnini et al. 2009). In one study, the effect of the probiotic bacteria, Lactobacillus plantarum (Lp) on infected diabetic ulcers, was examined. Topical application of Lp on diabetic ulcers for 30 days induced healing. This effect was observed in almost half of the treated diabetic patients. However, this was not significantly different from healthy treated control suggesting that probiotic treatment is effective in treating diabetic ulcers, but its effect does not vary between diabetic and non-diabetic individuals. It is therefore tempting to speculate that gut microfloral bacteria controls the innate immune responses towards normalizing harmful bacteria in an effort to protect its own environment and keep its own existence.

#### **5. Animal models suitable for investigating bile acids and probiotics effects on Type 1 diabetes**

During the process of drug development, various *in vivo*, *ex vivo*, *in situ* and *in silico* methods can be used. Each method has advantages and disadvantages, and so using more than one method can provide better confirmation of findings. *In silico* methods can provide an initial insight into a potential drug candidate with predicted high pharmacological activity and good stability, while *ex vivo* methods can provide more

aggregation and endothelial dysfunction (Hansen 2001). In T1D rats, taurine concentrations were found different in various organs (Goodman & Shihabi 1990; Hansen 2001; Reibel et al. 1979). Taurine concentrations in kidney and liver were low, while they were higher in heart and skeletal muscle. One important diabetic complication, platelet hyperaggregation, has been normalized by the alteration of bile acids composition through the addition of taurine (Franconi et al. 1995). Another complication is T1D retinopathy which have shown significantly less taurine levels in the retina, compared with that in healthy rats (Vilchis & Salceda 1996). Diabetic nephropathy are other major complication of T1D. Taurine consumption has shown to reduce chronic diabetic nephropathy in T1D rats (Trachtman & Sturman 1996). Other diabetic complications can also be reduced or even prevented by the addition of taurine. These include high glucose induced apoptosis in human vascular endothelial cells (Di Wu et al. 1999) and impaired

Even though the composition of gut microflora has been reported to be different in T1D patients, it may be difficult to quantify or qualify such a difference. Gut microflora interacts closely with the body immune system and has shown to control the immune response to various inflammatory stimuli. The mechanism of action of probiotics could be one or more of the following. Firstly, by competitive exclusion, where gut microfloral bacteria resist colonization of other 'foreign' bacteria. Secondly, by barrier formation where the microflora forms a physical barrier reducing bacterial translocation by forming a wall surrounding the outside part of the gut enterocytes. Thirdly, gut bacteria can produce bacteriocins and change the pH to create a harsher environment for other invading bacteria to settle in the gut. Fourthly, gut microflora can influence the immune system through its effect on gut enterocytes (quorum sensing) and the innate and adaptive

It is a common conception that the efficiency of the immune system is compromised in diabetic patients resulting in prolonged healing of infections and diabetic ulcers (Steed et al. 1996). This is also brought about by the higher rates of bacterial infections reported in diabetes and higher rate of antibiotic use (Goldberg & Krause 2009; Paccagnini et al. 2009). In one study, the effect of the probiotic bacteria, Lactobacillus plantarum (Lp) on infected diabetic ulcers, was examined. Topical application of Lp on diabetic ulcers for 30 days induced healing. This effect was observed in almost half of the treated diabetic patients. However, this was not significantly different from healthy treated control suggesting that probiotic treatment is effective in treating diabetic ulcers, but its effect does not vary between diabetic and non-diabetic individuals. It is therefore tempting to speculate that gut microfloral bacteria controls the innate immune responses towards normalizing harmful

bacteria in an effort to protect its own environment and keep its own existence.

**5. Animal models suitable for investigating bile acids and probiotics effects** 

During the process of drug development, various *in vivo*, *ex vivo*, *in situ* and *in silico* methods can be used. Each method has advantages and disadvantages, and so using more than one method can provide better confirmation of findings. *In silico* methods can provide an initial insight into a potential drug candidate with predicted high pharmacological activity and good stability, while *ex vivo* methods can provide more

endothelium-dependent vasodilatation in diabetic mice.

immune system (Gareau et al. 2010; Walker 2008a).

**on Type 1 diabetes** 

information about a drug's interaction with living tissue, and are more cost-effective compared with *in vivo* animal models (Qin et al. 2010). *In situ* methods can better predict drug absorption compared with *ex vivo* models but *in vivo* models can provide more comprehensive pharmacokinetic profiles and give a better understanding of drug-tissue interactions (Zanchi et al. 1998). I*n vivo* studies are usually carried out where drug therapeutic formulations are administered to animals in order to investigate short and long term safety, to explore various clinical effects and to study different physicochemical parameters before confirming suitability of the formulation to a disease condition(s). Various animal models are used to represent various diseases.

Although there is a surplus of animal models (spontaneous and induced) to study T1D, there is no ideal or standard model for studying the effect of bile acids and probiotics on T1D. Rats lack gall bladder which means bile is not stored before secretion but rather is secreted immediately after food intake. However, this does not seem to stop researches from using rats as an animal model of T1D (Al-Salami et al. 2008e). Rats, mice and hamsters have been used to study bile acids and probiotics applications in T1D, however, future research is needed, to compare the effect of bile acids and probiotics on T1D, using different animal models.

An ideal animal model should represent a specific medical condition in terms of disease development, pathophysiology, biological disturbances and short & long term complications.

If we are to create a better model of human T1D, we should carefully consider the disease effect on the following:


The current therapeutics for T1D are inadequate, which necessitate further drug development and *in vivo* studies. Clinical translation of T1D pathophysiology and clinical manifestations, from animal to human, has been limited and rather difficult. This is because very little is known about T1D; the extent of heterogeneity, polymorphism, genetic distance, the exact site of initial immune response (gut or pancreas), and diabetogenic antigens. Creating a suitable animal model for T1D requires the ability to accurately translate the findings to human. These findings include therapeutic efficacy (prevention/treatment), safety and PK/PD profiles. There are various animal models for T1D, with the nonobese diabetic (NOD) mouse being the 'standard' one. Other models are induction models of rats, mice and hamsters using alloxan or streptozotocin to destroy pancreatic beta cells and induce T1D. The NOD mouse represents the best spontaneous model for a human autoimmune disease, in particular, T1D. NOD mouse model allows the investigation of various immunointerventions that can be used in human T1D. Similar to T1D in human, NOD mice have higher levels of macrophages, dendritic cells, CD4+ and B cells.

The induction of T1D in NOD mouse can be achieved through environmental conditions, mimicking the development of T1D in human. However, the development of T1D in NOD mouse takes place quickly and can produce a significant inflammatory condition that may over-respond to immunomanipulation and exaggerate the effect of a treatment. Also, the

Potentials and Limitations of Bile Acids and Probiotics in Diabetes Mellitus 373

acids and their analogues are now recognized as having major therapeutic applications in the treatment of cholelithiasis, as transport promoters for other substances, in potentiating the action of other substances (analgesic, antiviral, hypoglycaemic) and as hypoglycaemic and hypolipidemic agents. In one study, lithocholic acid concentration was higher after diabetes development which resulted in gallstone formation (Chijiiwa 1990). This indicates that diabetes directly altered bile composition. However, the exact mechanism by which

One hypothesis linking bile acid disturbance with the initiation of diabetes development, is through the over-production of lithocholic acid, brought about by disturbances in the gut microflora (De Leon et al. 1978; Kokk et al. 2005; Meinders et al. 1981a; Meinders et al. 1981b). Diabetes mellitus has been associated with unbalanced secretion of bile (cholelithiasis). In addition, many studies have linked changes in bile composition to the changes in the composition of the gut microflora (Kokk et al. 2005; Mikov et al. 2004; Mikov

Potential therapeutic use of bile acids in T1D can be achieved through two main applications; as hypoglycaemic agents and as absorption-enhancing agent to insulin

Monoketocholic acid (MKC) (Figure 1) is a stable semisynthetic primary bile acid (cholic acid analogue) with low toxicity that has been shown to enhance the nasal absorption of insulin in rats (89). In addition, MKC has been shown to exert a effect in its own right when

<sup>H</sup> <sup>O</sup> <sup>O</sup> <sup>H</sup>

Permeation enhancement through the tissue-solubilising effect of bile salts was found to be one of several mechanisms by which bile salts can facilitate drug absorption. Other mechanisms involve bile salts' effect on efflux and afflux protein transporters on the cell wall of various tissues including gut enterocytes, hepatocytes, nasal mucosa and others (Al-

Fig. 1. The chemical structure of 12-monoketocholic acid (MKC).

Salami et al. 2008c; Al-Salami et al. 2008d; Al-Salami et al. 2009a).

O

O H

O

administered by the oral route in alloxan-induced T1D rats (Mikov et al. 2007).

diabetes can alter bile acid composition remains unclear.

et al. 2005; Mikov et al. 2006; Mikov & Fawcett 2006b).

The OH group at C-12 in cholic acid is replaced

with a ketone group to enhance stability

delivery.

incidence of T1D is different between males and females in this model while the incidence is the same in males and females in human. This can further limit the applications and the findings of this animal model (Dieleman et al. 1997). Many therapeutics that showed good efficacy in this model failed to achieve similar results in T1D human subjects (Srinivasan & Ramarao 2007). Having said that and regardless of how different this model is, from the 'true' human TID, NOD mouse remains the most representative of human T1D. Interestingly, in a recently published study, the incidence of T1D was much higher, when the mice were maintained in a germ-free environment suggesting direct connection between gut microflora and the development of T1D (Li-Wen et al. 2007).

The suitable animal model for human T1D should ideally be easy to breed and handle, and can accommodate various medical conditions that may come about or be associated with T1D. Thus, extrapolation of its findings to human should be easily done, and with great accuracy and precision.

#### **6. The therapeutic applications of bile acids and probiotics in Type 1 diabetes**

In pathophysiology such as gall stone formations, inflammatory bowel disease and allergic reactions, the administration of probiotics significantly improves body physiology and reduces complications (Cary & Boullata 2010; Gourbeyre et al. 2011; Martin & Walker 2008; Morris et al. 2009; Stephani et al. 2011). In one study, the administration of bile acids and gliclazide to probiotic pre-treated diabetic animals showed efficacy and a significant reduction of diabetic complications (Al-Salami et al. 2008e; Al-Salami et al. 2008g).

The synthesis of bile acids is highly regulated by nuclear hormone receptors and other transcription factors, which ensure a constant supply of bile acids in a very changing metabolic environment. In healthy individuals, bile acids control their own haemostasis through feedback mechanisms involving phosphoenolpyruvate carboxykinase (PEPCK) and farnesoid X receptor alpha (FXR-alpha) nuclear receptors. Their direct effect on diabetes development remains debatable, but through the inhibition of PEPCK and FXR-alpha (via TGR5-D2 signalling pathways), bile acids also inhibits gluconeogenesis. Such mechanisms may seem to oppose that of insulin, which suggests direct effect on glucose haemostasis in healthy individuals. Inherited mutations that impair bile acid synthesis cause many human disorders including early childhood liver inflammation and failure. During the development of diabetes, bile acid synthesis is increased, the bile acid pool is expanded, and bile acid excretion is increased suggesting lack of adequate control over the feedback regulating bile acid haemostasis. Accordingly, several recent studies have investigated the role of and applications of bile acids in glucose haemostasis. Interestingly, where both factors, PEPCK and FXR-alpha fit remains under investigation. During the fasting state, hepatocytes produce more FXR-alpha suggesting that FXR-alpha production takes place in the absent of insulin (Zhang et al, 2004). In another study, when FXR-alpha was tested in diabetic animals, it was noticed to be lower than these in healthy, but when insulin was administered; it normalized such an effect (Duran-Sandoval et al, 2004). Overall, BAs have been reported to inhibit gluconeogenesis via downregulation of phosphoenolpyruvate carboxykinase (PEPCK) mRNA levels in a FXR-alpha-dependent and –independent manner (De Fabiani et al, 2003; Yamagata et al, 2004).

Apart from basic physiological functions like the elimination of cholesterol and the intestinal solubilisation (emulsification) of triacylglycerol, cholesterol and lipid, soluble vitamins, bile

incidence of T1D is different between males and females in this model while the incidence is the same in males and females in human. This can further limit the applications and the findings of this animal model (Dieleman et al. 1997). Many therapeutics that showed good efficacy in this model failed to achieve similar results in T1D human subjects (Srinivasan & Ramarao 2007). Having said that and regardless of how different this model is, from the 'true' human TID, NOD mouse remains the most representative of human T1D. Interestingly, in a recently published study, the incidence of T1D was much higher, when the mice were maintained in a germ-free environment suggesting direct connection between

The suitable animal model for human T1D should ideally be easy to breed and handle, and can accommodate various medical conditions that may come about or be associated with T1D. Thus, extrapolation of its findings to human should be easily done, and with

**6. The therapeutic applications of bile acids and probiotics in Type 1 diabetes**  In pathophysiology such as gall stone formations, inflammatory bowel disease and allergic reactions, the administration of probiotics significantly improves body physiology and reduces complications (Cary & Boullata 2010; Gourbeyre et al. 2011; Martin & Walker 2008; Morris et al. 2009; Stephani et al. 2011). In one study, the administration of bile acids and gliclazide to probiotic pre-treated diabetic animals showed efficacy and a significant

The synthesis of bile acids is highly regulated by nuclear hormone receptors and other transcription factors, which ensure a constant supply of bile acids in a very changing metabolic environment. In healthy individuals, bile acids control their own haemostasis through feedback mechanisms involving phosphoenolpyruvate carboxykinase (PEPCK) and farnesoid X receptor alpha (FXR-alpha) nuclear receptors. Their direct effect on diabetes development remains debatable, but through the inhibition of PEPCK and FXR-alpha (via TGR5-D2 signalling pathways), bile acids also inhibits gluconeogenesis. Such mechanisms may seem to oppose that of insulin, which suggests direct effect on glucose haemostasis in healthy individuals. Inherited mutations that impair bile acid synthesis cause many human disorders including early childhood liver inflammation and failure. During the development of diabetes, bile acid synthesis is increased, the bile acid pool is expanded, and bile acid excretion is increased suggesting lack of adequate control over the feedback regulating bile acid haemostasis. Accordingly, several recent studies have investigated the role of and applications of bile acids in glucose haemostasis. Interestingly, where both factors, PEPCK and FXR-alpha fit remains under investigation. During the fasting state, hepatocytes produce more FXR-alpha suggesting that FXR-alpha production takes place in the absent of insulin (Zhang et al, 2004). In another study, when FXR-alpha was tested in diabetic animals, it was noticed to be lower than these in healthy, but when insulin was administered; it normalized such an effect (Duran-Sandoval et al, 2004). Overall, BAs have been reported to inhibit gluconeogenesis via downregulation of phosphoenolpyruvate carboxykinase (PEPCK) mRNA levels in a FXR-alpha-dependent and –independent manner

Apart from basic physiological functions like the elimination of cholesterol and the intestinal solubilisation (emulsification) of triacylglycerol, cholesterol and lipid, soluble vitamins, bile

reduction of diabetic complications (Al-Salami et al. 2008e; Al-Salami et al. 2008g).

gut microflora and the development of T1D (Li-Wen et al. 2007).

great accuracy and precision.

(De Fabiani et al, 2003; Yamagata et al, 2004).

acids and their analogues are now recognized as having major therapeutic applications in the treatment of cholelithiasis, as transport promoters for other substances, in potentiating the action of other substances (analgesic, antiviral, hypoglycaemic) and as hypoglycaemic and hypolipidemic agents. In one study, lithocholic acid concentration was higher after diabetes development which resulted in gallstone formation (Chijiiwa 1990). This indicates that diabetes directly altered bile composition. However, the exact mechanism by which diabetes can alter bile acid composition remains unclear.

One hypothesis linking bile acid disturbance with the initiation of diabetes development, is through the over-production of lithocholic acid, brought about by disturbances in the gut microflora (De Leon et al. 1978; Kokk et al. 2005; Meinders et al. 1981a; Meinders et al. 1981b). Diabetes mellitus has been associated with unbalanced secretion of bile (cholelithiasis). In addition, many studies have linked changes in bile composition to the changes in the composition of the gut microflora (Kokk et al. 2005; Mikov et al. 2004; Mikov et al. 2005; Mikov et al. 2006; Mikov & Fawcett 2006b).

Potential therapeutic use of bile acids in T1D can be achieved through two main applications; as hypoglycaemic agents and as absorption-enhancing agent to insulin delivery.

Monoketocholic acid (MKC) (Figure 1) is a stable semisynthetic primary bile acid (cholic acid analogue) with low toxicity that has been shown to enhance the nasal absorption of insulin in rats (89). In addition, MKC has been shown to exert a effect in its own right when administered by the oral route in alloxan-induced T1D rats (Mikov et al. 2007).

The OH group at C-12 in cholic acid is replaced with a ketone group to enhance stability

Fig. 1. The chemical structure of 12-monoketocholic acid (MKC).

Permeation enhancement through the tissue-solubilising effect of bile salts was found to be one of several mechanisms by which bile salts can facilitate drug absorption. Other mechanisms involve bile salts' effect on efflux and afflux protein transporters on the cell wall of various tissues including gut enterocytes, hepatocytes, nasal mucosa and others (Al-Salami et al. 2008c; Al-Salami et al. 2008d; Al-Salami et al. 2009a).

Potentials and Limitations of Bile Acids and Probiotics in Diabetes Mellitus 375

(Matthaei et al. 1986). Accordingly, protein transporters have shown strong association with

Gliclazide is used in Type 2 diabetes (T2D) to stimulate insulin production but it also has beneficial extrapancreatic effects which makes it potentially useful in T1D. In fact, some T2D patients continue to use gliclazide even after their diabetes progresses to T1D since it provides better glycemic control than insulin alone. Gliclazide has three main structural

**8. The effect of co-administration of gliclazide on bile acids & probiotics** 

features, an aromatic ring, a sulphonylurea group and an azabicyclic ring (Figure 2).

Fig. 2. The chemical structure of gliclazide with three main groups: aromatic ring,

In a recent study investigating the applications of bile acids and probiotics in T1D, the bile acid analogue, MKC, was administered i.v. (four groups) and orally (four groups) to healthy, diabetic, probiotic pretreated healthy and probiotic pretreated diabetic rats. The pharmacokinetic parameters of MKC after i.v. administration were found to be similar in all four groups suggesting no significant differences in pharmacokinetic parameters between healthy and diabetic rats irrespective of probiotic pretreatment. Cmax (maximum concentration), AUC (area under the curve) and F (bioavailability) values after oral administration to untreated healthy rats were also found similar to corresponding values in untreated diabetic rats suggesting similar mechanisms of absorption and systemic distribution of MKC. MKC also showed clear evidence of enterohepatic recycling with

sulphonylurea moiety and azabicyclooctyle ring.

diabetes development and progression as well as diabetic complications.

### **7. The interaction between protein transportors, bile acid composition and diabetes developement**

Bile acids effect on T1D development and progression may also be through their effect on protein transporters, since many transporters have their expression and functionality altered in T1D (Al-Salami et al. 2008c). The exact mechanism associating the change in transporters, bile acids composition and diabetes development, is still unknown but there are few assumptions to explain such an interaction. The first assumption is that T1D starts on the first few months of life with a direct insult in the gut, initiating a disturbance in the gut microflora and a consequent disturbed bile flow. This results in an altered bile feedback mechanisms and a change in the expression of protein transporters responsible for bile enterohepatic recirculation. This results in an inflammatory condition that brings about T1D and beta cells destruction. The second assumption is that disturbance in protein transporters expression and functionality, caused by a genetic mutation, produces a disturbance in bile flow. This leads to disturbances in gut microflora initiating inflammation in the gut affecting beta cells and resulting in T1D. The third assumption is that the functionality of the immune system is altered (due to either an insult in the gut or genetic mutation). This alters the composition of gut microflora resulting in initiating of inflammation reaching the beta cells, as a case of mistaken identity. As a consequence of beta cell inflammation, bile acids synthesis and flow are disturbed resulting in exacerbation of the inflammation and worsening of symptoms. In all these assumptions, genetic susceptibility is expected, and contributes further to T1D development and progression. The above assumptions were based on the work of the authors as well as careful evaluation of the literature.

In recent publications, alterations in the functionality of some transporters have been linked to the development of diabetes; however, the exact mechanism remains not fully understood. Bile salts output in diabetic animals was extremely high compared with healthy, and the expression of Mdr2 was also high after STZ treatment (van Waarde et al. 2002). In another study, a mutation in Zinc transporter 8 (ZT8) located in beta cells, is implicated in the dysregulation of insulin transport and release, and an exacerbation of the inflammatory response leading to T1D. In this study, ZT8 was considered as an autoantigen resulting in the stimulation and production of beta cells autoantibodies and T1D development (Rungby 2010). Moreover, streptozotocin (STZ) had different but significant effect on the expression of Na/Cl/glucose cotransporters, and the administration of insulin reduced such an effect (Vidotti et al. 2008). Hyperglyemia itself directly reduced the activity of Mdr1 suggesting a clear association between pre-T1D hyperglycemia and disturbances in protein transporters (Tramonti et al. 2006). In another recent study, the effect of STZ on cation protein transporters was reported, interestingly, at different levels of protein synthesis; transcriptional and posttranscriptional depending on the type of the transporters affected (Grover et al. 2004). However, some studies suggest a diabetic influence is stronger on enzymatic activities than on protein transporters with the enzymatic influence being the cause of exacerbation of inflammation and development of the disease (Py et al. 2002). The impairment of protein transporters functionality, reported in the diabetic animals can take place either by reduced protein expression or reduced action. When glucose protein transporters in the blood brain barrier were studied under chronic hyperglycemia, their concentrations remain constant but functionality and glucose intake were impaired (Mooradian & Morin 1991). However, under acute hyperglycemia induced by STZ, their concentration decreased suggesting different response at different stages of the disease

Bile acids effect on T1D development and progression may also be through their effect on protein transporters, since many transporters have their expression and functionality altered in T1D (Al-Salami et al. 2008c). The exact mechanism associating the change in transporters, bile acids composition and diabetes development, is still unknown but there are few assumptions to explain such an interaction. The first assumption is that T1D starts on the first few months of life with a direct insult in the gut, initiating a disturbance in the gut microflora and a consequent disturbed bile flow. This results in an altered bile feedback mechanisms and a change in the expression of protein transporters responsible for bile enterohepatic recirculation. This results in an inflammatory condition that brings about T1D and beta cells destruction. The second assumption is that disturbance in protein transporters expression and functionality, caused by a genetic mutation, produces a disturbance in bile flow. This leads to disturbances in gut microflora initiating inflammation in the gut affecting beta cells and resulting in T1D. The third assumption is that the functionality of the immune system is altered (due to either an insult in the gut or genetic mutation). This alters the composition of gut microflora resulting in initiating of inflammation reaching the beta cells, as a case of mistaken identity. As a consequence of beta cell inflammation, bile acids synthesis and flow are disturbed resulting in exacerbation of the inflammation and worsening of symptoms. In all these assumptions, genetic susceptibility is expected, and contributes further to T1D development and progression. The above assumptions were

**7. The interaction between protein transportors, bile acid composition and** 

based on the work of the authors as well as careful evaluation of the literature.

In recent publications, alterations in the functionality of some transporters have been linked to the development of diabetes; however, the exact mechanism remains not fully understood. Bile salts output in diabetic animals was extremely high compared with healthy, and the expression of Mdr2 was also high after STZ treatment (van Waarde et al. 2002). In another study, a mutation in Zinc transporter 8 (ZT8) located in beta cells, is implicated in the dysregulation of insulin transport and release, and an exacerbation of the inflammatory response leading to T1D. In this study, ZT8 was considered as an autoantigen resulting in the stimulation and production of beta cells autoantibodies and T1D development (Rungby 2010). Moreover, streptozotocin (STZ) had different but significant effect on the expression of Na/Cl/glucose cotransporters, and the administration of insulin reduced such an effect (Vidotti et al. 2008). Hyperglyemia itself directly reduced the activity of Mdr1 suggesting a clear association between pre-T1D hyperglycemia and disturbances in protein transporters (Tramonti et al. 2006). In another recent study, the effect of STZ on cation protein transporters was reported, interestingly, at different levels of protein synthesis; transcriptional and posttranscriptional depending on the type of the transporters affected (Grover et al. 2004). However, some studies suggest a diabetic influence is stronger on enzymatic activities than on protein transporters with the enzymatic influence being the cause of exacerbation of inflammation and development of the disease (Py et al. 2002). The impairment of protein transporters functionality, reported in the diabetic animals can take place either by reduced protein expression or reduced action. When glucose protein transporters in the blood brain barrier were studied under chronic hyperglycemia, their concentrations remain constant but functionality and glucose intake were impaired (Mooradian & Morin 1991). However, under acute hyperglycemia induced by STZ, their concentration decreased suggesting different response at different stages of the disease

**diabetes developement** 

(Matthaei et al. 1986). Accordingly, protein transporters have shown strong association with diabetes development and progression as well as diabetic complications.
