**1. Introduction**

Diabetes mellitus is a metabolic disorder classified as Type 1 (T1D) or Type 2 (T2D). T1D is an autoimmune disorder characterized by the destruction of the -cells of the pancreas resulting in a partial or complete lack of insulin production and the inability of the body to control glucose homeostasis (Akerblom et al. 2002). T1D is also known as juvenile-onset diabetes because it manifests at a young age (Bruno et al. 2005). As it requires the patient to inject insulin to supplement the partial or complete lack of insulin production by the pancreas, it is also called insulin-dependent diabetes mellitus (IDDM). T2D, formerly known as noninsulin-dependent diabetes mellitus (NIDDM) or adult-onset diabetes, is a metabolic disorder with onset most common in middle age and later life (Campbell 1991). T2D may be controlled by diet and exercise and, unlike T1D, does not always require the use of insulin (Campbell 2004). However, the term "noninsulin-dependent" is a misnomer since many patients require insulin therapy at some time in the course of their disease. T2D is often associated with obesity, hypertension and insulin resistance and can result in the complete destruction of beta-cells of the pancreas leading to T1D (Campbell 2004; Weiss & Caprio 2006). The prevalence of T1D and T2D are on the rise worldwide, which has generated a strong drive towards developing preventative measures as well as cure. Recent data published by the International Diabetes Federation highlighted the severity of diabetes epidemic. Data show that the disease is currently affecting 246 million people worldwide, with 46% of all those affected in the 40-59 age group. Previous figures underestimated the scope of the problem, while even the most pessimistic predictions fell short of the current figure. It is predicted that the total number of people living with diabetes will increase to 380 million within twenty years if no new and substantially more effective drugs are produced (Moore et al. 2003a; Rosenbloom et al. 1999). On 2007, the health costs of diabetes have exceeded 200 billion dollars only in the US. This adds to the cost generated from higher rate of hospitalization, higher mortality rate, and impaired performance of workers with diabetes. This has generated a strong drive towards developing preventative measures as

Potentials and Limitations of Bile Acids and Probiotics in Diabetes Mellitus 367

responsible for the solubilisation of non-polar lipids such as cholesterol and fat-soluble vitamins. Twenty years later it was proposed that bile salts were simultaneously absorbed into the ileal mucosa. Heaton and Morris confirmed that active transport of bile salts occurs

Primary bile acids are synthesized in hepatocytes from endogenous or dietary cholesterol. They are then conjugated to glycine or taurine to form primary conjugated bile acids. In the small intestine, the conjugated bile acids are metabolised by the gut microflora into secondary bile acids before being reabsorbed in the process of enterohepatic recirculation (Ridlon et al. 2006). Approximately 90-95 % of bile acids secreted into the gut is reabsorbed from the intestine back into the circulation via bile acid transporters, while about 400-800 mg/day is excreted from the body in the faeces (Roberts et al. 2002). The bile acid transporters are mainly the sodium-dependent taurocholate cotransporting polypeptide (NTCP), sodium-independent organic anion transporting protein (OATP), the bile salt export pump (BSEP) (Ballatori et al. 2005a; Higgins & Gottesman 1992; Mao & Unadkat 2005), the organic cation transporter polypeptide (OCTP) and the apical sodium-dependent bile salt transporter (ASBT) (Bodo et al. 2003; Zelcer et al. 2003a; Zelcer et al. 2003b; Zollner et al. 2003). Conjugated bile acids are transported by ASBT, whereas unconjugated bile acids are transported by OATP and by passive diffusion. Conjugated bile acids are transported by intracellular transport mechanisms within hepatocytes to the canalicular poles and secreted

Cholic acid is an important precursor for the synthesis of steroids and chenodeoxycholic acid, and of recently has been investigated and applied in biliary calculus (cholelith) therapy. To optimise the stability and minimise toxicity of cholic acid, a more stable semisynthetic analogue MKC has been designed and synthesized. This is done on cholic acid through replacing the hydroxyl group on carbon atom 12 with a ketone group. Generally, the hydroxyl groups on the carbon atoms, C7 and C12 are replaced by hydrogen to enhance stability and reduce side effects. However, despite bile acids being endogenous compounds, manufacturing stable analogues can be challenging. The challenges include: 1. The need for selective protection of 2 hydroxyl groups which is done by acylation. 2. The choice of a suitable reagent to transform the remaining hydroxyl groups as

Although enzymatic dehydroxylation of cholic acid may easily overcome these challenges, chemical reactions involving suitable reagents is still favoured especially for industrial production (Mikov & Fawcett 2006a). 3 hydroxyl groups (C3, C7 and C12) are targeted for acylation. The type of reaction will depend on the type of the bond and its configurational arrangement in the molecule. C3-OH is equatorial thus can be removed through estrification while with C7 and C12 axial groups, oxidation is sufficient. In addition to exploring the

Today it is well known that bile is a complex fluid containing water, electrolytes and other organic molecules including bile salts, cholesterol, phospholipids and bilirubin that flows from the bile duct into the small intestine (Al-Salami et al. 2007). The main endogenous bile acids are primary (cholic and chenodeoxycholic acids) and secondary (deoxycholic and lithocholic acids). Approximately 1 L of bile is secreted by the liver daily. Bile has a pH of 7.8-8.6 and is nearly isotonic with blood. It is secreted from the liver into small ducts that join to form the common hepatic duct. Bile salts are anionic water-soluble products of cholesterol metabolism. Bile salts can form micelles 4-7 nm in diameters which contain fatty

but only in the ileum (Heaton 1971; Lowbeer et al. 1970).

into the canalicular lumen by BSEP (Asamoto et al. 2001; Mita et al. 2006).

potential effect of bile acids, they can also be used as absorption enhancers.

appropriate.

well as cure for the disease and its complications. Diabetes is a disease that incorporates various metabolic disturbances such as impaired glucose haemostasis, blood dyscrasias and hyperlipidemia. Major disturbances also include slower gut movement (gastroparesis) and microfloral overgrowth (especially of fermentation bacteria and yeasts due to the slightly more acidic gut contents) (Al-Salami et al. 2007; Husebye 2005). Improving diabetes complications, reducing prevalence and restoring normal physiological patterns should significantly optimise diabetes treatment and the quality of life for diabetic patients.

Side effects associated with diabetes therapy include hypoglycemia, toxin build up in the gut, and lactic acidosis. These remain major issues and cause of death especially in the presence of compromised liver and kidney functions. So despite strict glycemic control, the disease and its complications remain a growing health concern. Diabetic patients suffer complications due to disturbed physiological and biochemical processes associated with the disease including disturbed bile acids production and microfloral composition (Barbeau et al. 2006; Ogura et al. 1986; Peng & Hagopian 2007; Rozanova et al. 2002; Slivka et al. 1979a; Thomson 1983). Thus the use of bile acids and probiotics in diabetes treatment may improve glycemia as well the ameliorate complications. A major improvement would be the discovery of treatments for diabetes that avoid and even replace the absolute requirement for injected insulin. Recent studies in a rat model of Type 1 diabetes show that a multitherapeutic approach incorporating bile acids and probiotics, as adjunct therapy, exerted better control over glycemia and resulted in ameliorating complications, than when each treatment was administered alone (Al-Salami et al. 2008a; Al-Salami et al. 2008b; Al-Salami et al. 2008e; Al-Salami et al. 2009b). Accordingly, improving diabetes complications, reducing prevalence and restoring normal physiological patterns should significantly optimise diabetes treatment and the quality of life of diabetic patients.

Bile has been used as a therapeutic agent since ancient times. The use of bear gall bladder in treating fever, liver diseases and eye infections has been an ancient phenomenon practiced by many civilizations including the Chinese. Recent studies have showed the therapeutic effects of bear bile in treating gallstones and liver diseases. Bear bile contains substantial amount of ursodeoxycholic acid (UDCA) and chenodeoxycholic acid (CDCA) (Bachrach & Hofmann 1982a; Bachrach & Hofmann 1982b), which recent reports have shown them to also be present in pig bile. Current Chinese medicine uses extracts from pig bile for constipation, jaundice, whooping cough and asthma. Pig bile has also been found to have anti-inflammatory, anticonvulsant and analgesic effects. The applications of bile acids to certain diseases as therapeutic agents have been greatly explored by the ancient Greeks in the sixth century B.C. The ancient Greeks proposed the *Doctrine of Four Humours* or *body fluids* which included yellow bile, black bile, blood and mucus or phlegm. Health is a result of a balanced mixture of the Four Humours (krasis) whereas disease is due to an excess of one of the Four Humours and an imbalance (dyskrasis) of the body fluids (Heaton 1971). Bile therapeutic applications have been explored further by Galen in the second century A.D., and bile was used to facilitate the excretion of stools as a laxative. In 1863 Hoppe-Seyler demonstrated even though bile salts are the major active component in bile, little bile acids is detected in the feces. He proposed bile acids be reabsorbed from the intestine and that bile salts are the major constituents and also proposed continuous recirculation of bile salts, now known as enterohepatic recycling. Heinrich Otto Wieland (1877-1957) won the Nobel Prize in chemistry in 1927 *for his investigations of the constitution of the bile acids and related substances.* In 1940, Roepke and Mason demonstrated that micelle formation was

well as cure for the disease and its complications. Diabetes is a disease that incorporates various metabolic disturbances such as impaired glucose haemostasis, blood dyscrasias and hyperlipidemia. Major disturbances also include slower gut movement (gastroparesis) and microfloral overgrowth (especially of fermentation bacteria and yeasts due to the slightly more acidic gut contents) (Al-Salami et al. 2007; Husebye 2005). Improving diabetes complications, reducing prevalence and restoring normal physiological patterns should

Side effects associated with diabetes therapy include hypoglycemia, toxin build up in the gut, and lactic acidosis. These remain major issues and cause of death especially in the presence of compromised liver and kidney functions. So despite strict glycemic control, the disease and its complications remain a growing health concern. Diabetic patients suffer complications due to disturbed physiological and biochemical processes associated with the disease including disturbed bile acids production and microfloral composition (Barbeau et al. 2006; Ogura et al. 1986; Peng & Hagopian 2007; Rozanova et al. 2002; Slivka et al. 1979a; Thomson 1983). Thus the use of bile acids and probiotics in diabetes treatment may improve glycemia as well the ameliorate complications. A major improvement would be the discovery of treatments for diabetes that avoid and even replace the absolute requirement for injected insulin. Recent studies in a rat model of Type 1 diabetes show that a multitherapeutic approach incorporating bile acids and probiotics, as adjunct therapy, exerted better control over glycemia and resulted in ameliorating complications, than when each treatment was administered alone (Al-Salami et al. 2008a; Al-Salami et al. 2008b; Al-Salami et al. 2008e; Al-Salami et al. 2009b). Accordingly, improving diabetes complications, reducing prevalence and restoring normal physiological patterns should significantly

Bile has been used as a therapeutic agent since ancient times. The use of bear gall bladder in treating fever, liver diseases and eye infections has been an ancient phenomenon practiced by many civilizations including the Chinese. Recent studies have showed the therapeutic effects of bear bile in treating gallstones and liver diseases. Bear bile contains substantial amount of ursodeoxycholic acid (UDCA) and chenodeoxycholic acid (CDCA) (Bachrach & Hofmann 1982a; Bachrach & Hofmann 1982b), which recent reports have shown them to also be present in pig bile. Current Chinese medicine uses extracts from pig bile for constipation, jaundice, whooping cough and asthma. Pig bile has also been found to have anti-inflammatory, anticonvulsant and analgesic effects. The applications of bile acids to certain diseases as therapeutic agents have been greatly explored by the ancient Greeks in the sixth century B.C. The ancient Greeks proposed the *Doctrine of Four Humours* or *body fluids* which included yellow bile, black bile, blood and mucus or phlegm. Health is a result of a balanced mixture of the Four Humours (krasis) whereas disease is due to an excess of one of the Four Humours and an imbalance (dyskrasis) of the body fluids (Heaton 1971). Bile therapeutic applications have been explored further by Galen in the second century A.D., and bile was used to facilitate the excretion of stools as a laxative. In 1863 Hoppe-Seyler demonstrated even though bile salts are the major active component in bile, little bile acids is detected in the feces. He proposed bile acids be reabsorbed from the intestine and that bile salts are the major constituents and also proposed continuous recirculation of bile salts, now known as enterohepatic recycling. Heinrich Otto Wieland (1877-1957) won the Nobel Prize in chemistry in 1927 *for his investigations of the constitution of the bile acids and related substances.* In 1940, Roepke and Mason demonstrated that micelle formation was

significantly optimise diabetes treatment and the quality of life for diabetic patients.

optimise diabetes treatment and the quality of life of diabetic patients.

responsible for the solubilisation of non-polar lipids such as cholesterol and fat-soluble vitamins. Twenty years later it was proposed that bile salts were simultaneously absorbed into the ileal mucosa. Heaton and Morris confirmed that active transport of bile salts occurs but only in the ileum (Heaton 1971; Lowbeer et al. 1970).

Primary bile acids are synthesized in hepatocytes from endogenous or dietary cholesterol. They are then conjugated to glycine or taurine to form primary conjugated bile acids. In the small intestine, the conjugated bile acids are metabolised by the gut microflora into secondary bile acids before being reabsorbed in the process of enterohepatic recirculation (Ridlon et al. 2006). Approximately 90-95 % of bile acids secreted into the gut is reabsorbed from the intestine back into the circulation via bile acid transporters, while about 400-800 mg/day is excreted from the body in the faeces (Roberts et al. 2002). The bile acid transporters are mainly the sodium-dependent taurocholate cotransporting polypeptide (NTCP), sodium-independent organic anion transporting protein (OATP), the bile salt export pump (BSEP) (Ballatori et al. 2005a; Higgins & Gottesman 1992; Mao & Unadkat 2005), the organic cation transporter polypeptide (OCTP) and the apical sodium-dependent bile salt transporter (ASBT) (Bodo et al. 2003; Zelcer et al. 2003a; Zelcer et al. 2003b; Zollner et al. 2003). Conjugated bile acids are transported by ASBT, whereas unconjugated bile acids are transported by OATP and by passive diffusion. Conjugated bile acids are transported by intracellular transport mechanisms within hepatocytes to the canalicular poles and secreted into the canalicular lumen by BSEP (Asamoto et al. 2001; Mita et al. 2006).

Cholic acid is an important precursor for the synthesis of steroids and chenodeoxycholic acid, and of recently has been investigated and applied in biliary calculus (cholelith) therapy. To optimise the stability and minimise toxicity of cholic acid, a more stable semisynthetic analogue MKC has been designed and synthesized. This is done on cholic acid through replacing the hydroxyl group on carbon atom 12 with a ketone group. Generally, the hydroxyl groups on the carbon atoms, C7 and C12 are replaced by hydrogen to enhance stability and reduce side effects. However, despite bile acids being endogenous compounds, manufacturing stable analogues can be challenging. The challenges include:


Although enzymatic dehydroxylation of cholic acid may easily overcome these challenges, chemical reactions involving suitable reagents is still favoured especially for industrial production (Mikov & Fawcett 2006a). 3 hydroxyl groups (C3, C7 and C12) are targeted for acylation. The type of reaction will depend on the type of the bond and its configurational arrangement in the molecule. C3-OH is equatorial thus can be removed through estrification while with C7 and C12 axial groups, oxidation is sufficient. In addition to exploring the potential effect of bile acids, they can also be used as absorption enhancers.

Today it is well known that bile is a complex fluid containing water, electrolytes and other organic molecules including bile salts, cholesterol, phospholipids and bilirubin that flows from the bile duct into the small intestine (Al-Salami et al. 2007). The main endogenous bile acids are primary (cholic and chenodeoxycholic acids) and secondary (deoxycholic and lithocholic acids). Approximately 1 L of bile is secreted by the liver daily. Bile has a pH of 7.8-8.6 and is nearly isotonic with blood. It is secreted from the liver into small ducts that join to form the common hepatic duct. Bile salts are anionic water-soluble products of cholesterol metabolism. Bile salts can form micelles 4-7 nm in diameters which contain fatty

Potentials and Limitations of Bile Acids and Probiotics in Diabetes Mellitus 369

Recent studies have shown that the inflammation which leads to the destruction of β-cells is initiated in the gut (Devendra et al. 2004). It is likely to occur within the first three months of life (Notkins & Lernmark 2001) due to different diabetic-causing xenobiotics (diabetogenics) that include gluten (Akerblom et al. 2002), cow milk protein (Barbeau et al. 2007), viruses such as rubella (Vaarala 2006), and food-toxins such as alloxan, streptozotocin and N-nitroso compounds (Vaarala 2006; Ziegler et al. 2003). Although the pathogenesis of T1D remains unclear, the generally accepted explanation is that T1D is a chronic autoimmune disease triggered in genetically susceptible individuals by a primary insult initiated in the gut (Ghosh et al. 2004). T2D develops in adult life probably due to environmental factors (Moore et al. 2003b) that lead to tissue desensitization to insulin. Continuous stimulation of betacells through hyperglycemia or certain types of antidiabetic drugs such as sulphonylureas can lead to tissue exhaustion and eventual cessation of insulin production due to tissue

The associated-disturbances in the compositions of bile and gut microflora are reported in the literature. However whether the changes in bile and microfloral compositions are caused by diabetes, or diabetes develops as a result of disturbed bile 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

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

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

cardiovascular, tissue necrosis and ulcerations, and metabolic disturbances.

**3. Pathogenesis and risk factors of Type 1 diabetes** 

damage which results in the development of T1D (Fajans 1987).

remains to be determined.

acids, monoglycerides and phospholipids. These micelles solubilise lipids and transport them across biological membranes (Hamada et al. 2006; Leng et al. 2003).

In the past, bile acids were considered to have three basic physiological functions (Kuhajda et al. 2006a; Kuhajda et al. 2006b; Mikov & Fawcett 2006b):


However, recent studies have expanded the role of bile acids to include endocrine signalling to regulate glucose, lipid and their own homeostasis and influence energy expenditure and gut microfloral composition (48, 53, 88).

This chapter aims to explore the changes in gut physiology and metabolic pathways which are associated with diabetes. It also aims to identify current and potential applications of bile acids and probiotics in the prevention and treatment of the disease.

#### **2. Glucose regulation and insulin secretion**

Glucose is a major source of energy with the normal range (normoglycemia) being 3.5-7.8 mmol/l (Cubeddu & Hoffmann 2002). When the body is at absolute rest (the basal state), glucose consumption is equal to its production (Overkamp et al. 1997; Zisser et al. 2007). When glucose is absorbed into the circulation and the body has no immediate need for energy, glucose is stored in the liver and muscles as glycogen (Overkamp et al. 1997). In healthy individuals, glycogen synthesis (glyconeogenesis) in tissues is stimulated by insulin. When the amount of glucose in the blood gets low, glycogen breaks down in the liver to glucose (glycogenolysis). In healthy individuals, feedback processes ensure that glucose levels are under homeostatic control by balancing glyconeogenesis and glycogenolysis. The liver can also convert lactate to glucose via a process known as gluconeogenesis to further supply the required glucose to the blood when levels are low. Glyconeogenesis, glycogenolysis and gluconeogenesis are controlled by anabolic hormones released from the Islets of Langerhans in the pancreas such as glucagon (released from the -cells) and insulin (released from the β-cells). These hormones bind to specific receptors to trigger a chain of reactions that control glucose homeostasis. GLUT-2 (mainly in beta-cells) and GLUT-4 (mainly in skeletal muscles) are the dominant glucose transporters. In general, insulin activates to become fully functional pores that are able to transport glucose molecules into tissues (Rosa et al. 2011; Stuart et al. 2009).

The pancreas produces large quantities of insulin which it stores in intracellular secretary granules (Al-Salami et al. 2007). Upon stimulation from rising levels of glucose, these granules release their insulin into the mesenteric veins (Juhl et al. 2002; Just et al. 2008). Insulin secretion is different in healthy and diabetic individuals. In healthy individuals, there are two phases of insulin secretion; first phase insulin secretion (FPIS) which starts immediately after the initial stimulus of raised glucose levels and second phase insulin secretion (SPIS) which starts shortly after FPIS, and has a shorter duration but greater magnitude. FPIS occurs from -cells of the pancreas as a direct response to high influx of extracellular glucose. In T1D patients, FPIS and SPIS do not exist since there is a complete lack of insulin production while, in T2D patients, FPIS is impaired and further exposure to glucose results in a reduction in insulin secretion in SPIS due to the desensitization of -cells to glucose.
