**9. The effect of gut microflora and diet on inflammation**

There is a great conclusion regarding the importance of gut microflora, made by Sir Henry Shaw (1818–1885): 'I have finally come to the conclusion that a good reliable set of bowels is worth more to a man than any quantity of brains'.

Many autoimmune and inflammatory diseases have shown positive response to probiotic and prebiotic treatments (Sherman et al. 2009; Tlaskalova-Hogenova et al. 2011). These diseases include acute gastroenteritis, antibiotic-associated diarrhoea and colitis, inflammatory bowel disease, type 1 diabetes, irritable bowel syndrome and necrotizing enterocolitis. The composition of the intestinal microflora may also affect mammalian

Potentials and Limitations of Bile Acids and Probiotics in Diabetes Mellitus 379

changes with diet and also as we age (Rebole et al. 2010; Respondek et al. 2008; Yen et al. 2011). In one study, a high fat diet was associated with higher endotoxaemia and a lowering of bifidobacterium species in mice cecum (Cani et al. 2008). In a follow up study, the administration of prebiotics, in particular, oligofructose, to mice given high fat diet, restored the reduced quantity of bifidobacterium. This also resulted in reducing metabolic endotoxaemia, the inflammatory tone and slowing the development of diabetes. In this study and compared with control mice on chow diet, high fat diet significantly reduced intestinal Gram negative and Gram positive gut bacteria, increased endotoxaemia and diabetes-associated inflammation. However, when diabetic mice on high fat diet were given oligofructose, metabolic normalization took place including the quantity of gut bifidobacteria. In these mice, multiple correlation analyses showed that endotoxaemia negatively correlated with bifidobacteria quantity. By the same token, bifidobacterium quantity significantly and positively correlated with improved glucose tolerance, glucoseinduced insulin secretion and normalised inflammatory tone (decreased endotoxaemia and plasma and adipose tissue proinflammatory cytokines) (Cani et al. 2007). In general, the level of microfloral diversity and gut bifidobacteria in human, relate to health status and

Compromised gut movement associated with diabetes can result in substantial bacterial and yeast overgrowth which is postulated to disturb bile acids composition and exacerbate the diabetes-associated inflammation (Cani et al. 2009; Fox et al. 2010). Diabetes inflammation and bile acids disturbances can cause chemical unbalance that has been linked to poor tissue sensitivity to insulin (Maki et al. 1995), rise in the levels of reactive radicals in the blood (Jain et al. 2002), poor enterohepatic recirculation and negatively affecting liver detoxification and performance (Oktar et al. 2001; Quraishy et al. 1996). Accordingly, future diabetes therapy should not only focus on rectifying glucose imbalance but also in targeting the disturbances in bile acids composition and the inflammation cascade initiated in the gut. This can be achieved through normalizing the composition of bile acids and microflora, gut immuneresponse and microflora-epithelial interactions towards maintaining normal biochemical reactions and healthy body physiology. Physiological features of human development including the innate and adaptive immunity, immune tolerance, bioavailability of nutrients, and intestinal barrier functions, are directly related to the composition and functionality of the human microflora. This includes the percentages of what is currently known as good and bad gut microflora. Good microflora includes two main species, Lactobacillius and Bifidobacteria. Microflora modifications may take place due to antibiotics consumption, prebiotic and probiotics administration and the use of drugs which affect gastric motility resulting in changes in gastric pH and gut-emptying rate. These modifications have been shown to be significantly profound in diabetic subjects resulting in the reduction of the percentage of good bacteria, the increase of the percentage of bad bacteria and yeasts and the consequent increase in the percentage of toxic bile salts such as lithocholic acid. This can also contribute to the higher incidence of gall stones and liver necrosis reported in diabetic patients. Accordingly, probiotics can introduce missing microbial components with known beneficial functions for the human host, while prebiotics can enhance the proliferation of beneficial microbes or probiotics, resulting in sustainable changes in the human microflora. Symbiotic relationship between probiotics and prebiotic administration is expected to exert a synergistic effect and in the right dose, may normalize and even reverse dysbiosis-

both decrease with age (Hopkins & Macfarlane 2002).

associated complications.

physiology outside the gastrointestinal tract. Recent studies have shown significant changes in gut microfloral and bile acid compositions in T1D (Jaakkola et al. 2003; Siow et al. 1991; Slivka et al. 1979b; Uchida et al. 1979; Uchida et al. 1985). Thus, it is clear that our symbiotic microflora award many metabolic capabilities that our mammalian genomes lack (Zaneveld et al. 2008), and so therapeutics that target microfloral modulation may prove rewarding. When the newborn baby leaves the germ free uterus, she/he enters a highly contaminated extra-uterus environment. This requires the activation of her/his immune system to prevent infection. Over the period of the first year, the newborn's intestinal microflora develops and its composition becomes her/his gut microfloral fingerprint! Gut microflora has been shown to play a major rule in controlling the inflammatory response of the host immune system through direct and indirect bacteria-bacteria and bacteria-host interactions. These interactions include physical and metabolic functions of the gut microfloral bacteria, which protect the intestinal tract from foreign pathogenic bacteria, eliminate the presence of unwanted bacteria through producing bacteriocins and other chemicals, and inform the gut epithelium and the host immune system about whether a local inflammatory response is needed (Shi & Walker 2004; Walker 2008b). Gut microflora can control the host immune system through four main actions. The induction of IgA secretion to protect against infection, triggers localized inflammatory responses, neutralizing T-helper (Th) cell response and also contributing to the induction or inhibition of generalized mucosal immune responses. Recent studies have shown that in autoimmune diseases and gut inflammation disorders, there is a significant disturbances in the ratios of Th cells such as the increase in the Th-2/Th-1 ratio associated with inflammatory bowel diseases, which has been linked to exacerbation of the gut inflammation and the development of the disease. In recent studies, gut-associated dendritic cells in the lamina propria can extend their appendices reaching the gut mucosa and using their Toll-like receptors (TLR) 2 and 4, to sample bacterial metabolites (Rescigno et al. 2001; von & Nepom 2009a). This may result in dendritic cells releasing certain cytokines that stimulate the activation of naive Th-0 into active Th- cells such as 1, 2 and 3/1 (von & Nepom 2009b; Walker 2008b). Interestingly, some microfloral bacteria can actually cross enterocytic microfolds and interact with antigen presenting immune cells in mesenteric lymph nodes to activate naive plasma cells into IgA-producing B cells (Macpherson & Uhr 2004). IgA coats the intestinal mucosa and control further bacterial penetration thus protecting the host from potential pathogenic bacteria. Even more interestingly, gut microflora bacteria have shown ability to not only initiate an inflammatory response but also to control and inhibit such a response. Some microfloral bacteria or their metabolites can interact with the intracellular receptor TLR-9, to which the bacteria activates T cells through the production of potent anti-inflammatory cytokines such as IL-10 (Rachmilewitz et al. 2004). Microfloral bacteria can also produce small molecules that can enter intestinal epithelial cells to inhibit activation of nuclear factor kappa-light-chainenhancer of activated beta-cells (NFkB) (Neish et al. 2000). Moreover, prolonged exposure to bacterial endotoxins, in particular, LPS (which interacts with TLR 2 and 4) can activate intracellular anti-inflammatory associated proteins that result in an overall antiinflammatory effect (Otte & Podolsky 2004). Such gut bacterial-host interactions are critical in maintaining a balanced and effective immune response to various infections while maintaining control over prolonged or chronic inflammation and reducing the overstimulation of the host immune system.

Recent evidence suggests that a particular gut microfloral community may favour occurrence of the metabolic diseases. It is well know that the composition of gut microflora

physiology outside the gastrointestinal tract. Recent studies have shown significant changes in gut microfloral and bile acid compositions in T1D (Jaakkola et al. 2003; Siow et al. 1991; Slivka et al. 1979b; Uchida et al. 1979; Uchida et al. 1985). Thus, it is clear that our symbiotic microflora award many metabolic capabilities that our mammalian genomes lack (Zaneveld et al. 2008), and so therapeutics that target microfloral modulation may prove rewarding. When the newborn baby leaves the germ free uterus, she/he enters a highly contaminated extra-uterus environment. This requires the activation of her/his immune system to prevent infection. Over the period of the first year, the newborn's intestinal microflora develops and its composition becomes her/his gut microfloral fingerprint! Gut microflora has been shown to play a major rule in controlling the inflammatory response of the host immune system through direct and indirect bacteria-bacteria and bacteria-host interactions. These interactions include physical and metabolic functions of the gut microfloral bacteria, which protect the intestinal tract from foreign pathogenic bacteria, eliminate the presence of unwanted bacteria through producing bacteriocins and other chemicals, and inform the gut epithelium and the host immune system about whether a local inflammatory response is needed (Shi & Walker 2004; Walker 2008b). Gut microflora can control the host immune system through four main actions. The induction of IgA secretion to protect against infection, triggers localized inflammatory responses, neutralizing T-helper (Th) cell response and also contributing to the induction or inhibition of generalized mucosal immune responses. Recent studies have shown that in autoimmune diseases and gut inflammation disorders, there is a significant disturbances in the ratios of Th cells such as the increase in the Th-2/Th-1 ratio associated with inflammatory bowel diseases, which has been linked to exacerbation of the gut inflammation and the development of the disease. In recent studies, gut-associated dendritic cells in the lamina propria can extend their appendices reaching the gut mucosa and using their Toll-like receptors (TLR) 2 and 4, to sample bacterial metabolites (Rescigno et al. 2001; von & Nepom 2009a). This may result in dendritic cells releasing certain cytokines that stimulate the activation of naive Th-0 into active Th- cells such as 1, 2 and 3/1 (von & Nepom 2009b; Walker 2008b). Interestingly, some microfloral bacteria can actually cross enterocytic microfolds and interact with antigen presenting immune cells in mesenteric lymph nodes to activate naive plasma cells into IgA-producing B cells (Macpherson & Uhr 2004). IgA coats the intestinal mucosa and control further bacterial penetration thus protecting the host from potential pathogenic bacteria. Even more interestingly, gut microflora bacteria have shown ability to not only initiate an inflammatory response but also to control and inhibit such a response. Some microfloral bacteria or their metabolites can interact with the intracellular receptor TLR-9, to which the bacteria activates T cells through the production of potent anti-inflammatory cytokines such as IL-10 (Rachmilewitz et al. 2004). Microfloral bacteria can also produce small molecules that can enter intestinal epithelial cells to inhibit activation of nuclear factor kappa-light-chainenhancer of activated beta-cells (NFkB) (Neish et al. 2000). Moreover, prolonged exposure to bacterial endotoxins, in particular, LPS (which interacts with TLR 2 and 4) can activate intracellular anti-inflammatory associated proteins that result in an overall antiinflammatory effect (Otte & Podolsky 2004). Such gut bacterial-host interactions are critical in maintaining a balanced and effective immune response to various infections while maintaining control over prolonged or chronic inflammation and reducing the

overstimulation of the host immune system.

Recent evidence suggests that a particular gut microfloral community may favour occurrence of the metabolic diseases. It is well know that the composition of gut microflora changes with diet and also as we age (Rebole et al. 2010; Respondek et al. 2008; Yen et al. 2011). In one study, a high fat diet was associated with higher endotoxaemia and a lowering of bifidobacterium species in mice cecum (Cani et al. 2008). In a follow up study, the administration of prebiotics, in particular, oligofructose, to mice given high fat diet, restored the reduced quantity of bifidobacterium. This also resulted in reducing metabolic endotoxaemia, the inflammatory tone and slowing the development of diabetes. In this study and compared with control mice on chow diet, high fat diet significantly reduced intestinal Gram negative and Gram positive gut bacteria, increased endotoxaemia and diabetes-associated inflammation. However, when diabetic mice on high fat diet were given oligofructose, metabolic normalization took place including the quantity of gut bifidobacteria. In these mice, multiple correlation analyses showed that endotoxaemia negatively correlated with bifidobacteria quantity. By the same token, bifidobacterium quantity significantly and positively correlated with improved glucose tolerance, glucoseinduced insulin secretion and normalised inflammatory tone (decreased endotoxaemia and plasma and adipose tissue proinflammatory cytokines) (Cani et al. 2007). In general, the level of microfloral diversity and gut bifidobacteria in human, relate to health status and both decrease with age (Hopkins & Macfarlane 2002).

Compromised gut movement associated with diabetes can result in substantial bacterial and yeast overgrowth which is postulated to disturb bile acids composition and exacerbate the diabetes-associated inflammation (Cani et al. 2009; Fox et al. 2010). Diabetes inflammation and bile acids disturbances can cause chemical unbalance that has been linked to poor tissue sensitivity to insulin (Maki et al. 1995), rise in the levels of reactive radicals in the blood (Jain et al. 2002), poor enterohepatic recirculation and negatively affecting liver detoxification and performance (Oktar et al. 2001; Quraishy et al. 1996). Accordingly, future diabetes therapy should not only focus on rectifying glucose imbalance but also in targeting the disturbances in bile acids composition and the inflammation cascade initiated in the gut. This can be achieved through normalizing the composition of bile acids and microflora, gut immuneresponse and microflora-epithelial interactions towards maintaining normal biochemical reactions and healthy body physiology. Physiological features of human development including the innate and adaptive immunity, immune tolerance, bioavailability of nutrients, and intestinal barrier functions, are directly related to the composition and functionality of the human microflora. This includes the percentages of what is currently known as good and bad gut microflora. Good microflora includes two main species, Lactobacillius and Bifidobacteria. Microflora modifications may take place due to antibiotics consumption, prebiotic and probiotics administration and the use of drugs which affect gastric motility resulting in changes in gastric pH and gut-emptying rate. These modifications have been shown to be significantly profound in diabetic subjects resulting in the reduction of the percentage of good bacteria, the increase of the percentage of bad bacteria and yeasts and the consequent increase in the percentage of toxic bile salts such as lithocholic acid. This can also contribute to the higher incidence of gall stones and liver necrosis reported in diabetic patients. Accordingly, probiotics can introduce missing microbial components with known beneficial functions for the human host, while prebiotics can enhance the proliferation of beneficial microbes or probiotics, resulting in sustainable changes in the human microflora. Symbiotic relationship between probiotics and prebiotic administration is expected to exert a synergistic effect and in the right dose, may normalize and even reverse dysbiosisassociated complications.

Potentials and Limitations of Bile Acids and Probiotics in Diabetes Mellitus 381

the gut (bile and pH tolerability) and long term safety. Lactobacillus rhamnosus, Lactobacillus acidophilus and Bifidobacterium lactis show good bile and pH tolerability under normal conditions of pH (1.5-8) and bile acid concentration (0.8 – 3 %) (Table 1), in addition to long term safety (Franz & Bode 1973; Hedenborg & Norman 1984; Hedenborg &

Bile acids and their derivatives can act as absorption enhancers where they are capable of promoting mucosal and systemic drug absorption. Bile acids and their derivatives can increase drug bioavailability, allowing therapeutic doses to be administered by several routes. Bile acids as therapeutic agents have the potential to produce beneficial effects in improving primary biliary cirrhosis and primary sclerosing cholangitis. Bile acids can also control endocrine signalling and enzymatic activities in various disorders. This includes inflammatory diseases (such as diabetes) and cholestatic liver disease in cystic fibrosis. Permeation of a drug through a biological membranes by passive diffusion is influenced by the drug's solubility and molecular weight, the thickness of both, the mucous and the cytoplasmic membrane, while drug diffusibility is influenced by permeability, surface area and the concentration gradient (Higgins & Gottesman 1992; Maki et al. 2003; Mao &

Bile salts (conjugated bile acids) are known to increase the permeation of many drugs. They increase the permeability of the mucosal membrane by breaking down mucous and disrupting cells, thus widening the tight junctions between these cells. This enhances penetration of drugs via the paracellular route. Bile salts can also improve transcellular absorption by increasing drug solubility and dissolution rate. Bile salts can form micelles which increase the permeability of the mucosal membrane by overcoming resistance at the aqueous diffusion layer. They also enhance drug delivery by interacting with membrane

lipids and proteins that affect membrane fluidity and the rate of drug trafficking.

Fig. 3. Protein transporters in the mucosal and serosal sides of the gut enterocytes.

**11. Bile acids as absorption enhancers in Type 1 diabetes therapy** 

Norman 1985).

Unadkat 2005; Neubert et al. 1987).
