and the Cellular Energy Cycles — A Novel Perspective on Type 2 Diabetes Treatment

None of the currently available therapeutic interventions for type 2 diabetes mellitus address the intracellular metabolism of glucose through the main energy pathways of the cell. Thiamine (vitamin B1) is a water-soluble vitamin and essential normal dietary component. When modified in the body to the pyrophosphate derivative, it acts as a coenzyme for pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase & transketolase which are required for the utilization and consumption of glycolytic and hexose monophosphate pathway intermediates & form an integral part of intracellular and glucose metabolism. Thiamine deficiency decreases the activities of these enzymes, leading to imbalances in the metabolic pathways. The effects of these imbalances are more pronounced in diabetes mellitus where renal dysfunction produces mild thiamine deficiency.This indepth review presents a novel perspective on, the cellular energy cycles ,thiamine dependant enzymes,pharmacotherapeutics of type 2 diabetes especially thiamine and their impact on type 2 diabetes treatment.Thiamine, with its well established safety record, easy accessibility and affordability could be an invaluable adjunct for our type 2 diabetic population and help to improve the quality of their lives by giving them some respite from the complications of type 2 diabetes and perhaps reduce the need of more expensive oral hypoglycaemic agents required by them. to noticeable and an inverse linkage between thiamine therapy and vWF was apparent in this group as compared to placebo, suggested noticeable benefit with reduction in the risk factors of type 2 diabetes. Significant changes in other serum and urinary biomarkers profile were also observed in type 2 diabetics following thiamine therapy in a simultaneously carried out proteomic study. Three thiamine dependant enzymes PDE3, PDE1β, AKGDHE1 and Transketolase were determined to be dysfunctional at baseline in type 2 microalbuminuric diabetic patients in comparison to normal healthy controls, and improved in both activity and gene expression with high dose thiamine therapy While importantly no hepatic or renal adverse effects were encountered prior, during therapy or as a residual effect, post washout thus fortifying the previously established human safety track record of thiamine. [ 200 ]. We hope that these findings would contribute to knowledge regarding the role of thiamine therapy at 300mg/day dosage on biochemical profile and molecular aspects of those vital thiamine dependant enzymes and help in providing improved, safe and more effective treatment for type 2 diabetic patients with incipient nephropathy, dyslipidemia with expected decrease risk of heart disease and kidney failure.


Introduction
Cellular survival is dependant upon the energy pathways ingrained within them. Their comprehension is imperative in understanding the role of their component enzymes in type 2 diabetes treatment and the inextricable linkage of a few of them to thiamine. The Glycolytic pathway is an ancient metabolic, cytosolic pathway that converts glucose into pyruvate under anaerobic conditions and further into lactate or ethanol. The free energy released from this forms high energy compounds ATP and NADH.Under aerobic conditions CO2 and substantially more ATP is produced [1]. The pathway of glycolysis comprises of 2 clear divisions ( Fig   1). After glycolysis, further aerobic processing of glucose is conducted through the Kreb Cycle, synonymous with tricarboxylic acid or citric acid cycle (Fig 2). Intracellularly the mitochondria serve as site of citric acid cycle and oxidative phosphorylation activities.
The overall chemical reaction of the tricarboxylic acid cycle is: Acetyl-CoA + 3NAD + FAD + GDP + P + 2H O 2CO + 3NADH + FADH + GTP + 2H + HSCoA (1) s high energy compounds ATP and NADH.Under aerobic conditions CO2 and substantially more ATP is produc he pathway of glycolysis comprises of 2 clear divisions (Fig1). After glycolysis,where glucose is broken down int vate only releasing fractional amounts of ATP, further aerobic processing of glucose is conducted through the Kr e, synonymous with tricarboxylic acid or citric acid cycle (Fig 2). Intracellularly the mitochondria serve as site of acid cycle and oxidative phosphorylation activities.   Treatment of Type 2 Diabetes 24 unstructured linkers [3] (Fig.3). Similarly, each E3BP subunit consists of a single LBD (referred to as L3), the E3-binding domain (E3BD) and the noncatalytic inner core domain. It is presumed that the lipoyl bearing domains LBDs (L1, L2, and L3) and 60 subunits of the transacetylase seem to form a free circulation of lipoyl groups among which the acetyl groups are freely exchanged [18] and shuttle between the active sites of the three catalytic components of the PDC during the oxidative decarboxylation cycle [19]. Unspecified copies of each PDC regulatory enzyme pyruvate dehydrogenase kinases and pyruvate dehydrogenase phosphatases are also strung non-covalently to the core by the LBD2 [5,20].
The active site synchronization over a distance of 20 Angstroms via proton wire through an acidic tunnel in the protein, keeps the active sites in an alternating activation state [22 ]. Phosphorylation of the heterotetrameric (α2 β2) E1p component is essential for the inactivation of the human PDC which occurs at 3 serine residues of the alpha subunit. Two of these sites are located in the conserved phosphorylation loop A [6] which forms one wall of the active site channel and helps to anchor ThDP to its active site.Site 3 is in the phosphorylation loop B which provides coordination to magnesium is chelated by the ThDP potassium. Phosphorylation of any of the 3 sites inactivates E1p and drastically reduces the affinity for pyruvate [24]. Disordered loops of E1p arise from phosphorylation and result in downregulation of the PDC activity. Binding of the cofactor ThDP induces ordering of both the loops which then can mediate decarboxylation and reductive acetylation of the pyruvate. Phosphorylation of PDC is crucial in regulating carbohydrate and lipid metabolism [14,25]. Starvation and diabetes increase phosphorylation that inactivates PDC, leading to impaired glucose oxidation [26,27]. On the other hand prevention of PDC phosphorylation by specific PDK inhibitor, dichloracetate increases reactive oxygen species levels in the mitochondria leading to cellular apoptosis and the inhibition of tumour growth [28,29]. Therefore the regulation of PDC flux by reversible phosphorylation is a potential target for obesity and cancer [30,31].Finally the expression of PDK2and PDK4 is down regulated by insulin in the long term [32,33]. In the animal model, downregulation of skeletal muscle pyruvate dehydrogenase in the rat model before and after the onset of diabetes mellitus has been observed [34]. Dephosphorylation/activation of the PDC is ascribed to two Mg and Ca dependant genetically and biochemically distinct isoforms of pyruvate dehydrogenase phosphatase PDP heterodimeric (PDP1&PDP2), which are important regulators of PDC activity. PDP1 has both a catalytic (PDPc) subunit bound to the inner mitochondrial membrane and a regulatory (PDPr) subunit [35]. Both PDP1 components are targeted by insulin which enhances PDPc activity and lessens PDPr negative control resulting in enhanced overall PDP1 efficiency.These effects are at the core of insulin signaling of PDH [36]. PDP2, recently discovered in rat tissues consists of a catalytic subunit insensitive to Ca, 10 fold less sensitive to Mg than PDP c is also considered a target in insulin signaling [37,38]. In humans too, down regulation of PDP in obese subjects is a malfunction that signals insulin resistance [39].

Diseases produced by defective PDC
As the PDC has prime significance in intermediary metabolism, mutations in the genes encoding for PDCsubunits produce severe clinical phenotypes [40]. Congenital defects in E1p in the X linked gene lead to lactic acidemias, encephalopathies, neuronal dysfunction in infancy [40]. Mutations in the E2, E3BP cause primary biliary cirrhosis leading to liver failure [41,42], autoimmune hepatitis [43] and neurodegenerative conditions such as Alzheimer's disease. Combined enzyme deficiencies of α-ketoacid dehydrogenase complexes pyruvate dehydrogenase complex, BCKDC and ketoglutarate dehydrogenase complexes have been observed due to genetic changes in human E3 [44] resulting in lactic acidemias and maple syrup urine disease [45][46][47]. Other anamolies of the PDC include autoantibodies leading to paediatric biliary cirrhosis [47].Additionally, the aberrant down-regulation of pyruvate dehydrogenase complex activity by reversible phosphorylation has been shown to be contributory to hyperglycemic states observed in type-2 diabetes [25], increasing the chances of pyruvate dehydrogenase complex as a therapeutic target for a 150 million people affliction i.e. diabetes). Failure of functioning of the pyruvate dehydrogenase complex and specially of its E1p subunit due to lack of thiamine vitamin B1 would therefore inevitably lead to poor handling of glucose and its substrates and could manifest as deleterious effects in type 2 diabetics. The human 2 ketoglutarate dehydrogenase complex while extensively studied has not yet been reconstructed in vitro and reliance on other mammal models persists [5,48] (Fig 4).

Structure of alphaketoglutarate dehydrogenase complex
This 4 to 10 mega Dalton supramolecular complex is organized around a polyhedral form of a cubic core of 24/60 lipoate bearing dihydrolipoyl succinyltransferase E2 subunits (8 trimers) arranged with octahedral (432) symmetry [5] associated with non covalently attached multiple copies of dihydrolipoamide E1k and dihydrolipoamide E3K individually held via its E1/E3 binding domains which serve as scaffolds for the E2 core. There is also biochemical evidence of E3 binding to the aminoacid terminal region of E1 terminal allowing for separation of a stable E1-E3 submolecular complex from the E2 core [49].Also attached are regulatory kinase and phosphatase units [50]. Further lipoyl bearing domains LBDs of the E2 core are attached serving as swing arms impart substrate chanelling by sequentially visiting the different active sites in each of the three E1, E2 and E3 catalytic components [51] to transfer acyl groups to the active site of E2 leading to oxidative decarboxylation of the alpha ketoacids [51] [55,56]. It contains a thiamine diphosphate cofactor and catalyzes thiamine diphosphate dependant decarboxylation of 2 oxoglutarate and subsequent reductive acylation of the oxidized lipoyl moiety LBD (lip-LBD-S2) which is covalently bound to the E2 component dihydrolipoamide succinyl transferase [5]. Thiamine diphosphate is tightly but not covalently bound to the 2oxoglutarate dehydrogenase component [57] ThDP remains an essential cofactor and alphaketoglutarate dehydrogenase complex in the form of homo dimers alpha2, homo tetramers alpha 4 or heterotetramers alpha 2 beta 2 contain ThDP binding pockets that constitute two or four active sites for this enzyme which operate independently without an obligatory alternating mechanism in the E1b component [58] and overall activity is abolished at 50% phosphorylation (1 of 2 sites) within each active channel similar to PDC [59].
2. Dihydrolipoamide S-succinyltransferase E2k core has 24 /60 copies containing lipoyl active site as well as active sites for E1 and E3 subunits based on similar mammalian PDC structural studies and molecular wt of 64.5 KDa [5]. It is encoded in gene DLST located on chromosome 14 q24.2-q24.3 with a length of 21815 base pairs [60]. This inner core plays an essential role in mediating the E1 catalyzed decarboxylation of 2 oxoglutarate and reductive acylation of the lipoyl moiety and E3 catalyzed reoxidation of the dihydrolipoyl moiety.
3. Located in the mitochondrial lumen, Dihydrolipoamide dehydrogenase E3k or E3component a flavoprotein (dimer) has 12 copies, a sequence length of 28796 aminoacids and is 54.15kDa in weight. It is encoded in the DLD gene localized to 7q31-q32 [61], its function is to catalyze the transfer of electrons from dihydrolipoamide to NAD+and bears close  [62] KGDHC catalyzes a vital step in the Krebs cycle, which is also a step in the metabolism of the potentially excitotoxic neurotransmitter glutamate. It allows amino acids to enter the citric acid cycle and produce energy; this is a reversible reaction in which glucose which enters the cycle can leave it to make amino acids thus linking amino acid pathways to the citric acid cycle. It also participates in lysine degredation and tryptophan metabolism. Alpha-KGDH is vital for maintaining NADH supply to the respiratory chain and is limited only when alpha-KGDH is also inhibited by ROS. In addition being a key target, it is also able to generate ROS during its catalytic function which is regulated by the NADH/NAD+ratio [63]. Its cofactors are TPP bound to E1, lipoic acid covalently bound to lysine on E2 which accepts the hydroxyethyl carbanion from TPP as an acetyl group, coenzyme A which is substrate for E2 and accepts the acetyl group from it, FAD bound to the E3 subunit reduced by lipoamide and NAD which is substrate for E3 and reduced by FADH2 [64]. Basic short term regulation of KGDHC is through adenosine diphosphate ADP, P (i) and Ca2+; these positive effectors increase manifold the affinity of ketoglutarate dehydrogenase complex to alpha-ketoglutarate. While KGDHC inhibitors are NADH, adenosine triphosphate, succinyl-CoA, and thioredoxin protects KGDHC from self-inactivation during catalysis [65]. Alpha-KGDH is also sensitive to oxidative stress and a number of metabolites modify the activity of KGDHC, including inactivation by 4-hydroxynonenal. In the human brain, comparison of KGDHC activity to other enzymes of energy metabolism like aconitase, phospho-fructokinase and the electron transport complexes shows it to be lower than all of them. Therefore impairment of KGDHC function is likely to disturb brain energy metabolism and result in brain disease [66]. In Wernickes encephalopathy there is AKGDH and thiamine deficiency associated with increased oxidative stress markers, lipid peroxidation resulting in neuronal cell death in pons, thalamus and cerebellum [67,69]. In general, the clinical manifestations of KGDHC deficiency relate to the severity of the deficiency. A range of disorders have been recognized: varying from psychomotor retardation in childhood, to intermittent neuropsychiatric disease with ataxia and other motor disabilities, such as Friedreich's and other spinocerebellar ataxias [70], as well as neural diseases where mental deficits are also visible such as Parkinson's disease, and Alzheimer's disease (AD) [70]In Parkinsons Disease which has been deeply investigated, KGDHC Activity is reduced, coupled to elevated levels of monoamine oxidase B [71] and cytosolic accumulation of cytochrome c which inturn activates other pathways, including cell death cascades and enzyme inhibition which alters Ca2+homeostasis [72] The KGDHC enzyme is further a target for ubiquitinationdependent degradation in mitochondria by binding of Siah2, the RING finger ubiquitinprotein isopeptide ligase 2, encoded by gene siah2 [73]. Diabetes mellitus, thiamine dependent megaloblastic anaemia and sesorineural deafness associated with deficient alpha ketoglutarate dehydrogenase activity have also been reported [74]. There exist 2 wings,oxidative and reductive of the pentose phosphate pathway (Fig 5). The oxidation steps, utilizing glucose-6phosphate (G6P) as the substrate, occur at the beginning of the pathway and generate 2 moles of NADPH. The reactions catalyzed by glucose-6-phosphate dehydrogenase (G6PD) and 6phosphogluconate dehydrogenase are essential for the conversion of hexoses to pentoses [75].

Functions of the Pentose Phosphate Pathway in Normal and Diseased Conditions:
The Pentose phosphate pathway is primarily energy forming, and non mitochondrial with only a cytoplasmic enzymatic presence entrusted to utilizing 6 carbon sugars, and producing in turn 5 carbon sugars for the synthesis of neucleotides, nucleic acids and reducing equivalents in the form of NADPH. The pentose phosphate pathway is a metabolic redox estimator and regulates transcription during the anti-oxidant response, as a shift from primary carbon metabolism, is fastest in oxidative stress 77 . NADPH cofactor serves as reducing equivalent in the endoplasmic reticulum lumen for fatty acid and steroid biosynthesis in hepatic and, adipose tissue, adrenal cortex 78 . High levels of PPP enzymes are in neutrophils and macrophages as they utilize NADPH to produce ROS to destroy engulfed microbes in a process termed as respiratory burst 79 . G6PD deficiency effects red blood cell viability dependent on PPP generated NADPH , a glutathione reducer, the absence of which results in hemolysis seen with certain drugs and diseases like malaria which cause oxidative stresss 80 . Cancer cells are known to access successfully the glucose flux in the pentose phosphate pathway supporting NADPH and reactive oxygen species production and glutathione reduction 81 responding to both incremental and decremental reactive oxygen species 82 . Electron leakage from the mitochondrial electron transport remains essential (through the action of ribonucleotide reductase) in generating deoxyribonucleiotides from nucleotides as well producing ROS in collusion with oncogenes 83 and molecular oxygen 84 promoting genetic damage in normal cells and therapy resistance in cancerous cells 85 .Malignant cells also use reduced glutathione 81 or NADPH to combat oxidative stress and to support the oxidation of fatty acids in detached cells 86 . Transketolase is the premier cytosolic enzyme of the reductive pentose phosphate pathway. Its 3 genes TKT, Transketolase like TKTLI and Transketolase like TKTL2 encode for proteins with transketolase activity.All of them participate in the reductive pentose pathway reactions catalyzing transfer of a 2 carbon fragment from a ketose donor to an aldose (acceptor substrate) 87 . The non-oxidative reactions of the pentose phosphate pathway are mainly functioning to produce ribose 5 phosphate, and equally significantly to convert dietary 5 carbon sugars into both 6 (fructose-6-phosphate) and 3 (glyceraldehyde-3-phosphate) carbon sugars which can then be utilized by the pathways of glycolysis [76].

Functions of the pentose phosphate pathway in normal and diseased conditions
The Pentose phosphate pathway (PPP) is primarily energy forming, and non mitochondrial with only a cytoplasmic enzymatic presence entrusted to utilizing 6 carbon sugars, and producing in turn 5 carbon sugars for the synthesis of neucleotides, nucleic acids and reducing equivalents in the form of NADPH. The pentose phosphate pathway is a metabolic redox estimator and regulates transcription during the anti-oxidant response, as a shift from primary carbon metabolism, is fastest in oxidative stress [77]. NADPH cofactor serves as reducing equivalent in the endoplasmic reticulum lumen for fatty acid and steroid biosynthesis in hepatic and, adipose tissue, adrenal cortex [78]. High levels of PPP enzymes are in neutrophils and macrophages as they utilize NADPH to produce ROS to destroy engulfed microbes in a process termed as respiratory burst [79]. G6PD deficiency effects red blood cell viability dependent on PPP generated NADPH, a glutathione reducer, the absence of which results in hemolysis seen with certain drugs and diseases like malaria which cause oxidative stresss [80]. Cancer cells are known to access successfully the glucose flux in the pentose phosphate pathway supporting NADPH and reactive oxygen species production and glutathione reduction [81] responding to both incremental and decremental reactive oxygen species [82]. Electron leakage from the mitochondrial electron transport remains essential (through the action of ribonucleotide reductase) in generating deoxyribonucleiotides from nucleotides as well producing ROS in collusion with oncogenes [83] and molecular oxygen [84] promoting genetic damage in normal cells and therapy resistance in cancerous cells [85].Malignant cells also use reduced glutathione [81] or NADPH to combat oxidative stress and to support the oxidation of fatty acids in detached cells [86]. Transketolase is the premier cytosolic enzyme of the reductive pentose phosphate pathway. Its 3 genes TKT, Transketolase like TKTLI and Transketolase like TKTL2 encode for proteins with transketolase activity.All of them participate in the reductive pentose pathway reactions catalyzing transfer of a 2 carbon fragment from a ketose donor to an aldose (acceptor substrate) [87].
TKT Structure: Transketolase (TK) is a homodimer [95] (Fig 6) and the least structurally complicated member of thiamine diphosphate (ThDP)-dependent enzymes group containing PDHC & OGDHC [96]. Each monomer consists of three distinct regions the N terminal or PP binding region, the middle or pyrimidine binding region and C terminal region [87]. The first 2 regions are associated with coenzyme binding while the role of the third remains unknown [85,97].
Thiamine Binding Site: TKT has two active centres with one THDP molecule attached to a binding motif [98,99] and a bivalent cation (Ca affinity more than Mg [100]) tightly bound at each centre by noncovalent interactions [101]. Thiamine binding site is located within a deep furrow which allows only the C2 atom of the thiazolium ring to be exposed to the donor substrate [101]. A highly conserved starter sequence glycine-aspartate-glycine GDG and concluding sequence asparagine-asparagine (NN) represent this site between residues 154 and 185 [101]. Further the interactions of the non-covalently bound coenzyme ThDP-magnesium with the protein component are at five critical sites containing arginines (Arg 101, Arg 318, Arg 395, Arg 401 and Arg 474and Asp155) [101] contribute to dimer formation, stability or catalytic activity [102,96]. The dimerization process involves initial binding of magnesium to the aspartate in the starter sequence which inturn interacts with the pyrophosphate molecule of the thiamine diphosphate through hydrogen bonding [101], followed by one transketolase monomer engaging the pyrophosphate moiety and the other with the thiazolium and pyrimidine rings of ThDP [88,97]. The importance of this interaction is reflected in the noticeable refractoriness in Wernickes encephalopathy to thiamine treatment alone in hypomagnesemic alcoholics [103]. This enzyme has a 2 stage catalytic cycle central to which is the TPP molecule, initiated by the deprotonation in its thiazolium ring due to interaction with Glu 418 of apotransketolase.

Role of transketolase in disease and therapy
Transketolase enzyme genetic variants and depreciated enzyme activities have been noted in neurodegenerative diseases like Wernickes Korsakoff syndrome and Alzheimers disease [104]. Upregulation of the TKT L1 gene has been found in a number of malignant disorders resulting in enhanced total transketolase activity and cellular proliferation in human colon cancer [105], thyroid [106], cervical [107], ovarian cancer [108], nephroblastoma and adenocarcinoma. Its increased expression is found to be a potential diagnostic biomarker for breast cancer [109] and prognostic biomarker for nasopharyngeal [110] and laryngeal squamous cell carcinoma [111]. The reason may lie in the role of tranketolase in the reductive pentose pathway which remains a source a carbons such as in ribose required for neucleotide synthesis, NADPH and reduced glutathione in addition to aromatic acids and fatty acids required for cellular growth in general and explosive growth in particular. Transketolase has begun to emerge as a target in the cellular immune response in multiple sclerosis [112]. Human transketolase can be used in structure-based drug design as target for inhibition in the treatment of cancer [113] and in the search for new transketolase inhibitors as non permanently charged thiamine analogs,which are substrates for the thiamine activator thiamine pyrophosphokinase. These pyrophosphate analogs antagonize the ability of transketolase in vitro [113]. In diabetes mellitus type 2 experimental model, the role of transketolase in the reductive pentose pathway and its activation by administration of lipid soluble thiamine derivative benfotiamine is well documented and undeniable [114] and further clinical research is ongoing.

Pharmacotherapeutics of type 2 diabetes
Treatment is done using 4 categories of oral antidiabetic drugs. These act by stimulating insulin release from pancreatic B cells. Sulfonylureas may also act by decreasing hepatic insulin clearance [115]. They increase insulin concentration often failing to improve first phase insulin release in response to a glycemic challenge. There is secondary failure and tachyphylaxis to sulfonylurea therapy following prolonged use. Their adverse effects are hypoglycaemia, GIT disturbances, cholestatic jaundice, agranulocytosis, aplastic and hemolytic anemia, generalized hypersensitivity and dermatological reactions [116]. There is also a debate on associated cardiovascular mortality -due to blockage of KATP channels of the hearts and vascular tissues [117]. Second generation sulfonylurea glimepiride is useful as single therapy in previously drug naïve patients and also in combination with non-secretagogue medication [118]. Glimepiride may be linked to lower incidence of hypoglycaemia [119] and may improve insulin sensitivity [120]. It also has an insulin sparing action [121].

D-phenylalanine derivatives:
Netaglinide is the latest insulin secretagogue to become available. It selectively enhances early insulin release providing excellent meal time glucose control while reducing total insulin exposure [122] iv.

Biguanides:
These agents don't cause hypoglycemia and are thus called euglycemic agents. Current proposed mechanisms of biguanides include glycolysis simulation in tissues, reducing glucose absorption from GIT with increased glucose to lactate conversion, reduced hepatic and renal gluconeogenesis, in the GI tract and reduction of plasma glucagon levels [123]. Most frequent toxicity are gastrointestinal (anorexia,nausea,vomiting,abdominal discomfort and diarrhea). It is contra indicated in patients with hepatic disease or in conditions predisposing to tissue anoxia because of risk of lactic acidosis [124].

v.
Thiazolidinediones (glitazones): They are also considered to be euglycemic and are effective in 70% users. Three drugs have been used clinically from this group (Troglitazone, Rosiglitazone and Pioglitazone). Troglitazone a severely hepatotoxic and its removal from public use is well known. These are selective agonists for nuclear peroxisome proliferator -activated receptor -gamma (PPAR GAMMA) whose activation enhances insulin responsive genes that regulate carbohydrate and protein metabolism [125] vi.

Alpha Glucosidase Inhibitors:
Competitive inhibitors of intestinal alpha glucosidases namely acarbose and miglitol decrease the post meal digestion and assimilation of simple and complex carbohydrates such as starch and disaccharides [126]. These are effective also in prediabetic individuals and successfully restored β cells function. Therefore, diabetes prevention may be a further indication for their usage [127].

Currently available
The Incretin hormones released by the gut, gastric inhibitory peptide (GIP) and Glucagon like peptide1 (GLP-1) (liraglutide) stimulate insulin secretion upon nutrient entry into the gut, suppression of glucagon release,slow gastric emptying and decrease food intake [128,129]. Therefore, they have an antidiabetogenic potential. Incretin mimetics e.g. Exenatide LAR from exendin 4 is currently in use and most resistant to DPP4 degredation. GIP has also been shown enhancing β cell proliferation and inhibiting apoptosis in islet cell lines [130,131]. Additionally functional GIP receptors have been identified on adipocytes and shown to stimulate glucose transport, accelerating fatty acid synthesis and stimulating lipoprotein lipase activity in animal models [131,133,134]. Several novel GIP analogues have been developed which act as stronger GIP agonists, showing resistance to degradation by Dipeptidyl Peptidase-4 (DIPP-4) [135] and demonstrating increased insulinotropic and blood glucose lowering activity [135]. Dipeptidyl peptidase Inhibitors (vildagliptin & sitagliptin) suppress breakdown of Glucagon like peptide1 (GLP-1) show great potential and are undergoing clinical testing. Antihyperglycemic synthetic analogs of amylin a hormone which are produced by the pancreas to lower blood sugar levels are available in injectable form and require close monitoring. Dapagliflozin a renal glucose reabsorption inhibitor reduces glycemic reabsorption independent of insulin, promises to be a new drug for type 2 diabetes treatment [136]. Testosterone replacement therapy in diabetic hypogonadal men decreases insulin resistance [137] probably by protective effect on pancreatic beta cells through its action on inflammatory cytokines [138].

Experimental new drugs
A vanadium and allixin based drug [139] and macrophage migration inhibitory factor MIF blocking inhibitory synthetic oral drug reducing blood sugar levels was tried in the mouse model and found to be effective in both the type 1&2 diabetc model [140]. Lisofylline, a fat metabolism inhibitor which prevents buildup of ceramide a by product of fat metabolism in mouse skeletal muscle decreased the insulin resistance and thus appears to be a novel new approach for type 2 diabetes [141]. It also has the ability to protect insulin producing cells by inhibiting cytokines produced by immune cells leading to apoptosis and cellular dysfunction and is thus effective in type1 diabetes [142]. LXR agonists have shown potential and require further testing in human and model systems [143]. Growth factors and protein kinase C inhibitors may act as innovative therapies for diabetic retinopathy [144].

Surgical interventions
Recently a type of gastric bypass surgery has been successful in normalizing blood sugar in a small number of normal to moderately obese type 2 diabetics [145,146]. This surgery may possibly reduce death rate by 40% from all causes in morbidly obese people [147].

Micronutrient approaches to treatment of diabetic complications
People with diabetes have reduced antioxidant capacity which lays the basis for usage of antioxidant vitamins such as β carotene or vitamin C or E. A reduced level of ascorbic acid (Vitamin C) leaves the body more at mercy of the detrimental effects of aldose reductase, an enzyme responsible for many diabetic complications, such as cataracts and peripheral neuropathy [148]. Quercetin is another powerful aldose reductase inhibitor. It has been shown to inhibit aldose reductase by upto 50% [149]. Vitamin E is a free radical scavenger. It may play a preventive role in diabetic retinopathy by decreasing DAG levels, normalizing protein kinase C activation, normalizing blood flow in retinal and renal microvasculature and restoring NO mediated endothelium dependent relaxation [150,151]. Renal and retinal vascular flows and responses were normalized in individuals who had diabetes of less than 10 years duration with high dose oral vitamin E therapy given for short periods while unchanged glycaemic control was observed [152].
Magnesium and chromium deficiency have been associated with poor diabetic control, insulin resistance, macro vascular disease and hypertension [153] and decreased glucose tolerance respectively [154]. Reduction of neuronal damage in diabetics by inhibiting glutamate dehydrogenase via vitamin B6 therapy has also been observed [155].N-Reduced glutathione precursor NAcetyl Cysteine is a gene expression and cellular metabolism modulating antiox-idant and its role in prevention of β cell oxidatory damage by acting as NFKB (a genetic regulator) inhibitor and subsequent deintensification of inflammatory responses is well documented [156]. Trace element vanadyl sulfate that behaves like insulin normalized hyperglycemic levels in diabetic animals and decreased the insulin need by upto 75% [157]. In human with Type 2 diabetes, low doses of vanadyl sulfate enhanced insulin responsive glucose uptake, glycogen production and decreased endogenous glucose formation. This resulted in reduced lipid oxidation and plasma free fatty acids levels [158]. Alpha lipoic Acid has powerful antioxidant activity, insulinomimetic action and provides protection from insulin resistance linked diabetic stress while improving glucose utilization [159]. Hyperglycemia reduction in diabetic rats was observed along with improvement in GSH levels with selenium therapy [160].Calcium AEP has benefited both type 1 and type 2 diabetics as it is alpha cell membrane integrity factor required for cellular membrane function. The hormone dehydroepiandrosterone (DHEA) undergoes a decrease in levels with aging that many researchers have linked to impair glucose metabolism. It was found to be as effective in reducing body fats and maintaining insulin responsiveness as exercise [161]. Thiamine is also now showing potential as therapy for type 2 diabetes. Thiamine (termed aneurin or antineuritic vitamin initially) was the premier discovery of the B vitamins and thus ranked vitamin B1 (Fig 7). It has relative temperature, acid stability and water solubility containing a pyrimidine ring and a thiazole nucleus linked with a methylene bridge. Thiamine is an essential micronutrient with a dietary reference intake (DRI) for normal healthy subjects of 1.1 mg/day for females and 1.3 mg/day for males [162]. Found in range of foodstuffs such as cereal grains. Its rich sources are brown rice, bran, oat meal, flax, poultry, egg yolks, beef, pork, liver, nuts, fruits and vegetables such as oranges, asparagus, kale, cauliflower, potatoes [163].UK law demands compulsary fortification of flour with thiamin of not less than 0.24mg/100g flour to replace losses during milling. In Pakistan no compulsory fortification is done and the general public consumes milled white flour which is easily available and probably thiamine deficient. Thiamine is naturally found in 4 forms in varying degrees of phosphorylation in TMP thiamine monophosphate, TPP thiamine pyrophosphate or diphosphate and TTP=thiamine triphosphate. It is commercially available as salt in its mononitrate HCl (also natural byproduct) and relatively inaccessible semi lipid soluble form S-acyl derivative benfotiamine and truly lipid soluble thiamine disulphide derivatives sulbutiamine and fursultiamine. Out of these, Thiamine HCl is the water soluble, easily accessible and commonly used vitamin supplement available with the trade name Benerva.

Thiamine absorption in normal conditions
Thiamine is released from its administered form by phosphatase and pyrophosphatase in the proximal part of the small intestine, following which absorption occurs mainly from this site with some from the stomach and the colon; thiamine absorbed in the colon may originate from intestinal microflora. Its absorption is hindered by alcohol consumption and folic acid deficiency [164].High affinity organic anion transporters THTR1 [165], THR2 for thiamine and reduced folate transporter RFC-1 transports both folic acid thiamine monophosphate (TMP) intracellularly [166,168] at normal physiological concentrations. At high expression levels RFC1 also transports TPP out of the cells [167]. At higher concentrations thiamine crosses cell membranes in its open unionized form of the thiazolium ring even by passive diffusion.THTR2 is placed on the luminal surface of the gastrointestinal epithelial cells and THTR1 is on the basolateral surface mainly but not exclusively [169]. THTRI is expressed widely in human tisseues with particular high expression in skeletal muscles, placenta, heart liver and kidney [168,170].Mutations in the SLC19A2 (D93H, S143F and G172D) cause malfunctioning of the thiamine transporter THTR1, thiamine deficiency and thiamine responsive megaloblastic anaemia (TRMA) [171,172]. THTR2 is widely expressed most abundantly in placenta, kidney and liver [173]. Also highly expressed RFC-1, is in human tissues including mitochondrial membranes [168,174]. It has affinities for TMP and TPP of 26µM and 32µM respectively [167,168]. Cellular efflux is the probable reason for the presence of thiamine in plasma and cerebrospinal fluid [175][176][177].Thiamine in the glomerular filtrate is reabsorbed by the renal brush border membrane high affinity transporters where influx is increased by an outward directed H+gradient [178] RFC-1 is expressed on the apical and basolateral surface of the proximal tubular epithelial cells [179]; it may mediate the reuptake of TMP and provide a solution to the normal absence of TMP in the urine. Proton antiport membrane transport may operate in both intestinal and renal proximal tubular thiamine uptake [180]

Assessment of thiamine status
Erythrocytes contain approximately 90% of total thiamine in the blood and therefore conventionally their transketolase levels have generally been considered to be the measure of thiamine status in the body [181].Thiamine deficiency is assessed conventionally by measuring the percentage below complete saturation of the thiamine dependant enzyme transketolase (TK) in RBCs-"thiamine effect". The normal value of the thiamine effect in human subjects is in the range 0-15%, mild deficiency is 15-25% and severe thiamine deficiency >25% [182]. Latest research has however questioned its reliability as thiamine transporters THTR1 and RFC1 in erythrocytes are upregulated in thiamine deficieny and RBC TK levels are not decreased in tandem [183]. Furthermore it doesn't account for changes in TK expression in RBC and other precursor cells. The expression of TK is decreased in thiamine deficiency [184]. Currently assessment of mononuclear TK activity and plasma thiamine concentration determination using HPLC flourimetric determination with respect to normal healthy controls gives greater insight into thiamine status [185][186][187]. More recently in capillary enzyme reaction and capillary electrophoresis methods are emerging as potential alternative monitoring and determining techniques for thiamine in samples [188].

Thiamine metabolism within the cells
When TMP enters cells by RFC-1 it is hydrolyzed to thiamine by phosphatases [168]. Thiamine deficiency decreased the activity of TPPK [189] and was implicated in decreased hepatic levels of TPP with normal levels of thiamine in STZ diabetic rats [190].Within mitochondria TPP is slowly hydrolyzed to TMP by phosphatases which may leave the mitochondria via the same transporter. High concentrations of thiamine monophosphate inhibit thiamine pyrophsphokinase activity noncompetitively [191] and inhibit the entry of TPP into the mitochondria competitively [189]. A small amount of TPP is further phosphorylated to thiamine triphosphate (TTP) by thiamine pyrophosphate kinase and hydrolyzed to TPP by TPPphosphatase [192] [ 168]. Plasma half life is relatively short (2days) [193] but its tissue half life is approximately 9-18 days [194]. Thiamine is stored largely in skeletal muscle and the highly perfused organs such as heart, brain, liver and kidneys [163]. Subcellularly only 10% of total TPP is available for binding to transketolase most of it is associated with the mitochondria [185].Thiamine and its acid metabolites are are excreted primarily in the urine [195].

Pharmacodynamics of thiamine
Thiamine diphosphate binds to a evolutionarily highly conserved domain located in a deep cleft in the active sites of the thiamine dependant enzymes resulting in the activity of these enzymes [196]. The physiological function of thiamine is mainly fulfilled by TPP (TDP). Structurally the basis of thiamine action and activation of all ThDP-dependent enzymes lies in thiamine catalysis and deprotonation of the thiazolium ring and contribution of the aminopyrimidine side chain in this effect [197][198] while the pyrimidine ring with its dual proton donor and acceptor capability functioning as a proton transfer system. On the basis of these chemical alterations TPP functions as coenzyme for mitochondrial enzymes pyruvate dehydrogenase (PDH [199] and α ketoglutarate dehydrogenase [200] of the citric acid cycle.

Symtoms of severe thiamine deficiency
Thiamine derivatives and thiamine dependant enzymes are universally present in all cells of the body thus a thiamine deficiency would seem to affect all organ systems especially the heart and the nervous system due to their high oxidative metabolism as witnessed in its severest form as beriberi (dry, wet or infantile) [195]. Symptoms occur rarely include tachycardia, warmth, flushing, irritability, sweating, nausea, restlessness and allergic reactions. Pharmacokinetic interactions at the level of drug metabolism include microsomal enzyme induction by prolonged anticonvulsant pheytoin resulting in decreased plasma levels of thiamin in patients with seizure disorders such as epilepsy. The water soluble thiamine HCl form is safe in humans in oral doses less than or equal to several hundred milligrams via oral route. A UK EVM found that a small clinical trial in Alzheimers patients revealed no adverse effects of thiamineHCl at daily oral intakes of 6000 to 8000mg for five to six months. A randomized double blind placebo controlled trial was conducted in India for therapy of primary dysmenorrhea, a daily oral dose of 100mg thiamine was given to 556 females for 60-90 days and no adverse effects were noted.In extremely rare cases of allergic sensitivity were noted solely in patients using thiamine by the parenteral route and were probably due to the injection vehicle and it not been reported to be carcinogenic or mutagenic. No known genetic microsomal variations increase susceptibility to thiamine toxicity [214].

Thiamine and diabetes
Experimental evidence suggests that thiamine transport maybe abnormal in diabetes.In experimental diabetes, these was diminished intestinal absorption of thiamine and TMP. Mild deficiency of thiamine in diabetes may induce increased expression of THR1 as found in frank thiamine deficiency. In streptozocin induced diabetic rats with supportive insulin therapy to regulate hyperglycemia, 54% decreased plasma thiamine concentration was reported in contrast to normal controls [190]. This was induced in the diabetic state despite high dietary intake (9 fold) in excess of DRI for rats. The primary cause was marked increased renal clearance of thiamine which was increased by 8 fold. In streptozotocin-induced diabetic rats, there was decreased transketolase expression and activity in renal glomeruli, liver, skeletal muscle and RBCs after 12 weeks of diabetes was found associated with progressive increase in the renal clearance of thiamine and increased albuminuria with duration of diabetes, suggesting that abnormal renal handling of thiamine may occur early in the process of impairment of renal function in diabetes [190]. In experimental diabetes, similar low plasma thiamine concentration was associated with low TK activity and expression in renal glomeruli.Reduced activity of PDH was also noticed due to thiamine depletion. Similar impairment of thiamine-related metabolism may occur in the diabetic retina and peripheral nerves predisposing these tissues to the adverse effects of hyperglycaemia.

Effect of thiamine therapy in diabetes: On glycemic control in experimental and animal model
Thiamine therapy was found to decrease hyperglycemia in cirrhosis, insulin resistance of muscle and inadequate insulin secretion by β cells. In thiamine responsive megaloblastic anaemia too hyperglycemia is linked to impaired insulin secretion due to mutated high affinity thiamine transporter. Therapeutic intervention by thiamine in both cases is likely to involve improved β cell metabolism and insulin secretion. This effect was not noticed in permanent insulin deficiency of the STZ diabetic rat model where most of the pancreatic β cells are damaged or destroyed and resultantly no improvement in glycemic control is observed. It is not yet known if thiamine or benfotiamine improve glycemic control in type 2 diabetic animal model.

Mild thiamine deficiency in diabetics and improved post therapy thiamine status in clinical studies
Mild thiamine deficiency has been observed in diabetics in different international studies.There is paucity of data on thiamine and thiamine dependant enzyme status in clinical diabetes mellitus. In Japan a study of 46 diabetic patients (7 type 1, 39 type 2) with moderate glycemic control (glycated hemoglobinA1c 9%) found lower diabetic RBC TK activity in 79% of patients and a concomitant decrease in thiamine level in 76% of diabetics. Oral thiamine supplementation 3-80mg/day increased thiamine levels (20 patients) and TK activity (15 patients). In a larger Israel study of 100 type 2 diabetic patients (glycated HbAic 9.2%), TK activity was lower than the minimum normal range in 18% of diabetics. A smaller Italian study of 10 type 1 diabetic children with normal renal function found plasma thiamine concentration to be decreased by 34% with respect to normal healthy controls and was normalized in a placebo controlled intervention with lipophilic thiamine derivative benzoxymethyl thiamine (50mg/day).

Intervention of high dose thiamine therapy in biochemical dysfunction in diabetes and the prevention of microvascular dysfunction, neuropathy, dyslipidemia complications
Microvascular disease (nephropathy, retinopathy and neuropathy) a common debilitating manifestation of chronic diabetes mellitus, has no effective therapy. Hyperglycaemia in diabetic subjects is an essential element for development of both microvascular and macrovascular complications risk factor DCCT 2003. High doses of thiamine and its derivative Sbenzoylthiamine monophosphate (Benfotiamine) are proposed as a new therapy to counteract biochemical dysfunction leading to the development of microvascular complications [114].
High dose thiamine and Benfotiamine may counter the development of microvascular complications by activation of the reductive pentosephosphate pathway. Interestingly by activation of the hexosamine pathway the glucose-mediated induction of lipogenic enzymes, glycerophosphate dehydrogenase (GPDH), fatty acid synthase (FAS) and acetyl-CoA carboxylase, was stimulated in liver and adipocytes (Fig 8). In turn, this diverts metabolic flux away from the hexosamine pathway, decreased lipogenesis and correct diabetic dyslipidaemia as shown below (Fig 8).
Drugs such as cerivastatin decreased total and LDL cholesterol, triglycerides, microalbuminuria and increased HDL cholesterol in type 2 diabetic patients. However, normal levels of these metabolite were not achieved. Interestingly pharmacologically combined therapy of vit B1, B6 and B12 did not augur well in diabetics having diabetic nephropathy and substantial adverse outcomes associated with high dose vitamin B6, B9 and B12 co-supplementation in patients with advanced diabetic nephropathy was brought to light Recently concluded Diabetic Intervention with Vitamins to Improve Nephropathy (DIVINE) study produced an unexpected accelerated decline in renal function. The reasons could have been multipronged ranging from toxic accumulation of folate and B12 in patients of diabetic nephropathy with low GFR or competitive inhibition of TMP and TPP transport at the level of RFC1 transporter by high dose folate at key sites such as the kidney and vascular cells thus adversely affecting sharing of thiamine between tissues rich in thiamine and those deficient in it [183].

Summary
Thus final summarization of these studies indicates that high dose thiamine repletion may decrease the risk of micro and macrovascular disease and counter incipient nephropathy in diabetes. The effect of thiamine occurred independent of control of hyperglycaemia, blood pressure and statin/fibrate therapy, suggesting that high dose thiamine therapy may produce improvements in the prevention of dyslipidaemia and diabetic nephropathy in addition to those produced by current therapy for control of hyperglycaemia, blood pressure, cholesterol and lipids. Since dyslipidemia and microalbuminuria are reversible in type 2 diabetic patients, it is possible that high dose thiamine therapy might improve renal function and metabolic control through reduction in biochemical dysfunction and improvement in thiamine dependant enzyme activities in diabetic patients with existing dyslipidaemia and microalbuminuria.
However, it appears that there may be noticeable variations in these parameters on the basis Treatment of Type 2 Diabetes 42 of geographical, racial and pharmacogenetic factors. So the need of the hour was an indepth study as a double blind placebo controlled clinical trial to study the effect of high dose thiamine therapy on biochemical profile and activities of thiamine dependant enzymes in type 2 diabetic patients in our multiracial population in Pakistan.

Therapeutic implications
Based on the data above, the first ever randomized, double blinded, placebo controlled clinical intervention trial registered with the World Health Organization involving high dose B1 therapy was conducted by Dr.Saadia ShahzadAlam of the Pharmacology Deptt (Co-Principal Investigator 1) of Federal Postgraduate Medical Institute Lahore for a period of 5 months to study the effect of high dose thiamine therapy on biochemical profile and activities of thiamine dependant enzymes on type 2 diabetics in the Pakistani population. This trial was also pioneering internationally on the subject of diabetic nephropathy and the effect of thiamine supplementation on it. 40 type 2 micoalbuminuric diabetic patients at the Diabetes Clinic of Shaikh Zayed Hospital Lahore were administered 300mg/day (100mg tablets Administration of 300mg B1 TDS) / placebo for 3 months followed by a 2 month washout period. The results of this trial were quite interesting and have been published internationally, plasma thiamine levels of both thiamine and placebo groups were significantly depleted as compared to normal controls. There were significant baseline derangements of incipient diabetic nephropathy (microalbuminuria), glycemic control parameters FBS and glycated hemoglobin, lipid profile including total cholesterol, HDL, LDL, triglycerides and VLDL in type 2 microalbuminuric diabetics as compared to healthy individuals. Following 3 months 300 mg/day thiamine administration there was significant improvement of urinary albumin excretion, and preservation of glomerular filtration rate suggested that these occurred due to thiamine replenishment and decreased glycated hemoglobin and LDL cholesterol levels were observed in the washout period as a delayed effect. Additionally following thiamine therapy significant reduction in plasma levels of sVCAM-1, noticeable and an inverse linkage between thiamine therapy and vWF was apparent in this group as compared to placebo, suggested noticeable benefit with reduction in the risk factors of type 2 diabetes. Significant changes in other serum and urinary biomarkers profile were also observed in type 2 diabetics following thiamine therapy in a simultaneously carried out proteomic study. Three thiamine dependant enzymes PDE3, PDE1β, AKGDHE1 and Transketolase were determined to be dysfunctional at baseline in type 2 microalbuminuric diabetic patients in comparison to normal healthy controls, and improved in both activity and gene expression with high dose thiamine therapy While importantly no hepatic or renal adverse effects were encountered prior, during therapy or as a residual effect, post washout thus fortifying the previously established human safety track record of thiamine. [ 200 ]. We hope that these findings would contribute to knowledge regarding the role of thiamine therapy at 300mg/day dosage on biochemical profile and molecular aspects of those vital thiamine dependant enzymes and help in providing improved, safe and more effective treatment for type 2 diabetic patients with incipient nephropathy, dyslipidemia with expected decrease risk of heart disease and kidney failure.