**2. The mitochondrial catalytic repertoire**

**Thiamine and the Cellular Energy Cycles: A Novel Perspective on Type 2 Diabetes Treatment** 

*Pharmacology Department, Federal Postgraduate Medical Institute, Lahore, Pakistan* 

*Department of Microbiology and Molecular Genetics, University of the Punjab, Lahore, Pakistan* 

**ABSTRACT** 

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

**INTRODUCTION**

ellular 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. This is an ancient metabolic, cytosolic pathway that converts glucose into pyruvate under anaerobic conditions (i.e. it doesn't require much oxygen) 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

. The pathway of glycolysis comprises of 2 clear divisions (Fig1). After glycolysis,where glucose is broken down into pyruvate only releasing fractional amounts of ATP, 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

 **Fig 1 A & B: A Schematic Pathway of glycolysis from glucose to pyruvate.and its connection to the reductive pentose pathway and citric acid cycle. Adapted from Michael W. King, Ph.D / IU School of Medicine / miking at iupui.edu / © 1996–2011.**

**Figure 1.** (A) Phase1 (Priming Phase) of Embden Meyerhoff Pathway; (B) Phase 2 (Energy Yielding Phase) of Embden Meyerhoff Pathway; A & B: A Schematic Pathway of glycolysis from glucose to pyruvate.and its connection to the re‐

Adapted from Michael W. King, Ph.D / IU School of Medicine / miking at iupui.edu / © 1996–2011.

**A B**

**, Samreen Riaz2**

**Saadia Shahzad Alam1**

the need of more expensive oral hypoglycaemic agents required by them.

citric acid cycle and oxidative phosphorylation activities.

24 Treatment of Type 2 Diabetes

**Fig. 1 A) Phase1 (Priming Phase) of Embden Meyerhoff Pathway** 

ductive pentose pathway and citric acid cycle.

**Figure 2.** Krebs cycle (www. library.thinkquest.org)

 **1B Phase 2 (Energy Yielding Phase) of Embden Meyerhoff Pathway** 

*1*

*2*

1

**C**

**The pyruvate dehydrogenase complex:** Both prokaryotic and eukaryotic species carry among others, conglomeration of proteins into a mega, specifically arranged multienzyme structural complex termed (a "metabolon").

**Figure 3.** Protein-protein Interactions in the Native human PDC. Adapted from Brautigham (2006)


**c.** Possible arrangement of E2p and E3BP components in a 40/20 core.Shown is a dodecahe‐ dral arrangement of 20 heterotrimers composed of 2 E2p proteins (purple) and one E3BP (green)(Brautigham 2008). Of these enzyme complexes of the metabolon, the pyruvate dehydrogenase complex is highly evolutionarily conserved mitochondrial α-ketoacid dehydrogenase complex, along with the branched-chain α-ketoacid dehydrogenase complex (BCKDC), and the α-ketoglutarate dehydrogenase complex (KGDC) [4, 5]. The complex has 3 main components with multiple subunits and multiple names.(Fig 3)

The heterotetramer **PDEI p PYRUVATE DEHYDROGENASE (EC1.2.4.1)** comprises of 2 alpha and 2 beta subunits [6]. Its alpha 1 subunit is designated as PDE1A-2,(pyruvate dehydrogenase (lipoamide) alpha2). Its gene PDHA2 is located on chromosome 4 having length of 1383 bp/460 aa [7, 8] Whereas the alpha 2 subunit is designated asPDH E1-A type1 (i.e.) synonym PHE1A. Its gene PDHA1 is located on chromosome X which has length of 15922 bps [9] and a mol.wt of 160KDa. The Pyruvate dehydrogenase E1 component subunit beta, or pyruvate dehydrogenase (lipoamide) beta mitochondrial, synonym, PDE1- B, has gene located on chromosome 3 having length of 6198 bp [10-12]. The key function of the complex E1alpha subunit containing the active site is to be the rate limiting enzyme, unidirectionally funneling intermediate metabolites from glucose breakdown to either the oxidative metabolic pathways or fatty acid and cholesterol synthesis [13]. **PDE2p** contains **dihydrolipoyl transacetylase enzyme activity (EC2.3.1.12)** encoded by DLAT Dihydrolipoa‐ mide acetyl tranferase gene, present on human chromosome 11 band q23.1. It has mol wt 200 KDa [14]. Interestingly, this long arm region of chromosome 11 often presents with translocations in cellular genetic abnormalities [15]. **PDE3/GCSL/LAD/PHE3 (EC 1.8.1.4)** component contains the dihydrolipoyl dehydrogenase activity. E3 activity is encoded by the DLD located on chromosome 7 and length 28799 [16]. It has a mol.wt of 110 KDa. This protein has four different sites: the flavin adenine dinucleotide binding site, the nicotina‐ mide adenine dinucleotide binding site, the centre site and the interface site. The protein forms a homodimer with the FAD and NAD binding regions on one unit and the inter‐ face domain of the other unit forming the active centre [17].

#### **2.1. Structural association of the 3 units**

The human pyruvate dehydrogenase multi enzyme complex (PDC) is a nuclear encoded mitochondrial matrix 9.5 megadalton catalytic organization of copies of three catalytic components i.e. heterodimeric pyruvate dehydrogenase (E1p 30copies) (thiamine diphos‐ phate (ThDPdependant), homodimeric dihydrolipoyl transacetylase (E2p12 copies) and dihydrolipoamide dehydrogenase dimer (E3) (FAD containing) residing in the inner mitochondrial membrane [4](Fig. 3). The (E1p) and E3subunits surround a 60-meric dodecahedral core of 40 copies of E2p and 20 copies of a monomeric non catalytic component, E3-binding protein (E3BP), which specifically tethers E3 dimers to the pyru‐ vate dehydrogenase complex [18]. Each E2p subunit contains two consecutive lipoic acidbearing domains (LBDs), termed as L1 and L2, one subunit binding domain (SBDp) which binds E1p and the inner-core/catalytic domain containing the E2 p active site responsible for the self assembly of the core which connects with the other independent domains by 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 do‐ main. 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]. Unspeci‐ fied copies of each PDC regulatory enzyme pyruvate dehydrogenase kinases and pyru‐ vate dehydrogenase phosphatases are also strung non-covalently to the core by the LBD2 [5, 20].

**c.** Possible arrangement of E2p and E3BP components in a 40/20 core.Shown is a dodecahe‐ dral arrangement of 20 heterotrimers composed of 2 E2p proteins (purple) and one E3BP (green)(Brautigham 2008). Of these enzyme complexes of the metabolon, the pyruvate dehydrogenase complex is highly evolutionarily conserved mitochondrial α-ketoacid dehydrogenase complex, along with the branched-chain α-ketoacid dehydrogenase complex (BCKDC), and the α-ketoglutarate dehydrogenase complex (KGDC) [4, 5]. The complex has 3 main components with multiple subunits and multiple names.(Fig 3) The heterotetramer **PDEI p PYRUVATE DEHYDROGENASE (EC1.2.4.1)** comprises of 2 alpha and 2 beta subunits [6]. Its alpha 1 subunit is designated as PDE1A-2,(pyruvate dehydrogenase (lipoamide) alpha2). Its gene PDHA2 is located on chromosome 4 having length of 1383 bp/460 aa [7, 8] Whereas the alpha 2 subunit is designated asPDH E1-A type1 (i.e.) synonym PHE1A. Its gene PDHA1 is located on chromosome X which has length of 15922 bps [9] and a mol.wt of 160KDa. The Pyruvate dehydrogenase E1 component subunit beta, or pyruvate dehydrogenase (lipoamide) beta mitochondrial, synonym, PDE1- B, has gene located on chromosome 3 having length of 6198 bp [10-12]. The key function of the complex E1alpha subunit containing the active site is to be the rate limiting enzyme, unidirectionally funneling intermediate metabolites from glucose breakdown to either the oxidative metabolic pathways or fatty acid and cholesterol synthesis [13]. **PDE2p** contains **dihydrolipoyl transacetylase enzyme activity (EC2.3.1.12)** encoded by DLAT Dihydrolipoa‐ mide acetyl tranferase gene, present on human chromosome 11 band q23.1. It has mol wt 200 KDa [14]. Interestingly, this long arm region of chromosome 11 often presents with translocations in cellular genetic abnormalities [15]. **PDE3/GCSL/LAD/PHE3 (EC 1.8.1.4)** component contains the dihydrolipoyl dehydrogenase activity. E3 activity is encoded by the DLD located on chromosome 7 and length 28799 [16]. It has a mol.wt of 110 KDa. This protein has four different sites: the flavin adenine dinucleotide binding site, the nicotina‐ mide adenine dinucleotide binding site, the centre site and the interface site. The protein forms a homodimer with the FAD and NAD binding regions on one unit and the inter‐

face domain of the other unit forming the active centre [17].

The human pyruvate dehydrogenase multi enzyme complex (PDC) is a nuclear encoded mitochondrial matrix 9.5 megadalton catalytic organization of copies of three catalytic components i.e. heterodimeric pyruvate dehydrogenase (E1p 30copies) (thiamine diphos‐ phate (ThDPdependant), homodimeric dihydrolipoyl transacetylase (E2p12 copies) and dihydrolipoamide dehydrogenase dimer (E3) (FAD containing) residing in the inner mitochondrial membrane [4](Fig. 3). The (E1p) and E3subunits surround a 60-meric dodecahedral core of 40 copies of E2p and 20 copies of a monomeric non catalytic component, E3-binding protein (E3BP), which specifically tethers E3 dimers to the pyru‐ vate dehydrogenase complex [18]. Each E2p subunit contains two consecutive lipoic acidbearing domains (LBDs), termed as L1 and L2, one subunit binding domain (SBDp) which binds E1p and the inner-core/catalytic domain containing the E2 p active site responsible for the self assembly of the core which connects with the other independent domains by

**2.1. Structural association of the 3 units**

26 Treatment of Type 2 Diabetes

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. Phosphory‐ lation 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, dichlorace‐ tate 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 impor‐ tant 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].

#### **2.2. 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 dehydro‐ genase 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 [45-47]. 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 hyper‐ glycemic states observed in type-2 diabetes [25], increasing the chances of pyruvate dehydro‐ genase 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 reconstruct‐ ed in vitro and reliance on other mammal models persists [5, 48](Fig 4).

**Figure 4.** Representative Model for Human 2 Ketoglutarate Dehydrogenase Complex: All figures of molecular struc‐ tures were created with the program PyMol (DeLano Scientific, San Carlos, CA). Jun Li. The Journal of Biological Chemistry, 2007;282, 11904-913.

#### **2.3. Structure of alphaketoglutarate dehydrogenase complex**

**2.2. Diseases produced by defective PDC**

28 Treatment of Type 2 Diabetes

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 dehydro‐ genase 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 [45-47]. 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 hyper‐ glycemic states observed in type-2 diabetes [25], increasing the chances of pyruvate dehydro‐ genase 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 reconstruct‐

**Figure 4.** Representative Model for Human 2 Ketoglutarate Dehydrogenase Complex: All figures of molecular struc‐ tures were created with the program PyMol (DeLano Scientific, San Carlos, CA). Jun Li. The Journal of Biological

Chemistry, 2007;282, 11904-913.

ed in vitro and reliance on other mammal models persists [5, 48](Fig 4).

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].The complex has 3 main enzymatic components with multiple subunits & copies and varied names: oxoglutarate dehydrogenase (lipoamide); EC: 1.2.4.2 (E1k), dihydrolipoamide S-succinyltrans‐ ferase; EC:2.3.1.61 (E2k) and dihydrolipoamide dehydrogenase; EC:1.8.1.4 (E3k) [52].


structural and functional approximation to the PDE3 component of pyruvate dehydro‐ genase and its full complex contains 6 dimers [5].

The alphaketoglutarate dehydrogenase complex EC 1.2.4.2 also termed as oxoglutarate dehydrogenase complex, acts on alphaketo-glutarate/2 oxoglutarate a key intermediate in the krebs cycle converting to succinyl co A, produces NADH and CO2 in an irreversible reaction [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-6 phosphate (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 6 phosphogluconate dehydrogenase are essential for the conversion of hexoses to pentoses [75].

structural and functional approximation to the PDE3 component of pyruvate dehydro‐

The alphaketoglutarate dehydrogenase complex EC 1.2.4.2 also termed as oxoglutarate dehydrogenase complex, acts on alphaketo-glutarate/2 oxoglutarate a key intermediate in the krebs cycle converting to succinyl co A, produces NADH and CO2 in an irreversible reaction [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-6 phosphate (G6P) as the substrate, occur at the beginning of the pathway and generate 2 moles

genase and its full complex contains 6 dimers [5].

30 Treatment of Type 2 Diabetes

### **A) Digrammatic Representation of the Oxidative Stage of Hexose Monophosphate Shunt**

#### **B) Reductive or Non Oxidative Stage of the Hexose Monophosphate Shunt**

**Fig. 5: (A): Digrammatic Representation of the Oxidative Stage of Hexose Monophosphate Shunt and (B) Reductive Stage of the Hexose Monophosphate Shunt Figure 5.** (A): Digrammatic Representation of the Oxidative Stage of Hexose Monophosphate Shunt and (B) Reductive Stage of the Hexose Monophosphate Shunt

**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 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].

metabolic redox estimator and regulates transcription during the anti-oxidant response, as a shift from primary carbon

#### metabolism, is fastest in oxidative stress77. NADPH cofactor serves as reducing equivalent in the endoplasmic reticulum lumen for fatty acid and steroid biosynthesis in hepatic and, adipose tissue, adrenal cortex78. High levels of PPP **2.4. Functions of the pentose phosphate pathway in normal and diseased conditions**

enzymes are in neutrophils and macrophages as they utilize NADPH to produce ROS to destroy engulfed microbes in a process termed as respiratory burst79. 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 stresss80. Cancer cells are known to access successfully the glucose flux in the pentose phosphate pathway supporting NADPH and reactive oxygen species production and glutathione reduction81 responding to both incremental and decremental reactive oxygen species82. 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 oncogenes83 and molecular oxygen84 promoting genetic damage in 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

normal cells and therapy resistance in cancerous cells85.Malignant cells also use reduced glutathione81 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.

6

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 partici‐ pate in the reductive pentose pathway reactions catalyzing transfer of a 2 carbon fragment from a ketose donor to an aldose (acceptor substrate) [87].

Adapted from Kochetov2005, Lindquist 1992

**Figure 6.** Schematic View of Transketolase Dimer Showing its Different Components. The 3 components are colour dif‐ ferentiated: N terminal domain, light blue, middle domain, light brown & C terminal domain yellow.The bound cofac‐ tor ThDP is shown as a CPK model and Ca++ion in green

**Transketolase:** synonymous with TKT1 &TK is composed of and encoded by the TKTgene located on chromosome 3 (30390 bp) [89-91]. **Transketolase like protein 1:** named as TKT2, TKR, TK 2, Transketolase 2, Transketolase-related protein has molwt of 60-70 KDaltons depending on splice variation encoded by the TKTL1 gene located on chromosomeX Length: 25052 bp [92, 93]. **Transketolase like protein 2** termed TK is composed of 913 aminoacids encoded by gene TKTL2 located on chromosome 4 having length of 2742 bp [94].

**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 pyri‐ midine 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.

#### **2.5. Role of transketolase in disease and therapy**

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 partici‐ pate in the reductive pentose pathway reactions catalyzing transfer of a 2 carbon fragment

**Figure 6.** Schematic View of Transketolase Dimer Showing its Different Components. The 3 components are colour dif‐ ferentiated: N terminal domain, light blue, middle domain, light brown & C terminal domain yellow.The bound cofac‐

from a ketose donor to an aldose (acceptor substrate) [87].

32 Treatment of Type 2 Diabetes

Adapted from Kochetov2005, Lindquist 1992

tor ThDP is shown as a CPK model and Ca++ion in green

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 ana‐ logs,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.

#### **2.6. Pharmacotherapeutics of type 2 diabetes**

Treatment is done using 4 categories of oral antidiabetic drugs.

	- **i.** Biguanides
	- **ii.** Thiazolidinediones (glitazones)

#### **2.7. Insulin secretagogues**

**i.** Sulfonylureas:

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, agranulocyto‐ sis, 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].

**ii.** Meglitinides:

Like the sulfonylureas, meglitinides also stimulate insulin secretion.

**iii.** 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:

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 ana‐ logs,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.

**1.** Insulin secretagogues: Sulfonylureas, meglitinides, D-phenylalanine derivatives

**3.** Those decreasing carbohydrate absorption from the gut: Alpha Glucosidase inhibitors.

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, agranulocyto‐ sis, 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

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

Treatment is done using 4 categories of oral antidiabetic drugs.

**2.6. Pharmacotherapeutics of type 2 diabetes**

**ii.** Thiazolidinediones (glitazones)

**2.** Those reducing insulin resistance:

**i.** Biguanides

34 Treatment of Type 2 Diabetes

**2.7. Insulin secretagogues i.** Sulfonylureas:

has an insulin sparing action [121].

**iii.** D-phenylalanine derivatives:

Like the sulfonylureas, meglitinides also stimulate insulin secretion.

**ii.** Meglitinides:

exposure [122]

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). Troglita‐ zone 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].
