Current Reviews, 2005, 1, 287-298 287

The Potential Role of (Vitamin B1) in Diabetic Complications

Paul J. Thornalley*

Department of Biological Sciences, University of Essex, Central Campus, Wivenhoe Park, Colchester, Essex CO4 3SQ, United Kingdom

Abstract: Accumulation of triosephosphates arising from high cytosolic concentrations in hyperglycemia is one likely or potential trigger for biochemical dysfunction leading to the development of diabetic complications. This may be prevented by disposal of excess triosephosphates via the reductive pentosephosphate pathway. This pathway is impaired in experimental and clinical diabetes by mild . The expression and activity of the thiamine-dependent enzyme, transketolase - the pacemaking enzyme of the reductive pentosephosphate pathway, is consequently decreased. Correction of thiamine deficiency in experimental diabetes by high dose therapy with thiamine and the thiamine monophosphate prodrug, Benfotiamine, restores disposal of triosephosphates by the reductive pentosephosphate pathway in hyperglycemia. This prevented multiple mechanisms of biochemical dysfunction: activation of kinase C, activation of the hexosamine pathway, increased glycation and oxidative stress. Consequently, the development of incipient diabetic nephropathy, neuropathy and retinopathy were prevented. Both thiamine and Benfotiamine produced other remarkable effects in experimental diabetes: marked reversals of increased diuresis and glucosuria without change in glycemic status. High dose thiamine also corrected dyslipidemia in experimental diabetes – normalizing cholesterol and triglycerides. Dysfunction of b-cells and impaired glucose tolerance in thiamine deficiency and suggestion of a link of impaired glucose tolerance with dietary thiamine indicates that thiamine therapy may have a future role in prevention of type 2 diabetes. More immediately, given the emerging multiple benefits of thiamine repletion, even mild thiamine deficiency in diabetes should be avoided and thiamine supplementation to high dose should be considered as adjunct nutritional therapy to prevent dyslipidemia and the development of vascular complications in clinical diabetes.

Keywords: Thiamine, Benfotiamine, Diabetes, Diabetic Complications, Dyslipidemia, Cholesterol, Beta-cell and .

THIAMINE: NUTRITIONAL ASPECTS AND MILD thiamine deficiency, however, since it does not take account THIAMINE DEFICIENCY IN DIABETES of changes in TK expression in RBC precursor and other cells; The expression of TK is decreased in thiamine Thiamine (vitamin B1) is an essential micronutrient with deficiency [10]. Assessment of TK activity and plasma a dietary reference intake (DRI) for normal healthy adult thiamine concentration, with respect to normal healthy human subjects of 1.1 mg per day for females and 1.3 mg per control values, gives greater insight into thiamine status. day for males [1;2] (Fig. (1)). Thiamine supplements are usually only given to subjects suffering symptoms of severe There is a paucity of data on thiamine and thiamine- thiamine deficiency – Wernicke-Korsakoff syndrome dependent enzyme status in clinical diabetes mellitus. A (encephalopathy and associated psychosis) [3] and beriberi study of 21 type 1 and 12 type 2 diabetic patients in Norway [4]. This is associated with severe malnutrition, HIV-AIDS, found a 27% decrease of thiamine concentration in whole inadequate intake, uptake and dysfunctional metabolism of blood samples of type 1 patients and no significant decrease thiamine in clinical alcoholism, and increased excretion of in type 2 patients, with respect to normal controls [11]. A thiamine in renal dialysis of subjects with end stage renal study in Japan of 46 diabetic patients (7 type 1, 39 type 2) disease [5-8]. Wernicke-Korsakoff syndrome and beriberi with moderate glycemic control (mean glycated hemoglobin are not symptoms associated with clinical diabetes mellitus HbA1c 9%) found that TK activity of RBCs was lower than although a mild, asymptomatic thiamine deficiency may be the normal range minimum in 79% of diabetic patients and prevalent. the concentration of thiamine in blood plasma was lower Thiamine deficiency is assessed conventionally by than the normal range minimum in 76% of diabetic patients. measuring the percentage below complete saturation of the In patients with abnormally low plasma thiamine thiamine-dependent enzyme transketolase (TK) in red blood concentration, the thiamine effect was 24 ± 16%. In 24 cells (RBCs) – the “thiamine effect”. The normal value of patients given oral thiamine supplements of 3 - 80 mg/day, the thiamine effect in human subjects is in the range 0 –15%, normal plasma thiamine concentrations were achieved in 20 mild thiamine deficiency is 15 – 25%, and severe thiamine patients and normal RBC TK activity in 15 patients [12]. In a deficiency >25% [9]. This is not a satisfactory assessment of study of 100 type 2 diabetic patients in Israel with HbA1c of 9.2%, TK activity of RBCs was lower than the minimum normal range in 18% of diabetic patients [13]. A small study in Italy of 10 type 1 diabetic children with normal renal *Address correspondence to this author at the Department of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, Essex CO4 3SQ, function found that plasma thiamine concentration was U.K.; Tel/Fax: +44 1206 873010; E-mail: [email protected] deceased 34%, with respect to normal healthy controls, and

1573-3998/05 $50.00+.00 © 2005 Bentham Science Publishers Ltd. 288 Current Diabetes Reviews, 2005, Vol. 1, No. 3 Paul J. Thornalley

OH OH NH2 CH3 a. CH3 NH2 – HO N N + SH N N S HO– CH3 N H O

CH3 N Thiamine Thiamine (thiazolium cation) (acyclic form cation)

O O – b. O p O– O p O NH CH NH2 CH 2 3 3 OH Non-specific OH esterase N N N N S SH (-benzoic acid ) CH N B O CH3 N H O 3 O Cyclisation Benfotiamine O (–OH–) O p O– CH3 NH2 OH + N N S

CH3 N Thiamine monophosphate

Fig. (1). Thiamine and Benfotiamine. a. Solution species of thiamine. b. Metabolism of Benfotiamine. was normalised in a placebo-controlled intervention with the maintenance insulin therapy to moderate hyperglycemia, the lipophilic thiamine derivative benzoxymethyl-thiamine (50 plasma thiamine concentration was decreased 54%, with mg/day) [14]. respect to normal controls [15] (Fig. (2)). This was induced in the diabetic state despite dietary intake of thiamine 9-fold Mild thiamine deficiency is prevalent in experimental in excess of the DRI for rats [16]. The primary cause of the diabetes. In streptozotocin-induced (STZ) diabetic rats with thiamine deficiency was a marked increased renal clearance

Fig. (2). Effect of high dose thiamine and Benfotiamine therapy on plasma thiamine status in STZ diabetic rats and controls. a. Plasma thiamine concentration and b. plasma thiamine monophosphate concentration. Key: C, control; CT70, control + 70 mg/kg thiamine; CB70, control + 70 mg/kg Benfotiamine; D, diabetic; DT7, diabetic + 7 mg/kg thiamine; DT70, diabetic + 70 mg/kg thiamine; DB7, diabetic + 7 mg/kg Benfotiamine; DB70, diabetic + 70 mg/kg Benfotiamine. Data are mean ± SEM (n = 6 – 13). From [15]. The Potential Role of Thiamine (Vitamin B1) Current Diabetes Reviews, 2005, Vol. 1, No. 3 289 of thiamine. The renal clearance of thiamine increased 8-fold [17]. There was an associated decreased activity of TK in renal glomeruli, and RBCs, attributed to decreased expression of TK [15;17]. There was also decreased thiamine concentration in the liver and of adult STZ diabetic rats [18] – although in our study (TPP) but not thiamine concentration was decreased in the liver of STZ diabetic rats [15;17]. The decrease in TK activity of RBCs developed after 12 weeks of diabetes concomitant with a progressive increase in the renal clearance of thiamine and increased albuminuria with duration of diabetes [17]. After only 3 - 4 weeks of diabetes, pregnant STZ rats did not develop decreased TK activity in RBCs - although their pups had significantly decreased TK activity of RBCs. This was corrected by maternal thiamine therapy during gestation (ca. 20 – 25 mg/kg/day) [19]. The development of thiamine deficiency with increased renal clearance and albuminuria in STZ diabetic rats suggests that abnormal renal handling of thiamine may be an early feature of impairment of renal function in diabetes.

MEMBRANE TRANSPORTERS OF THIAMINE AND METABOLISM TO THIAMINE MONOPHOSPHATE AND THIAMINE PYROPHOSPHATE. DYS- FUNCTIONAL THIAMINE METABOLITE TRANS- PORT AND METABOLISM IN DIABETES Thiamine is an organic cation and utilizes high affinity organic cation transporters to cross cell membranes at normal physiological concentrations. At high concentrations, thiamine may cross cell membranes by passive diffusion of the thiazolium ring-opened, unionized form (Fig. (1a)). The for thiamine transporters are members of the SLC (solute carrier) 19A family with each encoding with approximately 25% overall amino-acid identity. Three Fig. (3). Membrane transport of thiamine, thiamine members of this family have been identified: SLC19A2 and monophosphate and thiamine pyrophosphate. (a) Intestinal SLC19A3 - encoding thiamine transporters THTR1 [20] and epithelium, (b) tissues, and (c) renal proximal tubule. The main THTR2 [21]; and SLC19A1 – encoding the reduced directions of thiamine metabolite flow are shown. Mitochondrial transporter RFC-1 which transports folic acid and also flows and TPP binding to enzymes in the intestinal and renal transports thiamine monophosphate (TMP) into cells [22;23]. proximal tubules are omitted for clarity. At high expression levels, RFC-1 transports TPP out of cells [24]. Thiamine is mainly absorbed in the proximal part of the efflux of TMP is the probable explanation for the presence of small intestine with some absorption also in the stomach and TMP in blood plasma and cerebrospinal fluid [15;36;37] colon; thiamine absorbed in the colon may originate from (Fig. (3b)). Thiamine in the glomerular filtrate is reabsorbed by renal brush border membrane high affinity transporters intestinal microflora. THTR2 is the transporter on the + luminal surface of gastrointestinal epithelial cells and where influx was increased by an outward directed H THTR1 is on the basolateral surface mainly but not gradient [38]. RFC-1 is expressed on the apical and exclusively [25]; THTR1-mediated uptake occurs in the basolateral surface of proximal tubular epithelial cells [39]; it colon [26]. Transport of thiamine is ATP-dependent with a may mediate the re-uptake of TMP and explain why TMP is not present normally in the urine (Fig. (3c)). Proton antiport KM of 0.8 – 2.5 mM [20;26;27] (Fig. (3a)). THTR1 is expressed widely in human tissues with particular high membrane transport may operate in both intestinal and renal expression in , placenta, heart, liver, and proximal tubular thiamine uptake [40]. [20;28]. in SLC19A2 (D93H, S143F, and Experimental evidence suggests that thiamine and TMP G172D) cause malfunctioning of the thiamine transporter transport may be abnormal in diabetes. In experimental THTR1, thiamine deficiency and thiamine-responsive diabetes, there was decreased intestinal absorption of megaloblastic syndrome (TRMA) [29-32]. THTR2 is thiamine and TMP [41]. Mild deficiency of thiamine in widely expressed, with the most abundant expression diabetes may induce increased expression of THTR1 – as observed in placenta, kidney, and liver [33]. The KM for found in frank thiamine deficiency [42]. Experimental thiamine transport by THTR2 was 27 nM [25]. RFC-1 is diabetes has recently also been associated with decreased widely expressed in human tissues – including in the expression of RFC-1 [43]. Mild thiamine deficiency in mitochondrial membrane [23;34]. It has affinities for TMP diabetes may, therefore, lead to induced expression of tissue and TPP of 26 mM and 32 mM, respectively [23;35]. Cellular THTR1 and THTR2 transporters to improve tissue 290 Current Diabetes Reviews, 2005, Vol. 1, No. 3 Paul J. Thornalley

OH CH NH2 3 + N N S phosphatase (EC 3..1.3.-) Thiamine O CH3 N Thiamine O P OH CH3 O– NH2 Benfotiamine + ATP N N S Thiamine pyrophosphate kinase CH (EC2.7.6.2) 3 N AMP Thiamine monophosphate (TMP)

O O O P O P OH

CH3 NH2 O O Nucleoside triphosphatase (EC 3.6.1.15) + N N S

CH3 N Thiamine pyrophosphate (TPP) ATP Thiamine triphosphatase Thiamine pyrophosphate kinase (EC 2.7.6.2) and (EC 3.6.1.28) Thiamine diphosphate kinase (EC 2.7.4.15) ADP O O O O P O P O P OH CH O– O– O– NH2 3 + N N S

CH3 N Fig. (4). Mammalian metabolism of thiamine. scavenging of the available thiamine and decreased TK of the pentosephosphate pathway [49] and for the expression of RFC-1 transporter activity to retain tissue TPP. mitochondrial enzymes, pyruvate dehydrogenase (PDH) [50] Having entered cells by THTR1 and THTR2, thiamine is and a -ketoglutarate dehydrogenase (a KDH) [51], of the converted to TPP by thiamine pyrophosphokinase (TPPK). citric acid cycle. TPP is also a co-factor of branched chain a - Human TPPK has high expression in the testes, small keto-acid dehydrogenase, which had decreased activity and intestine and kidney with moderate expression in brain, liver, expression in the liver of STZ diabetic rats [52]. TTP is a placenta and spleen [44]. When TMP enters cells by RFC-1, cofactor for neuronal protein with unusual it is hydrolysed to thiamine by phosphatases [23]. Thiamine phosphorylation of histidine residues [53] and may have a deficiency decreased the activity of TPPK [45] and this was role in the regulation of neurotransmitter signaling [54;55]. implicated in decreased hepatic levels of TPP with normal TPP is hydrolysed to TMP and to thiamine by phosphatases levels of thiamine in STZ diabetic rats [15]. TPP enters [23]. Subcellular localization studies suggest that only 10% mitochondria by a carrier mediated process, probably via of total cellular TPP is in the available to bind to TK RFC-1 [46]. Within mitochondria, TPP is slowly hydrolysed with most cellular TPP is associated with mitochondria [56]. to TMP which may leave mitochondria via the same Thiamine deficiency of human cells in culture induced transporter (Fig. (3b)). High concentrations of TMP inhibit decreased activities and expression of TK and PH but not TPPK activity non-competitively (Ki = 17 mM) [47] and a KDH [10]. The latter enzyme has high affinity and low inhibit the entry of TPP into mitochondria competitively (Ki exchange of the TPP co-factor and is therefore resistant to = 150 mM) [46]. A small amount of TPP is further the effects of thiamine deficiency. Thiamine deficiency in phosphorylated to thiamine triphosphate (TTP) by thiamine vivo induced decreased activities of TK and PH [57]. Since pyrophosphate kinase and hydrolysed to TPP by TPP TK has a short half-life when unsaturated with TPP phosphatase [48] (Fig. (4)). (approximately 25 min) [58], decreased tissue concentrations The physiological functional of thiamine is mainly of TPP are expected to lead to a prompt decrease in TK fulfilled by TPP. TPP is a co-factor for the cytosolic enzyme expression. The Potential Role of Thiamine (Vitamin B1) Current Diabetes Reviews, 2005, Vol. 1, No. 3 291

EFFECT OF THIAMINE DEFICIENCY ON and dyslipidaemia – particularly high triglycerides and small, PANCREATIC BETA-CELL FUNCTION AND dense LDL – have also been linked to the development of GLYCEMIC CONTROL vascular complications. Tight control of blood glucose and blood pressure with aggressive intervention to counter There was no improvement of glycemic control in STZ dyslipidaemia decreases the risk of microvascular diabetic rats by high dose thiamine or Benfotiamine therapy complications but this is not always achievable – particularly [17;59-61]. Thiamine therapy has been shown to decrease because of limitations of current drug therapy [71]. High hyperglycemia in cirrhosis [62] where hyperglycemia is dose therapy with thiamine and the thiamine derivative linked to of muscle and inadequate insulin Benfotiamine (S-benzoylthiamine monophosphate (Fig. secretion by b-cells [63] and thiamine-responsive (1b)) are now proposed as a novel therapy to counter megaloblastic anemia (due to mutated high affinity thiamine biochemical dysfunction leading to the development of transporter) where hyperglycemia is linked to impaired microvascular complications [15;17;60;72]. insulin secretion [64]. Remedial intervention by thiamine in both cases is likely to involve improved b-cell metabolism In the last decade, significant advances in understanding and insulin secretion. This is not available in the permanent of the link of hyperglycemia to microvascular complications insulin deficiency of the STZ-diabetic rat model where most have been made. A key observation was that biochemical pancreatic b-cells have been destroyed and this is probably dysfunction linked to complications development is initiated why thiamine and Benfotiamine usually give no by increased cytosolic glucose concentration in endothelial improvement in glycaemic control. It is not known if cells and mesangial cells with high expression of the glucose thiamine or Benfotiamine improves glycaemic control in an transporter GLUT1 [73]. Cytosolic hyperglycemia leads to model of type 2 diabetes. the accumulation of triosephosphates and closely related glycolytic intermediates which then initiate multiple Since mild thiamine deficiency may be prevalent in pathways of biochemical dysfunction: increased formation of diabetes, particularly with nephropathy, the effect of diacylglycerol de novo and activation of protein kinase C thiamine deficiency on b-cell function is of interest. Isolated b (PKCb), activation of the polyol pathway, activation of the pancreatic islets of thiamine deficient rats had decreased hexosamine pathway, increased flux through the basal secretion of insulin and decreased glucose- and glycerophosphate shuttle and mitochondrial dysfunction with tolbutamide-induced insulin secretion [65]. In thiamine- increased oxygen radical production, and increased deficient rats, there was increased fasting glucose decreased formation of methylglyoxal and related formation of fasting insulin concentration [66]. There was also impaired advanced glycation endproducts (AGEs) [74] (Fig. (5)). insulin secretion with impaired glucose tolerance (IGT) and Other reinforcing biochemical pathways are brought into increased plasma glucagon concentration in an oral glucose play: activation of vascular NADPH oxidase by activated tolerance test [67]. It is of interest that the Hoorn Study, a PKC, and activation and uncoupling of endothelial nitric study of glucose tolerance in 2196 human subjects, 50-75 oxide synthase (eNOS) by increased oxygen radical years old without diabetes, dietary fibre intake was inversely production [75;76], activation of poly(ADP-ribose)poly- associated with fasting glucose, and fibre intake correlated merase (PARP) – in response to oxidative (and probably strongly with thiamine intake. Thiamine intake had a strong glycation) damage to DNA [77], and AGE receptor (RAGE)- association with 2-h postprandial glucose concentrations mediated vascular cell activation [78]. Triosephosphate which was independent of fibre intake and fasting glucose. accumulation is the trigger for these processes [74] and a This led to the conclusion that part of the association strategy to reverse this could alleviate multiple pathways of between fibre intake and glucose tolerance was attributable biochemical dysfunction. to dietary thiamine intake [68]. Three laboratories – my own, that of Porta and a These studies suggest that thiamine deficiency impair - b collaborative network of Hammes, Brownlee and others, cell function and thiamine deficiency may contribute to IGT have examined the possibility that activation of the reductive in the human population. Further epidemiological analysis pentosephosphate pathway by high dose thiamine and and intervention trials with thiamine or Benfotiamine may Benfotiamine might divert triosephosphates and fructose-6- establish a basis for prevention of type 2 diabetes by high phosphate to ribose-5-phosphate and relieve biochemical dose thiamine derivative therapy. dysfunction in hyperglycemia. This was found for human RBCs in vitro [79]. When bovine aortal endothelial cells INTERVENTION OF HIGH DOSE THIAMINE were incubated under hyperglycemic conditions in vitro, THERAPY IN BIOCHEMICAL DYSFUNCTION IN there was increased membrane-localized PKC activity, DIABETES AND THE PREVENTION OF activation of PARP [80] increased cellular concentrations of MICROVASCULAR COMPLICATIONS the hexosamine pathway intermediate UDP-N- Microvascular disease (nephropathy, retinopathy and acetylglucosamine, increased intracellular AGEs and neuropathy) develops in diabetic patients over a period of activation of the oxidant sensitive transcription factor NF- 10-15 years. It is a common and disabilitating complication kB. All these responses were prevented by Benfotiamine of diabetes mellitus with no effective therapy. [60;81]. Hyperglycemia also delayed replication and Hyperglycemia is a risk factor for the development of increased von Willebrand factor secretion by bovine aortal microvascular complications in both type 1 and type 2 endothelial cells which was prevented by thiamine [82;83]. diabetic subjects [69;70]. Hypertension (typically diastolic Both thiamine and Benfotiamine prevented decreased hypertension in type 1 patients and systolic hypertension in replication, increased and AGE accumulation type 2 patients) and high systolic-diastolic pulse pressure, induced by hyperglycemia in bovine retinal pericytes and 292 Current Diabetes Reviews, 2005, Vol. 1, No. 3 Paul J. Thornalley

Fig. (5). Metabolic dysfunction linked to the development of vascular complications of diabetes. Reversal by high dose thiamine therapy. human umbilical vein endothelial cells in vitro [72;83]. It correction of decreased nerve conduction velocity in diabetic therefore seemed that high dose thiamine and Benfotiamine controls. AGE accumulation was also decreased [61]. In a may counter the development of microvascular clinical study of 24 diabetic patients, a double blind, complications in experimental diabetes in vivo. placebo-controlled trial with Benfotiamine (80 mg/day)- Thiamine and Benfotiamine were given orally at high pyridoxine (180 mg/day)-cyanocobalamin (0.5 mg/day) for 2 dose (7 and 70 mg/kg/day) to STZ diabetic rats with weeks and then half this dose for a further 10 weeks, there maintenance insulin therapy for 24 weeks. Both thiamine and was a significant improvement in nerve conduction velocity Benfotiamine prevented the development of incipient with the therapy [84]. In a 6-week open trial with 36 patients nephropathy, as judged by the prevention of receiving doses up to 4 times higher than indicated above, microalbuminuria. Benfotiamine also prevented hyper- there was significant improvement in pain, vibration and filtration. TK activity and expression was decreased in renal current perception on the peroneal nerve [85]. glomeruli of STZ diabetic rats. High dose thiamine and These studies show that thiamine repletion with thiamine Benfotiamine therapy normalised TK expression and activity and Benfotiamine may prevent the development of diabetic and thereby increased the conversion of triosephosphates to microvascular complications in vivo. ribose-5-phosphate. This was associated with decreased activity of membrane and cytosolic protein kinase C, INTERVENTION OF HIGH DOSE THIAMINE decreased protein glycation and oxidative stress. This was THERAPY IN BIOCHEMICAL DYSFUNCTION IN achieved without change in hyperglycemic status and DIABETES AND REVERSAL OF DIABETIC glycemic control [17]. DYSLIPIDEMIA Retinopathy developed in STZ diabetic Wistar rats over Diabetes mellitus is associated with a 2–3 fold increased 36 weeks, as judged by the 3-fold increase in formation of risk of coronary heart disease (CHD) in men and a 3–5 fold retinal acellular capillaries. High dose therapy with increase in women, relative to the non-diabetic population. Benfotiamine (80 mg/kg/day) normalised the number of CHD risk determinants in the diabetic population include retinal acellular capillaries in STZ diabetic rats and hence hyperglycemia, in insulin resistance, prevented the development of retinopathy [60]. systolic hypertension, low-grade inflammation, increased High dose therapy with thiamine (70 mg/kg/day) and triglycerides, cholesterol and plasminogen activator Benfotiamine (100 mg/kg/day) prevented the development of inhibitor-1 [86;87]. Dyslipidaemia is a crucial feature in neuropathy in STZ diabetic Wistar rats, as judged by the diabetic CHD where increased levels of very low density The Potential Role of Thiamine (Vitamin B1) Current Diabetes Reviews, 2005, Vol. 1, No. 3 293

Fig. (6). Effect of high dose thiamine and Benfotiamine therapy on the development of microalbuminuria in streptozotocin-induced diabetic rats and normal healthy controls. a. Thiamine dosing study. b. Benfotiamine dosing study. Key: hollow bars – controls, and from left to right – control, and control + 70 mg/kg thiamine (a) or Benfotiamine (b); solid bars – diabetics, and from left to right – diabetic, diabetic + 7 mg/kg thiamine (a) or Benfotiamine (b), and diabetic + 70 mg/kg thiamine (a) or Benfotiamine (b). Data are mean ± SEM (n = 5 – 9). lipoprotein-1 (VLDL-1) particles initiate the development of achieved by prevention of thiamine depletion and decreased small dense low density lipoprotein (LDL) and high density TK activity in the liver of diabetic rats. There was a lipoprotein (HDL) particles that pose the main atherogenic concomitant decrease in hepatic UDP-N-acetylglucosamine threat [88]. Increased lipoprotein secretion by the liver, and synthase activity. Thiamine also normalised preceded by a switch from lipid oxidation to lipogenesis and food intake of diabetic rats. A lower dose of thiamine (7 increased lipoprotein synthesis, appears to be key to the mg/kg) and the Benfotiamine (7 and 70 mg/kg) were development of dyslipidaemia. The hepatic hexosamine ineffective. High dose thiamine therapy prevented diabetic pathway has been implicated in signaling for de novo dyslipidaemia in experimental diabetes probably by lipogenesis by the liver. Overexpression of suppression of food intake and hexosamine pathway glutamine:fructose-6-phosphate amidotransferase (GFAT), signaling. Other factors may also be involved. Benfotiamine the rate-limiting enzyme in the hexosamine pathway, in the was ineffective. Thiamine has also been shown to decreased liver of transgenic mice was associated with hyperlipidaemia aortic smooth muscle cell proliferation induced by [89]. The glucose-mediated induction of lipogenic enzymes, hyperglycemia and hyperinsulinemia in vitro [91]. glycerophosphate dehydrogenase (GPDH), fatty acid synthase (FAS), and acetyl-CoA carboxylase, was stimulated OTHER PHYSIOLOGICAL EFFECTS OF HIGH in liver and by activation of the hexosamine DOSE THERAPY WITH THIAMINE AND BENFOTI- pathway [90]. If flux through the hepatic hexosamine AMINE pathway is important in lipogenesis in diabetes, high dose Therapeutic intervention with high dose thiamine and therapy with thiamine and Benfotiamine is expected to Benfotiamine in STZ diabetic rats produced further counter this effect (Fig. (6)). remarkable pharmacological effects. In STZ diabetic rats with maintenance insulin therapy, Both high dose thiamine and Benfotiamine decreased high dose therapy with thiamine therapy (70 mg/kg) glucosuria and diuresis of STZ diabetic rats in a dose- prevented diabetes-induced increases in plasma cholesterol dependent manner [15]. For glucosuria, thiamine and and triglycerides in diabetic rats but did not reverse the Benfotiamine gave decreases of 63 and 72% at the 70 mg/kg diabetes-induced decrease of HDL [91] (Fig. (8)). This was dose, with respect to the diabetic control. The mechanism is 294 Current Diabetes Reviews, 2005, Vol. 1, No. 3 Paul J. Thornalley

Fig. (7). Metabolic mechanism for the suppression of hepatic lipogenesis in diabetes by thiamine.

Fig. (8). Reversal of increased plasma cholesterol and triglycerides in streptozotocin-induced diabetic rats by high dose thiamine therapy. a. Non-HDL cholesterol and b. triglycerides. Key: C, control; CT70, control + 70 mg/kg thiamine; CB70, control + 70 mg/kg Benfotiamine; D, diabetic; DT7, diabetic + 7 mg/kg thiamine; DT70, diabetic + 70 mg/kg thiamine; DB7, diabetic + 7 mg/kg Benfotiamine; DB70, diabetic + 70 mg/kg Benfotiamine. Data are mean ± SEM (n = 6 – 13). unknown but decreased glucosuria may be linked to that both thiamine and Benfotiamine increased the renal decreased washout of glucose by the concomitant decreased reuptake of glucose. Glucose re-absorption in the kidney diuresis and effects on renal glucose transporters. Since occurs mainly via sodium-glucose co-transporter (SGLT). plasma glucose concentration was not changed, this suggests The insulin-stimulated (GLUT4) is also The Potential Role of Thiamine (Vitamin B1) Current Diabetes Reviews, 2005, Vol. 1, No. 3 295 expressed in the ascending limb of the loop of Henle and is however, that although Benfotiamine has a significant also induced by vasopressin [36;92;93]. Thiamine and advantage over thiamine in delivery of thiamine (and TMP) Benfotiamine-induced decrease in glucosuria may be linked in normal healthy rats, this advantage is lost in diabetes (Fig. to changes in SGLT and vasopressin-stimulated increase in (2)). It is conceivable that increased expression of high GLUT4 activity. For diuresis, thiamine and Benfotiamine affinity thiamine transporters and the decreased expression gave decreases of 60 and 70% at the 70 mg/kg dose, with of RFC-1 in diabetes accounts for this. Whether the same respect to the diabetic control. Decreased diuresis may be happens in clinical diabetes is not known. Moreover, high linked to reversal of diabetes-induced activation of protein dose thiamine but not Benfotiamine therapy reversed kinase C by high dose thiamine and Benfotiamine [17] and diabetic dyslipidemia – a differential response attributed to consequent reversal of inhibition of water re-uptake by more effective delivery of thiamine to the liver in the aquaporins in renal collecting duct cells [94;95]. High dose postprandial period by high dose thiamine than high dose thiamine therapy (but not Benfotiamine therapy) also Benfotiamine. The high tissue concentrations of TMP normalised food consumption. This suggests a complex achieved with high dose Benfotiamine therapy may also be mechanism of action of high dose thiamine therapy that may of concern. High concentrations of TMP inhibit TPPK be linked to more efficient absorption and/or use of nutrients activity non-competitively (Ki = 17 mM for the porcine than in STZ diabetic rats with and without high dose enzyme) [47] and inhibit the entry of TPP into mitochondria Benfotiamine therapy. Decreased food consumption by STZ competitively (Ki = 150 mM) [46]. This may decrease the diabetic rats with high dose thiamine therapy may be linked availability of TPP in high dose Benfotiamine therapy and to affects of thiamine metabolites, TPP and thiamine limit the effectiveness of Benfotiamine and other vehicles of triphosphate (TTP), on dopamine signaling in brain related to TMP delivery. Other lipophilic derivatives of thiamine, such sensory-specific satiety [55;96]. Chronic increased dopamine as benzoylthiamine [100] and benzoxymethylthiamine [14], secretion by TPP and TTP may down regulate the feeding are available for evaluation. It is not clear, currently, incentive response. Oral dosing of thiamine produced higher therefore, if high dose Benfotiamine therapy offers a amounts of protein-associated thiamine metabolites in the significant advantage over high dose thiamine therapy for the brain of mice than dosing with Benfotiamine in the critical prevention of diabetic microvascular and macrovascular post-prandial period [97]. This may be why thiamine had a complications. Further studies, including clinical studies, satiation effect but Benfotiamine did not. Increased will be required to examine this. Nevertheless, the expended energy in thiamine- and Benfotiamine-treated rats prevention of diabetic complications in experimental may be due to increased physical activity and/or increased diabetes by high dose thiamine and Benfotiamine suggests thermogenesis but these were not characterised. that biochemical responses to thiamine repletion are important in decreasing the risk of developing diabetic CONCLUDING REMARKS complications. Given the continuing toll of microvascular complications and cardiovascular disease in the diabetic The emergence of high dose thiamine and Benfotiamine population, I suggest that even mild thiamine deficiency in therapy for the prevention of diabetic complications is diabetes should be avoided and thiamine supplementation to consistent with the reversal of biochemical dysfunction high dose should be considered as adjunct nutritional therapy linked to the development of diabetic complications in a to counter diabetic dyslipidaemia and vascular unifying mechanism [74]. Activation of the reductive complications. pentosephosphate pathway with consumption of triosephosphates removes one of the metabolic pathways ACKNOWLEDGEMENTS increasing electron transport into complex II of mitochondria driving mitochondrial dysfunction and oxidative stress [98]. I thank the Juvenile Diabetes Research Fund (USA), A further feature of high dose thiamine therapy is that it Diabetes UK and the Wellcome Trust (U.K.) for support for corrects thiamine deficiency in diabetes. Diabetic patients my research and the past and current members of my appear to be at increased risk of mild thiamine deficiency, research team for contributions to research on the prevention which is probably linked to decline in renal function. At the of diabetic complications by high dose thiamine and outset of studies on the prevention of diabetic complications Benfotiamine. by thiamine, there seemed to be a clear justification for the use of Benfotiamine rather than thiamine since ABBREVIATIONS administration of Benfotiamine (100 mg) to healthy human ACC = Acetyl-CoA carboxylase subjects had a 5-fold higher bioavailability than an equivalent dose of thiamine [99]. Benfotiamine is a delivery AGEs = Advanced glycation endproducts vehicle for TMP and is probably absorbed by the RFC-1 DHAP = Dihydroxyacetonephosphate transporter; Benfotiamine-treated normal, healthy rats had high concentrations of TMP in plasma and tissues [15]. It has DRI = Dietary reference intakes strong ionic character at physiological pH and a very low eNOS = Endothelial nitric oxide synthase partition coefficient [100]. Non-specific esterases in blood plasma and tissues cleave the benzoyl ester and then the FAS = Fatty acid synthase thiazolium ring closes spontaneously to form TMP (Fig. F-1,6-bis-P = Fructose-1,6-bisphosphate (2b)). TMP is hydrolysed to thiamine such that Benfotiamine therapy also increases plasma and tissue thiamine F-6-P = Fructose-6-phosphate concentrations [15]. Studies with STZ diabetic rats suggest, GA3P = Glyceraldehyde-3-phosphate 296 Current Diabetes Reviews, 2005, Vol. 1, No. 3 Paul J. Thornalley

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Received: 10 January, 2005 Accepted: 28 March, 2005