Skip to main content
PMC home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2011 Jun 21.
Published in final edited form as: Stem Cells Dev. 2009 Jan-Feb;18(1):37–46. doi: 10.1089/scd.2007.0255

Detection of Transketolase in Bone Marrow–Derived Insulin-Producing Cells: Benfotiamine Enhances Insulin Synthesis and Glucose Metabolism

Seh-Hoon Oh 1, Rafal P Witek 1, Si-Hyun Bae 1, Houda Darwiche 1, Youngmi Jung 1, Liya Pi 1, Alicia Brown 1, Bryon E Petersen 2
PMCID: PMC3118870  NIHMSID: NIHMS303002  PMID: 18393672

Abstract

Adult bone marrow (BM)-derived insulin-producing cells (IPCs) are capable of regulating blood glucose levels in chemically induced hyperglycemic mice. Using cell transplantation therapy, fully functional BM-derived IPCs help to mediate treatment of diabetes mellitus. Here, we demonstrate the detection of the pentose phosphate pathway enzyme, transketolase (TK), in BM-derived IPCs cultured under high-glucose conditions. Benfotiamine, a known activator of TK, was not shown to affect the proliferation of insulinoma cell line, INS-1; however, when INS-1 cells were cultured with oxythiamine, an inhibitor of TK, cell proliferation was suppressed. Treatment with benfotiamine activated glucose metabolism in INS-1 cells in high-glucose culture conditions, and appeared to maximize the BM-derived IPCs ability to synthesize insulin. Benfotiamine was not shown to induce the glucose receptor Glut-2, however it was shown to activate glucokinase, the enzyme responsible for conversion of glucose to glucose-6-phosphate. Furthermore, benfotiamine-treated groups showed upregulation of the downstream glycolytic enzyme, glyceraldehyde phosphate dehydrogenase (GAPDH). However, in cell where the pentose phosphate pathway was blocked by oxythiamine treatment, there was a clear downregulation of Glut-2, glucokinase, insulin, and GAPDH. When benfotiamine was used to treat mice transplanted with BM-derived IPCs transplanted, their glucose level was brought to a normal range. The glucose challenge of normal mice treated with benfotiamine lead to rapidly normalized blood glucose levels. These results indicate that benfotiamine activates glucose metabolism and insulin synthesis to prevent glucose toxicity caused by high concentrations of blood glucose in diabetes mellitus.

Introduction

Type 1 diabetes results when the β-cells of the pancreas are destroyed by T-cells of the autoimmune system. Insulin deficiency and elevated blood sugar levels characterize the disease, which is a leading cause of early mortality in adults. The increasing incidence of type 1 diabetes throughout the world has generated considerable interest in developing a treatment that would restore glucose responsive insulin secretion. Various endeavors to solve this challenge have been attempted including stimulating the endogenous regeneration of islets [1], transplantation of donor islets [2] or transplantation of in vitro–differentiated islet-like cells [3,4].

Cell therapy using stem cells and their progeny is a promising new approach that may be capable of addressing many unmet medical needs [5]. Bone marrow (BM)-derived cells have been shown to differentiate into various lineages, such as liver [6-9], pancreas [10,11], and lung [12,13]. Recent reports have described that insulin-producing cells (IPCs) may be induced from BM cells using in vitro culture systems, and that transplantation of BM-derived IPCs into hyperglycemic mice decreased circulating blood glucose levels allowing for maintenance of comparatively normal glucose homeostasis [11]. Furthermore, other cell types, such as hepatic oval cells [14], splenocytes [15], neoplastic liver cells [16], and embryonic stem cells [17-19] were differentiated into pancreatic endocrine hormone-producing cells. With the progression of stem cell research, new methods for the treatment of diseases such as diabetes mellitus may be possible.

High plasma glucose concentrations in diabetes accelerates the aging process and lead to complications that include blindness, renal failure, nerve damage, stroke, cardiovascular disease, and even delayed wound response. High-glucose concentrations are responsible for increased mitochondrial free radical production and subsequent inactivation of glyceraldehyde phosphate dehydrogenase (GAPDH), thereby diverting upstream metabolites from glycolysis into four major glucose-driven signaling pathways (polyol, hexosamine, diacylglycerol, and AGE pathway) that cause hyperglycemic damage [20,21]. Two of these upstream metabolites, fructose-6-phosphate and glyceraldehyde-3-phosphate, are also end products of the nonoxidative branch of the pentose phosphate pathway. These metabolites are produced by the thiamine-dependent transketolase (TK) enzyme [22], which occupies a pivotal place in metabolkic regulation, providing a link between the glycolytic and pentose phosphate pathways. Also, TK has a controlling role in the supply of ribose units for nucleoside biosynthesis in microorganisms. It has been shown that diabetic patients have subnormal erythrocyte TK activity [23]. S-benzoylthiamine monophosphate (benfotiamine), a lipid-soluble thiamine derivative, has a greater bioavailability than thiamine, thus allowing upregulation of TK activity [24]. In addition, high-dose benfotiamine therapy has been reported as a potential novel strategy for the prevention of clinical diabetic nephropathy [25] and retinopathy [26] by blocking oxidative stress generated by the three major pathways of biochemical dysfunction present in hyperglycemia.

Recently attention has focused on the possible use of stem cells and clinically useful medicines for the treatment of type 1 and 2 diabetic disorders. Here, we show that TK was activated in BM-derived IPCs cultured in high-glucose medium, thereby activating glucose metabolism. We found that benfotiamine activated glucokinase for conversion of glucose to glucose-6-phosphate, and furthermore, benfotiamine activated expression of insulin message as well as maximization of their insulin synthesis. The glycolytic enzyme GAPDH was activated in cluture with benfotiamine, but when IPCs were cultured with oxythiamine, the GAPDH decreased. In chemically induced hyperglycemic nonobese diabetic severe combined immunodefificency (NOD/scid) mice, the admininstration of benfotiamine alone did not affect blood glucose levels. However, when benfotiamine-treated BM-derived IPCs were transplanted into chemically induced hyperglycemic mice, normal blood glucose levels were observed. Furthermore, glucose challenge of normal mice treated with benfotiamine was shown to be rapidly normalizing blood glucose levels. The data provided in this work may lead to the development of a new treatment for diabetes through a stem cell/benfotiamine therapy combination.

Materials and Methods

Cell culture

All procedures involving amimals were conducted according to institutionally approved protocols and guidelines. the rat BM-derived IPCs were induced in a culture-conditioned system [11]. Rat insulinoma cell line, INS-1 was cultured in rosewell Park Memorial Insitute-1640 with 10% fetal bovine serum (FBS), and 50 μM 2-mercaptoethanol to maintain differentiation.

For analysis of insulin protein level, 80% confluent INS-1 cell and hand picked 150 clusters of BM-derived IPCs were for high-glucose challenge and insulin secretion into media. The INS-1 and BM-derived IPCs were cultured in serum-free medium containing 0.5% bovine serum albumin (BSA; Sigma, Louis, MO, USA) and 5.5 mM glucose to eliminate exogenous insulin from serum. the cells were incubated in this condition for 5 h at 37°C followed by washing twice with additional serum-free medium. High-glucose challenge of the cells was achieved by addition of serum-free media containing 25 mM glucose with S-benzoylthiamine monophosphate (benfotiamine; Sigma) or oxythiamine (Sigma) for 2 h at 37°C. Oxythiamine, a TK inhibitor, inhibits synthesis of ribose by blocking the pentose phosphate cycle [27].

Immunocytochemistry

For immunofluorescent staining on normal pancreas, non-cultured BM cells and BM-derived clusters wrere followed method described by Oh et al. [11]. Rabbit anti-rat insulin (1:100, Santa Cruz Biotechnology, Inc. Santa Cruz, CA, USA), and goat anti-rat C-peptide (1:100, Linco Research Inc., St. Charles, MO, USA) antibodies were used in this procedure. Alexa Fluor 488 donkey anti-rabbit and Alexa Flour 568 donkey anti-goat IgG, anti-goat (1:500, Invitrogen, Carlsbad, CA, USA) were used as secondary antibodies, respectively. DAPI (Vector Lab. Burlingame, CA, USA) was used for nuclear staining.

Detection of TK protein and mRNA

Bone marrow cells were cultured in low-glucose Dulbecco’s modified Eagle’s medium (DMEM) in the presence of 1% dimethyl sulfoxide (DMSO) for 1 and 3 days (D1 and D3), and changed to 10% FBS in DMEM with high glucose for 2, 4, 6, 8, and 10 days (D5, 7, 9, 11, and 13). Intracellular proteins were detected in cell lystes by separating the precipitated material on 12.5% dodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels, followed by silver staining (Sigma) or Coomassie staining.

Total RNA was isolated from the INS-1 cell and BM cells treated with 1% DMSO/DMEM low-glucose medium (day 1 and 3), DMEM high-glucose medium with 10% FBS (days 5, 7, 9, 11, and 13), or nonculture BM cells by the RNeasy kit (Qiagen, Valencia, CA, USA). Two microgram RNA was used for complementary DNA (cDNA) synthesis. Reverse transcriptase polymerase chain reaction (RT-PCR) products were directly sequenced using an AmliTaq cycle sequencing kit (Perkin-Elmer Setus, Branchburg, NJ, USA) for genetic confirmation.

Cell proliferation and glucose metabolite assay

MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma] assay was performed as described previously by Mosmann [28]. For the cell proliferation assay using MTT, the INS-1 cells cultured with benfotiamine (1, 100, and 10 μM) or oxythiamine (10 and 1 μM). The cells were cultured with benfotiamine and oxythiamine after 24 and 48 h, and then analyzed using spectrophotometer.

To determine the glucose concentration after glucose metabolite in cell cultured and subjected to high-glucose challenge, the INS-1 cells were cultured with benfotiamine or oxythiamine in serum-free high glucose (25 mM, 4,500 mg/dL) DMEM medium containing 0.5% BSA. The cells were cultured after 5 h, cultured conditioned media tested for glucose concentration using Glucose Assay kit (Sigma) by following protocol.

Northern blot analysis

Total RNA was extracted from phosphate-buffered saline–washed INS-1 cells following high-glucose challenge with benfotiamine or oxythiamine, using RNA-Bee (Tel-Test, Inc., Friendswood, TX, USA) and purified per the manufacturer’s guidelines. Samples of RNA (25 μg/lane) where used for northern analysis. Hybridization was performed with insulin or GAPDH cDNA that had been labeled with [α-32 P] dCTP using the Amersham random primer kit (Amersham, Arlington Heights, IL, USA).

Western blot analysis and ELISA

Protein extract from INS-1 cells and BM-derived IPCs following high-glucose challenge with benfotiamine or oxythiamine, and this was saved along with the conditioned media. Intracellular insulin was detected by cell extraction with lysis buffer and western blotting, as detailed by Oh et al. [11]. Secretion of insulin into cultured media was detected by enzyme-linked immunosorbent assay (ELISA). ELISA was performed on the conditioned media to determine insulin secretion using the Rat insulin ELISA kit, and following the manufacturer’s instructions (Crystal Chem Inc., Chicago, IL, USA).

Treatment of hyperglycemic mice with BM-derived IPCs transplantation and benfotiamine

Hyperglycemia was induced in 8-week-old male NOD/scid mice (The Jackson Laboratory, Bar Harbor, ME, USA) through intraperitoneal injection of 40 mg/kg of streptozotocin (STZ) once a day for 5 consecutive days as described by Oh et al. [11]. Blood glucose levels were determined using a standard blood glucose meter (One touch profile, Johnson and Johnson Co., Milpitas, CA, USA).

Under general anesthesia mice received a renal subcapsular transplant of 150 BM-derived insuling-producing clusters (approximately >1,000 cells in a cluster) (n = 4), or sham transplant of saline solution (n = 4) in the right subcapsular renal space. Blood glucose levels were monitoried every 2 days after transplantation for 90 days. Also, BM-derived IPCs transplanted and nontransplanted mice were tested for blood glucose level using treatment of benfotiamine. Transplanted and nontransplanted animals received benfotiaminre (40 mg/kg body weight) using the gavage needle. Blood glucose levels were screened before treatment and 3 h after treatment.

The normal male mice were examined for blood glucose level after glucose challenge with or without treatment of benfotiamine. The mice were divided into three groups (n = 3), and were fasted 5 h before experiments. One group treated only benfotiamine (40 mg/kg body weight). Second group received glucose (1 g/kg body weight). Final group received benfotiamine (40 mg/kg) and glucose (1 g/kg) using the gavage needle. Blood glucose levels of all mice were screened before and every 30 min until 3 h after treatment. All results are expressed as the mean ± SD. Statistical differences were analyzed using Student’s t-test. p Values of <0.01 were considered to denote statistical significance.

Results

BM-derived cells differentiate into IPCs

Induction of BM-derived IPCs was achieved as previously described using specific in vitro conditions [11]. Using the BM cells cultured conditions, small spheroid clusters began to form at approximately day 7 (Fig. 1A). At day 10, under the same conditions, the number and dimension of these spheroid cell clusters were markedly increased (Fig. 1B). These three-dimensional cell growths morphoglogically resemble islet-like clusters, as previously described in several reports [3,11,14,29].

FIG. 1.

FIG. 1

Open in a new tab

Bone marrow (BM)-derived cells differentiate into insulin-producing cells. Bone marrow–derived cells cultured under conditions inducing an endocrine cell phenotype. Small clusters begin to form at day 7 (A) and continue to expand forming a tightly organized mass of cells at day 10 (B). Immunofluorescent staining for detection of insulin and C-peptide was performed on BM-derived clusters after 10 days in culture. Freshly isolated, noncultured BM cells (C) reveals no insulin. Normal pancreatic islet were stained insulin (D; green), C-peptide (E; red) and merged image (F; yellow). Bone marrow–derived clusters were expressed insulin (G; green), C-peptide (H; red) and merged image (I; Yellow). The counter-stained with DAPI (blue). Original magnification for A and B: 20×, and C–F 40× objective.

Double immunofluorescent analysis was performed for proinsulin, insulin, and C-peptide in BM-derived clusters. Freshly isolated, non-cultured BM cells did not show expression of insulin (red; Fig. 1C). Normal rat pancreatic islets strongly expressed insulin (green; Fig. 1D) and C-peptide (red; Fig. 1E). Also, approximately >90% of the cells in the BM-derived clusters were found to express insulin (green; Fig. 1G) and C-peptide (red; Fig. 1H). Finally, pancreatic islets and BM-derived clusters show storage of proinsulin in the cytoplasm (yellow; Fig. 1F and I). These results confirm that in vitro culture conditions used in this study were capable of inducing both insulin expression and C-peptide processing similar to that seen in pancreatic islet β-cells. The data presented in figure one corroborates our prevous report that BM-derived clusters were expressing other islet proteins, such as glucagon, somatostatin, and pancreatic polypeptide, this clusters are capable of insulin sectetion during high-glucose challenge [11].

Investigation of TK levels within BM-derived IPCs

The protein levels in non-differentiated and differentiating BM-derived cells, or BM-derived IPCs were examined by the SDS-PAGE. Specially, when BM cells were cultured with high-glucose DMEM with 10% FBS at days 5 to 13, several unique proteins appeared to be strongly expressed. We determined a molecular weight of ~60 kDa based on separation, and then sent for sequencing by the Protein Chemistry Core Facility, Biotechnology Program at the University of Florida. The sequencing results revealed that there were three protein products. The sequences were analyzed utilizing the NCBI database. The sequences were analyzed utilizing the NCBI database. The proteins were found to be Rattus norvegicus TK, R. norvegicus unnamed protein product, and Bostaurus albumin. The albumin was determined to be a result of contamination during BM cell culture with 10% FBS. Further confirmation of TK induction by BM-derived IPCs and INS-1 cells was analyzed by comparing the TK gene expression via RT-PCR (Fig. 2A). TK messenger RNA (mRNA) was not detected in cells exposed to either low-glucose DMEM with 1% DMSO (D1 and D3) or high-glucose DMEM with 10% FBS (D5, D7). However, TK mRNA was detected after 9, 11, and 13 days of exposure to high-glucose culture condition. Additonally, freshly isolated BM (D0) and INS-1 cells showed expression of the TK gene. This supports the fact that the fresh BM cells include erythrocytes, which are known to express the TK gene [22]. These results indicate that TK may be controlling the function of BM-derived IPCs and INS-1 cells.

FIG. 2.

FIG. 2

Open in a new tab

Detection of transketolase from BM-derived IPCs. (A) RT-PCR analysis for expression of transketolase and insulin I genes in INS-1 cells, freshly isolated BM-derived cells (D0), cultured BM-derived cells (D1 to D13). (B) Effect of benfotiamine or oxythiamine on INS-1 cells proliferation using MTT assay. To determine the level of cell proliferation, the INS-1 cells were cultured in INS-1 culture medium, with benfotiamine (1 mM, 100 and 10 μM) or oxythiamine (10 and 1μM). The MTT assay was performed on cells cultured for 24 and 48 h. Data represent the mean ± SD of five independent experiments. *p < 0.05 and **p < 0.01 compared with each experiment of glucose concentration (C) Test of glucose levels in INS-1 cells culture-conditioned media. The samples were collected from the INS-1 cells cultured in high-glucose (4,500 mg/dL) medium, with benfotiamine (1 mM, 100 and 10 μM) or oxythiamine (10 and 1 μM). The cells were cultured for 5 h after which culture-conditioned media was collected and assayed for glucose concentration in meida. The dotted line indicated basal glucose concentration (4,500 mg/dL). Data represent the mean ± SD of five independent experiments. a–dMeans within columns with different superscripts are different (at least p < 0.05).

Effect of cell proliferation and glucose metabolite in benfotiamine

Transketolase enzyme has been shown to have a function in the pentose phosphate pathway as well as in nucleoside biosynthesis [24]. Thus, the effect of TK activation or inhibition on INS-1 proliferation was investigated. Figure 2B shows that INS-1 cells cultured with TK activator benfotiamine demonstrated no significant change in proliferation and that this effect is not dose dependent; however, treatment with the TK inhibitor oxythiamine resulted in a significant decrease in proliferation after 24 h in culture. Furthermore, in INS-1 cells cultured with benfotiamine or oxythiamine at 48 h, high does of benfotiamine (1 mM), oxythiamine (10 μm and 1 μm) significantly inhibited cellular proliferation, indicating that benfotiamine is not an activator of proliferation.

We also determined the metabolic regulation of glucose in INS-1 cells cultured with benfotiamine or oxythiamine (Fig. 2C). To test the glucose concentration from culture-conditioned media, INS-1 were cells cultured in high glucose (control; 4,500 mg/dL), with benfotiamine (1 mM, 100 and 10 μM) or oxythiamine (10 and 1 μM) for 5 h. When INS-1 cells were cultured with high glucose at 5 h, the culture-conditioned medium glucose concentration was 4,203 ± 129.94 mg/dL. The cells cultured with 10 μM benfotiamine in high glucose showed a significant decrease in glucose concentration (3,979 ± 22.19 mg/dL). However, oxythiamine significantly inhibtied glucose metabolism. These results indicate that benfotiamine may activate glucose metabolism for IPCs.

Insulin and GAPDH gene expression with activation or inhibition of TK

The expression of insulin and GAPDH mRNA in INS-1 cells cultured with benfotiamine or oxythiamine was analyzed by Northern blot. Figure 3 shows expression of insulin message in cells cultured with either the activator or inhibitor. The TK activator, benfotiamine, activated the insulin gene when added to culture at concentrations of 10 and 100 μM, and even as high as 1 mM. Conversely, when the TK inhibitor, oxythiamine, was added at 10 μM, insulin gene was downregulated. However, low concentrations of oxythiamine (1 μM) did not affect message levels. Furthermore, downstream of the metabolic pathway, treatment with befotiamine activated GAPDH message in a dose-dependent manner. When oxythiamine (10 μM) was used GAPDH was downregulated, but low concentrations of oxythiamine did not have an effect on GAPDH levels. These results indicate that activation of TK by benfotiamine could be playing a significant role in upregulation of both glucose metabolism and insulin protein synthesis.

FIG. 3.

FIG. 3

Open in a new tab

Northern blot analysis for experssion of insulin message in INS-1 cells cultured with benfotiamine or oxythiamine. Insulin and GAPDH mRNA levels in INS-1 cells cultured in high-glucose medium, with benfotiamine (1 mM, 100 and 10 μM) or oxythiamine (10 and 1 μM). 18S and 28S were used as a loading control. Data shown represent one of three experiments with similar results.

Protein analysis of INS-1 cells

Synthesis of insulin during glucose challenge was examined using Western blot analysis in INS-1 cells treated with benfotiamine or oxythimaine (Fig. 4). During glucose challenge, INS-1 cells cultured with benfotiamine activated insulin synthesis at 100, 10 μM; high concentrations of benfotiamine were not shown to activate insulin synthesis. Conversely, when oxythamine was present in the culture, insulin was not detected at high concentrations (10 μM) or shown to be inactivated by lower concentrations of oxythiamine (1 μM) (Fig. 4). Glucose transport protein, Glut 2 was not activated by benfotiamine dose dependently, but oxythiamine treatment shows inhibition of Glut 2. The conversion of glucose to glucose-6-phosphate, catalyzed by glucoskinase (GCK), was upregulated with 100, 10μM benfotiamine treatment. However, cell culture with oxythiamine (10 and 1 μM) shows inactivated of GCK. In addition to Western blot analysis, an ELISA assay was performed to quantify insulin secretion in the media. INS-1 cells cultured under high-glucose conditions secreted 450 ± 23 ng/mL of insulin into media over a 2-h period (Fig. 5A). INS-1 cells cultured with 100 or 10 μM benfotiamine secreted 550 ± 17 ng/mL and 620 ± 34 ng/mL of insulin, respectively. When cells were exposed to oxythimaine at a low concentration (1 μM) insulin secretion was greatly decreased (170 ± 32 ng/mL), and a higher level (10 μM) insulin levels were undetectable (Fig. 5A). Furthermore, when BM-derived IPCs were challenged with high-glucose condition, 145 ± 34 ng/mL of secreted insulin was detected. Also, culturing BM-derived IPCs in the presence of benfotiamine increased secreted insulin levels to 2978 ± 43 ng/mL. On the other hand, oxythiamine treatment did not yield secretion in insulin (Fig. 5B). These results indicate that benfotiamine increases insulin synthesis from IPCs through the pentose phosphate pathway.

FIG. 4.

FIG. 4

Open in a new tab

Western blot analysis of insulin protein expression in INS-1 cells cultured with benfotiamine or oxythiamine. Western blot analysis of insulin, glucose transporter (Glut-2) and glucokinase (GCK) measured following collection of cell lysates. INS-1 cells were cultured in high-glucose medium, with benfotiamine (1 mM, 100 and 10 μM) or oxythiamine (10 and 1 μM) for 2 h. Actin was used as a loading control. Data shown represent one of three experiments with similar results.

FIG. 5.

FIG. 5

Open in a new tab

Determination in insulin secretion into media following treatment with benfotiamine or oxythiamine. ELISA analysis of insulin secretions measured following collection of cell culture-conditioned media. (A) INS-1 cells cultured in high-glucose medium, with benfotiamine (1 mM, 100 and 10 μM) or oxythiamine (10 and 1 μM) for 2 h. Data represent the mean ± SD of four independent experiments. (B) BM-derived IPCs cultured in high-glucose medium, with benfotiamine (10 μM) or oxythiamine (10 μM) for 2 h. Data represent the mean ± SD of four independent experiments.

Effect of benfotiamine on BM-derived IPCs transplanted into hyperglycemic mice

The ability of IPCs to reverse hyperglycemia was examined in vivo using a STZ-induced hyperglycemia NOD/scid mouse model. Two groups of hyperglycemic mice were prepared; one group served as a control and the other was transplanted with BM-derived IPCs. The mice that received the transplant began normalizing their blood glucose levels within 2–3 days (Fig. 6A). Approximately 9 days after transplantation, their blood glucose levels were 216 ± 16 mg/dL and levels in nontransplanted animals were at 537 ± 45.8 mg/dL. Our results suggest that the BM-derived IPCs have a function in insulin secretion. However, BM-derived IPCs transplanted mice blood glucose levels were still high compared to normal mice (~100 mg/dL). We hypothesize that benfotiamine treatment rapidly leads to normalized blood glucose levels in transplanted mice following activation of glucose metabolism and also enhances insulin synthesis from BM-derived IPCs.

FIG. 6.

FIG. 6

Open in a new tab

Benfotiamine treatment of hyperglycemic mice transplanted with BM-derived IPCs. (A) Blood glucose levels of STZ-treated hyperglycemic NOD/scid mice transplanted with BM-derived IPCs. To ascertain the function, BM-derived IPCs were transplanted into chemically induced diabetic mice and subsequently the changes of blood glucose levels were determined. STZ treated mice were transplanted with saline (non-Tx; n = 4), or transplanted with ~150 BM-derived IPCs (BM-derived IPCs Tx; n = 4). As seen in the graph, nontreated mice between hyperglycemic and died. (B) Effect of benfotiamine on blood glucose level in benfotiamine treatment of transplanted BM-derived IPCs into hyperglycemic mice. The transplanted and nontransplanted mice were analyzed for blood glucose level before (white bars) and 3 h after (black bars) the benfotiamine treatment (arrows indicated treatment of benfotiamine in Fig. 6A). The data are represented as mean ± SD of blood glucose levels. *p < 0.01 of blood glucose levels compared before and after the treatment of benfotiamine. (C) Blood glucose level in glucose challenge with benfotiamine into normal mice. Glucose challenges were treated 1 g/kg body weight using gavage needle (black bars), benfotiamine alone (blank bars), and glucose with benfotiamine (dotted bars). The data are represented as mean ± SD of blood glucose levels. *p < 0.01 and **p < 0.001 compared with each time point in animals blood glucose level.

We examined the effect on blood glucose levels when animals were transplanted with BM-derived IPCs and treated with benfotiamine (Fig. 6A, arrow indicates treatment with benfotiamine; 40 mg/kg, n = 5). Non-transplanted animals, with or without benfotiamine treatment, maintained high blood glucose levels (467.1 ± 43.3 mg/dL and 446.4 ± 38.9 mg/dL respectively). These animals eventually died. However, hyperglycemic mice transplanted with BM-derived IPCs dropped their blood glucose levels to 185.1 ± 20 mg/dL. Furthermore, when these mice were subsequently treated with benfotiamine, their glucose levels further dropped to 127.8 ± 17.1mg/dL (Fig. 6B), significantly. In the animals not treated with benfotiamine, blood glucose levels rose to 167.4 ± 29.7 mg/dL after 21–96 days. Unfortunately, however, these transplanted mice had increased glucose levels and eventually died after 96 days. These data suggest that transplantation of BM-derived IPCs, combined with benfotiamine treatment, lead to increased insulin secretion from the transplanted cells.

In addition, we examined the effect of benfotiamine in normal mice alone and in conjunction with glucose challenge (Fig. 6C). Treatment with benfotiamine alone did not affected blood glucose levels; however, blood glucose levels were increased in mice after treatment with glucose (1 g/kg). Thirty minutes after treatment, mice showed an increase in blood glucose levels to 176.3 ± 18.8 mg/dL. Similarly, mice treated with benfotiamine prior to glucose challenge also increased blood glucose levels to 176.3 ± 1.1 mg/dL. The glucose challenged mice that did not receive benfotiamine maintained elevated levels of blood glucose 60 min after challenge (158.7 ± 6.2 mg/dL) (Fig. 6C). In contrast, glucose challenged mice treated with benfotiamine more rapidly normalized blood glucose levels (124.7 + 5.1 mg/dL, 60 min after challenge) (Fig. 6C). These data suggest that benfotiamine leads to increased glucose metabolism and insulin synthesis from normal IPCs or transplanted BM-derived IPCs.

Discussion

The data presented demonstrate that adult BM cells can differentiate into indocrine-like cells, capable of producing and secreting insulin under high-glucose conditions. The TK enzyme was detected in BM-derived IPCs under high-glucose culture conditions. When INS-1 cells and BM-derived IPCs were cultured with the TK activator benfotiamine, insulin expression was activated. Furthermore, INS-1 cells cultured with benfotiamine in high-glucose medium activated the glycolytic enzyme GAPDH as well as glucokinase, which coverts glucose to glucose-6-phosphate. When treated with benfotiamine, hyperglycemic mice transplanted with BM-derived IPCs clusters normalized their blood glucose levels. In addition, normal mice under glucose challenge treated with benfotiamine showed rapid normalization of blood glucose levels. Also, previous reports demonstrated that benfotiamine blocks three major pathway of hyperglycemic damage and prevent diabetic retinopathy and nephropathy [25,26]. Taken together, our results indicate that benfotiamine treatment inconjunction with transplantation of BM-derived IPCs may be a useful treatment for diabetic mellitus by activating glucose metabolism.

Several studies indicate that the pentose phosphate pathway controls insulin secretion through glucose phosphorylation [29-31]. When INS-1 cells were cultured with benfotiamine cell proliferation was not enhanced; however, oxythiamine was shown to inhibit cell proliferation by blocking the pentose phosphate pathway (Fig. 2B). During INS-1 cell culture for glucose metabolism, benfotiamine activated glucose metabolism as compared to control and oxythiamine treatment (Fig. 2C). Also, the glycolytic enzyme GAPDH was activated in INS-1 cells cultured with benfotiamine, another indication that benfotiamine enhanced glucose metabolism (Fig. 3). These data indicate that benfotiamine activated glucose metabolism, potentially alleviating the glucose toxicity caused by diabetic mellitus. Furthermore, it has been shown that superoxide partially inhibits the glycolytic enzyme GAPDH, thereby diverting upstream metabolites from glycolysis into the four major glucose-driven signaling pathways that cause hyperglycemic damage [20,21,32].

Previous reports have indicated that benfotiamine activates the thiamine-dependent pentose phosphate pathway enzyme TK, which converts excess fructose-6-phophate and glyceraldehyde-3-phosphate [26]. However, our data show that benfotiamine also activates the conversion of excess glucose to glucose-6-phosphate (Fig. 4). These data indicate that benfotiamine results in activation of GCK for glucose metabolism, allowing high glucose levels to be brought down to normal levels in diabetes mellitus and thereby preventing diabetic complication. Quantitation of glucose-6-phosphate levels as well as glucose uptake studies in the IPCs will be required to ensure that this process in increasing glucose metabolism and insulin synthesis.

When INS-1 cells were cultured with benfotiamine, the insulin gene was upregulated (Figs. 3 and 4), supporting the notion that benfotiamine plays a controlling role in IPCs with regard to insulin biosynthesis through glucose metabolism. When IPCs were cultured with oxythiamine, TK activity and insulin gene expression was suppressed (Figs. 3-5). These data indicate that TK mediates insulin synthesis and secretion through the pentose phosphate pathway after glucose challenge. In Figure 5, benfotiamine activated insulin secretion in the INS-1 cells and BM-derived IPCs; however, the results indicate that the level of insulin activation is different between the two cell types. There are at least two possible interpretations on the results: (1) The INS-1 cells are producing insulin, so that total amount of insulin in the media might over saturate the sensitivity of the assay. In the BM-derived IPCs, there is only a fraction of the cells producing insulin, so the total amount of insulin in the media does not overwhelm the assay and the addition of benfotiamine gives rise to a more significant reading. (2) The BM-derived IPCs are heterogeneous population and therefore it is possible that benfotiamine might have effects not only on insulin-secreting cells but also on other cell types within these BM-derived IPCs. However, the mechanism by which benfotiamine regulates insulin synthesis (i.e., directly or indirectly) is still unclear and will require further characterization.

Previous reports have described that mesenchymal cells, also known as multipotent adult progenitor cells, isolated from adult BM can be kept in culture for an extended period of time and can differentiate into both hepatic and neural lineages [33,34]. Our data demonstrate that adult BM cells can differentiate into endocrine cells capable of producing and secreting physiologically active insulin after upregulation of TK under high-glucose culture conditions. Recently several studies have shown that embryonic stem cells have the ability to become β-like IPCs [17-19]. However, Fujikawa et al. [19] observed that embryonic stem cell–derived IPCs transplanted into hyperglycemic mice from tumors, deriving possibly from contamination of undifferentiated embryonic stem cells or the mature IPCs, and were able to revert back to an embryonic phenotype.

Previously we reported that manipulation of BM cells to IPCs can be accomplished with relative ease [11]. The differentiation method, as presented, requires only 10 days from BM isolation to the formation of IPC clusters, thus providing an accessible cell source and a simple method for the cellular treatment of diabetes. Bone marrow–derived IPCs transplanted into chemically induced hyperglycemic mice were shown to have normal blood glucose levels for 90 days [11]. Additionally, when benfotiamine was used in glucose challenged mice, their blood glucose level rapidly normalized when compared to glucose challenge alone (Fig. 6C). These results indicate that the mechanism by which blood glucose levels are reduced upon treatment with benfotiamine is the upregulation of TK [26].

In conclusion, these findings provide evidence that BM-derived IPCs and INS-1 cells are activated to produce insulin through upregulation of the TK enzyme. Therefore benfotiamine potentially prevents diabetic complications by mediating activation of glucose metabolism. The BM-derived IPCs can function as IPCs both in vivo and in vitro; however, it may be clinically relevant that these cells may not be fully mature β-cells, thereby alleviating the activation of the immune system. Additional characterization of BM-derived IPCs willl be required to determine it this is indeed the case. Our results suggest that BM cells should be a powerful tool for the study of pancreatic development and function, as well as offering a new potential treatment for type 1 diabetes.

Acknowledgements

B.E.P. is inventor of a patent(s) related to this technology and may benefit from royalties paid to the University of Florida related to its commercialization. National Institute of Health grants DK60015 and DK58614 awarded to B.E.P. funded this research.

References

  • 1.Guz Y, Nasir I, Teitelman G. Regeneration of pancreantic beta cells from intra-islet precursor cells in an experimental model of diabetes. Endocrinology. 2001;142:4956–4968. doi: 10.1210/endo.142.11.8501. [DOI] [PubMed] [Google Scholar]
  • 2.Shapiro AM, Lakey JR, Ryan EA, Korbutt GS, Toth E, Warnock GL, Kneteman NM, Rajotte RV. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med. 2000;343:230–238. doi: 10.1056/NEJM200007273430401. [DOI] [PubMed] [Google Scholar]
  • 3.Bonner-Weir S, Taneja M, Weir GC, Tatarkiewicz K, Song KH, Sharma A, O’Neil JJ. In vitro cultivation of human islets from expaned ductal tissue. Proc Natl Acad Sci USA. 2000;97:7999–8004. doi: 10.1073/pnas.97.14.7999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Halvorsen T, Levine F. Diabetes mellitus-cell transplantation and gene therapy approaches. Curr Mol Med. 2001;1:273–286. doi: 10.2174/1566524013363951. [DOI] [PubMed] [Google Scholar]
  • 5.Weissman IL. Translating stem and progenitor cell biology to the clinic: barriers and opportunities. Science. 287:1442–1446. doi: 10.1126/science.287.5457.1442. 200. [DOI] [PubMed] [Google Scholar]
  • 6.Petersen BE, Bowen WC, Patrene KD, Mars WM, Sullivan AK, Murase N, Boggs SS, Greenberger JS, Goff JP. Bone marrow as a potential source of hepatic oval cells. Science. 1999;284:1168–1170. doi: 10.1126/science.284.5417.1168. [DOI] [PubMed] [Google Scholar]
  • 7.Theise ND, Badve S, Saxena R, Henegariu O, Sell S, Crawford JM, Krause DS. Derivation of hepatocytes from bone marrow cells in mice after radiation-induced myeloablation. Hepatology. 2000;31:235–240. doi: 10.1002/hep.510310135. [DOI] [PubMed] [Google Scholar]
  • 8.Oh SH, Miyazaki M, Kouchi H, Inoue Y, Sakaguchi M, Tsuji T, Shima N, Higashio K, Namba M. Hepatocyte growth factor induces differentiation of adult rat bone marrow cells into a hepatocyte lineage in vitro. Biochem Biophys Res Commun. 2000;279:500–504. doi: 10.1006/bbrc.2000.3985. [DOI] [PubMed] [Google Scholar]
  • 9.Oh SH, Witek RP, Bae SH, Zheng D, Jung Y, Piscaglia AC, Petersen BE. Bone marrow is the source of hepatic oval cells for hepatocytes differentiation in the 2AAF/PHx liver regeneration model. Gastroenterology. 2007;132:1077–1087. doi: 10.1053/j.gastro.2007.01.001. [DOI] [PubMed] [Google Scholar]
  • 10.Ianus A, Holz GG, Theise ND, Hussain MA. In vivo derivation of glucose-competent pancreatic endocrine cells from bone marrow without evidence of cell fusion. J Clin Invest. 2003;111:843–50. doi: 10.1172/JCI16502. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Oh SH, Muzzonigro TM, Bae SH, LaPlante JM, Hatch HM, Petersen BE. Adult bone marrow-derived cells trans-differentiating into insulin-producing cells for the treatment of type I diabetes. Lab Invest. 2004;84:607–617. doi: 10.1038/labinvest.3700074. [DOI] [PubMed] [Google Scholar]
  • 12.Kotton DN, Ma BY, Cardoso WV, Sanderson EA, Summer RS, Williams MC, Fine A. Bone marrow-derived cells as progenitors of lung alveolar epithelium. Development. 2001;128:5181–5188. doi: 10.1242/dev.128.24.5181. [DOI] [PubMed] [Google Scholar]
  • 13.Hashimoto N, Jin H, Liu TJ, Chensue SW, Phan SH. Bone marrow-derived progenitor cells in pulmonary fibrosis. J Clin Invest. 2004;113:243–252. doi: 10.1172/JCI18847. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Yang L, Li S, Hatch MH, Ahrens K, Cornelius JG, Petersen BE, Peck AB. In vitro trans-differentiation of adult hepatic stem cells into pancreatic endocrine hormone-producing cells. Proc Natl Acad Sci USA. 2002;99:8078–8083. doi: 10.1073/pnas.122210699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Kodama S, Kuhtreiber W, Fujimura S, Dale EA, Faustman DL. Islet regeneration during the reversal of autoimmune diabetes in NOD mice. Science. 2003;302:1223–1227. doi: 10.1126/science.1088949. [DOI] [PubMed] [Google Scholar]
  • 16.Tuch BE, Szymanska B, Yao M, Tabiin MT, Gross DJ, Holman S, Swan MA, Humphrey RK, Marshall GM, Simpson AM. Function of a genetically modified human liver cell line that stores, processes and secretes insulin. Gene Ther. 2003;10:490–503. doi: 10.1038/sj.gt.3301911. [DOI] [PubMed] [Google Scholar]
  • 17.Lumelsky N, Blondel O, Laeng P, Velasco I, Ravin R, McKay R. Differentiation of embryonic stem cells to insulin-secreting structures similar to pancreatic islets. Science. 2001;292:1389–1394. doi: 10.1126/science.1058866. [DOI] [PubMed] [Google Scholar]
  • 18.Soria B, Roche E, Berná G, León-Quinto T, Reig JA, Martín F. Insulin-secreting cells derived from embryonic stem cells normalize glycemia in streptozotocin-induced diabetic mice. Diabetes. 2000;49:157–162. doi: 10.2337/diabetes.49.2.157. [DOI] [PubMed] [Google Scholar]
  • 19.Fujikawa T, Oh SH, Pi L, Hatch HM, Shupe T, Petersen BE. Teratoma formation leads to failure of treatment for type I diabetes using embryonic stem cell-derived insulin-producing cells. Am J Pathol. 2005;166:1781–1791. doi: 10.1016/S0002-9440(10)62488-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Du XL, Edelstein D, Rossetti L, Fantus IG, Goldberg H, Ziyadeh F, Wu J, Brownlee M. Hyperglycemia-induced mitochondrial superoxide overproduction activates the hexosamine pathway and induces plasminogen activator inhibitor-1 expression by increasing Sp1 glycosylation. Proc Natl Acad Sci USA. 2000;97:12222–12226. doi: 10.1073/pnas.97.22.12222. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Brownlee M. Biochemistry and molecular cell biology of diabetic complications. Nature. 2001;414:813–820. doi: 10.1038/414813a. [DOI] [PubMed] [Google Scholar]
  • 22.Nicholas JT. Applications of transketolases in organic synthesis. Biotechnology. 2000;11:527–531. doi: 10.1016/s0958-1669(00)00140-3. [DOI] [PubMed] [Google Scholar]
  • 23.Saito N, Kimura M, Kuchiba A, Itokawa Y. Blood thiamine levels in outpatients with diabetes mellitus. J Nutr Sci Vitamionl (Tokyo) 1987;33:421–430. doi: 10.3177/jnsv.33.421. [DOI] [PubMed] [Google Scholar]
  • 24.Loew D. Phamacokinetics of thiamine derivatives especially of benfotiamine. Int J Clin Pharmacol Ther. 1997;34:47–50. [PubMed] [Google Scholar]
  • 25.Babaei-Jadidi R, Karachalias N, Ahmed N, Battah S, Thornalley PJ. Prevention of incipient diabetic nephropathy by high-dose thiamine and benfotiamine. Daibetes. 2003;52:2110–2120. doi: 10.2337/diabetes.52.8.2110. [DOI] [PubMed] [Google Scholar]
  • 26.Hammes HP, Du X, Edelstein D, Taguchi T, Matsumura T, Ju Q, Lin J, Bierhaus A, Nawroth P, Hannak D, Neumaier M, Bergfeld R, Giardino I, Brownlee M. Benfotiamine blocks three major pathways of hyperglycemic damage and prevents experimental diabetic retinopathy. Nat Med. 2003;9:294–299. doi: 10.1038/nm834. [DOI] [PubMed] [Google Scholar]
  • 27.Boros LG, Puigjaner J, Cascante M, Lee WN, Brandes JL, Bassilian S, Yusuf FI, Williams RD, Muscarella P, Melvin WS, Schirmer WJ. Oxythiamine and dehydroepiandrosterone inhibit the nonoxidative synthesis of ribose and tumor cell proliferation. Cancer Res. 1997;57:4242–4248. [PubMed] [Google Scholar]
  • 28.Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983;65:55–63. doi: 10.1016/0022-1759(83)90303-4. [DOI] [PubMed] [Google Scholar]
  • 29.Ramiya VK, Maraist M, Arfors KE, Schatz DA, Peck AB, Cornelius JG. Reversal of insulin-dependent diabetes using islets generated in vitro from pancreatic stem cells. Nat Med. 2000;63:278–282. doi: 10.1038/73128. [DOI] [PubMed] [Google Scholar]
  • 30.Escolar JC, -Paris Hoo R, Castex C, Sutter BC. Effect of low temperatures on glucose-induced insulin secretion and glucose metabolism in isolated pancreatic islet of the rat. J Endocrinol. 1990;125:45–51. doi: 10.1677/joe.0.1250045. [DOI] [PubMed] [Google Scholar]
  • 31.Verspohl EJ, Breuning I, Ammon HP. Effect of CCK-8 on pentose phosphate shunt activity, pyridine nucleotides, and glucokinase of rat islets. Am J Physiol. 1989;256:E68–73. doi: 10.1152/ajpendo.1989.256.1.E68. [DOI] [PubMed] [Google Scholar]
  • 32.Hawthorne GC, Alberti KG. The effect of high glucose and high insulin concentration on pentose phosphate shunt enzymes and malic enzyme in cultured human endothelial cells. Horm Metab Res. 1988;20:645–647. doi: 10.1055/s-2007-1010906. [DOI] [PubMed] [Google Scholar]
  • 33.Jiang Y, Jahagirdar BN, Reinhardt RL, Schwartz RE, Keene CD, Ortiz-Gonzalez XR, Reyes M, Lenvik T, Lund T, Blackstad M, Du J, Aldrich S, Lisberg A, Low WC, Largaespada DA, Verfaillie CM. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature. 2002;418:41–49. doi: 10.1038/nature00870. [DOI] [PubMed] [Google Scholar]
  • 34.Reyes M, Dudek A, Jahagirdar B, Koodie L, Marker PH, Verfaillie CM. Origin of endothelial progenitors in human postnatal bone marrow. J Clin Invest. 2002;109:337–346. doi: 10.1172/JCI14327. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES