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© 2004 The American Society for Nutritional Sciences J. Nutr. 134:3257-3263, December 2004

Biochemical and Molecular Actions of Nutrients

Soluble Fibroin Enhances Insulin Sensitivity and Glucose Metabolism in 3T3-L1 Adipocytes1,2

Chang-Kee Hyun, Il-Yong Kim and Susan C. Frost*,3

School of Bioscience and Food Technology, Handong Global University, Pohang, South Korea and * Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, Gainesville, FL

3To whom correspondence should be addressed. E-mail: sfrost{at}ufl.edu.


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Type 2 diabetes is characterized by hyperglycemia and hyperinsulinemia, features of insulin resistance. In vivo treatment of ob/ob mice with hydrolyzed fibroin reverses these pathological attributes. To explore the mechanism underlying this effect, we used the murine, 3T3-L1 adipocyte cell line, which has been used extensively to model adipocyte function. Chronic exposure of 3T3-L1 adipocytes to insulin leads to a 50% loss of insulin-stimulated glucose uptake. Chronic exposure to different preparations of fibroin partially blocked the response to insulin but also increased the sensitivity of control cells to the acute action of insulin. The latter effect was most robust at physiologic concentrations of insulin. Fibroin did not prevent the insulin-induced downregulation of the insulin receptor or the tyrosine kinase activity associated with the receptor. Further, fibroin had no effect on the activity of the insulin-sensitive downstream kinase, Akt. Interestingly, fibroin accelerated glucose metabolism and glycogen turnover independent of insulin action. In addition, fibroin upregulated glucose transporter (GLUT)1, which increased its expression at the cell surface and enhanced GLUT4 translocation. Together, these phenomena may underlie the improvement in diabetic hyperglycemia noted in vivo in response to fibroin.

KEY WORDS: • insulin resistance • 3T3-L1 adipocytes • GLUT1 • GLUT4 • fibroin

Diabetes and its complications are a major cause of morbidity and mortality in the United States. The prevalence of diabetes over the past 30 years has greatly increased and now affects 12% of the adult population (1). Two major types of diabetes have been described, referred to as type I diabetes (formerly called insulin-dependent diabetes) and type 2 diabetes (formerly called noninsulin-dependent diabetes). Type 1 diabetes results from the destruction of pancreatic ß-cells leading to absolute insulin deficiency. Type 2 diabetes results from insulin resistance with only relative insulin deficiency. Most patients with this latter form of diabetes are obese, and obesity itself causes some degree of insulin resistance (2). Glycemic control is considered the key to preventing and/or delaying the long-term complications of type 2 diabetes, including damage to blood vessels (3) and peripheral nerves (4), which increases the risk of heart attack, stroke, blindness, and kidney failure. Both pharmacologic (5) and nutritional (6) supplements have been used to regulate blood glucose. In the present study, we tested the effect of soluble fibroin protein on the insulin sensitivity of 3T3-L1 adipocytes.

Silk fibroin consists of heavy (H) and light (L) chain polypeptides of ~390 and 25 kDa, respectively (7). These are linked by a single disulfide bond between cysteine residues in the C-termini of each of these proteins. In addition, a 3rd polypeptide (P25) associates with the H-L complex primarily by hydrophobic interactions. The 2 smaller proteins appear to function as chaperones for the secretion process although they are contained in the final water-insoluble fiber that comprises the silk cocoon. The complete amino sequence of the heavy chain has now been deduced from the 17-kbp fibroin gene (GenBank AF226688) isolated from Bombyx mori (8). Most of the sequence is of low complexity and forms 2377 repeats of a GlyX dipeptide motif. The residue X is Ala in 64% of the repeats, Ser in 22%, Tyr in 10%, Val in 3%, and Thr in 1.3% of the repeats. Because of its structural features, fibroin has been used as a cellular matrix (9), to immobilize enzymes (10), and as an oral adjuvant (11). Biological effects have also been reported. For example, lowered cholesterol levels were observed in fibroin-fed rats (12) and anti-HIV activity was detected with sulfated fibroin treatment (13). In addition, fibroin peptides were shown to prevent DNA damage (14).

In 1995 Nahm and Oh (15) demonstrated that fibroin hydrolysates decreased both blood glucose and insulin in ob/ob mice. The mechanism underlying this antidiabetic activity was not explored. The purpose of the present study was to test the effect of fibroin in 3T3-L1 adipocytes to better understand its molecular action.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
    Materials. Unless specifically mentioned, reagents were purchased from Sigma-Aldrich. Antibodies against glucose transporter (GLUT)1,4 GLUT4, and the insulin receptor were prepared in our laboratory and evaluated as previously described (16–18). Antibodies against phospho-Akt were purchased from Santa Cruz (ser473) or Cell Signaling (thr308). Antibodies against phosphotyrosine were purchased from Transduction Laboratories.

    Fibroin preparations. Fibroin from cocoons of the silkworm, B. mori, was supplied by Aminogen. Sericin, the other major silk protein, was removed as previously described (14). Briefly, 50 g of cocoon material was cut into small pieces and boiled in 2.5 L of 5% Na2CO3 (i.e., 50 g/L) for 1 h and then passed through filter paper. The residue, which is mainly fibroin, was washed 3 times with hot distilled H2O to remove any remaining sericin. The fibroin was then solubilized by heating 35 g of the residue in 0.346 L of distilled water and 0.28 L of ethanol containing 226.4 g CaCl2 at 90°C for 1 h. The solution was dialyzed against distilled H2O for 2 d and then lyophilized. This preparation constituted fibroin protein (FP). To prepare the hydrolysate, fibroin was dissolved in an 80-fold volume of HCl (2 mol/L) and boiled for 4 h. The brown-colored hydrolysate was neutralized to pH 7.4 with NaOH (2 mol/L) and dialyzed against distilled water for 1 d and lyophilized. The efficiency of hydrolysis was ~75%. The unhydrolyzed fibroin was removed by centrifugation through a centrifugal filter membrane with a molecular weight cutoff of 5000. This preparation is referred to as the fibroin hydrolysate (FH). To separate bioactive fractions from the hydrolysate, ~0.3 g dissolved in water was applied to a Sephadex G-25 column (2 x 110 cm) and eluted with water at a flow rate of 0.6 mL/min. Three fractions were noted: Fraction I (mw ~ 5000); Fraction II (mw ~ 3000); and Fraction III (mw ~ 1000). Fraction III contained ~30% of the total peptide eluted from the column and is referred to as peptide fraction III (FIII). Further analysis of this fraction by Sephadex G-15 chromatography indicated that it was composed of at least 4 peptide fragments. These fragments were not individually collected or further analyzed because of limited quantities. The amino acid compositions of FP and FIII have been reported (14). Of note is the high concentration of glycine and alanine (73 and 80%, respectively, for FP and FIII).

    Cell culture and induction of insulin resistance. 3T3-L1 fibroblasts were grown and differentiated as previously described (19). 3T3-L1 adipocytes were treated or not with FP, FH, or FIII at specific concentrations (0.1–10 g/L) for the times noted in the figure legends. To induce insulin resistance, cells were chronically exposed to insulin (10 nmol/L) for 12 h in the presence or absence of fibroin. Insulin resistance was also induced with 500 pmol/L tumor necrosis factor (TNF){alpha} (R&D Systems, 410-MT) for 96 h in the presence or absence of fibroin. Before the glucose transport assay or in the subcellular fractionation studies, cells were washed 3 times with 3.0 mL Krebs Ringer phosphate buffer containing glucose (5mmol/L) and bovine serum albumin (BSA; 1g/L; Sigma, #7050) at 40-min intervals over a 2-h period

    Glucose transport assay. Cytochalasin B–inhibitable glucose transport was assayed as previously described (17). In some experiments, the concentration of insulin in short-term incubation (acute) was varied from 0.01 to 1000 nmol/L.

    Insulin receptor immunoprecipitation and detection. The insulin receptor was immunoprecipitated as previously described (18). Proteins released from Protein A Sepharose beads were separated by 7.5% SDS-PAGE followed by transfer to nitrocellulose. Blots were screened for total and tyrosine-phosphorylated receptor.

    Analysis of Akt. To test for the involvement of Akt, cells were extracted in buffer containing NaCl (140 mmol/L), Tris-base (20 mmol/L), EDTA (1 mmol/L), NaF (100 mmol/L), Na2PO4 (10 mmol/L), Na3VO4 (1 mmol/L), phenylmethylsulfonyl fluoride (1 mmol/L), protease inhibitor cocktail (Sigma), NP-40 (10 g/L), pH 7.5. The extract was mixed at 4°C for 30 min and clarified by centrifugation at 16,000 x g at 4°C for 10 min. Proteins in the clarified supernatant were separated by SDS-PAGE and transferred to nitrocellulose. Samples were not heated because this caused aggregation of Akt. The concentration of total Akt was compared against the phosphorylated pool (serine473 and threonine308) by Western blot analysis.

    Membrane isolation. Total membranes were prepared as described by Kitzman et al. (20) and subcellular membranes were isolated from cells as previously described (21). Proteins were separated by SDS-PAGE and transferred to nitrocellulose for Western blot analysis.

    Western blotting. For GLUT1, GLUT4, and the insulin receptor, the Western blotting procedure was described previously (17,18,20). For the detection of pAkt, the procedure varied from the published procedure in that the primary antibody was diluted in Tris-buffered saline-Tween (TBS-T) containing BSA (50 g/L) and incubation was carried out overnight as 4°C. The secondary antibody was also diluted in TBS-T with BSA. Analysis of bands was performed by video densitometry (Visage, Millipore) in the linear range of the film based on the optical density of the integrated area of the bands.

    Northern blot analysis. mRNA was analyzed as previously described (22).

    Statistical analysis. Numerical data are reported as means ± SEM with the number of replicates indicated in the figure legends. Each graphical point represents an independent data set whether assayed for time or treatment. For comparison between 2 groups with 1 independent variable, significance was assessed using the unpaired Student’s t test (Sigma Plot, version 8). For >2 independent variables, 1-way ANOVA was applied (using the GLM procedure of SAS, vs 8.2) setting {alpha} at 0.05. Post-hoc analysis of treatment groups was conducted using Student-Newman-Keuls test for multiple comparisons.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
    Effect of fibroin on glucose uptake and metabolism. Previous studies showed that the glucose transport system in 3T3-L1 adipocytes is exquisitely sensitive to the addition of acute pharmacologic doses of insulin (1 µmol/L) (19). In the present experiments, insulin addition increased glucose uptake by 21-fold (Fig. 1). Confirming our earlier observation (17), chronic exposure to a lower concentration of insulin (10 nmol/L) reduced insulin-sensitive glucose uptake by 50% (P < 0.00001). We, and others (23), have defined this as insulin-induced insulin resistance.



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  		FIGURE 1 Soluble fibroin enhances insulin-dependent glucose uptake and blocks insulin resistance in 3T3-L1 adipocytes. 3T3-L1 adipocytes were exposed to 5 g/L FP, FIII, or FH for 24 h in the absence (Panel A) or presence (Panel B) of chronic exposure to insulin (10 nmol/L) for the final 12 h. Cells were then washed and glucose transport activity measured in the absence or presence of acute exposure to insulin (1 µmol/L). Each data point in a given experiment was done in duplicate. The duplicates were then averaged and compiled with the means of replicate experiments (n). For controls, n = 7; for FP, n = 5; for FIII, n = 3, for FH, n = 4. The data represent the mean of cytochalasin B-inhibitable uptake ± SEM. Panel A: ANOVA; F7,28 = 206.04, P < 0.0001. Panel B: ANOVA, F7,25 = 104.57, P < 0.0001. Means in a graph without a common letter differ, P < 0.05.

 
With this model system, we tested the effect of chronic exposure to fibroin protein (FP), peptide fraction III (FIII), and fibroin hydrolysate (FH) on insulin-stimulated glucose transport in control (Fig. 1A) and insulin-resistant cells (Fig. 1B). In control cells, no preparation of fibroin had an effect on basal glucose uptake. However, each preparation of fibroin significantly enhanced insulin-stimulated glucose transport activity compared with the absence of any fibroin (FIII = FH > FP). In subsequent experiments in which the effect of fibroin was tested under conditions of acute insulin exposure, the use of FIII and FH was favored and they were used interchangeably. In cells chronically exposed to insulin (insulin-resistant cells), again no preparation of fibroin affected basal glucose uptake. Again, all 3 preparations significantly blocked the loss of glucose uptake in response to chronic insulin exposure (FP >> FH = FP). FP completely prevented the insulin-induced loss of insulin-stimulated glucose transport activity. In subsequent experiments in which the effect of fibroin was tested under conditions of chronic insulin exposure, FP was employed preferentially. The effects of each of the tested fibroin preparations were specific because neither albumin nor {gamma}-globulin (tested at the same concentration) had any effect on insulin sensitivity or interference with the development of insulin resistance (data not shown). The minimal concentration of fibroin required for these effects, regardless of preparation, was 1 g/L (data not shown).

Because physiologic concentrations of insulin are in the subnanomolar range (24), we tested the effect of FIII on insulin sensitivity at lower concentrations of insulin. Chronic exposure to FIII not only increased the maximal insulin-dependent transport activity, but also the sensitivity of the cells toward insulin as indicated by the fibroin-induced shift in the dose-response curve (Fig. 2A). By plotting the ratio of transport rates in the presence and absence of FIII, at each concentration of insulin, we showed that fibroin was most effective in promoting insulin-stimulated glucose uptake at insulin levels ranging from 0.1 to 1.0 nmol/L (Fig. 2B).



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  		FIGURE 2 Peptide fraction III increases the sensitivity of glucose transport in 3T3-L1 adipocytes to acute exposure to insulin. Panel A. Cells were treated with FIII (5 g/L) for 24 h. Cells were washed and transport activity was measured in the presence or absence of insulin. The concentration of insulin in mol/L is indicated on a log scale (see Panel B). The data represent the mean of cytochalasin B-inhibitable uptake ± SEM, n = 2. Error bars that are not visible are contained within the symbol. Asterisks indicate a difference from controls, ***P < 0.0001; **P < 0.001; *P < 0.01. Panel B. Using the data from Panel A, the ratio of transport activity in FIII- treated vs. control cells was plotted against the concentration of insulin.

 
Next, we examined the time required for the fibroin effect. In cells chronically exposed to FIII, with at little as 12 h of exposure, there was a significant effect on glucose insulin-stimulated glucose uptake (Fig 3A). This was followed by a steady rise for 30 h. During this phase, FIII had no effect on basal glucose uptake. At the 36- and 48-h time points, insulin-stimulated glucose uptake increased significantly in the presence of fibroin. However, this effect was in large part the result of a significant increase in "basal" glucose uptake at the 36- and 48-h time points. Because glucose deprivation increases basal glucose uptake (20), we examined the effect of refeeding at the 24-h time point. Indeed, the "activation" of glucose uptake was significantly blocked by this treatment (Fig. 3B).



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  		FIGURE 3 Peptide fraction III specifically increases basal glucose uptake in 3T3-L1 adipocytes. Panel A: Cells were treated with FIII (2.5 g/L) for the indicated times. Glucose uptake was measured at the times indicated in the absence or presence of insulin (1 µmol/L). The data represent the mean of cytochalasin B-inhibitable uptake ± SEM, n = 2–6. Panel B: Cells were treated with FIII as in Panel A. Some cells were refed at 24 h with fresh medium and fibroin. At 48 h, glucose uptake was measured in the absence or presence of insulin. Data represent the mean ± SEM, n = 2. Error bars that are not visible are included within the symbol. Panel A: ANOVA; F13,47 = 1687.02, P < 0.0001. Panel B: ANOVA; F7,15 = 1078.35, P < 0.0001. Means in a graph without a common letter differ, P < 0.05.

 
To further examine this phenomenon, we measured the concentration of glucose remaining in the medium from cells exposed or not to fibroin. As expected, the glucose concentration fell over time in medium overlaying control cells (Fig. 4A). By 48 h, glucose was essentially depleted. Note that at each time point during the 48-h time course, the glucose concentration in medium overlaying cells exposed to FIII was significantly reduced relative to controls. The percentage difference between the control and FIII-treated cells increased over time. As expected, refeeding at 24 h delayed the loss of glucose at subsequent time points. Yet, FIII-treated cells continued to have significantly less extracellular glucose than control cells. Because previous experiments revealed that glucose deprivation increases glycogen turnover in 3T3-L1 adipocytes (25), we also measured glycogen content as an indicator of glucose utilization. After 24 h of exposure to FIII, the concentration of cellular glycogen was not significantly different from controls (Fig. 4B). However, at 48 h of exposure, the fibroin-treated cells contained only 22% of the glycogen content of control cells (at 48 h); this loss could be prevented in part by refeeding.



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  		FIGURE 4 Peptide fraction III increases glucose utilization in 3T3-L1 adipocytes. Panel A: Cells were exposed or not to FIII (2.5 g/L) for the times indicated. At 24 h, some cells were refed with fresh medium with or without FIII. Glucose concentration in the medium was assayed at the points indicated. These data represent the mean ± SEM, n = 2. Because each time point is comprised of both control and FIII-treated cells, we applied Student’s t test at each time point for data comparison. Asterisks indicate a difference between control and FIII-treated cells, ***P ≤ 0.0001; **P ≤ 0.005; *P ≤ 0.03. Panel B: Cells were treated with or without FIII (2.5 g/L) for 24 or 48 h; some cells were refed at 24 h. Total glycogen was measured as previously described (25) and reported as glucose released from hydrolyzed glycogen. These data represent the mean ± SEM, n = 2. ANOVA; F5,11 = 43.57, P = 0.0001. Means in the graph without a common letter differ, P < 0.05.

 
In addition to chronic exposure to insulin, tumor necrosis factor{alpha} (TNF{alpha}) was implicated in the development of insulin resistance (26–30). This phenomenon appears to be related to general cachexia, which leads to the de-differentiation of adipocytes and thus loss of GLUT4 expression. To determine whether fibroin interferes with this process, we examined the effect of FP on insulin-stimulated glucose uptake in 3T3-L1 adipocytes chronically exposed to TNF{alpha} (Fig. 5). Several days of TNF{alpha} treatment were required to elicit insulin-resistant glucose transport. Alone, chronic exposure to TNF{alpha} reduced acute, insulin-sensitive transport by nearly 60%. FP, which completely prevents the development of insulin-induced insulin resistance (see Fig. 1), was significantly less effective in blocking the insulin-resistance induced by TNF{alpha}.



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  		FIGURE 5 Fibroin protein does not block TNF{alpha}-mediated insulin resistant glucose uptake in 3T3-L1 adipocytes. Cells were incubated for 96 h in the presence or absence of FP (2.5 g/L) and/or TNF{alpha} (500 pmol/L). Medium was changed every 24 h. Cells were washed and transport measured in the absence or presence of insulin as described in Figure 1. Data represent the mean of cytochalasin B-inhibitable uptake ± SEM, n = 2. ANOVA; F7,15 = 564.92, P < 0.0001. Means in the graph without a common letter differ, P < 0.05.

 
    Effect of fibroin on insulin signaling. Previously, we showed that chronic exposure to insulin reduces insulin binding by ~20% in the presence or absence of glucose (17). Based on the argument that only 10% of the receptor pool has to be occupied for 100% of the insulin-dependent glucose uptake (31), we concluded at that time that the decrease in insulin-dependent glucose uptake after chronic exposure to insulin was not due to loss of insulin binding. This was also observed by Garvey et al. (23) in cultured adipocytes. We concluded that insulin resistance must be due to effects at a postbinding or postreceptor site. Thus, to further explore the effect of fibroin on insulin resistance, we next examined insulin receptor autophosphorylation (Fig. 6A). Chronic exposure to insulin resulted in a 50% loss in the total pool of receptors (relative density of 25.4 ± 1.8 vs. 11.1 ± 1.3 for control and chronically exposed cells, respectively, as quantified in Fig. 6B). Even more striking was the 80% reduction in insulin-dependent autophosphorylation (relative density of 22.7 ± 2.8 vs 4.4 ± 0.7 for control and chronically exposed cells, respectively). Although we might have expected an increase in the receptor pool or an increase in insulin-dependent autophosphorylation in response to chronic fibroin, we observed no changes.



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  		FIGURE 6 Fibroin hydrolysate does not affect insulin-induced downregulation of receptor number or catalytic activity in 3T3-L1 adipocytes. Panel A: Cells were treated with FH (5.0 g/L) when indicated for a total of 24 h. Insulin (10 nmol/L) was added 12 h into the incubation. Cells were washed extensively and acutely exposed to insulin (1 µmol/L) added in Krebs Ringer phosphate for 10 min. After washing, cells were scraped in a buffer and a total membrane fraction was isolated. After extraction, the insulin receptor was immunoprecipitated from equal protein (3.5 mg) and collected with Protein A Sepharose beads The complexes were released with sample dilution buffer, separated by SDS-PAGE, and analyzed by Western blotting for total and phosphorylated insulin receptor. Panel B: Data represent the densitometric analysis of 3 independent experiments plotted as the mean of the relative densities ± SEM. Only the effect of insulin is shown because fibroin had no effect on either receptor content or autophosphorylation. **Different from controls, P < 0.001.

 
We next focused on an event downstream of insulin receptor phosphorylation, insulin-dependent Akt phosphorylation. Others showed that there are 2 phosphorylation sites in Akt that provide insulin-sensitive phosphorylation: Ser473 and Thr308 (32). As expected, acute insulin exposure over a range of concentrations stimulated Akt phosphorylation at both Ser473 and Thr308 without affecting the total pool (Fig. 7A). Although there appeared to be a preference for threonine phosphorylation at higher insulin concentrations (Fig. 7B), chronic exposure to fibroin had no effect on the extent of phosphorylation. Others showed that chronic exposure to insulin decreased the ability of acute exposure to insulin to stimulate Akt phosphorylation by 50% (32). Although we were able to confirm this observation, once again, chronic exposure to fibroin treatment had no effect on this desensitization (data not shown).



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  		FIGURE 7 Fibroin hydrolysate does not affect insulin-dependent or -independent Akt phosphorylation in 3T3-L1 adipocytes. Panel A: Cells were treated or not with FH (5 g/L) for 24 h. Cells were rinsed in Krebs Ringer phosphate and exposed to specific concentrations of insulin as indicated for 10 min. Cells were then washed and extracted for Akt. Equal protein was analyzed by SDS/PAGE-Western blot to measure total Akt, p-Akt473, and p-Akt308. Panel B: Data from Panel A were analyzed by densitometry and are representative of duplicate experiments. Asterisks indicate a difference in threonine vs. serine phosphorylation, **P ≤ 0.005; *P ≤ 0.05.

 
    Effect of fibroin on GLUT expression distribution. The ultimate target of insulin action is its ability to stimulate glucose uptake, as we observed. Glucose uptake is mediated by 2 transport proteins in 3T3-L1 adipocytes, i.e., GLUT1 and GLUT4. Thus, we next analyzed the expression and distribution of these 2 GLUT isoforms in response to chronic exposures to fibroin and insulin. As observed by us and others, acute exposure to insulin alters the distribution of both GLUT1 and GLUT4 (Fig. 8A). The relative amount of GLUT4 in the plasma membrane (the PM fraction) increased by 2.7-fold, with a consequent loss from the intracellular pool (the LDM fraction). The relative amount of GLUT1 in the PM increased (by ~1.5-fold) in response to acute exposure to insulin, which also arose from the LDM. Chronic treatment with FH had little effect on the level of GLUT4 in the PM in the absence of acute exposure to insulin, but a further increase in GLUT4 relative to insulin alone (~1.4-fold). In contrast, FH treatment increased the relative amount of GLUT1 in both the PM and LDM fraction in the absence of acute exposure to insulin (~1.7- and 1.1-fold, respectively). The biosynthetic pool (the high-density membranes from the biosynthetic compartment) also showed a substantial increase (data not shown). Acute addition of insulin again increased the amount of GLUT1 in the PM fraction relative to the addition of insulin alone (~1.5-fold). Finally we examined the effect of fibroin on GLUT1 mRNA expression (Fig. 8B). In these experiments, chronic insulin exposure was used as a positive control because insulin stimulates the transcription of GLUT1 (33,34). Although not as robust as chronic exposure to insulin, FH exposure increased the expression of GLUT1 by 2-fold.



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  		FIGURE 8 Fibroin hydrolysate increases expression of GLUT1 in 3T3-L1 adipocytes Panel A: Cells were treated or not with FH (2.5 g/L) for 24 h. Cells were washed and exposed to insulin (1 µmol/L) for 10 min. Membrane fractions were collected and analyzed by SDS/PAGE and Western blotting for GLUT1 and GLUT4. These data are representative of triplicate experiments. Panel B: Cells were exposed to insulin (100 nmol/L) for 12 h or FH (2.5 g/L) for a total of 24 h. Total RNA was collected and Northern blot analysis was performed using a GLUT1 cDNA probe prepared by PCR from total RNA. The blot is representative of duplicate experiments. C = control; I = insulin; F = FH.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
In these studies, we pursued the mechanism by which fibroin, the major protein in silk, exerts its hypoglycemic effects in vivo. Our data show that fibroin has multiple effects on adipocyte metabolism. In 3T3-L1 adipocytes, fibroin significantly increased insulin-sensitive glucose uptake particularly in response to physiologic concentrations of insulin. In addition, fibroin blocks the development of insulin resistance in response to chronic insulin exposure but not that in response to TNF{alpha}. Fibroin also increased glucose metabolism, as reflected by the rapid loss of glucose from the medium. This ultimately led to glucose deprivation as noted by the more rapid turnover of glycogen in fibroin-treated cells compared with controls. We surmise that accelerated glucose metabolism in the absence of acute insulin is mediated by the increase in GLUT1 expression at the cell surface. With short-term (acute) incubation with insulin, we observed an additional increase in both GLUT1 and GLUT4 at the cell surface after fibroin exposure (relative to its absence), which likely contributed to the reversal of insulin-induced insulin resistance.

In defining the mechanism that underlies the effects of fibroin on insulin sensitivity and glucose metabolism, we examined several steps in the insulin-signaling cascade. First, we showed that chronic insulin exposure affects insulin receptor expression. Although this has been observed at high concentrations of insulin, this result was unexpected on the basis of the limited reduction in insulin binding noted after exposure to only 10 nmol/L insulin in previous studies (17,23). Subsequent insulin-stimulated autophosphorylation was diminished even further, suggesting that the catalytic activity of the receptor was compromised, independent of decreased expression. Surprisingly, fibroin did not influence receptor expression or alter the catalytic activity in either the presence or absence of chronic exposure to insulin.

One of the downstream events in the signaling cascade is the activation of phosphoinositide 3-kinase (PI3-kinase), which ultimately results in the translocation of GLUT4 (35–39). As shown by others (36,40), insulin-sensitive glucose uptake is completely blocked by wortmannin, a PI3-kinase inhibitor. Wortmannin blocked the increase in glucose uptake stimulated by the acute addition of insulin in either the presence or absence of fibroin (data not shown). Although this suggests that fibroin may augment signaling events downstream of PI3 kinase, fibroin had little effect on Akt phosphorylation in the absence or presence of insulin (either acute or chronic exposure).

It was hypothesized that adipocytes with higher metabolic activity in vivo exhibit enhanced insulin sensitivity (41). A number of the currently used hypoglycemic drugs appear to function in this way (5). For example, metformin increases glucose uptake in a variety of extrahepatic tissues (42). In a small percentage of individuals, metformin actually induces lactic acidosis, a potentially fatal condition (43). Yet, metformin reverses the insulin-induced reduction in tyrosine-specific insulin receptor phosphorylation, which is in contrast to the action of fibroin. Thiazolinediones also increase insulin sensitivity in tissues, acting as agonists for the nuclear transcription factor, peroxisomal proliferator-activated receptor-{gamma} (PPAR{gamma}) (44). Although this transcription factor is important in adipogenesis (45), its activation also leads to enhanced expression of GLUT4 in adipose tissue (46) and enhanced translocation of GLUT4 in muscle (47). Although we did not measure the expression of PPAR{gamma}, the hallmarks of fibroin action are not consistent with this mechanism.

The fibroin-induced increase in GLUT1 was intriguing because chronic exposure to insulin has a similar effect. Not only does insulin increase the transcription of GLUT1, it also increases the efficiency by which GLUT1 mRNA is translated (48). The investigators in that study showed that insulin inactivates the translational suppressor eukaryotic initiation factor 4E-binding protein through the mammalian target of rapamycin (mTOR), which leads to enhanced association of GLUT1 mRNA with polyribosomes. Interestingly, dominant negative mutants of PI3 kinase or Akt block the increase in GLUT1 translation, suggesting a connection between mTOR and elements of the immediate insulin-signaling cascade. Although fibroin does not appear to function via a PI3 kinase–dependent path, it may alter GLUT1 synthesis by activating the mTOR path through PI3 kinase–independent signaling events.

In conclusion, we showed that fibroin increases glucose uptake and metabolism in 3T3-L1 adipocytes, suggesting that the hypoglycemic effect of this natural product in ob/ob mice is a result of increased extrahepatic glucose uptake. We speculate that the increased glucose uptake is primarily a result of increased GLUT1 expression, which results in an increase in GLUT1 protein at the cell surface. There is also a modest increase in GLUT4 in the PM in response to fibroin, which does not appear to result from increased synthesis. At this point in time, the signaling events that lead to these changes are unknown.


   ACKNOWLEDGMENTS
 
The authors thank Xiao Wei Gu for her excellent technical assistance.


   FOOTNOTES
 
1 Presented in part at the 63rd Session of the American Diabetes Association, June 2003, New Orleans, LA [Hyun, C.-K. & Frost, S. C. (2003) Fibroin blocks the development of insulin resistance in 3T3-L1 adipocytes. Diabetes 52 (suppl. 2): 1244-P. Back

2 Funded in part by a grant from the National Institutes of Health (DK45035) to S.C.F. Back

4 Abbreviations used: BSA, bovine serum albumin; FH, fibroin hydrolysate; FIII, fibroin peptide fraction III; FP, fibroin protein; GLUT, glucose transporter; LDM, low-density membranes from endosomes; mTOR, mammalian target of rapamycin; PI3-kinase, phosphoinositide 3-kinase; PM, plasma membrane; PPAR{gamma}, peroxisomal proliferator-activated receptor-{gamma}; TBS-T, Tris-buffered saline-Tween; TNF{alpha}, tumor necrosis factor-{alpha}. Back

Manuscript received 13 October 2003. Initial review completed 22 January 2004. Revision accepted 27 September 2004.


   LITERATURE CITED
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 

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