BACKGROUND
human insulin-like growth factor-i  plays a critical role in cellular differentiation, proliferation, growth and apoptosis  <cit> .
it is a single polypeptide chain of  <dig> amino acid residues and is encoded by a single gene on chromosome  <dig>  with a molecular weight of  <dig> da  <cit> .
it has 50% amino acid sequence homology with insulin.
hence, higf-i has been proposed as an alternative therapeutic agent to treat diabetic mellitus, especially for those patients with defects in insulin receptors.
apart from diabetes mellitus, the efficacy of higf-i in the treatment of growth disorders as well as insulin resistance has been studied  <cit> .
in late  <dig>  recombinant higf-i  was approved by the united states food and drug administration  as a therapeutic agent for the growth hormone insensitivity syndrome   <cit> .
recombinant higf-i was first synthesized by recombinant dna techniques in  <dig>  to date, commercial rhigf-i is mainly produced in different organisms  <cit> .
problems of these processes include low expression levels, high equipment and production costs, incorrect post-translational modifications as well as potential contamination with human pathogens.
with the advance in genetic engineering, rice is recently recognized as a promising alternative for the production of safe and economical biopharmaceutical in large quantities  <cit> .
rice offers the advantage of producing large quantity of proteins in terms of cost, product safety, scalability and authenticity  <cit> .
rice is known to exclude any noxious chemicals such as nicotine and toxic alkaloids in tobacco as well as having low allergenicity.
large amount of recombinant proteins can be synthesized at one time as a single rice plant can produce over  <dig>  grains.
besides, as rice is self-pollinated, simple regulatory rules like isolation distances can be set up to prevent cross-pollinating and out crossing of the transgenic trait.
in an attempt to achieve high-yield expression of foreign genes in plants, the coding sequence of heterologous gene has to be modified to plant-preferred codons.
previous studies have showed that codon usage biases are strongly correlated with gene expression levels  <cit> .
highly expressed genes preferentially use a subset of "optimal" codons which correspond to the most abundant trnas, leading to enhanced translation accuracy and efficiency  <cit> .
moreover, proteins yields can be increased if the protein is directed to specific compartments in order to prevent degradation by the proteolytic system of the cells.
in a plant cell , an amino-terminal signal peptide can direct proteins to the secretory pathway, including the endoplasmic reticulum , the golgi complex and hydrolytic compartments , or to secretion from the cell.
it has been found that secretory proteins could be accumulated to high level than those expressed in cytosol  <cit> .
some studies found that proteins yields can be further enhanced if the protein is retained in the er lumen using the lys-asp-glu-leu  c-terminal tetrapeptide  <cit> .
protein levels were 6- <dig> times higher in cells transformed with the construct containing kdel than that without kdel  <cit> .
in the present study, we hypothesized that functional rhigf-i could be produced in transgenic rice grains.
codons of cdna of higf-i was modified to plant-preferred sequences  <cit>  and introduced into rice by agrobacterium-mediated transformation.
glutelin signal peptide and kdel tetrapeptide were included for expression enhancement of recombinant proteins.
biological activity of rhigf-i was confirmed by the induction of membrane ruffles and the increase in glucose transport in rat skeletal muscle cells .
in vivo study demonstrated that transgenic rhigf-i from rice reduced blood glucose level in streptozotocin induced diabetic and zucker fatty rats.
these findings confirmed that biologically active rhigf-i could be produced in the rice-based system.
RESULTS
expression of rhigf-i in transgenic rice seeds
to increase the expression level of higf-i, glutelin signal peptide  and er-retention signal kdel were added to the chimeric constructs.
three chimeric gene constructs  were designed and transferred into rice by agrobacterium-mediated transformation .
southern blot analysis confirmed the integration of constructs  into the rice genome .
as the restriction enzyme bamhi cuts only once in all expression constructs, the number of bands represented the number of copies of the transgenes introduced into rice.
most of the transformants contained  <dig> to  <dig> copies of the transgenes, while no signal was detected in wild type plant.
total seed protein extracts from different transformants were examined by tricine sds-page and western blot.
a distinct band of about  <dig>  kda was observed in transformants si and sik , whereas no signals were detected in transformant i and wild type rice plant .
these results suggested that glutelin sp was essential for the expression of the transgenes.
ruffling effect of commercial and rice-produced rhigf-i
growth factors such as igf-i and insulin are known to cause reorganization of actin filament leading to membrane ruffles  <cit> .
using the rat skeletal muscle cells , we compared the effect on actin remodeling of rhigf-i from commercial source and transgenic rice.
we found that the morphological change induced by  <dig> nm of commercial rhigf-i was equivalent to that of  <dig> nm of insulin .
to study the in vitro function of rice-produced rhigf-i, seed crude protein extracts of various amount from different transformants  and commercial rhigf-i  were added to l6myc cells.
membrane ruffles were found only when si protein was added , while no morphological changes were observed when l6myc cells were treated with i, sik  or wild type rice .
as depicted in figure 3c, the ruffling effect induced by si protein was directly proportional to the amount of the protein added.
extract containing  <dig> mg of si protein caused membrane ruffling similar to that of  <dig> nm of commercial rhigf-i .
glucose uptake assay
the metabolic effect of rhigf-i was studied by measuring the glucose uptake in l6myc cells.
only si protein was tested as it possessed biological activity as demonstrated by membrane ruffle experiments.
different concentrations of commercial rhigf-i  and si protein  were added to l6myc cells.
insulin  was used as positive control.
as depicted in figure  <dig>  both commercial rhigf-i and si proteins stimulated glucose uptake in a dose-dependent manner.
five mg of si protein was found to cause comparable glucose uptake as  <dig> nm of commercial rhigf-i .
no significant difference in glucose uptake was observed when basal or wild type rice seed proteins were added.
in vivo feeding test
to determine in vivo glucose lowering effect, different animal models of diabetes, namely streptozotocin-induced diabetic rats  and zucker diabetic fatty rats  were fed with transgenic rhigf-i rice .
sprague-dawley  rats were used as control .
plasma glucose levels of sd rats fed with si rice were similar to those fed with wild type rice .
in zucker fatty rats  and stz-induced diabetic rats , ingestion of si rice was associated with lower plasma glucose levels when compared with those fed with wild type rice.
significant differences were observed at  <dig> hours  and  <dig>  hours  for zucker fatty rats, while for stz-induced diabetic rats, significant differences occurred at  <dig>  hours  and  <dig> hours .
the results indicated that si rice reduced blood glucose in zucker fatty and stz-induced diabetic rats, though it had no effect on blood glucose level in normal sd rats.
starch contents of rice grains
total starch analysis was performed to confirm the endogenous starch contents of si transgenic rice and wild type rice.
the od reading of si and wild type rice were  <dig>  ±  <dig>  vs.  <dig>  ±  <dig> , p =  <dig> ) respectively .
the result suggested that both transgenic and wild type rice grains had similar starch content.
discussion
this is the first report to demonstrate the expression of native rhigf-i in transgenic rice grains.
various strategies were employed in the design of the chimeric constructs to enhance the stable expression of the human recombinant proteins in plant.
firstly, the codons of human igf-i gene were modified to plant-preferred sequence.
secondly, sequences of two plant seed storage proteins, lysine-rich protein  from winged bean and methionine-rich 2s albumin from paradise nut  <cit>  were included in constructs for high protein expression and stable accumulation  <cit> .
thirdly, as subcellular targeting is another crucial factor in determining the expression of foreign proteins, rice glutelin signal peptide  and er-retention sequence kdel were employed for stable accumulation of rhigf-i in transgenic rice grains.
western blot analysis showed that only constructs containing sp and kdel  were expressed in transgenic rice grains .
we postulated that the addition of sp promotes the inclusion of candidate proteins si and sik into the secretory pathway of the endoplasmic reticulum   <cit> .
after the cleavage of sp in the er, the si protein, in the absence of further protein sorting sequences, went through the golgi apparatus and was finally secreted out of the cell in the intercellular space for storage.
in contrast, the sik protein was re-directed and retained in the er by the presence of kdel sequence.
the kdel tetrapeptide contributes to protein localization by interacting with a receptor that recycles between the golgi apparatus and the er  <cit> .
the oxidizing environment and the presence of molecular chaperones in er promote protein assembly and folding.
furthermore, few proteases are present in er for degradation of proteins.
hence, both si and sik proteins were not degraded by protease and could be stably accumulated.
our results were consistent with the increased accumulation of vicilin, a pea vacuole storage protein in transgenic alfalfa leaves when the kdel signal was included in c-terminus  <cit> .
despite being expressed in the transgenic rice grain, sik protein failed to induce membrane ruffling in l6myc cells.
this results suggested that sik protein was devoid of biological activity of igf-i.
in native human igf-i protein, there are no glycosylation site.
a possible explanation of the difference in biological activity between si and sik proteins could be related to the glycosylation of candidate protein.
the presence of the kdel sequence has been shown to affect the extent of glycosylation in the er and golgi apparatus  <cit> .
it is feasible that the presence of the kdel sequence in sik proteins might enhance the glycosylation of candidate proteins.
further study on the presence of glycosylation in sik will be required to confirm the hypothesis.
other possible explanation includes the misfolding of candidate protein while in the er.
the increase in sik protein in the er may lead to er stress, resulting in over-expression of er chaperones, luminal binding protein  and the protein folding catalyst, protein disulphide-isomerase  <cit> .
previous studies had reported that over-expression of bip had inhibitory effect on protein translation, folding and deposition of storage proteins into rice endosperm  <cit> .
our results were consistent with previous study in which the fluorescence intensity of green fluorescent protein with kdel was reduced as a result of protein misfolding  <cit> .
the membrane ruffling and glucose uptake in rat skeletal muscle cells  were used to evaluate the biological activity of transgenic rhigf-i.
growth factors including igf-i are known to cause rapid actin remodeling leading to membrane ruffles  <cit> .
the igf-i has been shown to be about  <dig> -  <dig> times more potent than insulin in causing membrane ruffles  <cit> .
in the present study,  <dig> nm of commercial rhigf-i was found to have similar effect in membrane ruffles as compared to  <dig> nm of insulin in l6myc.
importantly, rice-produced rhigf-i  was found to induce similar membrane ruffling as commercial rhigf-i and the effect was dose-dependent .
in term of membrane ruffles, one gram of si rice grain possessed equivalent activity to  <dig>  μm of commercial rhigf-i.
for glucose uptake study,  <dig> mg of si protein was found to be equivalent to  <dig> nm of commercial rhigf-i .
hence, it was estimated that one gram of si rice grain contained  <dig>  μm of functional rhigf-i.
taken together, these results indicated that the properties of rice-produced rhigf-i were quite similar to those of the native igf-i and insulin on membrane ruffles and glucose uptake.
schoenle et al.
previously showed that treatment with igf-i reduced both insulin and blood glucose levels in patients with severe insulin resistance  <cit> .
it has been postulated that igf-i might enhance insulin secretion and sensitivity  <cit> , stimulate β cell regeneration  <cit>  as well as prevent β cell from apoptosis  <cit>  in patients with diabetes.
to evaluate whether the rice-derived rhigf-i could also lower blood glucose level, an oral rice tolerance test  was developed based upon the methodology of oral glucose tolerance test.
as starch contributes 80% of rice grain, glucose released after digestion will lead to higher blood glucose level, which is similar to the effect of glucose intake.
we found that si could efficiently lower blood glucose levels in zucker and stz-induced rats , whereas it did not have significant effect in normal sd rats  when compared with wild type rice.
blood glucose levels only showed significant differences after  <dig> hours in zucker fatty rats and  <dig>  hours in stz rats.
this time delay may be due to the release of rice-derived rhigf-i after digestion of si rice grain powder.
when feeding the three types of rats with equal amount of commercial rhigf-i, no significant differences were found in blood glucose levels , suggesting that commercial rhigf-i might be totally degraded as it went through the gastrointestinal tract in rats.
on the other hand, rice grain might help protect the rice-derived rhigf-i from the digestive system in rats.
this effect might be attributed to the location of rhigf-i accumulated in rice grain.
seed protein is normally deposited in storage organelles known as protein bodies  <cit> .
studies found that protein bodies can withstand the harsh acidic environment of the digestive tract in rats, making it possible for inducing antigen-specific immune responses by oral administration of rice-based mucosal vaccine  <cit> .
to scrutinize whether the blood glucose lowering effect was solely resulted from the action of the rhigf-i in si rice, total starch contnet of si and wild type rice grains was assessed by enzymatic digestion with α-amylase and amyloglucosidase .
as depicted in supplemental figure s <dig>  there was no significant difference in endogenous starch contents between si and wild type rice grains.
our results confirmed that the blood glucose lowering effect was solely due to the action of rice-derived rhigf-i.
recent study demonstrated that rice-codon-modified rhigf- <dig> could also be expressed in transgenic rice grains.
oral administration of the codon-modified rhigf-i could reduce blood glucose in diabetic mice by enhancing plasma rhigf-i level.
the rhigf-i produced by xie et al.
was a fusion protein of  <dig> kda, with a c-terminal er luminal binding protein, but not native rhigf-i  <cit> .
in the present study, native rhigf-i with about  <dig>  kda  could successfully be expressed and its biological functions were similar to those of commercial rhigf-i, as indicated in both in vitro  and in vivo  analysis.
hence, our approach might provide an alternative methodology in producing native rhigf-i.
using a different approach, rhigf-i was expressed in rice leaves  <cit> .
further extraction and purification of the protein from leaves would be required before the protein could be utilized.
the expression of rhigf-i in rice grain in the present study represents a better way of producing functional rhigf-i that possesses insulin-like biological functions for clinical applications.
the limitation of this report is that it is only a preliminary study to test the feasibility of higf-i expression in transgenic rice.
the in vivo study we performed was an acute effect of the rice-derived rhigf-i on rats.
long-term study will be carried out to see whether the rhigf-i possesses glucose lowering effect and its side effects in diabetic rats.
besides, the level of igf-i will be measured to see whether the glucose lowering effect is attributed to the rice-derived rhigf-i.
moreover, as the rice-derived rhigf-i was neither isolated nor purified, attempts will be put on purification of the rice-derived rhigf-i for possible clinical uses.
CONCLUSIONS
we have successfully expressed native rhigf-i in rice grains using transgenic approach.
plant-optimized higf-i cdna was incorporated in different expression constructs and introduced into rice by agrobacterium-mediated transformation.
one gram of si rice grain possessed the maximum activity level equivalent to  <dig>  μm of commercial rhigf-i.
in vitro studies showed that rice-derived rhigf-i could induce membrane ruffling and enhance glucose uptake in l6myc cells.
oral administration of transgenic rhigf-i rice confirmed that it reduced plasma glucose levels in stz-induced and zucker diabetic fatty rats, while it did not have significant effect in normal sd rats.
taken together, our findings suggested that biologically active rhigf-i could be produced in transgenic rice.

 human insulin-like growth factor-i plays a critical role in cellular differentiation, proliferation, growth and apoptosis. it is a single polypeptide chain of amino acid residues and is encoded by a single gene on chromosome with a molecular weight of da. it has 50% amino acid sequence homology with insulin. hence, higf-i has been proposed as an alternative therapeutic agent to treat diabetic mellitus, especially for those patients with defects in insulin receptors. apart from diabetes mellitus, the efficacy of higf-i in the treatment of growth disorders as well as insulin resistance has been studied. in late recombinant higf-i was approved by the united states food and drug administration as a therapeutic agent for the growth hormone insensitivity syndrome. recombinant higf-i was first synthesized by recombinant dna techniques in to date, commercial rhigf-i is mainly produced in different organisms. problems of these processes include low expression levels, high equipment and production costs, incorrect post-translational modifications as well as potential contamination with human pathogens. with the advance in genetic engineering, rice is recently recognized as a promising alternative for the production of safe and economical biopharmaceutical in large quantities. rice offers the advantage of producing large quantity of proteins in terms of cost, product safety, scalability and authenticity. rice is known to exclude any noxious chemicals such as nicotine and toxic alkaloids in tobacco as well as having low allergenicity. large amount of recombinant proteins can be synthesized at one time as a single rice plant can produce over grains. besides, as rice is self-pollinated, simple regulatory rules like isolation distances can be set up to prevent cross-pollinating and out crossing of the transgenic trait. in an attempt to achieve high-yield expression of foreign genes in plants, the coding sequence of heterologous gene has to be modified to plant-preferred codons. previous studies have showed that codon usage biases are strongly correlated with gene expression levels. highly expressed genes preferentially use a subset of "optimal" codons which correspond to the most abundant trnas, leading to enhanced translation accuracy and efficiency. moreover, proteins yields can be increased if the protein is directed to specific compartments in order to prevent degradation by the proteolytic system of the cells. in a plant cell, an amino-terminal signal peptide can direct proteins to the secretory pathway, including the endoplasmic reticulum, the golgi complex and hydrolytic compartments, or to secretion from the cell. it has been found that secretory proteins could be accumulated to high level than those expressed in cytosol. some studies found that proteins yields can be further enhanced if the protein is retained in the er lumen using the lys-asp-glu-leu c-terminal tetrapeptide. protein levels were 6- times higher in cells transformed with the construct containing kdel than that without kdel. in the present study, we hypothesized that functional rhigf-i could be produced in transgenic rice grains. codons of cdna of higf-i was modified to plant-preferred sequences and introduced into rice by agrobacterium-mediated transformation. glutelin signal peptide and kdel tetrapeptide were included for expression enhancement of recombinant proteins. biological activity of rhigf-i was confirmed by the induction of membrane ruffles and the increase in glucose transport in rat skeletal muscle cells. in vivo study demonstrated that transgenic rhigf-i from rice reduced blood glucose level in streptozotocin induced diabetic and zucker fatty rats. these findings confirmed that biologically active rhigf-i could be produced in the rice-based system.  expression of rhigf-i in transgenic rice seeds to increase the expression level of higf-i, glutelin signal peptide and er-retention signal kdel were added to the chimeric constructs. three chimeric gene constructs were designed and transferred into rice by agrobacterium-mediated transformation. southern blot analysis confirmed the integration of constructs into the rice genome. as the restriction enzyme bamhi cuts only once in all expression constructs, the number of bands represented the number of copies of the transgenes introduced into rice. most of the transformants contained to copies of the transgenes, while no signal was detected in wild type plant. total seed protein extracts from different transformants were examined by tricine sds-page and western blot. a distinct band of about kda was observed in transformants si and sik, whereas no signals were detected in transformant i and wild type rice plant. these results suggested that glutelin sp was essential for the expression of the transgenes. ruffling effect of commercial and rice-produced rhigf-i growth factors such as igf-i and insulin are known to cause reorganization of actin filament leading to membrane ruffles. using the rat skeletal muscle cells, we compared the effect on actin remodeling of rhigf-i from commercial source and transgenic rice. we found that the morphological change induced by nm of commercial rhigf-i was equivalent to that of nm of insulin. to study the in vitro function of rice-produced rhigf-i, seed crude protein extracts of various amount from different transformants and commercial rhigf-i were added to l6myc cells. membrane ruffles were found only when si protein was added, while no morphological changes were observed when l6myc cells were treated with i, sik or wild type rice. as depicted in figure 3c, the ruffling effect induced by si protein was directly proportional to the amount of the protein added. extract containing mg of si protein caused membrane ruffling similar to that of nm of commercial rhigf-i. glucose uptake assay the metabolic effect of rhigf-i was studied by measuring the glucose uptake in l6myc cells. only si protein was tested as it possessed biological activity as demonstrated by membrane ruffle experiments. different concentrations of commercial rhigf-i and si protein were added to l6myc cells. insulin was used as positive control. as depicted in figure both commercial rhigf-i and si proteins stimulated glucose uptake in a dose-dependent manner. five mg of si protein was found to cause comparable glucose uptake as nm of commercial rhigf-i. no significant difference in glucose uptake was observed when basal or wild type rice seed proteins were added. in vivo feeding test to determine in vivo glucose lowering effect, different animal models of diabetes, namely streptozotocin-induced diabetic rats and zucker diabetic fatty rats were fed with transgenic rhigf-i rice. sprague-dawley rats were used as control. plasma glucose levels of sd rats fed with si rice were similar to those fed with wild type rice. in zucker fatty rats and stz-induced diabetic rats, ingestion of si rice was associated with lower plasma glucose levels when compared with those fed with wild type rice. significant differences were observed at hours and hours for zucker fatty rats, while for stz-induced diabetic rats, significant differences occurred at hours and hours. the results indicated that si rice reduced blood glucose in zucker fatty and stz-induced diabetic rats, though it had no effect on blood glucose level in normal sd rats. starch contents of rice grains total starch analysis was performed to confirm the endogenous starch contents of si transgenic rice and wild type rice. the od reading of si and wild type rice were ± vs. ±, p = ) respectively. the result suggested that both transgenic and wild type rice grains had similar starch content. discussion this is the first report to demonstrate the expression of native rhigf-i in transgenic rice grains. various strategies were employed in the design of the chimeric constructs to enhance the stable expression of the human recombinant proteins in plant. firstly, the codons of human igf-i gene were modified to plant-preferred sequence. secondly, sequences of two plant seed storage proteins, lysine-rich protein from winged bean and methionine-rich 2s albumin from paradise nut were included in constructs for high protein expression and stable accumulation. thirdly, as subcellular targeting is another crucial factor in determining the expression of foreign proteins, rice glutelin signal peptide and er-retention sequence kdel were employed for stable accumulation of rhigf-i in transgenic rice grains. western blot analysis showed that only constructs containing sp and kdel were expressed in transgenic rice grains. we postulated that the addition of sp promotes the inclusion of candidate proteins si and sik into the secretory pathway of the endoplasmic reticulum. after the cleavage of sp in the er, the si protein, in the absence of further protein sorting sequences, went through the golgi apparatus and was finally secreted out of the cell in the intercellular space for storage. in contrast, the sik protein was re-directed and retained in the er by the presence of kdel sequence. the kdel tetrapeptide contributes to protein localization by interacting with a receptor that recycles between the golgi apparatus and the er. the oxidizing environment and the presence of molecular chaperones in er promote protein assembly and folding. furthermore, few proteases are present in er for degradation of proteins. hence, both si and sik proteins were not degraded by protease and could be stably accumulated. our results were consistent with the increased accumulation of vicilin, a pea vacuole storage protein in transgenic alfalfa leaves when the kdel signal was included in c-terminus. despite being expressed in the transgenic rice grain, sik protein failed to induce membrane ruffling in l6myc cells. this results suggested that sik protein was devoid of biological activity of igf-i. in native human igf-i protein, there are no glycosylation site. a possible explanation of the difference in biological activity between si and sik proteins could be related to the glycosylation of candidate protein. the presence of the kdel sequence has been shown to affect the extent of glycosylation in the er and golgi apparatus. it is feasible that the presence of the kdel sequence in sik proteins might enhance the glycosylation of candidate proteins. further study on the presence of glycosylation in sik will be required to confirm the hypothesis. other possible explanation includes the misfolding of candidate protein while in the er. the increase in sik protein in the er may lead to er stress, resulting in over-expression of er chaperones, luminal binding protein and the protein folding catalyst, protein disulphide-isomerase. previous studies had reported that over-expression of bip had inhibitory effect on protein translation, folding and deposition of storage proteins into rice endosperm. our results were consistent with previous study in which the fluorescence intensity of green fluorescent protein with kdel was reduced as a result of protein misfolding. the membrane ruffling and glucose uptake in rat skeletal muscle cells were used to evaluate the biological activity of transgenic rhigf-i. growth factors including igf-i are known to cause rapid actin remodeling leading to membrane ruffles. the igf-i has been shown to be about - times more potent than insulin in causing membrane ruffles. in the present study, nm of commercial rhigf-i was found to have similar effect in membrane ruffles as compared to nm of insulin in l6myc. importantly, rice-produced rhigf-i was found to induce similar membrane ruffling as commercial rhigf-i and the effect was dose-dependent. in term of membrane ruffles, one gram of si rice grain possessed equivalent activity to μm of commercial rhigf-i. for glucose uptake study, mg of si protein was found to be equivalent to nm of commercial rhigf-i. hence, it was estimated that one gram of si rice grain contained μm of functional rhigf-i. taken together, these results indicated that the properties of rice-produced rhigf-i were quite similar to those of the native igf-i and insulin on membrane ruffles and glucose uptake. schoenle et al. previously showed that treatment with igf-i reduced both insulin and blood glucose levels in patients with severe insulin resistance. it has been postulated that igf-i might enhance insulin secretion and sensitivity, stimulate β cell regeneration as well as prevent β cell from apoptosis in patients with diabetes. to evaluate whether the rice-derived rhigf-i could also lower blood glucose level, an oral rice tolerance test was developed based upon the methodology of oral glucose tolerance test. as starch contributes 80% of rice grain, glucose released after digestion will lead to higher blood glucose level, which is similar to the effect of glucose intake. we found that si could efficiently lower blood glucose levels in zucker and stz-induced rats, whereas it did not have significant effect in normal sd rats when compared with wild type rice. blood glucose levels only showed significant differences after hours in zucker fatty rats and hours in stz rats. this time delay may be due to the release of rice-derived rhigf-i after digestion of si rice grain powder. when feeding the three types of rats with equal amount of commercial rhigf-i, no significant differences were found in blood glucose levels, suggesting that commercial rhigf-i might be totally degraded as it went through the gastrointestinal tract in rats. on the other hand, rice grain might help protect the rice-derived rhigf-i from the digestive system in rats. this effect might be attributed to the location of rhigf-i accumulated in rice grain. seed protein is normally deposited in storage organelles known as protein bodies. studies found that protein bodies can withstand the harsh acidic environment of the digestive tract in rats, making it possible for inducing antigen-specific immune responses by oral administration of rice-based mucosal vaccine. to scrutinize whether the blood glucose lowering effect was solely resulted from the action of the rhigf-i in si rice, total starch contnet of si and wild type rice grains was assessed by enzymatic digestion with α-amylase and amyloglucosidase. as depicted in supplemental figure s there was no significant difference in endogenous starch contents between si and wild type rice grains. our results confirmed that the blood glucose lowering effect was solely due to the action of rice-derived rhigf-i. recent study demonstrated that rice-codon-modified rhigf- could also be expressed in transgenic rice grains. oral administration of the codon-modified rhigf-i could reduce blood glucose in diabetic mice by enhancing plasma rhigf-i level. the rhigf-i produced by xie et al. was a fusion protein of kda, with a c-terminal er luminal binding protein, but not native rhigf-i. in the present study, native rhigf-i with about kda could successfully be expressed and its biological functions were similar to those of commercial rhigf-i, as indicated in both in vitro and in vivo analysis. hence, our approach might provide an alternative methodology in producing native rhigf-i. using a different approach, rhigf-i was expressed in rice leaves. further extraction and purification of the protein from leaves would be required before the protein could be utilized. the expression of rhigf-i in rice grain in the present study represents a better way of producing functional rhigf-i that possesses insulin-like biological functions for clinical applications. the limitation of this report is that it is only a preliminary study to test the feasibility of higf-i expression in transgenic rice. the in vivo study we performed was an acute effect of the rice-derived rhigf-i on rats. long-term study will be carried out to see whether the rhigf-i possesses glucose lowering effect and its side effects in diabetic rats. besides, the level of igf-i will be measured to see whether the glucose lowering effect is attributed to the rice-derived rhigf-i. moreover, as the rice-derived rhigf-i was neither isolated nor purified, attempts will be put on purification of the rice-derived rhigf-i for possible clinical uses.  we have successfully expressed native rhigf-i in rice grains using transgenic approach. plant-optimized higf-i cdna was incorporated in different expression constructs and introduced into rice by agrobacterium-mediated transformation. one gram of si rice grain possessed the maximum activity level equivalent to μm of commercial rhigf-i. in vitro studies showed that rice-derived rhigf-i could induce membrane ruffling and enhance glucose uptake in l6myc cells. oral administration of transgenic rhigf-i rice confirmed that it reduced plasma glucose levels in stz-induced and zucker diabetic fatty rats, while it did not have significant effect in normal sd rats. taken together, our findings suggested that biologically active rhigf-i could be produced in transgenic rice. 
the provision of large quantity of recombinant proteins will promote further research on the therapeutic potential of rhigf-i. in vitro functional analysis showed that the rice-derived rhigf-i was effective in inducing membrane ruffling and glucose uptake on rat skeletal muscle cells. in the present study, an efficient plant expression system was developed to produce biologically active recombinant higf-i in transgenic rice grains. oral meal test with rice-containing rhigf-i acutely reduced blood glucose levels in streptozotocin-induced and zucker diabetic rats, whereas it had no effect in normal rats. we found that only glutelin signal peptide could lead to successful expression of higf-i and one gram of higf-i rice grain possessed the maximum activity level equivalent to micro molar of commercial rhigf-i. our findings provided an alternative expression system to produce large quantities of biologically active rhigf-i. to enhance the stability and yield of rhigf-i, the endoplasmic reticulum-retention signal and glutelin signal peptide were used to deliver rhigf-i to endoplasmic reticulum for stable accumulation. it is essential for cell proliferation and has been proposed for treatment of various endocrine-associated diseases including growth hormone insensitivity syndrome and diabetes mellitus. human insulin-like growth factor-i is a growth factor which is highly resemble to insulin.
 human insulin-like growth factor-i is a growth factor which is highly resemble to insulin. it is essential for cell proliferation and has been proposed for treatment of various endocrine-associated diseases including growth hormone insensitivity syndrome and diabetes mellitus. in the present study, an efficient plant expression system was developed to produce biologically active recombinant higf-i in transgenic rice grains.  the plant-codon-optimized higf-i was introduced into rice via agrobacterium-mediated transformation. to enhance the stability and yield of rhigf-i, the endoplasmic reticulum-retention signal and glutelin signal peptide were used to deliver rhigf-i to endoplasmic reticulum for stable accumulation. we found that only glutelin signal peptide could lead to successful expression of higf-i and one gram of higf-i rice grain possessed the maximum activity level equivalent to micro molar of commercial rhigf-i. in vitro functional analysis showed that the rice-derived rhigf-i was effective in inducing membrane ruffling and glucose uptake on rat skeletal muscle cells. oral meal test with rice-containing rhigf-i acutely reduced blood glucose levels in streptozotocin-induced and zucker diabetic rats, whereas it had no effect in normal rats.  our findings provided an alternative expression system to produce large quantities of biologically active rhigf-i. the provision of large quantity of recombinant proteins will promote further research on the therapeutic potential of rhigf-i. oryza sativa l., plant bioreactortransgenic plantrecombinant proteinprotein targetingkdeligf-i 