JP2000503531A - Products derived from milk substantially free of beta casein from non-human mammals and related uses - Google Patents

Abstract

  (57) [Summary] The present invention relates to milk-derived products that are substantially free of beta-casein from non-human mammals. The invention also relates to the use of such products, especially for diets, and more particularly to the use of such products for early infants in the prevention of insulin-dependent diabetes.

Description

BACKGROUND OF THE INVENTION Technical Field of the Invention The induced fresh products and associated uses invention the beta casein from non-human mammal from milk substantially free is substantially the beta casein from non-human mammal Dairy-derived products, which do not contain dairy products. The invention also relates to the use of such products, especially with respect to its application, in diets in the prevention of insulin-dependent diabetes, in particular for early infants. BACKGROUND OF THE INVENTION It is well known that the art of obtaining products for early infants, especially food products, starts with non-human milk, such as milk, sheep and goat milk. The basic component of milk is characterized in the basic glossary by casein, which represents a group of proteins obtainable from milk sediment at acid pH and room temperature, in particular at pH 4.6 and 20 ° C. Casein represents about 80% of total milk protein and about 40% p / v of human milk. Casein can be subdivided into three main groups: alpha, beta and kappa. There is also a fourth group, represented by gamma casein, derived by removing the first 22 amino acids from beta casein. Thus, for the present invention, gamma casein could be considered as a part of beta casein. Beta casein represents about 25% p / v of total casein present in cow milk, compared to about 70% p / v in human milk. Bovine beta casein is known and characterized for several genetic variances, including A1, A2, A3, B, C, D and E. For the industrial production of milk, the genetic variance A1 of milk has mainly been preferred for increasing milk production. This dispersion, which contains more proteins than others, has been obtained from a variety of selected animals, especially cattle. Genetic data sequence analysis identified the amino acid sequence at positions 63-68 corresponding to the human beta casein 54-59 sequence for bovine beta casein A1. A similar situation was found for dispersion A2. Both the A1 and A2 variances of beta casein show sequence homology (at least 90%) within that region with the insulin-producing cell specific protein (GLUT2) in the pancreas. According to the inventor, sequences 63-68 of A1 and A2 beta casein, and more generally, other types of analogous sequences of casein such as A1, A2, A3, B, C, D and E, have An immune response is elicited through the production of anti-beta casein antibodies and lymphocytes that recognize such sequences. For the newborn and the first month of life infancy, the diet containing casein of these immunogens, due to the mechanism of molecular homology with the homologous sequence of beta casein, has been shown to respond to GLUT2 in insulin-producing cells of the pancreas. May cause a specific immune response. Under such a hypothesis, with the aim of obtaining a bovine milk product substantially free of non-human beta casein, and more particularly, the lack of such sequence homology results in an immunogenic result with respect to the GLUT2 protein. Studies were conducted to obtain beta casein-containing products from non-human mammals that did not. SUMMARY OF THE INVENTION The present invention relates to a milk-derived product or milk itself that is substantially free of non-human beta casein with immunogenic properties as specified in the prior art. Another object of the present invention is a milk-derived product or milk itself comprising at least one modified beta casein from a non-human mammal without the immunogenic properties described above. Another object of the present invention is the use of such products for diets. Another object of the present invention is the use of a product from milk or milk itself which is substantially free of non-human mammal beta casein for early infant diets for the prevention of insulin dependent diabetes. Further objects of the present invention will become clear from the detailed description of the invention. DETAILED DESCRIPTION OF THE INVENTION In the accompanying description, important amino acid sequences according to the invention are underlined. The term "substantially" refers to the presence of a substance in an amount ranging from 0 to 10% by weight. The amino acid sequences that are beneficial to the present invention are described below. As described above, according to the present inventor, insulin is exposed to milk due to molecular homology between the amino acid sequences of A1 and A2 of beta casein and the specific sequence of GLUT2 protein found in insulin producing cells. There is a correlation between the development of dependent diabetes. Such a sequence was confirmed as follows: Pro-Gly-Pro-Ile-His-Asn for A1 beta casein (where the underlined sequence is SEQ ID NO: 1) is a larger fragment: Ser- It is inserted into the Leu-Val-Tyr-Pro- Phe- Pr o-Gly-Pro-Ile-His -Asn ( SEQ ID NO: 3) (inserted in). As already mentioned, such sequences are also present in gamma casein. Other sequences for immunogenic peptides of beta casein that differ from those described above are exemplified. Cattle (bos taurus) derived from bovine (cow) beta casein (63-68), Pro-Gly- Pro-Ile- Pro -Asn ( sequences underlined here is SEQ ID NO: 2) is larger fragment: Ser -Leu-Val-Tyr-Pro-Phe- Pro-Gly-Pro-Ile-Pro- Asn (SEQ ID NO: 4). Beta casein (63-68) from bos indicus: Pro-Gly-Pro-Ile-Pro -Asn (sequence of SEQ ID NO: 2 underlined). As a comparison, human beta casein has the following sequence (48-59): Ser-Leu-Val-Tyr-Pro-Phe-Val-Glu-Pro-Ile-Pro-Tyr (SEQ ID NO: 6). A peptide fraction (54-59) suitable for the present invention was identified as Val-Glu-Pro-Ile-Pro (where the underlined sequence is SEQ ID NO: 5). The peptide sequence of GLUT2, a glucose transporter in insulin producing beta cells in the pancreas, is as follows. (409-420) Ser-Phe-Phe-Glu-lle-Gly- Pro-Gly-Pro-Ile-His -Trp (412-423) Glu-lle-Gly- Pro-Gly-Pro-Ile-His -Trp -Phe-Met-Val (414-425) Gly- Pro-Gly-Pro-Ile-His- Trp-Phe-Met-Val-Ala-Glu The inventors have sequenced A1, B and C beta casein and gamma casein, Pro-Gly-ro-Ile-His (SEQ ID NO: 1) and larger fragments containing it, similar sequences of beta caseins A2, A3 and E, Pro-Gly-Pro-Ile-Pro (SEQ ID NO: 2) ) Elicit an immune response to beta casein, suggesting that cross reactivity directs the immune response to the GLUT2 homologous sequence, causing destruction of the insulin-producing cells. Thus, the production of milk or generally food products comprising the non-immunogenic beta-casein for delivery to the diet is a prophylactic guide to insulin-dependent diabetes, especially for newborns and early infants. All caseins that do not contain the sequence Pro-Gly-Pro-Ile-His (SEQ ID NO: 1) or Pro-Gly-Pro-Ile-Pro (SEQ ID NO: 2) are not considered to be pathogenic and are therefore according to the invention Can be used to produce dietary products. The casein is modified with some or all amino acids present in the sequence; beta casein that does not contain such a sequence (eg, it has been removed); Beta casein modified to be replaced with a sequence of beta casein; all modifications are well known in the art of genetic engineering and classical biological techniques of cross selection as described in WO 93/04171. This can be done by applying technology. The milk obtained containing the casein modified as described above can be dispensed as such, since the casein contained therein can be separated and used to produce food and drug products, Alternatively, it can be processed by known methods. In particular, such casein-containing products can be used for administration to early infants and as a diet for the prevention of subsequent insulin-dependent diabetes. In the product according to the invention, A1 and / or A2 and / or other immunogen beta casein, in particular the sequence Pro-Gly-Pro-Ile-His (SEQ ID NO: 1) or Pro-Gly-Ile-Pro (SEQ ID NO: 1) It is preferred that those accompanied by 2) are not present in more than 10% by weight in the final product. The food product of the present invention is a variant in which the immunogen casein is replaced by the casein of the present invention, for example, for proteins such as pasta, milk and milk-derived products and proteins which are added to all foods already on the market. be able to. Also part of the invention are synthetic proteins, such as those derived from vegetables and / or soy. In accordance with the teachings of the present invention, beta casein with the amino acid sequence Pro-Gly-Pro-Ile-His (SEQ ID NO: 1) or Pro-Gly-Pro-Ile-Pro (SEQ ID NO: 2), especially for early infants It is possible to produce a diet or drug product that is substantially free of, or with such an arrangement, is less than 10% of the final weight of the product. It is also possible to produce food products or milk according to the following alternatives. -Beta casein is less than 10% by weight, or beta casein comprising the amino acid sequence Gly-Pro-Ile-His (SEQ ID NO: 7) or Gly-Pro-Ile-Pro (SEQ ID NO: 8) is 10% by weight Less substantially-beta casein comprising the amino acid sequence Gly-Pro-Ile-His (SEQ ID NO: 7) or Gly-Pro-Ile-Pro (SEQ ID NO :); and Incorporating peptides derived by hydrolyzing animals, vegetables and / or synthetic proteins lacking their mixture (FR 86-00325, WO 94/06306, WO p (02 539)); Beta casein comprising the amino acid sequence Gly-Pro-Ile-His (SEQ ID NO: 7) or Gly-Pro-Ile-Pro (SEQ ID NO: 8) is less than 10% by weight, and these sequences and mixtures thereof Animals and / or vegetables and / or synthetic proteins lacking Incorporating a peptide obtained by hydrolysis;-Does not genetically produce a protein having the amino acid sequence Gly-Pro-Ile-His (SEQ ID NO: 7) or Gly-Pro-Ile-Pro (SEQ ID NO: 8) Beta casein lacking the amino acid sequence in a protein obtained from an animal species;-milk naturally lacking beta casein because it is produced by a genetically modified animal according to patent WO 93/04171 -Milk comprising human beta casein obtained from a genetically engineered microorganism or animal as described in the above-mentioned patents; the protein fraction is obtained from chemical-physical processing of the milk and also lyophilized casein. Can be derived, for example, by differential solubility, liquid-liquid extraction, membrane separation, chromatographic separation as described in patents FR 86-00325 and WO 92/00017. Integration has been produced using yeast, bacteria, fungi or transgenic animals and with one of the well-known cloning methods such as those described in patent WO 93/04171, It can be performed using recombinant beta casein. The method for removing beta casein from milk is a chromatography method described below. Residual alpha and kappa casein are obtained by means of such a process, starting from acidic casein and separating beta casein by means of a two-step chromatographic means. The method can be optimized using already useful knowledge in the field. Such a method involves the use of an ion exchange resin as a base phase, such as Sepharose ^R from Pharmacia, with concentrating in a column. The mobile phase is constituted by buffer A comprising: sodium acetate at a concentration not lower than -10 mM; urea at a concentration not lower than -2 M; pH between -5 and 6. Acidic casein can be dissolved in buffer A at a pH below 6 with the addition of the specific reducing agent DTT (ditiotreeitol, dithiothreitol). The mixture is left under stirring for 24 hours, thus bringing it to a pH between 5 and 6, and placing it in the column. The beta casein fraction does not interact with the resin and elutes in 0 M NaCl. Therefore, it is not necessary to perform the step of increasing the ion concentration. The method is conveniently accomplished by simply eluting with buffer B at a concentration of no less than -10 mM sodium acetate; no more than -2 M urea; -0.8 M NaCl; a pH between -5 and 6; It involves the simple separation of beta casein from other fractions that would collect in the composition (isocratic). The fractionation removes urea and other impurities by subsequent diafiltration, and after concentration, collects the casein by acid precipitation and lyophilization of the resulting wet product. BRIEF DESCRIPTION OF THE FIGURES FIG. 1: Chromatogram for the first load of Example 1. Figure 2: Chromatographic peak for beta casein. Figure 3: Lack of beta casein in the chromatogram. Example 1 Separation of beta casein from acidic casein and collection of residual alpha and kappa casein fractions. Preparation 200 g acidic casein + 3000 ml buffer C: 20 mM sodium acetate. 4M urea. 10 mM ditiotreeitol DTT pH7. The casein is slowly dissolved in the buffer while maintaining buffer A at pH 7 with 2M NaOH for each addition. Leave under stirring at 5 ° C. for about 12 hours. The solution is filtered through a pre-filter Mi11ex AP-50 (Millipore). Wash the prefilter with 1000 ml of buffer C and collect. Bring 4000 ml of load to pH 5.5 with HCl 6M and adjust the ionic strength (2.2 mS) to 4.5 mS. Preparative chromatography- FPLC Waters 600 controller-Developer: Perkin Elmer UV / VIS spectrophotometer Lambda 3B 280 nm-Column: XK 50 Pharmacia (maximum pressure 3 bar) φ 5 cm, height 100 cm-Resin: S -Sepharose Pharmacia Height 85 cm, Volume 1670 ml-Eluent: Buffer A Sodium acetate 20 mM urea 4 M pH 5.5, Ionic strength 1.5 mS Buffer B Sodium acetate 20 mM urea 4 M pH 5.5 Sodium chloride 1 M Ionic strength 57.1 mS All buffers were filtered using a Millex prefilter AP-50 coupled in combination with a 0.45 μm Millipak (Millipore). -Temperature: room temperature-Conditioning: about 8000 ml Buffer A: 97% Buffer B: 3% Mixed ionic strength: 4.5 mS Flow: 30 ml / min P = 42 PSI Time: 4 hours, 25 minutes-Loading: buffer 200 g acidic casein dissolved in C (4000 ml total volume) Flow: 20 ml / min P = 50 PSI Time: 3 hours, 20 minutes-Elution: Approx. 8000 ml of the first stage (isocratic) Buffer A: 97% buffer Solution B: 3% flow: 30 ml / min P: 42 PSI Time: 4 hours, 25 minutes First stage (isocratic) approx. 9000 ml Buffer A: 20% Buffer B: 80% flow: 30 ml / min P = 42 PSI time: 5 hours Example 2-Control test The amount of the product of Example 1 was tested by chromatography to assess the absence of beta casein in the second stage isocratic. Such a lack was confirmed by the chromatogram in FIG. By comparison, FIG. 2 shows the peak for the presence of beta casein, which is only derived from the first stage elution, while FIG. 1 shows the chromatogram for the first load. Analytical chromatography- FPLC Perkin Elmer Biocompatible Binary Pump 250-Developer: Perkin Elmer LC95 280 nm-Column: Mono S HR Pharmacia-Loop: 100 μl-Eluate: Buffer A ^* Sodium acetate 20 mM urea 6 M pH 5 Buffer B ^* Sodium acetate 20 mM Urea 6 M Sodium chloride 1 M pH 5-Temperature: Room temperature-Flow: 1 ml / min-Conditioning: 8'00 "Buffer A ^* 100% Buffer B ^* : 0%-Elution: Gradient 5'00 "buffer B ^* 50% buffer B ^* : 50% (increase of B ^* 1% min) Incratic 2'00" buffer A ^* 50% buffer B ^* : 0% incratic 5 ' 00 "Buffer A ^* 0% Buffer B ^* : 100% Example 3 The product of Example 1 is purified from urea by the following method of diafiltration. −Ultrafiltration SGI −Cellulose membrane S-10 10,000 Da Amicon −Dialysis buffer: Demineralized water Sodium acetate 10 mM pH-7 Ionic strength 0.8 mS Total volume 250 1 (5 washes) Permeation flow: 32 + 37 1 / h − Temperature: 10 ° C.—product concentration up to 501, 201 The product was tested as follows to demonstrate lack of urea. Urea test-UV method (Boehringer Mannheim)-Spectrophotometer: Lambda 3B 340 nm (Perkin Elmer) Solution 1 = Triethanolamine buffer, pH 8.2, oxoglutarate, NADH solution 2 = Urease (Urase) solution 3 = Glutamate dehydrogenase Christ model Beta 1-16 Freeze urea-free products using the instrument Drying took place.

[Procedure of Amendment] Article 184-8, Paragraph 1 of the Patent Act [Submission date] January 7, 1998 (1998.1.7) [Correction contents]                                 The scope of the claims 1. Non-human beta-case used in diets to prevent insulin-dependent diabetes A milk-derived diet or drug product substantially free of ins, or Milk itself. 2. Used in diets to prevent insulin-dependent diabetes, GLUT2 protein Beta from non-human mammals resulting in immunogenicity in terms of molecular homology to the protein A milk-derived diet or drug product that is substantially free of casein, or Or milk itself. 3. Used in diets to prevent insulin dependent diabetes, protein GLU Bases from non-human mammals that are molecularly homologous to T2 and are consequently immunogens Animals, vegetables, their composites and mixtures thereof, which are substantially free of casein Milk-derived diet with the addition of the non-immunogen beta casein selected from Or drug product, or milk itself. 4. Used in the diet for the prevention of insulin-dependent diabetes, sequence: Pro-Gly-Pr o-Ile-His (SEQ ID NO: 1) or Pro-Gly-Pro-Ile-Pro (SEQ ID NO: 2) or it And a sequence comprising: Ser-Leu-Val-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-His-Asn Column No. 3) or Ser-Leu-Val-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-Asn (SEQ ID NO: No. 4) or a milk-derived diet substantially free of casein. Or the drug product, or the milk itself. 5. Used in diets to prevent insulin-dependent diabetes mellitus, sequence: Pro-G1y-Pr Does not give o-Ile-His (SEQ ID NO: 1) or Pro-Gly-Pro-Ile-Pro (SEQ ID NO: 2) Milk-derived diet or drug product comprising casein, or milk By itself, The casein -Some or all amino acids in the sequence are modified; The sequence has been removed, -The sequence has been replaced by a homologous sequence in human beta casein and related mixtures; Selected from 6. Used in diets to prevent insulin-dependent diabetes, sequence: Val-Glu-Pr o-Ile-Pro (SEQ ID NO: 5) or a longer sequence containing it: Ser-Leu-Val-Tyr-Pro -Phe-Val-Glu-Pro-Ile-Pro-Tyr (SEQ ID NO: 6). Diet or drug product derived from milk, or milk itself. 7. 2. The immunogen beta casein comprising less than 10% by weight of the immunogen beta casein. 6. The product according to any of 6. 8. Used in diets to prevent insulin-dependent diabetes, amino acid sequence: Gl Tan with y-Pro-Ile-His (SEQ ID NO: 7) or Gly-Pro-Ile-Pro (SEQ ID NO: 8) Since the amino acid sequence was produced by an animal species that does not genetically produce Diet or drug derived from milk, comprising casein lacking rows The product, or the milk itself. 9. Used in diets to prevent insulin-dependent diabetes, genetically modified Milk that is naturally lacking beta casein because it was produced by animals. 10. Used in diets to prevent insulin-dependent diabetes and genetically engineered Milk containing human beta casein obtained from a microorganism or animal obtained. 11. Amino acid sequences are transformed through the application of techniques such as genetic engineering and biological cross selection. A method for obtaining the diet product of claim 5, wherein the diet product is modified. 12. Manufacture of a diet or drug product for the prevention of insulin-dependent diabetes Casein or pep without molecular homology to protein GLUT2 Use of tide fragments. 13. Manufacture of a diet or drug product for the prevention of insulin dependent diabetes Use of a beta casein or a peptide fragment according to claim 12 for the treatment. 14． Manufacture of a diet or drug product for the prevention of insulin dependent diabetes Use of a casein according to any of claims 5-8 for the treatment. 15. Manufacture of a diet or drug product for the prevention of insulin dependent diabetes Use of a milk according to any of claims 9 to 10 for the use of.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C07K 14/47 ZNA A61K 37/16 (81) Designated country EP (AT, BE, CH, DE, DK, ES , FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN , TD, TG), AP (KE, LS, MW, SD, SZ, UG), UA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU , AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, HU, IL, IS, P, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU , SD, SE, SG, SI, SK, TJ, TM, TR, TT, UA, UG, US, UZ, VN

Claims (1)

[Claims] 1. For the manufacture of a diet or drug product for the prevention of insulin-dependent diabetes Beta casein which does not show molecular homology with the protein GLUT2 or Peptide fragments. 2. Non-human beta-case used in diets to prevent insulin-dependent diabetes A milk-derived diet or drug product substantially free of ins, or Milk itself. 3. Used in diets to prevent insulin-dependent diabetes, GLUT2 protein Beta from non-human mammals resulting in immunogenicity in terms of molecular homology to the protein A milk-derived diet or drug product that is substantially free of casein, or Or milk itself. 4. Used in diets to prevent insulin dependent diabetes, protein GLU Bases from non-human mammals that are molecularly homologous to T2 and are consequently immunogens Animals, vegetables, their composites and mixtures thereof, which are substantially free of casein Milk-derived diet with the addition of the non-immunogen beta casein selected from Or drug product, or milk itself. 5. Used in the diet for the prevention of insulin-dependent diabetes, sequence: Pro-Gly-Pr o-Ile-His (SEQ ID NO: 1) or Pro-Gly-Pro-Ile-Pro (SEQ ID NO: 2) or it And a sequence comprising: Ser-Leu-Val-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-His-Asn Column No. 3) or Ser-Leu-Val-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-Asn (SEQ ID NO: No. 4) or a milk-derived diet substantially free of casein. Or the drug product, or the milk itself. 6. Used in the diet for the prevention of insulin-dependent diabetes, sequence: Pro-Gly-Pr Does not give o-Ile-His (SEQ ID NO: 1) or Pro-Gly-Pro-Ile-Pro (SEQ ID NO: 2) Milk-derived diet or drug product comprising casein, or milk By itself, The casein -Some or all amino acids in the sequence are modified; The sequence has been removed, -The sequence has been replaced by a homologous sequence in human beta casein and related mixtures; Selected from 7. Used in diets to prevent insulin-dependent diabetes, sequence: Val-Glu-Pr o-Ile-Pro (SEQ ID NO: 5) or a longer sequence containing it: Ser-Leu-Val-Tyr-Pro -Phe-Val-Glu-Pro-Ile-Pro-Tyr (SEQ ID NO: 6). Diet or drug product derived from milk, or milk itself. 8. Claim 1-comprising the immunogen beta casein in an amount less than 10% by weight. 8. The product according to any one of items 7. 9. The product according to any one of claims 2 to 8, and vegetables, animals and / or vegetables. The sequence: Pro-Gly-Pro-Ile-His (SEQ ID NO: 1) or Pro-Gly Vegetables, animals and / or lacking -Pro-Ile-Pro (SEQ ID NO: 2) and mixtures thereof Or those incorporating peptides derived from the hydrolysis of synthetic proteins. 10. Used in diets to prevent insulin-dependent diabetes, amino acid sequence: Gly-Pro-Ile-His (SEQ ID NO: 7) or Gly-Pro-Ile-Pro (SEQ ID NO: 8) The above amino acids are produced by animal species that do not genetically produce proteins. Diet or drug derived from milk, comprising casein lacking the sequence Agent product, or milk itself. 11. Used in diets to prevent insulin-dependent diabetes and is genetically modified Milk that is naturally devoid of beta-casein because it is produced by a bred animal. 12. Used in diets to prevent insulin-dependent diabetes and genetically engineered Milk containing human beta casein obtained from a microorganism or animal obtained. 13. An ion exchange resin is used as the stationary phase, and the mobile phase is Sodium acetate at a concentration not less than -10 mM Concentration of urea not less than -2M A pH between -5 and 6; A method for extracting beta casein from milk, which is a buffer A comprising Such a method is based on acid casei previously dissolved in buffer A having a pH not lower than 6. Dithiothreitol was added to the mixture, and the whole substance was stirred for 24 hours. And thus brought to a pH between 5 and 6 and loaded into the column, Eluting at a concentration of 0 M NaCl; isolating other fractions containing beta casein And collect Sodium acetate at a concentration not less than -10 mM Concentration of urea not less than -2M -0.8M NaCl A pH between -5 and 6; Of urea and other impurities by diafiltration. After removal and concentration, the casein is collected by acid precipitation and lyophilization of the wet product. The process. 14． Amino acid sequences are processed through the application of techniques such as genetic engineering and biological hybrid selection. A method for obtaining a diet product according to claim 6, wherein the diet product is modified. 15. 2. The beta casein or claim 3 according to claim 1 for prevention of insulin-dependent diabetes. Or use of peptide fragments. 16. The dietary diet according to claim 2 to 10 for prevention of insulin-dependent diabetes. Or use of drug products. 17． Use of milk according to claims 11-12 for the prevention of insulin dependent diabetes .