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Six Months of Gluten-Free Diet Do Not Influence Autoantibody Titers, but Improve Insulin Secretion in Subjects at High Risk for Type 1 Diabetes

Internal Medicine, Diabetes and Endocrinology Unit, San Raffaele Vita-Salute University Hospital and Scientific Institute, 20132 Milan, Italy

Address all correspondence and requests for reprints to: Emanuele Bosi, M.D., Internal Medicine, Diabetes and Endocrinology Unit, San Raffaele Institute, Via Olgettina, 60, 20132 Milan, Italy. E-mail: bosi.emanuele{at}hsr.it.

	Abstract

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Removal of gluten from the diet can attenuate the intensity of autoimmunity and reduces the incidence of diabetes in the nonobese diabetic mouse. In this study, we tested whether a gluten-free diet could reduce autoimmunity in human preclinical type 1 diabetes. A trial consisting of 6 months of a gluten-free diet followed by another 6 months of normal gluten-containing diet was performed in 17 first-degree relatives with at least 2 antibodies among islet cell antibodies, glutamic acid decarboxylase autoantibodies, protein tyrosine islet antigen-2 autoantibodies, and insulin autoantibodies. Treatment effect was measured as autoantibody titers and acute insulin response to iv glucose tolerance test. Two subjects dropped out for lack of compliance to diet restrictions. Of the remaining 15 subjects, 3 developed diabetes. Autoantibody titers did not show significant changes after 6 months of gluten-free diet and again after return to normal diet. Acute insulin response to iv glucose tolerance test significantly increased in 12 of 14 subjects after the first 6 months of gluten deprivation (P = 0.04) and decreased in 10 of 13 subjects during the following 6-month period of normal diet (P = 0.07). Insulin sensitivity (homeostasis model assessment-insulin resistance) nonsignificantly improved after the gluten-free diet and subsequently decreased (P < 0.005) after 6 months of normal diet. These findings indicate that 6 months of gluten deprivation do not influence humoral autoimmunity, but may have a beneficial effect on preservation of ß-cell function in subjects at risk for type 1 diabetes.

	Introduction

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THE CLINICAL ONSET of type 1 diabetes is preceded by a long latency during which the islet-specific autoimmune process starts and progressively matures, leading to a progressive destruction of the pancreatic ß-cell mass, insulin insufficiency, and ultimately hyperglycemia (1). This asymptomatic preclinical phase, which may last from a few months to several years, represents an ideal window during which therapeutic interventions can be implemented to delay or prevent the onset of clinical diabetes.

Dietary manipulation is a candidate approach to reduce diabetes incidence (2). Although such manipulation is expected to be most beneficial when applied before the development of autoimmunity, dietary agents may also perpetuate autoimmunity (3). The most common dietary manipulation used in clinical medicine is gluten deprivation, which is the basic treatment of celiac disease but can also be beneficial in other disorders acting through a reduction of the protein load to be processed at the intestine level. The removal of gluten proteins from diet can also be considered as an approach to reduce the risk of progression to type 1 diabetes based on several lines of evidence: 1) Type 1 diabetes and celiac disease are both autoimmune and frequently associated disorders, occurring mostly in infancy or adolescence with a preferential association with the HLA-DR3 allele (4). 2) Antibodies to tissue transglutaminase (TGCA), the most sensitive marker of celiac disease, are frequently detected in type 1 diabetes at the time of clinical diagnosis (5). 3) When the two diseases coexist, diabetes almost invariably precedes, and infrequently follows, the onset of celiac disease and initiation of gluten-free diet (4). 4) Several reports indicate an attenuation of autoimmune phenomena in celiac patients after diet therapy (6), and conversely, autoimmune manifestations associated with celiac disease are directly related to duration of exposure to diet gluten (7). 5) Gluten-free diet has been shown to reduce diabetes incidence in both Bio-Breeding rat (8) and nonobese diabetic mouse (9) and reduce signs and symptoms in patients with rheumatoid arthritis (10). Altogether, these data prompted us to hypothesize that a gluten-free diet could reduce diabetes-associated autoimmunity and as a consequence, the risk of developing type 1 diabetes (11). To determine whether a gluten-free diet reduces diabetes-associated autoimmunity, we measured islet autoantibodies before and after 6 months of gluten-free diet and again after 6 months of normal diet in islet autoantibody-positive first-degree relatives of patients with type 1 diabetes. The end-point of the study was a reduction of the humoral autoimmune response, evaluated as antibody titer, and residual insulin secretion, evaluated as acute insulin response to iv glucose tolerance test (IVGTT).

	Subjects and Methods

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Identification of subjects at risk and screening strategy

Subjects at risk were first-degree relatives of patients with type 1 diabetes recruited through a regionwide campaign of information and screening performed in collaboration with the local Patient Associations and Diabetes Societies within Lombardy and surrounding areas of northern Italy (12). This campaign was an extension of the previous San Raffaele Hospital-based family study and followed its proposed two-step antibody screening strategy (13): first step, based on glutamic acid decarboxylase autoantibodies (GADA) and protein tyrosine islet antigen-2 autoantibodies (IA-2A) measurement in whole capillary blood samples collected at home and sent to our laboratory by post with no refrigeration (14); second step, in those found positive to either GADA or IA-2A consisting in the measurement of GADA, IA-2A, insulin autoantibodies (IAA), and islet cell antibodies (ICA) on a second serum sample. From an initial 601 screened relatives, 17 (2.8%) had two or more antibodies and were considered at high risk to develop type 1 diabetes and therefore eligible for intervention. All were proposed to enter the trial and all accepted. Six were male, and the median age was 16 yr (range, 2–29 yr). None of the subjects were positive for TGCA. Both protocols of antibody screening and gluten deprivation treatment were submitted to and approved by the Ethical Committee of the San Raffaele Hospital Scientific Institute (Milan, Italy). Informed written consent was obtained from all subjects entering the trial.

Gluten-free diet protocol

The experimental protocol consisted of 6 months of gluten-free diet followed by 6 months of normal diet. All subjects participating in the study were specifically educated on diet restrictions and allowances, according to the protocol usually administered to patients with celiac disease, and they were provided with all commercially available gluten-free food preparations (Plasmon S.p.A., Milan, Italy). Compliance to diet was monitored on the basis of reporting by individual self and other family members.

Autoimmune and metabolic monitoring

Venous blood samples for autoantibody testing were obtained at the beginning of the study (time 0) and every 3 months during the 12-month intervention period (3, 6, 9, and 12 months). IVGTT was performed at the beginning of the study, at the end of the 6-month period of gluten deprivation, and again after 6 months of normal diet (time 0, 6, and 12 months).

Metabolic testing

IVGTT was performed as a 3-min infusion, and acute insulin response to glucose was calculated both as the sum of 1 + 3 min and as the area above baseline from 0 to 10 min ( AUC), according to an internationally standardized protocol (15). Insulin sensitivity (or insulin resistance) was estimated using the homeostasis model assessment-insulin resistance (HOMA-IR) and calculated as follows: baseline insulin concentration (microunits per milliliter) x baseline glucose concentration (millimoles per liter)/22.5 (16). Serum insulin was measured with a Microparticle Enzyme Immunoassay using a commercial kit (Abbott Laboratories, Abbott Park, IL).

Autoantibody measurements and criteria for change evaluation

Screening for GADA and IA-2A on capillary blood samples was performed as previously described (14); on subsequent serum samples, GADA (17), IA-2A (18), IAA (19), IgA and IgG TGCA (20) were measured by radiobinding and immunoprecipitation; ICA was measured by immunofluorescence (21); and IgA and IgG gliadin antibodies (AGA) were measured by immunoenzymatic method using a commercial kit (Pharmacia Inc., Peapack, NJ). Criteria used for defining changes in autoantibody titers were at least a doubling for increasing titers and at least a halving for decreasing titers from time 0 to 6 months or time 6 months to 12 months.

Statistical analysis

Comparisons between time 0 and 6 months and between 6 and 12 months were performed using the nonparametric Wilcoxon signed rank test.

	Results

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At baseline, for the 17 individuals with 2 or more islet autoantibodies, ICA were detected in 16 subjects, GADA in 16, IA-2A in 12, and IAA in 8; 7 subjects had all 4 antibodies, 4 had 3, and 6 had 2. In 9 subjects, baseline acute insulin response to IVGTT evaluated as 1 + 3 min was less than 50 mU/liter, a value approximately equivalent to the first centile in other studies (22, 23).

During the first 6 months of gluten deprivation, two subjects reported compliance failure very early and discontinued participation. Of the remaining 15 subjects, 3 developed diabetes: 1 during the first 6-month period of gluten deprivation, 1 during the second 6-month period of normal diet, and 1 immediately after completion of the study. IVGTT was not performed after diabetes onset in these subjects.

Autoantibody titers did not show any significant trend over the entire 12-month study period (Fig. 1). Using the criteria of doubling or halving of autoantibody titer as change, 10 of 15 subjects had a rise or fall in islet autoantibody titer at the end of the gluten-free diet period, and 8 of 14 after the subsequent 6-month period of normal diet. Of 45 autoantibody events measurable at the end of the gluten-free diet period, increases were observed in 8 (3 GADA, 4 IA-2A, and 1 IAA), decreases were observed in 6 (2 GADA, 1 IA-2A, and 3 IAA), and 31 remained unchanged. Of 42 autoantibody events measurable at the end of the normal diet period, increases were observed in 2 (1 GADA, 1 IAA), decreases were observed in 5 (2 GADA, 3 IA-2A), and 35 remained unchanged. In three relatives, IAA decreased to levels below the threshold of detection, and in one relative IAA became positive at the end of the gluten-free period; no other seroconversions were observed during this period. IgA and IgG TGCA and IgA AGA remained below the threshold for positivity throughout the study. IgG AGA were detected at high titer in one subject and at low titer in another and did not change during the study period.

View larger version (26K): [in this window] [in a new window]   Figure 1. Course of islet autoantibodies in subjects at risk for type 1 diabetes during 6 months of gluten-free diet and subsequent 6 months of normal gluten-containing diet. The thresholds for positivity were 3 U for GADA, 1 U for IA-2A, and 3 U for IAA.

View larger version (23K): [in this window] [in a new window]   Figure 2. First phase insulin response to iv glucose, expressed as 1 + 3 min plasma insulin concentration, and insulin sensitivity/resistance, expressed as HOMA-IR.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

In the majority of relatives, autoantibody titers remained substantially stable, and the changes that were observed during the 6 months of gluten deprivation and the subsequent 6 months of return to normal diet did not appear to be treatment related, because they were in both directions in each of the diet study periods. Changes in autoantibody levels consistent with gluten being the antigen, which drives the autoimmunity as in celiac disease, were not found in any of the relatives. These findings are similar to those recently reported in islet autoantibody-positive children placed on a gluten-free diet for a period of 12 months (24, 25). Therefore, although gluten is not excluded from a role in the early stages of islet autoimmunity, it is unlikely to be responsible for its perpetuation.

In view of the absence of an effect on humoral autoimmunity, the improved insulin secretion was intriguing. Although there is no untreated control group, the changes in insulin secretion after IVGTT seem treatment-related because the trend was inverted after return to normal diet. The overall change was small, however; of the nine subjects with insulin secretion below the first centile at the beginning of the study, during the 6 months of gluten deprivation, one developed diabetes, seven had an increase of insulin secretion, but only two of them recovered above the first centile threshold. During the following 6 months of normal diet, one subject developed diabetes, and all of the others showed a decline of insulin secretion. One of these developed diabetes soon after completion of the study. Interestingly, the trend observed for insulin secretion was paralleled by that of insulin sensitivity as estimated by HOMA-IR.

The mechanism by which gluten removal from the diet could improve insulin secretion and insulin sensitivity is unclear. These subjects do not have nutritional abnormalities as seen in celiac disease, and therefore effects as a result of nutritional improvement do not seem likely. The glycemic index of gluten-free and gluten-containing foods is similar (26). However, it is feasible that the different type of carbohydrates in the diet may influence insulin response to glucose (27). It is also possible that islet autoimmune mechanisms may have been ameliorated without any appreciable effect on autoantibody titer.

Because this was an intervention in nondiabetic individuals, it was interesting to find a 100% acceptance and high compliance by the relatives of the protocol. Only two did not comply, and diet was relatively well tolerated. On one side, this was surprising provided the high content of gluten in the standard Italian diet, but on the other side, it emphasizes the motivation in these relatives to thwart diabetes risk, and, indeed, the majority of them requested to be maintained on this exclusion diet for a longer period of time.

	Acknowledgments

 
We are grateful to Plasmon S.p.A. for providing gluten-free food preparations, Marco Manzoni and Daniela Guiducci for assistance in the initial stages of the screening campaign, Lucilla Monti for insulin measurement, and Karen Toussoun and Maria Gioia Pich for clinical assistance.

	Footnotes

 
This work was supported by PREVEFIN project, Italian Ministry of Health; 1999–2000 cofunded project Italian MURST; special grant, University of Milan.

Abbreviations: AGA, Gliadin antibodies; AUC, incremental area under the curve; GADA, glutamic acid decarboxylase autoantibodies; HOMA-IR, homeostasis model assessment-insulin resistance; IA-2A, protein tyrosine islet antigen-2 autoantibodies; IAA, insulin autoantibodies; ICA, islet cell antibodies; IVGTT, iv glucose tolerance test; TGCA, tissue transglutaminase C autoantibodies.

Received July 29, 2002.

Accepted October 11, 2002.

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