Insulin, insulin antibodies and insulin autoantibodies

Recently we have been queried about the relationship of insulin antibodies (IA) and the development of either hypoglycemia or hyperglycemia in various persons with type 1 diabetes mellitus (T1DM). As opposed to IA, which are induced by any type of exogenous insulin, insulin autoantibodies (IAA) occur spontaneously in the plasma of some patients (predominantly children) prior to the diagnosis of T1DM and prior to exposure to exogenous insulin.^1,2 In general, IA are in much higher concentration than IAA.

A not uncommon inquiry is as follows: “A patient with T1DM is suffering recurrent and possibly severe hypoglycemia. Could this be due to insulin autoantibodies and/or the ‘insulin autoimmune syndrome’ (IAS; also known as Hirata disease)?”³ The other question is: “A patient with T1DM is taking his insulin but is in poor metabolic control. Could he be resistant to injected insulin? Should we measure his insulin levels?”

In terms of hypoglycemia that required hospital admission, often insulin and C-peptide were measured at the time of the hypoglycemic episode. Insulin present in the circulation could be endogenous or exogenous. However, the only source of C-peptide is the patient’s own pancreatic beta cells. So how are the C-peptide and insulin measurements interpreted in such clinical scenarios?

Insulin measurements

First, let’s address the question of the insulin measurement. Measuring insulin in insulin-treated patients is conceptually complex because many patients treated with insulin injections develop IA.⁴ This is true regardless of the type of insulin injected. Recombinant DNA insulins appear to be more immunogenic than animal insulins.⁵ Even exogenous human insulin is immunogenic.⁶ The literature reports IA frequencies of 78 percent to 97 percent in insulin-treated patients.^5,7,8 IA generally raise total insulin levels because IA act as binding proteins and the insulin immunoassays measure both bound and free insulin. The problem is that it is the “free” (unbound) insulin that is believed to be biologically active. Total insulin levels in IA-positive patients do not reflect the free insulin levels. Another consideration is the degree to which proinsulin is recognized in the insulin assay.

Normally insulin has no binding proteins in the circulation, so the “total” insulin concentration in the blood is identical to “bioactive” insulin. Unlike insulin-like growth factor I (IGF-I), where stable concentrations long-term are biologically desirable, for survival of the individual, insulin secretion needs to be regulated rapidly and precisely with secreted insulin cleared swiftly. Circulating insulin has a half-life of ~4 minutes⁹ and a duration of action (when given IV) of ~20 minutes. The half-life of C-peptide is somewhat longer, being near 10 minutes.⁹ So…why not measure “free” insulin? Indeed, we do measure free thyroxine (T4) routinely in assessing thyroid function.

Measuring free insulin

The problem with free insulin measurements is that these measurements are poorly standardized. The pre-analytical conditions can greatly affect this measurement.¹⁰ One paper concluded that samples for free insulin should be immediately treated with polyethylene glycol to remove antibody-bound insulin prior to analysis.¹¹ It is highly unlikely that this is done in routine clinical laboratories when only reference laboratories measure free insulin. The type of precipitation method for the measurement of free insulin can also alter the free insulin concentration by a factor of two!¹²

Without standardization, we are reluctant to endorse free insulin measurements as clinically useful in the routine evaluation of persons with T1DM and hypoglycemia or hyperglycemia. Certainly total insulin measurements in insulin-treated patients with IA are uninformative. Measuring C-peptide should be helpful in assessing whether insulin is being secreted endogenously. This is because injected insulins lack C-peptide and, therefore, there is no exogenous C-peptide to serve as an immunogen to stimulate C-peptide antibodies. Thus, C-peptide measurements are technically and clinically valid. Last, it is important to note that, unlike free insulin, we do not measure free T4 by precipitation methods after the removal of the protein-bound T4. Such methods are not accurate for the measurement of free T4, nor are such methods reproducible.

Insulin autoantibodies

Can IAA ever cause hypoglycemia? The answer is yes: IAS is a real condition. In IAS, IAA develop that can release excessive quantities of insulin, raising the free insulin concentration and abruptly producing hypoglycemia. At other times, hyperglycemia may develop from inadequate levels of free insulin.

Insulin autoimmune syndrome

IAS is a rare condition (except possibly in Japan where it is reported to be the third-most common cause of hypoglycemia¹³). Between 1970 and 2007, there were 380 reported cases of IAS, and most cases were reported from Japan.¹⁴ In the almost 20 years that we have measured IAA (and our method for IAA also detects IA and does not distinguish IAA from IA), we are aware of only one regional case of IAS. [Note: In our laboratory IAA (and IA) are measured by the immunoprecipitation of patient serum with polyethylene glycol after a one-week incubation with added A14-monoiodinated insulin.]

Some authors define IAS as occurring only in non-insulin treated individuals.¹⁵ Generally, IAS is not associated with T1DM, although the concurrence of these disorders has been reported.¹⁶ More commonly, IAS is seen in association with Graves disease and/or the use of drugs that can include sulfur atoms such as methimazole, alfa-mercaptopropionyl glycine, glutathione, or alpha lipoic acid (marketed as an anti-aging/dieting supplement).^17-20 Some investigators have reported insulin receptor autoantibodies in the serum of persons with the IAS.²¹  We are not aware of any commercial assays that measure insulin receptor autoantibodies.

Insulin antibodies

Can IA, which are acquired after treatment with insulin, cause hypoglycemia or hyperglycemia? While there are scattered case reports that IA can cause hypo- or hyperglycemia in insulin-treated patients,^22-24 overall there is little if any evidence that IA alone routinely cause dysglycemia. This was the conclusion of a 2005 Cochrane review.²⁵ Fifteen years earlier, researchers in Sweden reached the same conclusion.²⁶ Still a third paper from Bucharest published in 1990 found no relationship between IA and hypoglycemia.²⁷

Injected insulins

Further complicating insulin measurements in insulin-treated patients is the nature of the injected insulin (Table 1, Figure 1). For example, one insulin immunoassay only detects human insulin and does not detect recombinant-DNA-produced insulins with altered amino acid sequences.  Therefore, the nature of the injected insulin and insulin specificity of the immunoassay must be known. There is also the potential complication of endogenous production (the patient’s “human” insulin) and human insulin of exogenous origin. If IA are not present, having separate immunoassays for insulin, proinsulin, and all of the sequence-modified insulins could be beneficial. There may be a need to measure insulin in persons with diabetes if an insulinoma is suspected. Insulinoma has rarely been reported in patients with diabetes.^28,29 Possibly the earliest report of this is from 1958.³⁰

Hyperglycemia in persons with diabetes

In the case of T1DM patients with poor metabolic control (i.e., an elevated HbA1c), if IA exist (as is the case in the majority of insulin-treated patients), the level of exogenous insulin cannot be assessed. If IA are absent and insulin levels are being measured to assess control, the critical issue is having insulin assays that are capable of detecting the various species of exogenous insulin. While insulin resistance can develop in persons treated with insulin, the most common cause of poor metabolic control is inadequate insulin (including missed doses of insulin), mistimed insulin administration, excess caloric intake, and/or inadequate exercise.

In summary, IA are extremely common in insulin-treated patients. Thankfully, IA only rarely cause hypo or hyperglycemia. Measuring insulin in insulin-treated patients is very complex. If IA are present, we do not recommend measuring insulin. Measurements of free insulin are troublesome and their reliability is questionable. If IA are absent, measuring insulin is complicated by the various species of insulin that are injected and the ability of the insulin assay to detect non-human insulins.

REFERENCES

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William E. Winter, MD, DABCC, FACB, FCAP, is Professor of Pathology and Pediatrics at the University of Florida. Dr. Winter directs the Endocrine Autoantibody Laboratory that is part of the University of Florida Health Pathology Laboratories (UFHPL).

David Pittman serves as the manager of the University of Florida Health Pathology Laboratories (UFHPL), Endocrine Autoantibody Laboratory. Mr. Pittman is responsible for both the clinical and research activities of the laboratory unit including the Islet Cell Autoantibody (ICA) Core Laboratory for the Type 1 Diabetes TrialNet study, and thyroid autoantibody and TSH testing for the TEDDY study (The Environmental Determinants of Diabetes in Youth).

Laura M. Jacobsen, MD, is currently a second-year pediatric endocrinology fellow at the University of Florida. Dr. Jacobsen completed her medical school training at the University of Florida and her pediatric residency at the University of North Carolina in Chapel Hill.