Thyroid hormones and glycaemic indices in euthyroid, hyperthyroid, hypothyroid, all type 2 diabetics and non-diabetic subjects

Abnormal thyroid hormone levels have been reported in type 2, but the relationship between thyroid hormone levels and glycaemic indices: fasting plasma glucose (FPG), C-peptide (C-pep) and glycated haemoglobin (HbAlc), used in the management of type 2 is not well defined. This cross-sectional study examined the relationship between thyroid hormones and glycaemic indices in type 2. Positive correlations were observed between FPG and HbAlc in hypothyroid (r = 0.382, P = 0.011) and hyperthyroid group (r = 0.295, P = 0.012). FPG correlated with C-pep in hyperthyroid diabetics (r = 0.481, P < 0.001). HbAlc and TSH correlated positively in hypothyroid diabetics (r = 0.330: P = 0.031). HbAlc also correlated with T4 in hypothyroid diabetics (r = 0.379: P = 0.012). C-peptide and TSH correlated positively in hyperthyroid diabetics only (r = 0.279; P = 0.042). C-peptide also correlated with T3 and T4 in euthyroid diabetics (r = 0.231, P = 0.020; r = 0.248, P = 0.045), respectively. The presence of abnormal levels of thyroid hormones influenced glycaemic indices in type 2 population. This implies that thyroid hormones investigation can assist in proper diagnosis and management of diabetes mellitus.


Background
Thyroid disease and diabetes mellitus are common endocrine disorders encountered in clinical practice. Both diabetes mellitus and thyroid disorders mutually influence each other (American Diabetic Association 2014). Researches indicate that there is increased incidence of type 2 globally, highlighting increased incidence of childhood type 2 diabetes mellitus (Jin et al. 2011). Predictions for the future are that in 2030 the number of persons suffering type 2 and the percentage of individuals with the disease will be more than double (Wilds et al. 2004). In developing countries, increasing morbidity of type 2 is reported (Smithson 1998). The increasing incidence of type 2 is compounded by reports of the coexistence of thyroid dysfunction with diabetes (Udiong et al. 2015). Furthermore, disturbing is the report that the thyroid disorders may present with low or raised levels of thyroid hormones (Ghazali and Abbingesuku 2010;Gursoy and Turkel 1999;O' Meara et al. 1993). Glycaemic index levels are commonly used for diagnosis and management of diabetes mellitus without due attention to the influence of thyroid hormone levels on those indices such as fasting plasma glucose (FPG), glycated haemoglobin (HbA1c) and C-peptide. Existing reports have shown that the associations between thyroid hormone and glycaemic indices are altered in diabetes mellitus (Ghazali and Abbingesuku 2010;O' Meara et al. 1993), the general notion being that thyroid hormone levels fluctuate with FPG. The physiological and biochemical interrelationship between insulin and thyroid hormone and the influence of both insulin and iodothyronines on the metabolism of carbohydrates proteins and lipids have been extensively studied and recorded (Additional file 1). Endogenous production of glucose is enhanced by insulin and thyroid hormones since both FT 3 and FT 4 stimulate GLUT4 messenger RNA and protein expression in skeletal muscle and elevate glucose uptake (Kemp et al. 1997). In hyperthyroidism, increased level of thyroid hormones leads to increase GLUT4 and consequently increase hepatic output of glucose resulting in hyperglycaemia. Increased lipolysis which also occurs in hyperthyroidism results in increased levels of free fatty acid (FFA) (Maxon et al. 1975). Moreover, in hyperthyroidism, the non-oxidative disposal of glucose is enhanced resulting in overproduction of lactic acid which enters the Cori cycle and promotes further gluconeogenesis (Ghazali and Abbingesuku 2010). Hyperthyroidism is also associated with raised levels of growth hormone, glucagon and catecholamine which further impair glucose metabolism (Niki et al. 1992;Tosi et al. 2012). On the other hand, hypothyroidism also affects glucose metabolism through several mechanisms and reduced glucose production is reported. There is reduced rate of glucose production in hypothyroidism, a consequence of decreased production of GLUT4 (Kemp et al. 1997). Hypothyroidism has been recognized as insulin-resistant state due to impaired insulin-stimulated glucose utilization in peripheral tissues.
This information has not defined the interrelationship between the glycaemic indices and the relationship between the individual thyroid hormones (T 3 , T 4 and FT 3 ) and the different glycaemic indices, namely FPG, HbA1c and C-peptide/insulin. This study evaluates the relationship between thyroid hormone levels and glycaemic indices in type 2 diabetic subjects with euthyroid, hyperthyroid, hypothyroid hormone levels and in nondiabetic control subjects.

Study location/subject selection
This was a cross-sectional study involving one hundred and seventy type 2 diabetic subjects attending University of Calabar teaching hospital, University of Calabar Medical Centre, Luciama Memorial Specialist hospital and Mediscience laboratories Ltd. to monitor their glucose levels as the diabetic subjects. They were all residents of Calabar, Cross River State, Nigeria. All diabetic subjects were confirmed type 2 who previously had or were having fasting plasma glucose concentrations above 6.1 mmol/l and were receiving treatment for the ailment. Apparently healthy age (38-60 years) and sex match non-diabetic volunteers also residing in Calabar served as control subjects all of whom had fasting plasma glucose levels below 6.1 mmol/l. All subjects were briefed on the objectives of the study verbally, and informed consent was obtained. Very ill diabetics and patients with diabetic complications on admission were excluded from the study. The subjects under study were divided into 5 groups as follows: all type 2, euthyroid, hypothyroid, hyperthyroid type 2 diabetics and non-diabetic subjects. The parameters selected for thyroid profile were thyroid-stimulating hormone: TSH, triiodothyronine T 3 , thyroxine, T 4 and free thyroxine, FT 4 , which allowed assessment of TSH, FT 4 , T 4 and T 3 under the 5 groups studied.

Sample collection
Criteria for selection of type 2 diabetic subjects were: age of onset of diabetic as greater than 37 years, type of treatment the patient was receiving and their physician's diagnosis on the patient records such diabetic subjects had fasting C-peptide levels of 0.38 ng/ml or above and were responding to treatment. Both the diabetic and non-diabetic control subjects were instructed to fast overnight (at least 8 h), before blood samples were collected from them. Ten millilitres of venous blood was collected from each subject and dispensed as follows: 2 ml into fluoride oxalate bottle for plasma glucose estimation, 2 ml into EDTA bottle for glycated haemoglobin (HbA 1c ) and the remainder into a plain bottle from where serum was harvested and stored frozen at − 20°C until use for thyroid hormones and C-peptide assays.

Laboratory methods
ELISA reagent kits obtained from Dslab Inc., California, USA, were used for the estimation of thyroid-stimulating hormone (TSH), total triiodothyronine (T 3 ), total thyroxine (T 4 ), free thyroxine (FT 4 ) and C-peptide. Reagent kits obtained from UNICO Inc., USA, were used for the estimation of HbA1c, while Trinder glucose oxidase method (Trinder 1969) was used for the measurement of FPG.

Statistical analyses
Statistical analysis was performed using SPSS version 18.0. Pearson's correlation was used for correlation analyses. Quality control of procedures was done using control sera at low, normal and raised levels as provided by the reagents' manufacturers. The results obtained were expressed as mean ± SD. The level of significance was set at P < 0.05.

Results
FPG and HbA lc were positively correlated in all type 2 diabetic group (r = 0.211, P = 0.007) and in the hypothyroid type 2 diabetic subjects (r = 0.382, P = 0.011) and hyperthyroid groups (r = 0.295, P = 0.011). FPG also correlated positively with C-pep in all type diabetics (r = 0.224, P = 0.004) and hyperthyroid type 2 subjects (r = 0.481, P = < 0.001). There was no significant correlation between FPG and HbA lc or C-pep in the other group (Table 1). There were no significant correlations between FPG and all the thyroid hormones. HbA lc correlated positively with TSH in all type 2 (r = 0.211, P = 0.012) and in hypothyroid subjects (r = 0.330, P = 0.031), and there was positive association between HbA lc and T 4 in all types (r = 0.194, P = 0.038) and the hypothyroid groups (r = 0.379, P = 0.012). There were no observed correlations in other groups (Table 2). Table 3 shows a positive correlation between C-peptide and FT 4 only in the hyperthyroid group (r = 0.279, P = 0.042) and negative correlation between C-pep and T 4 in the euthyroid group. There were also significant positive correlations between C-peptide and T 3 in all type 2 and euthyroid diabetic groups. C-peptide did not correlate with TSH in any of the five groups of subjects.

Discussion
The coexistence of both low and raised levels of thyroid hormones in type 2 diabetic subjects I has been reported in many studies (Gursoy and Turkel 1999;O' Meara et al. 1993;Kemp et al. 1997;Maxon et al. 1975), but currently, glycaemic indices: FPG, HbA 1c and C-peptide levels, are frequently used for the diagnosis, evaluation and management of diabetic subjects without due attention to the possible effects of the coexistence of low or raised thyroid hormone levels alongside type 2 diabetes in those subjects.
In this study, FPG correlated positively with HbA lc in all type 2, hypothyroid and hyperthyroid groups. Glycation is said to be a non-enzymatic catalysed reaction (Udiong et al. 2007), and thus, our current findings suggest that Table 1 Correlation analysis between FPG and C-pep, HbA lc and thyroid profile in all type 2 diabetics, euthyroid, hypothyroid, hyperthyroid diabetic and non-diabetic control subjects FPG fasting plasma glucose, HbA lc glycated haemoglobin, C-pep C-peptide, TSH thyroid-stimulating hormone, FT 4 free thyroxine, T 4 total thyroxine, T 3 total triiodothyronine  altered thyroid hormone levels may play a role in glycation in type 2 since there was no significant association in both non-diabetic subjects and euthyroid type 2 diabetic subjects. But the positive correlations between FPG and HbA lc were observed in both diabetic subjects with low and raised thyroid hormone levels prompting a conclusion that the positive association between FPG and HbA lc is not driven by abnormal thyroid hormone levels; otherwise, the direction of correlation would have been opposite. The correlations observed in all type 2, hypothyroid diabetics would then depend on the FPG levels and the percentage of subjects in those groups that had raised glucose levels and the period, the hyperglycaemia lasted (Dimitriadis et al. 1985). FPG and C-peptide were positively correlated in all type 2 and hyperthyroid groups. There were no significant correlations between the two parameters in the euthyroid, hypothyroid diabetics and non-diabetic subjects. It may thus be reasoned that raised thyroid hormone levels enhance association between FPG and C-peptide and that the positive correlation found in all type 2 diabetic group is due to the presence of diabetes with raised thyroid hormone levels in that group. Raised thyroid hormone levels promote hyperglycaemia (Beer et al. 1989) and would aggravate existing hyperglycaemia in type 2. In type 2 diabetes subjects, the raised levels of FPG trigger increased secretion of C-peptide/insulin (Beer et al. 1989;Levin and Symth 1963). The correlation between FPG and C-peptide is enhanced by hyperthyroidism which increases glucose absorption in the gut (Levin and Symth 1963), alters pro-insulin processing (Matty and Seshadri 1963) and reduces the half-life of insulin by increasing the rate of degradation and enhancing release of biologically inactive insulin precursors (Arshad and Hussain 2013). FPG levels did not correlate with TSH, FT 4 , T 4 or T 3 levels in any of the 5 groups of subjects studied though positive correlation was reported between glucose and T 4 in streptozotocin-induced diabetic rats (Hage et al. 2011), and the disparity may be due to differences in the cause and intensity of diabetes in diabetic humans and induced diabetic rats. Our results imply that thyroid hormones levels are not influenced by glucose levels or vice versa. HbA lc did not correlate with C-peptide in all type 2, hypothyroid, hyperthyroid and euthyroid diabetic and also in non-diabetic controls. Rather, glycated haemoglobin correlated positivity with TSH and T 4 in all type 2 and hypothyroid diabetic groups. Raised TSH levels are associated with hypothyroidism and low TSH with hyperthyroid states (Lai et al. 2011) in non-thyroidal illness state such as type 2 mellitus; the findings are not different even in the presence of complicating factors.
Glycated haemoglobin also correlated positively with T 4 in all type 2 and those with low thyroid hormone levels similar to the relationship between HbA lc and TSH even though TSH levels would normally be expected to be inversely related to T 4 levels in primary thyroid disorders (Ghazali and Abbingesuku 2010). In type 2, where glucose, insulin and thyroid hormone levels are raised, there is also increased binding protein levels such thymoglobulin (TBG) level leading to raise total T 4 levels. Prolonged hyperglycaemia prompted by diabetes or hyperthyroidism will give rise to raise HbA lc and correlation with T 4 as observed in the study. Moreover, the rate of conversion of T 4 to T 3 is reduced or inhibited by raised levels of insulin found in type 2 resulting in raised T 4 levels (American Diabetic Association 2014; Jin et al. 2011). There were no significant correlations between HbA lc and TSH or T 4 in the euthyroid, hypothyroid and non-diabetic subjects. FT 4 and T 4 did not correlate with HbA lc in any of the 5 groups of subjects. This again shows that thyroid hormones do not influence glycation process, and hence, glycated haemoglobin results can be interpreted without reference to thyroid hormone levels.
C-peptide correlated positively with FT 4 in hyperthyroid diabetic group, positively with T 4 in the euthyroid Table 3 Correlation analysis between C-peptide and thyroid hormones in all type 2, euthyroid, hypothyroid and hyperthyroid type 2 diabetics and non-diabetic subjects C-pep C-peptide, TSH thyroid-stimulating hormone, FT 4 free thyroxine, T 4 total thyroxine, T 3 total triiodothyronine, r correlation coefficient, p probability, n sample size *Significant

Subjects
All diabetics and positively with T 3 in all type 2 and euthyroid group. The associations observed between C-peptide and FT 4 , T 4 and T 3 are similar. T 3 is biologically more potent than T 4 (Niki et al. 1992), but in type 2, FT 4 increases, while T 3 decreases. In spite of the disparity in activity, the observed association between T 4 , T 3 and C-peptide in diabetics with euthyroid hormones level shows that C-peptide levels are influenced by thyroid hormone levels. In type 2, insulin resistance leads to hyperglycaemia and increased C-peptide/insulin levels which may trigger thyroid hormone increase to enhance glucose metabolism, hence the correlation between C-peptide and the thyroid hormones (FT 4 and T 3 ). There was a positive correlation between C-peptide and T 3 in all type 2 group, but the association between C-peptide and FT 4 was not significant. The disparity may be due to the levels of and number of subjects in each of FT 4 and T 3 in the hyperthyroid group. Our study suggests that C-peptide levels are positively associated with thyroid hormones levels.

Conclusions
This study has shown that the presence of low or raised levels of thyroid hormones in the diabetic population influenced the levels of glycaemic indices in the general type 2 diabetic population. This implies that inclusion of thyroid hormonal profiling may be useful in proper diagnosis and management of diabetes mellitus.