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ORIGINAL CONTRIBUTIONS

Received: November 2^nd 2015

Judged: May 2^nd 2015

Association of inflammatory cytokines with bone turnover markers in type 1 diabetes

Mario Molina-Ayala,^a Ruth Carmina Cruz-Soto,^a Guadalupe Vargas-Ortega,^a Baldomero González-Virla,^a Aldo Ferreira-Hermosillo, ^a Julián Mac Gregor-Gooch,^b Victoria Mendoza-Zubieta^a

^aServicio de Endocrinología

^bDivisión de Medicina

Hospital de Especialidades, Centro Médico Nacional Siglo XXI, Instituto Mexicano del Seguro Social, Ciudad de México, México

Communication with: Victoria Mendoza Zubieta

Telephone: (55) 5627 6900, extension 21551

Email: vmendozazu@yahoo.com

Background: Diabetes mellitus (DM) adversely affects the skeleton and the physiological mechanisms implicated have not been explained sufficiently. Thus, the objective was to identify inflammatory cytokines (IL-1, IL-6 and TNF-alpha) in patients with T1DM and their association with markers of bone formation (sPINP) and markers of bone resorption (sCTX).

Methods: We studied 62 patients of 18 years of age or more with T1DM. We determined the values of HbA1c, vitamin D, inflammatory cytokines, as well as those of markers of bone formation and of markers of bone resorption.

Results: 49 patients were female with a mean age of 33.5 years. We found values of HbA1c > 7.5 in 83 %, vitamin D of 16 ng/mL. In patients with HbA1c >7.5 we found a positive correlation between TNF-alpha and sCTX (r = 0.43, p = 0.05), IL-6 and sCTX (r = 0.48, p = 0.037). With a model of simple linear regression between IL-6 and sCTX, it was found a beta coefficient of 23.8 with a p = 0.030 (95 % CI = 2-45.6), ie.: for every unit increase in IL-6 there is a sCTX increase of 23.8 pg/mL.

Conclusions: We found a positive association between TNF-alpha and IL-6 with the marker of bone resorption (sCTX) in the group of patients with HbA1c > 7.5. The loss of metabolic control was associated with TNF-alpha and IL-6.

Keywords: Type 1 diabetes mellitus; Bone density; Cytokines; Interleukins; Tumor necrosis factor-alpha

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Type 1 diabetes (T1D) is an autoimmune disease characterized by the destruction of beta cells in the pancreas.¹ Its incidence has increased globally by about 3% per year in children under five years, and it represents 10% of all cases of diabetes in Mexico.² The incidence of DM1 in children under 19 has increased from 3.6 to 6.2 per 100,000 enrollees.³

With the advent of insulin in 1921-1922, mortality due to acute complications such as diabetic ketoacidosis has been reduced dramatically. In addition, with advances in new insulin analogues, glucose monitoring technologies, and insulin infusion pumps, more patients with DM1 are able to achieve glycated hemoglobin (HbA1c) goals. This has significantly reduced microvascular complications and has considerably increased life expectancy.^4,5 Currently there is a need to detect other age-related complications, including cardiovascular disease, osteoporosis, and cognitive dysfunction.

In recent years, a number of epidemiological studies have shown that there is an increased incidence of osteopenia, osteoporosis, and increased risk of fractures in patients with DM1.⁶ Diabetes mellitus adversely affects the skeleton, and the pathophysiological mechanisms are still not fully understood. DM1 is associated with reduced bone mineral density (BMD), while DM2 has increased BMD; however, both are at increased risk of fracture. Patients with DM1 have one to two times higher risk of fractures at any skeletal site, and up to 6.9 times the risk of hip fracture.⁷ Diabetic patients have lower bone quality compared to nondiabetics.⁶

The bone remodeling process depends on a precise balance between the formation carried out by osteoblasts, and the resorption caused by osteoclasts. This balance is regulated by different molecular signaling pathways, involving systemic and local factors; interruption in any of these pathways alters bone turnover and bone quality.^6,7

In DM1, various mechanisms have been described that affect differentiation and activity of osteoblasts, encourage osteoclast differentiation, change bone quality, and cause greater bone loss and increased risk of fracture. These mechanisms include lack of insulin, deposits of advanced glycation end-products (AGEs) in the collagen, reduced serum levels of IGF-1 (insulin growth factor type 1), hypercalciuria, vitamin D, renal disease, microangiopathy, and inflammation mediated by inflammatory interleukins IL-1, IL-6, and tumor necrosis factor alpha (TNF-alpha).^8,9

Other autoimmune diseases that can coexist with DM1, such as hypo- or hyperthyroidism, hypogonadism, celiac disease, and insulin deficiency itself, prevent patients with DM1 from achieving peak bone mass in adulthood.^8-10

Markers of bone resorption and formation allow us to assess the bone remodeling process. IOF (International Osteoporosis Foundation) and IFCC (International Federation of Clinical Chemistry and Laboratory of Medicine) recommend the serum N-terminal propeptide of type I collagen (sPINP) as a marker of formation, and C-terminal telopeptide of type I collagen (sCTX) as a marker of resorption; according to both institutions, these are the most sensitive markers of bone turnover applicable to clinical use.¹¹

The aim of this study is to identify the type and concentration of inflammatory cytokines (IL-1, IL-6, and TNF-alpha) in patients with DM1 and their association with bone turnover markers sCTX and sPINP.

Methods

The Hospital de Especialidades of Centro Médico Nacional Siglo XXI has a Type 1 Diabetes Mellitus Clinic with approximately 350 patients, who receive follow-up with periodical clinical and biochemical evaluations. The protocol was approved by the Ethics Committee of the hospital. 62 patients with a diagnosis of DM1 over 18 years with regular follow-up (at least three visits per year) at the Type 1 Diabetes Mellitus Clinic of this center were included in this study. Patients previously signed a letter of informed consent. We excluded patients with renal impairment (KDQI 3), inflammatory diseases such as rheumatoid arthritis, systemic lupus erythematosus (SLE), treatment with glucocorticoids, hyperthyroidism, menopause, and patients with incomplete clinical evaluations.

Regarding the biochemical determinations, laboratory studies were obtained using a sample of 6 mL using BD Vacutainer (BD, Franklin Lakes, NJ, USA) and centrifuged at 3150 × g for 15 minutes, and the serum was divided into two aliquots. We analyzed glucose, total cholesterol, HDL-C, and triglycerides with a commercially available kit (Roche Diagnostics COBAS 2010, Indianapolis, USA) using photocolorimetry with a Roche Modular P800 spectrophotometer (2010 Roche Diagnostics, Indianapolis, USA). Samples of HDL-C were treated with enzymes modified with polyethylene glycol and dextran sulphate, and analyzed using the same photocolorimetry technique. Glycated hemoglobin (HbA1c) was evaluated by turbidimetric immunoassay (Roche Diagnostics COBAS 2010, Indianapolis, USA). Low-density lipoprotein cholesterol (LDL-C) was calculated if triglycerides were < 400 mg/dL,¹¹ with the Friedewald formula:

 

LDL-C (mg/dL) = CT mg/dL - (C-HDL mg/dL + Triglycerides mg/dL/5)

 

For the determination of cytokines, interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-10 (IL-10), and tumor necrosis factor alpha (TNF-alpha) were analyzed using BD cytometric Bead Array (CBA) using an inflammation flow cytometry kit (BD FACSAria, BD Biosciences, USA). The variation coefficient (VC) of IL-1 was 4-7%, for IL-6 it was 8-10%, for IL-10 it was 8-11% and for TNF-alpha it was 8-15%. The detection limit for each test was 1.7 pg/mL for IL-1, 1.5 pg/mL for IL-6, 2.3 pg/mL for IL-10, and 2.7 pg/mL for TNF-alpha.

As for bone turnover markers, the bone formation marker serum N-terminal propeptide of type I collagen (sPINP) and the bone resorption marker C-terminal telopeptide of type I collagen (sCTX) were analyzed in serum using the ELISA technique with the IDS-iSYS intac PINP kit (IDS, USA), with a detection limit < 1 ng/mL, VC 2.6-3%, and the Human C telopeptide of type I collagen, ICTP ELISA kit (MyBiosource, USA), with detection limit of 0.033 ng/mL, VC 2.1-4.9%.

For statistical analysis, data were analyzed using STATA version 11. The Shapiro Wilks test was used to establish the normal distribution of quantitative variables. To make associations between variables, the Chi-squared test, Mann-Whitney U, or t-test were used for quantitative variables. Results were expressed as mean and standard deviation or median and interquartile range (IR). Pearson or Spearman tests were used to establish correlations between variables. A logistic regression model was used to assess factors associated with bone resorption markers. A p < 0.05 was considered statistically significant.

Results

62 patients were included, with a median age of 33.5 years (24-46 years); 49 patients were female. HbA1c was 8.5% (7.7-10%) in 83% of patients with HbA1c > 7.5%. Creatinine clearance was 69.5 mL/min (61.2-89.5). All patients showed ranges of vitamin D deficiency (< 20 ng/mL) or insufficiency (20 to 29.9 ng/mL), with a median of 16 ng/mL (12.5-21.2). No patients showed vitamin D sufficiency (≥ 30 ng/mL).¹² Alkaline phosphatase was 82 U/L (69.5-98.5), calcium 9.4 mg/dL (9-9.7), and phosphorus 3.8 mg/dL (3.5-4.3). Cholesterol concentrations are shown in Table I. Positive correlation between TNF-alpha and sCTX was found in the group of patients with HbA1c > 7.5 (r = 0.43, p = 0.05); positive correlation was also found between IL-6 and sCTX (r = 0.48, p = 0.037) (Table II). After applying a simple linear regression model between sCTX and IL-6, a beta coefficient of 23.8 was found, with p = 0.030 (95% confidence interval [CI] 2-45.6), which means that for each increased unit of IL-6 there is an increase in CTX of approximately 23.8 pg/mL, which must be an increase in bone resorption.

Discussion

Both DM1 and DM2 have an increased risk of fracture, with a more severe impact in patients with type 1 diabetes. The prevalence of DM1 has increased worldwide and is associated with an increased risk of spine and hip fracture, the latter in an interval ranging from 6.4 to 6.9 times compared with individuals without diabetes.^7,13 With the increase in life expectancy of patients with DM1, a further increase in the number of fractures is expected in the future.¹⁴ 

The pathophysiology remains unclear at present. Bone remodeling is a dynamic process involving the bone-forming cells, osteoblasts, and the bone-resorbing cells, osteoclasts. Multiple signaling pathways maintain a precise balance of this process with the participation of local and systemic humoral factors.

In DM1, studies in animals and humans have found severe deterioration of bone formation with a reduction in differentiation and function of osteoblasts, mainly in patients without metabolic control.⁹

Factors not well-specified in DM1 produce a reduction in the osteoblastogenic signaling pathways Runx-2 and increased sclerostin secretion (by the osteocytes). Sclerostin is a negative regulator of the osteoblastogenic pathway Wnt, and its increase produces further deterioration of bone formation.⁹

Besides having a direct effect on the cells involved in bone remodeling, DM1 also affects the bone matrix, thus altering the quality of the bone. These effects are mediated by the formation of advanced glycosylation end-products (AGE), which are produced by the nonenzymatic glycation of proteins or lipids, and are involved in multiple complications of diabetes, including bone fragility.¹⁵

Some comorbidities that increase the risk of osteoporosis are more common in patients with DM1 (e.g., hypogonadism, certain disorders of intestinal malabsorption such as celiac disease, and hyperthyroidism). These comorbid conditions should be detected and treated early to prevent further deterioration of bone microarchitecture.^7,8

Vitamin D has a large role in health: its effect on bone metabolism is critical; it also acts on the cardiovascular system, on immunomodulation, neurological development, and cell proliferation. Vitamin D deficiency results in rickets in children and osteomalacia in adults. It also decreases osteoblast function, induces RANKL-mediated osteoclastogenesis, and causes increased bone loss; classically, vitamin D deficiency prevents normal mineralization of the osteoid. All patients in our study showed deficient and insufficient levels of vitamin D.¹²

DM1 is associated with increased cardiovascular disease, regardless of glycemic control. The inflammatory condition may be involved in both cardiovascular disease and bone metabolism disorders. IL-6 and TNF-alpha promote expression of RANKL, osteoclastogenesis, and increased bone resorption. Our results show a positive association of bone resorption markers with concentrations of IL-6 and TNF-alpha in patients with uncontrolled DM1 (HbA1c > 7.5).

Conclusions

Our work found a positive association between pro-inflammatory cytokines TNF-alpha and IL-6 with the bone resorption marker in the group of patients with HbA1c > 7.5. Metabolic control was associated with higher levels of the inflammatory cytokines TNF-alpha and IL-6 with increased bone resorption. We also found a correlation or positive association between IL-6 and the bone resorption marker sCTX (one unit increase in IL-6 increased sCTX by 23.8 pg/mL). DM1 is associated with increased bone fragility. The convergence of many factors including inflammatory status and vitamin D deficiency leads to poor bone quality with an increased risk of fractures, especially hip. These factors can be modified; however, further investigation of this problem is required to achieve better tools for evaluating and treating other factors associated with poor bone quality in these patients.

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