Diabetic skeletopathy of the Sprague-Dawley rat mandible in a chronic alcohol intake and antiretroviral therapy: An immunohistochemical and microfocus X-ray computed tomography study

Abstract

The prevalence of non-communicable diseases such as diabetes mellitus is increasing worldwide with Type 2 diabetes mellitus accounting for 90 to 95 per cent of all diabetes cases. Type 2 diabetes mellitus, excessive alcohol consumption, and co-infection with the Human Immunodeficiency Virus (HIV) are some of the most common chronic health and socioeconomic problems in South Africa. Individuals infected with HIV are enrolled in combination antiretroviral therapy (cART), adding further difficulty to the country’s health system. As type 2 diabetes is characterized by hyperglycemia and insulin resistance, chronic alcohol abuse increases caloric intake and leads to obesity, and later, to type 2 diabetes. Alcohol abuse also affects the ability to make good behavioural choices, increasing the likelihood of HIV infection and subsequent enrolment in a cART programme. Although cART is effective, it is associated with metabolic dysregulation with insulin resistance. These metabolic disorders exacerbate type 2 diabetes mellitus and increase the risk of osteoporosis and bone fractures. Type 2 diabetes mellitus is among the causes of osteoporosis and an increased risk of bone fractures. Research studies link type 2 diabetes, excessive alcohol consumption and the use of cART, independently, to osteoporosis and an increase in the risk of bone fracture. Transforming growth factors (TGF-β1) and bone morphogenetic proteins 3 (BMP-3) are notable cytokines that promote bone remodeling, proliferation, and differentiation of mesenchyme bone forming cells (osteogenesis). Alcohol, type 2 diabetes mellitus, and cART individually or together may have vii detrimental effects on these cytokines. While the singular effects of alcohol, type 2 diabetes mellitus or cART have been reported, their combined effect is unknown. This is despite the high prevalence of coexistence of diabetes, alcohol abuse and cART among individuals. The present study aimed to determine the impact of the interaction of alcohol and combination antiretroviral treatment (cART) on the diabetic Sprague Dawley male rat mandible. The rats were randomly distributed into 10 groups consisting of 3 control groups, 3 non-diabetic groups and 4 diabetic groups. The animals were placed in the following groups: (i) Untreated control group which received no treatment (n=8); (ii) Citrate buffer controls (CB), received a once of injection of citrate buffer (n=6); (iii) Gelatine group (GEL), received gelatine (n=8); (iv) Alcohol (ALC), consumed alcohol (n=8); (v) received combination antiretroviral treatment (cART) (n=8); (vi) ALC+cART, received alcohol and cART (n=7); (vii) DBT, diabetic group (n=7). The study included additional diabetic groups that received alcohol or cART or both as follows; (viii) diabetic animals that received alcohol (DBT+ALC) (n=6); (ix) diabetic animals received cART (DBT+cART) (n=7); and (x) diabetic animals that received both alcohol and cART (DBT+ALC+cART) (n=6). Gelatine was used as a mode of administration of the treatment cART treatment. Diabetes was induced by a diet enriched with 20% fructose a a once off sing STZ (40mg/kg.bw, ip) dose. The study begins by inducing and confirming induction of type 2 diabetes mellitus in the appropriate rat groups (DBT, DBT+ALC, DBT+cART and DBT+ALC+cART). Confirmation of diabetes was achieved by monitoring the fasting blood glucose levels, oral glucose tolerance tests (OGTT) viii and serum insulin levels. Serum levels of hormones that regulate bone health were then assessed. Afterwards, mandibular osteometry measurements were made. The trabecular microstructure parameters were assessed from microfocus X-ray computed tomography (Micro CT) to measure thickness (TbTh), spacing (TbSp), and number (TbN). The bones were tested for strength with a universal tensile tester and histologically evaluated for normal morphology. The expression of osteocytes, transforming growth factor beta-1 (TGF-1), and bone morphogenic protein-3 (BMP-3) was evaluated by immunohistochemistry. Diabetes was successfully induced as confirmed by, hyperglycemia, poor oral glucose tolerance and hypoinsulinemia in the groups that had diabetes (DBT, DBT+cART and DBT+ALC+cART). Serum parathyroid hormone and calcitonin concentrations were low in rats that were diabetic and taking and cART (DBT+cART) or diabetic rats taking both alcohol and cART (DBT+ALC+cART). Significantly higher levels of serum sclerostin were detected in rats subjected to various combinations of alcohol, CART or diabetes (ALC+cART, DBT+cART and DBT+ALC). However, the diabetic rats that received both alcohol and cART (DBT+ALC+cART) had normal sclerostin levels. Serum osteocalcin and catenin levels were also unaffected in this group. Diabetic rats on cART, with or without alcohol, showed decreased parathyroid hormone and calcitonin, but normal sclerostin, osteocalcin, and catenin. This suggests a specific disruption of parathyroid hormone and calcitonin regulation, while other compensatory mechanisms might maintain the levels of the other hormones. ix Diabetes adversely affected mandibular dimensions and was worsened by concurrent alcohol and cART. While the DBT+cART+ALC group had the highest stiffness, it was characterised by lower values for maximum force and break force, less displacement, and time to fracture, indicating stiff and brittle bones. This finding supports the hypothesis that concomitant cART and alcohol use in diabetes may result in weaker bones. The high BV/TV and TbN in the DBT, DBT+cART and DBT+cART+ALC did not translate to stronger bones, therefore, the bone was of lower quality. Diabetes (DBT), alcohol (ALC), diabetes with alcohol (DBT+ALC) or diabetics consuming alcohol and antiretroviral treatment (DBT+ALC+cART) exhibited significantly impaired osteocyte quantities. The high intensity of BMP3 immunostaining observed in the present study among the groups with diabetes, (DBT, DBT+ALC, DBT+ALC and DBT+cART+ALC) corroborates the diminished TRAP immunopositivity as BMP3 is antagonistic to osteoblasts. In the present study, both alcohol (ALC) and diabetes (DBT) or a combination of alcohol and diabetes (DBT+ALC) did not change the expression of TGFB1. Evaluating the intracellular expression of TGFB1 in a future study is recommended. Viewed altogether, these results show that when diabetes was confounded by combination antiretroviral treatment and alcohol consumption (DBT+ALC+cART), osteogenic cells were negatively impacted together with derangement of the hormone profile parathyroid hormone and calcitonin. This is coupled with osteometric changes and bone weakness. Therefore, diabetic patients should be advised to reduce alcohol intake and bone health should be monitored when combination antiretroviral therapy is employed.