Elsevier

Experimental Gerontology

Volume 72, December 2015, Pages 167-176
Experimental Gerontology

Aging and a long-term diabetes mellitus increase expression of 1 α-hydroxylase and vitamin D receptors in the rat liver

https://doi.org/10.1016/j.exger.2015.10.005Get rights and content

Highlights

  • 1α-Hydroxylase (1α-OHase) is highly expressed by liver monocyte/macrophage system.

  • Aging and long-term DM increase the number of 1α-OHase immunoreactive cells in the liver.

  • Aging and long-term DM increase the expression of VDR in hepatocytes.

  • VDR is strongly expressed in lipid droplets of hepatocytes.

Abstract

Diabetes mellitus (DM) is a metabolic disorder associated with serious liver complications. As a metabolic chronic disease, DM is very common in the elderly. Recent studies suggest ameliorating effects of vitamin D on metabolic and oxidative stress in the liver tissue in an experimental model of DM. The aim of this study was to investigate the expression of vitamin D receptors (VDRs) and 1α-hydroxylase, the key enzyme for the production of active vitamin D form (calcitriol) in the liver during long-term diabetes mellitus type 1 (DM1) in aging rats. We performed immunohistochemical analysis of liver expression of 1α-hydroxylase and VDRs during aging in long-term streptozotocin-induced DM1. 1α-Hydroxylase was identified in the monocyte/macrophage system of the liver. In addition to the nuclear expression, we also observed the expression of VDR in membranes of lipid droplets within hepatocytes. Aging and long-term DM1 resulted in significant increases in the number of 1α-hydroxylase immunoreactive cells, as well as the percentage of strongly positive VDR hepatocytes. In conclusion, the liver has the capacity for active vitamin D synthesis in its monocyte/macrophage system that is substantially increased in aging and long-term diabetes mellitus. These conditions are also characterized by significant increases in vitamin D receptor expression in hepatocytes. The present study suggests that VDR signaling system could be a potential target in prevention of liver complications caused by diabetes and aging.

Introduction

Diabetes mellitus (DM) is a chronic metabolic disease very common in the elderly (Chow et al., 2014) and represents a growing health problem (Wild et al., 2004). Oxidative stress and inflammation play important roles in mechanisms underlying aging and complications of diabetes (Halter et al., 2014), that are commonly associated with severe liver complications (George et al., 2012, Hamden et al., 2009). Recent studies suggested beneficial effects of vitamin D in age-associated pathologies related to cardiovascular diseases, DM, cancer and the autoimmune diseases (Haussler et al., 2011, Hayes, 2010, Najmi Varzaneh et al., 2013), as well as in reducing the effects of metabolic and oxidative stress in the liver during experimental DM (George et al., 2012).

Metabolic activation of vitamin D begins in the liver by synthesis of 25-hydroxyvitamin D3 (25(OH)D3), the major circulating form of vitamin D (Dusso et al., 2005, Haussler et al., 2013). The 25(OH)D3 is a substrate for the mitochondrial enzyme 25-hydroxyvitamin D3-1α-hydroxylase (1 α-hydroxylase, 1α-OHase, CYP27B1), that catalyzes conversion of 25(OH)D3 to calcitriol (1,25(OH)2D3), the most active vitamin D metabolite (Dusso et al., 2005, Haussler et al., 2013). Although 1α-OHase is primarily renal enzyme, it has also been detected in different extra-renal tissues such as the lymph nodes, skin, breast, prostate, colon and cells of the immune system (Townsend et al., 2005).

Calcitriol's actions are mediated through its interaction with vitamin D receptor (VDR), a member of the nuclear hormone receptor superfamily (Haussler et al., 2013, Nagpal et al., 2005). Liganded VDRs form heterodimers with the retinoid X receptors (RXRs), which subsequently bind to VDR-responsive elements in the promoter region of genes whose expression is directly controlled by vitamin D (Dusso et al., 2005, Haussler et al., 2013, Marshall, 2008). Calcitriol can also exert its bioactivity through a rapid non-genomic response system mediated by receptors associated with the plasma membrane (Dusso et al., 2005).

Although the presence of VDR in liver cells is confirmed (Barchetta et al., 2012, Ding et al., 2013, Gascon-Barre et al., 2003, Segura et al., 1999), the mechanisms underlying the ameliorating role of vitamin D in hepatic complications of DM (George et al., 2012) are poorly understood. The presence of 1α-OHase in the liver is known, but there are no data on the cellular distribution of the enzyme in the hepatic tissue.

The aim of our study was to determine the distribution and localization of VDR and 1α-OHase in the hepatic tissue of rats during long-term streptozotocin-induced DM1, and to investigate their association with aging and pathological changes in the liver cells caused by diabetes. In the present study, for the first time, we have demonstrated the presence of 1α-OHase in the monocyte–macrophage system of the liver, and varied strongly with aging and diabetes.

Section snippets

Ethics

The experimental protocol was approved by the Ethical Committee of the University of Split, School of Medicine. All experimental procedures followed the EU Directive (2010/63/EU).

Experimental animals

Thirty male 8-week-old Sprague–Dawley rats weighing ∼ 200 g were used. They were raised under controlled conditions (22.1 °C temperature and 12/12 h light schedule) at the University of Split's Animal Facility. Standard laboratory chow (4RF21 GLP, Mucedola srl, Settimo Milanese, Italy) and water were given ad libitum. The

Hepatocyte size and density

We observed significant changes of diameter and density of hepatocytes when comparing different age groups. The greatest hepatocyte-diameter and the lowest density were observed in c-3m group, on the contrary, the diameter was the smallest in c-14m group and hepatocyte density was the greatest in c-8m group. Nevertheless, when we compared diabetic and appropriate control groups, the differences in size and cellular density were not observed (Supplementary Fig. 1).

Expression of 1α-hydroxylase in the rat liver

Expression of 1α-OHase was

Discussion

We investigated the influence of aging and long term DM1 on the expression of vitamin D receptors and 1α-OHase in the rat liver. Immunohistochemical analysis demonstrated the presence of 1α-OHase in the population of cells that belong to monocyte–macrophage system of the liver. For the determination of the identity of 1α-OHase positive cells we used co-localizations of different markers. GFAP has been proven to be a marker of hepatic stellate cells (HSCs) (Buniatian et al., 1996). HSCs play

Conflict of interest

The authors have no conflicts of interests.

Acknowledgments

The study was funded by the Ministry of Science, Education and Sports, Republic of Croatia grant no. 216-2160528-0067 and the Croatian Science Foundation (HRZZ) grant no. 02.05./28.

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