Alterations of the expression levels of glucose, inflammation, and iron metabolism related miRNAs and their target genes in the hypothalamus of STZ-induced rat diabetes model

Diabetes, a systemic metabolic disease affects people all over the world. Diabetes can arise from different metabolic dysfunctions like insulin resistance, glucose intolerance, or from obesity [1]. In recent decades it has been revealed that the central nervous system (CNS), especially the hypothalamus is implicated in the development of diabetic conditions [2, 3]. The hypothalamic neurons in the nuclei (e.g. arcuate, ventromedial) respond to hyper- or hypoglycaemia to maintain circulating glucose concentration [4, 5]. Dysregulation of the hypothalamic glucose-sensing neurons leads to abnormal glucose metabolism [6].

The alterations of the neuron-glial cell interactions were also observed in the case of diet-induced metabolic changes and may contribute to the establishment of diabetes [7]. Fractalkine (FKN)/fractalkine receptor (CX3CR1) axis is crucial in the regulation of microglia, the immune cells of CNS [8]. FKN/CX3CR1 regulates the expression of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) promoting neuroinflammation. It has been described that FKN is involved in the development of hypothalamic inflammation, which may be one of the underlying causes of the disruption of energy balance [9, 10].

Iron may play a role in the pathogenesis of diabetes. Iron overload of the human body increases the risk of the establishment of metabolic disorders [11, 12]. Recently, it has been revealed that the serum level of the iron regulatory hormone hepcidin and ferritin correlates with the energy balance markers such as leptin [13, 14], which functions as a regulator of glucose-sensing neurons in the hypothalamus [15]. Moreover, insulin influences iron metabolism by affecting the transport of transferrin receptor 1 (TfR1) into the cell membranes and increasing ferritin (FTH) synthesis [16]. On the other hand, iron metabolism is regulated by inflammation as well as by FKN, which can increase the iron absorption of neurons and inhibit iron release through the receptor of hepcidin, the iron exporter ferroportin (FP) [17, 18].

The molecular mechanisms with which the glucose metabolism, inflammatory processes, and iron homeostasis of hypothalamus crosstalk, are still under investigation. The role of small non-coding RNA molecules, the miRNAs have been investigated in metabolic disorders [19]. The miRNAs affect the stability of the mRNA molecules and mainly inhibit their translation by decreasing the rate of protein synthesis of the target genes [20]. Numerous miRNAs have been described as the regulators of energy balance and iron homeostasis [21‐24].

In the present study, we investigated the molecular biological background and the possible links between diabetes, inflammation, and iron metabolism in the hypothalamus of an STZ-induced rat type 1 diabetes model.

Using the miRNA array, we analysed the expression of diabetes, inflammation, and iron metabolism related miRNAs. Determination of the effect of miRNAs altered by STZ treatment on the target genes was carried out at protein level.

Based on the findings, it is supposed that glucose metabolism, inflammation, and iron homeostasis of the hypothalamus are linked via the altered expression of common miRNAs as well as the increased expression of FKN, which contribute to the imbalance of energy homeostasis, the synthesis of pro-inflammatory cytokines and the iron accumulation and retention of the hypothalamus.

The CNS plays a crucial role in the regulation of glucose metabolism [2]. The hypothalamus of the CNS is implicated in the development of diabetes due to its glucose-sensing function [3]. In the hypothalamus, two types of glucose-sensing neurons can be found mainly in the hypothalamic nuclei, the arcuate (ARC) and the ventromedial (VMH) nuclei. Among the glucose-sensing neurons, the glucose-excited neurons are activated by hyperglycaemia, while hypoglycaemia activates glucose-inhibited neurons [49]. In the ARC the pro-opiomelanocortin (POMC) neurons are activated by elevated glucose level, while the neuropeptide Y/Agouti-related protein (NPY/AgRP) neurons are regulated by reduced blood glucose level [5]. VMH neurons control blood glucose level via influencing insulin secretion of the pancreas [4]. Dysregulation of the hypothalamic glucose control leads to abnormal glucose metabolism [6]. It changes the action of insulin on hepatic gluconeogenesis and causes insulin resistance in the hypothalamus that may conduct the development of peripheral insulin resistance [50]. Recent evidence has proven that the hypothalamus plays a special role in the development of diabetes mellitus [51].

We used the STZ-induced rat type 1 diabetes model [52] to investigate the action of hyperglycaemia on the glucose metabolism, inflammation, and iron metabolism related miRNAs in the hypothalamus as well as the mRNA and protein expressions of the target genes of these miRNAs. We also examined the effect of diabetic conditions on the regulation of iron homeostasis, iron transport and storage, and iron-dependent mitochondrial functions of the hypothalamus to reveal the interaction between diabetes and the disturbances of iron homeostasis.

MicroRNAs are short non-coding molecules working as key regulators of gene expression, usually by affecting the stability of the mRNA molecules. Therefore, they usually inhibit translation [20]. The role of miRNAs in the development of diabetes is under massive investigation [19]. Recent studies have described the action of several miRNA families (e.g. miR-17/92, miR143-145, miR-130, let-7, miR-200, miR-33, and miR-29) in the development of obesity and insulin resistance in adipose tissue, liver and skeletal muscle [24, 53, 54]. Hypothalamic miRNAs are supposed to have a role in the control of energy balance by modifying the expression of insulin receptor and leptin receptor acting as regulators of hypothalamic glucose metabolism [22, 23].

We found 18 miRNAs with altered expression levels in the hypothalamus of the STZ-treated animals, which act as the regulators of mRNAs involved in glucose metabolism, pro-inflammatory cytokine synthesis, and iron homeostasis suggesting a link between these processes in diabetes. The increasing level of miR-29a-3p and the elevated mRNA expression of PEPCK in the hypothalamus were correlated with the expression levels in the liver showing the systemic effect of STZ treatment (Additional file 1: Table S1, Fig. S1), which generated the development of diabetic condition [27]. The decreasing level of miR-200a-3p may influence the translation of INSR, LEPR mRNAs, as well as the iron importer TfR1 proving an interaction between diabetic condition and iron metabolism. We also found that increasing level of miR-194-5p resulted in a decreasing protein level of INSR and FP supporting another interaction with glucose and iron metabolism. The alterations in the expression level of miR-135b-5p, miR-21-5p, miR-200a-3p, miR-152-3p and miR-96-5p could modify hypothalamic glucose sensing, tolerance, uptake, and phosphorylation via affecting the stability of HXK-2, INSR, LEPR, GCK, GLUT4, IGFR1, and PEPCK mRNA molecules and as consequence miRNAs indirectly influence the protein expression levels of the aforementioned genes. The changes at protein level of HXK-2 and GCK in the hypothalamus of the STZ-treated rats predispose to enhanced glucose phosphorylation [55], while the elevated level of GLUT4 suggests that not only insulin signalling but miRNAs affected protein synthesis is also important in the expression of GLUT4 and glucose uptake. Moreover, the elevated level of inflammatory cytokines such as IL-6 can contribute to the increased GLUT4 level independently of insulin [56]. On the other hand, the raising protein level of LEPR may trigger the effect of leptin on lowering glucose concentration in the circulation [57, 58]. The level of miR-200a, known to act as an inhibitor of LEPR and INSR translation [23, 59], decreased showing a negative correlation with LEPR protein changing leptin sensitivity [60] but a positive correlation with the protein level of INSR. A possible reason for this latter result is the increased expression of an additional INSR inhibitory miRNA, miR-194-5p, leading to a decrease in the level of INSR protein. In addition, the alteration of iron metabolism especially the intracellular iron content also influences INSR expression [61]. This result proposes the decreased activity of the insulin signalling cascade [62].

Recent studies have revealed that hypothalamic inflammation contributes to the development of diabetes [9, 10, 63]. Increased activity of NFκB and JNK signalling pathways in the hypothalamus leads to the synthesis of IL-6 and TNF-α triggering neuroinflammation [64]. Hypothalamic IL-6, IL-1β and TNF-α pro-inflammatory cytokine mRNA levels were found to be positively correlated with miR-126a and miR-150 levels suggesting the activation of hypothalamic microglia and astrocytes and the development of inflammation in the STZ-treated rats. It has been described that not only cytokines, but the disruption of chemokines has an important role in the hypothalamic inflammatory process [9]. Fractalkine (FKN), which is crucial in the neuron-microglial crosstalk via the activation of microglial CX3CR1, has been implicated in the development of hypothalamic inflammation in obesity [10]. Our results show that the levels of four miRNAs, miR125b-5p, miR-195-5p, miR29b-3p and miR-503-5p were altered in the hypothalamus of the STZ-treated rats resulting in the elevation of FKN protein expression. The expression of CX3CR1 regulator miR-296-3p was found to be reduced showing a positive correlation with CX3CR1 mRNA as well as protein levels. However, reverse changes were observed in the protein expression of FKN and its receptor CX3CR1, the downstream signalling pathways may contribute to the overexpression of pro-inflammatory cytokines triggering inflammation.

Neuron-microglia communication is involved not only in the energy balance and inflammation but also in the regulation of iron homeostasis via the FKN/CX3CR1 axis [7, 17]. FKN and pro-inflammatory cytokines such as IL-6, increase cellular iron retention by elevating HAMP expression and triggering FP internalisation contributing to iron-mediated toxicity and neuronal cell death [17, 65, 66]. Our observations correlate with our previous results that the mRNA level of HAMP significantly increased, and FP was eliminated from the plasma membrane in the STZ-treated hypothalamus suggesting iron accumulation. One of the signalling pathways that may contribute to the elevated HAMP level is the BMPR/SMAD [66], also leptin/LEPR mediated signalling cascade works as a positive regulator of HAMP expression [14]. We found several miRNAs with altered expression acting as regulators of iron metabolism related genes. The miRNA, mRNA, and protein analyses of the diabetic hypothalamus revealed that the iron import via DMT-1, the iron export by FP, and the iron storage mediated by FTL were all influenced by miRNAs like miR-194-5p, miR-19a-3p, and miR-133a-3p suggesting the disturbance of hypothalamic iron homeostasis. It was revealed that the level of DMT-1 decreased suggesting the inhibitory effect of the regulatory miRNA on DMT-1 translation. Moreover, DMT-1 translation is also regulated by the intracellular iron content by IRPs; therefore, the decreasing level of DMT-1 supposes the increased intracellular iron concentration. Alternatively, Zip8 and Steap2 iron importers can take the role of DMT-1 in iron uptake of brain cells as they are not under the regulation of IRPs [67] and may contribute to the increased intracellular iron content causing iron overload. In the case of FP, the expression of regulatory miRNA showed an increasing level and the mRNA level decreased, both independent changes could contribute to the decreasing FP protein level. Indeed, we cannot exclude the effect of hepcidin on FP, but since there is no complete internalisation, the hepcidin may act in an internalisation-independent way, by inhibiting iron release via the iron exporter contributing iron retention of the cells [68, 69]. Moreover, the mRNA level of LF, a multipurpose glycoprotein, significantly decreased suggesting the deterioration of iron transport between neurons and glial cells, as well as the decreasing rate of ROS scavenging [70]. Normally, the TfR1 protein level should be downregulated by IRP by binding to the IRE element on the 3’end of the mRNA, upon intracellular iron accumulation but there are several reasons for the deregulation of iron metabolism. It has been described that TfR1 expression elevates at neuroinflammation. The possible reason for this is that inflammatory signals revoke the blocking effect of hepcidin on cellular iron uptake via TfR1, and therefore promote brain iron overload [71‐73].

The deregulation of IRP1 by pro-inflammatory cytokines may also contribute to the upregulation of TFR1 protein, downregulation of FP and intracellular iron deposition [74].

Our results support the development of iron accumulation and imbalance in the hypothalamus of STZ-treated animals as the cytosolic iron oxidizing and storage protein FTH level increased but the mitochondrial form of iron storage protein FTMT decreased. The increasing level of FTH can antagonise the reactivity of the labile iron pool and prevents iron-mediated oxidative stress [75]. The reason for the differential changes in FTL and FTH protein levels is that FTH becomes more abundant at inflammation [76], which also occurs in the diabetic condition in the brain. FTH expression is also triggered by inflammation e.g. IL-1, IL-6 and TNF-α pro-inflammatory cytokines via the NFκB signalling pathway [77, 78], which is regulated by FKN showing increased levels in our experiments. Different cell types of the hypothalamus express the two ferritins in different ratio: oligodendrocytes have equal amounts of both H and L subunits, whereas microglia express L-rich ferritin, and neurons have H-subunit abundant ferritin [79]. For elucidating the exact role of the different types of hypothalamic cells in the regulation of iron metabolism in diabetes further experiments are needed.

Moreover, it seems that the mitochondrial iron utilization was also altered: the protein level of FECH responsible for iron incorporation into heme decreased while the expression of NFS-1 working in the iron-sulphur cluster synthesis raised. The iron-sulphur clusters synthesised by the mitochondria convert IRP1 to aconitase to restore the normal expression of iron transporters and maintain the tricarboxylic acid (TCA) cycle [75, 80].

Based on our findings the STZ-induced diabetic condition affects hypothalamic glucose metabolism, inflammation, and iron homeostasis. The underlying molecular mechanisms mediating interactions between these processes are incompletely understood. It is supposed that they are linked together via the altered expression of common miRNAs as well as the increased expression of FKN. FKN acts as a regulator of inflammation and iron metabolism via controlling the downstream signalling cascades mediated by FKN/CX3CR1 interaction and it appears to work as a link between diabetes, inflammation, and iron metabolism. The dysregulation of hypothalamic iron homeostasis may contribute to the degeneration of neuronal cells and the imbalance of energy homeostasis. Our results raise the possibility that FKN could be a potential target of new therapies targeting both inflammation and iron disturbances in diabetic conditions.