Deletion of Bmal1 Impairs Pancreatic β-Cell Function via Mitochondrial Signaling Pathway

Several studies have demonstrated that brain and muscle Arnt-like protein-1 (Bmal1) acts as a core clock gene for maintaining normal cell function, including hepatocytes and cardiomyocytes. Loss of Bmal1 is associated with type 2 diabetes due to pancreatic β-cell failure. However, little information is available about its role and mechanism in pancreatic β-cell. To address this, we investigated the consequences of Bmal1 inhibition in an insulinoma cell line (INS-1) by using small interfering RNA (siRNA). We observed that knockout of Bmal1 impaired glucose-stimulated insulin secretion in β-cell. Meanwhile, the depletion of Bmal1 in β-cell caused an adverse change in mitochondrial membrane potential and mitochondrial architecture. Deletion of Bmal1 attenuated mRNA and protein expression of mitofusin 1 (Mfn1) and mitofusin 2 (Mfn2) and enhanced the expression of fission 1 (Fis1). In summary, the deletion of Bmal1 impaired β-cell function may be via the mitochondrial signaling pathway in INS-1 cells.


Introduction
Type 2 diabetes, a disease characterized by hyperglycaemia, has become one of the major chronic diseases that seriously endanger human health. Pancreatic β-cell dysfunction is a key factor in the development of type 2 diabetes [1]. However, the pathogenesis is not fully understood. Recent studies have shown that circadian clocks have an important role to play in diabetes [2].
Circadian clocks regulate various aspects of behavioral and metabolic processes in mammals [2]. This circadian system, composed of a master clock that locates in the hypothalamic suprachiasmatic nucleus (SCN) and various peripheral tissue clocks, is generated by a feedback loop formed by core clock genes including brain and muscle Arnt-like protein-1 (Bmal1), circadian locomotor output cycles kaput (Clock), Period (Per), and Cryptochrome (Cry). Bmal1 and Clock activate the transcriptional process of Per and Cry, while Per and Cry inhibit Clock/Bmal1 transactivation activity [3]. Interestingly, previous animal and human studies have linked abnormal Bmal1 expression to metabolic disease. For example, disruption of Bmal1 leads to diabetes, obesity, abnormal gluconeogenesis, and lipogenesis [4][5][6]. Moreover, certain haplotypes of the Bmal1 gene have been demonstrated to be associated with an increased risk of diabetes and hypertension in human studies [7,8]. Bmal1 depletion in mice enhances very-low-density lipoprotein (VLDL) assembly and secretion with the downregulation of src homology-2 domain-containing protein tyrosine phosphatase (Shp) and the upregulation of microsomal triglyceride transfer protein (Mtp). Meanwhile, Bmal1 deletion inhibits cholesterol secretion to the bile with the downregulation of Gata-binding protein 4 (Gata4) and ATP-binding cassette family G protein 5 and protein 8 (Abcg5 and Abcg8) [9]. Bmal1 regulates autophagy in cardiomyocytes via the mammalian target of the rapamycin (mTOR) signaling pathway [10]. Bmal1 knockout mice develop obesity [11]. However, the mechanism of Bmal1 in pancreatic β-cell is not well elucidated.
Mitochondria are required for normal β-cell function and are known to be responsible for cellular metabolism including adenosine triphosphate (ATP) production and cell apoptosis. Mitofusin 1 (Mfn1), mitofusin 2 (Mfn2), and fission 1 (Fis1), core mitochondrial dynamics proteins that localized on the mitochondrial outer membrane, are known as key regulators of mitochondrial networks [12][13][14][15]. It has been reported that mitochondrial dysfunction could impair glucose utilization and insulin secretion in β-cells [12,16]. Intriguingly, mitochondrial respiration displays daily oscillations [17]. Moreover, circadian clocks have been reported to regulate mitochondrial acetylation and various essential proteins involved in metabolic pathways [18]. Furthermore, mitochondrial deacetylation is demonstrated as Bmal1 dependent [19]. These data indicate that mitochondrial function is under circadian clock control.
In the present study, we evaluated the function of Bmal1 in INS-1 cells by using small interfering RNA (siRNA). We investigated whether Bmal1 could influence normal β-cell function and apoptosis. Our results suggest that the deletion of Bmal1 could impair β-cell function via the mitochondrial signaling pathway.

Bmal1 Deficiency Impaired Glucose-Stimulated Insulin
Secretion. To investigate the effect of Bmal1 on β-cell function, we performed a loss of function experiment. As shown in Figure 1, after the deletion of Bmal1, the mean insulin secretion in response to 2.8 mmol/L glucose was significantly decreased (p < 0:01). Moreover, insulin secretion in response to 16.7 mmol/L glucose was also significantly reduced. Therefore, Bmal1 is required for normal β-cell function (p < 0:001).

Bmal1 Deficiency Increased Cell Apoptosis.
To investigate the effect of Bmal1 on cell apoptosis, INS-1 cells were cultured in a normal glucose medium, with or without Bmal1 inhibition. Apoptotic cells were detected by the tunel assay. As shown in Figure 2, although not statistically significant, the total percentage of apoptotic cells had a trend to increase in cells lacking Bmal1 (p > 0:05).

Bmal1 Deficiency Attenuated Mitochondrial Membrane
Potential. MMP is an essential indicator of cellular metabolic activity. In addition, ROS, a core molecule related to oxidative stress, is generated by the potential difference across the inner mitochondrial membrane [20]. To investigate the effect of Bmal1 on MMP, we examined the alteration of MMP in βcells with or without Bmal1 inhibition by the JC-1 assay. As shown in Figure 3, in comparison with the normal control group, the intensity of green fluorescence emitted by the JC-1 monomer increased in cells treated with Bmal1 siRNA (p < 0:001), indicating that MMP was decreased. This result indicated reduced cellular activity in Bmal1-deficient β-cells.

Bmal1 Deficiency Altered Mitochondrial Architecture.
To investigate the effect of Bmal1 on mitochondrial architecture, we performed transmission electron microscopic analysis. Indeed, pathomorphological changes in the mitochondria of Bmal1 knockdown cells were observed by transmission electron microscopy. As shown in Figure 4, in the control group, most mitochondria were round or oval with intact membrane and a few tubular and vesicular cristae, whereas, in Bmal1-deficient β-cells, mitochondria were swollen with irregular shape and fracture and disappearance of mitochondrial cristae.  Figure 5: Bmal1 deficiency in β-cells altered core genes related to mitochondrial dynamics. Bmal1, mitofusin 1, mitofusin 2, and fission 1 mRNA were analyzed by qPCR. Expression levels were normalized to GAPDH mRNA, and each bar represents the mean ± SE of 3 individual experiments. NC: normal control group; siRNA-NC: INS-1 cells treated with negative control siRNA; siRNA-Bmal1: INS-1 cells treated with Bmal1 siRNA. A p value < 0.05 is considered as statistically significant. * p < 0:05, * * p < 0:01, and * * * p < 0:001.

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BioMed Research International with Bmal1 siRNA, Bmal1 mRNA expression was significantly reduced by 71.1% compared with that in the control group (p < 0:001) as shown in Figure 5. We then examined core molecules associated with mitochondrial dynamics and quality control including Mfn1, Mfn2, and Fis1 by quantitative real-time PCR. Interestingly, as shown in Figure 5, gene expression changes were observed in Bmal1-deficient β-cells. Mfn1 mRNA expression showed an obvious decrease trend (p = 0:07). Mfn2 mRNA expression was significantly reduced (p < 0:05), while Fis1 was remarkably increased (p < 0:01). Similar results were observed in western blot analysis. As shown in Figure 6, protein expression of Mfn1 and Mfn2 was obviously decreased, while Fis1 was significantly elevated (p < 0:01, p < 0:001, and p < 0:001, respectively).

Discussion
The important role of the circadian clock in diabetes and mitochondrial network has been recognized [2,21]. Mitochondrial proteome, acetylome, lipidome, morphology, and nutrient utilization and respiration exhibit circadian rhythms [21]. However, little is known about the required role of the clock gene Bmal1 in the pancreatic β-cell and mitochondrial signaling pathway.
To investigate the role of Bmal1 in β-cell function, we performed Bmal1-knockdown experiments. We observed that suppression of Bmal1 impaired glucose-stimulated insulin secretion in β-cell. This result is consistent with another previous study [22]. In addition, Bmal1 is a regulator of compensatory β-cell expansion and survival in mice [11]. Similar results have been observed in other tissues including myocardial stromal fibroblasts, macrophages, and glioblastoma stem cells [23][24][25]. These data indicate the important role of Bmal1 in maintaining normal cell function including β-cell.
Furthermore, MMP is a key indicator to determine cellular metabolic activity [26]. The present study revealed a decreased MMP in cells lacking Bmal1, indicating decreased β-cell function. Additionally, our previous work (data not shown) and another study showed an obvious increase of ROS accumulation and a decrease of ATP production in βcell when inhibiting Bmal1 expression [22]. This is particularly important that ROS clearance and the antioxidant capacity of β-cell are much lower than other tissues [27]. Thus, β-cell is more sensitive to oxidative stress. Indeed, an increased ROS level could cause deficient GSIS [28]. Moreover, mitochondrial ATP production is critical for glucosestimulated insulin secretion in β-cells [12,13,21]. These data indicate that Bmal1 deficiency is closely associated with βcell dysfunction.  BioMed Research International In addition, in the present study, ultrastructural changes including swollen mitochondria with irregular shape and cristae damage were observed in Bmal1 knockdown β-cell. Similar results have been observed in Bmal1-deficient hepatocytes [29] and cardiomyocytes [30]. Impaired mitochondrial morphology, reduced ATP synthesis, and accumulation of oxidative stress products can further cause cell damage and apoptosis [31]. Indeed, our results showed that β-cell apoptosis had a trend to increase in Bmal1knockdown β-cells in comparison with the control group.
However, the underlying mechanism remains unclear, and the role of Bmal1 in mitochondrial dynamics genes in β-cell is not studied. In the present study, we identified reduced Mfn1 and Mfn2 expression and increased Fis1 expression as a consequence of Bmal1 deficiency. Mitochondrial dysfunction is known as a core contributor to pancreatic β-cell failure in the pathogenesis of diabetes [32]. Indeed, Mfn1 and Mfn2 are required for mitochondrial remodeling and normal mitochondrial metabolism [14,33]. Cardiomyocytes lacking Mfn1 or Mfn2 result in mitochondrial fragmentation and display a remarkable defect in mitochondrial fusion and mitochondrial structure [34]. Fis1 has been shown to mediate mitochondrial quality control [35] and transmit an apoptosis signal from the mitochondria to the endoplasmic reticulum [36]. Inhibition of hFis1 function prevents mitochondrial fission [37], while overexpression of hFis1 induced mitochondrial fragmentation and impaired glucose-induced ATP, hyperpolarization, and GSIS [15,16]. Furthermore, pancreatic β-cell Mfn2 knockout had a defect in insulin secretion, mitochondrial fragmentation, and bioenergetic dysfunction [16]. Taken together, these data indicated the essential role of Bmal1 in preserving β-cell via mitochondrial dynamics genes.
In summary, we showed that defects in the Bmal1 function alter mitochondrial membrane potential and mitochondrial architecture in β-cell. Moreover, we revealed that Bmal1 regulated β-cell function via mitochondrial dynamics-related genes. These data provide new insights into understanding the role of the clock gene in preserving β-cell function.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
The authors have no conflicts of interest or any financial disclosures.