1. Introduction
Carbohydrates are the main source of energy in most animal diets [
1], and its properties such as digestion and absorption rate, viscosity, structural features, water-binding capacity and fermentation ability in the gastrointestinal tract are of critical importance in the effect of nutrition [
2]. It has been reported that the utilization of dietary carbohydrates and their effects on growth and nutrient deposition are very important [
3], and the dietary carbohydrate inclusion in several fish species appears to produce positive effects on growth and digestibility [
4,
5,
6].
Blunt snout bream (
Megalobrama amblycephala,
M. amblycephala) is one of the most important economic freshwater fish in China [
7]. According to the latest statistics from Food and Agriculture Organization (FAO), the total output of
M. amblycephala reached nearly 850 thousand tons in 2016, and the market demand is increasing due to its high economic value.
M. amblycephala is typical herbivorous feeding habit [
8], and its digestive function and disease resistance ability are related to high-fat and -glucose diets [
9]. Researches have shown that higher carbohydrate dietary formula (>34%) might result into glucose intolerance responses in
M. amblycephala [
10,
11,
12]. But the underlying molecular mechanism is still unclear. Our previous study provided a miRNA profiling in response to high starch treatment in
M. amblycephala, and showed that miRNAs might play crucial roles in glucose metabolism [
9]. In that study, 124 differentially expressed miRNAs (DEMs) were identified in liver tissue between fishes fed with normal and high starch diets and a noteworthy upregulated miRNA, miR-34a, was found in the DEMs list [
9]. There might be association between miR-34a upregulation and glucose metabolism.
miR-34a, a highly conserved, endogenous, small non-coding RNA, has emerged as a key biomarker in cellular senescence and tumor suppression through its interaction with
SIRT1 (Sirtuin 1) and
p53 (Tumor protein p53) [
13,
14]. miR-34a has additionally been implicated in diabetes as a target to prevent pancreatic β-cell death [
15,
16]. Several studies had reported that miR-34a was dysregulated in metabolic tissues in rodent models and human patients of obesity, type 2 diabetes (T2D) and Non-Alcoholic Fatty Liver Disease (NAFLD) [
17,
18,
19,
20,
21]. MiR-34a was highly elevated in liver in NAFLD and T2D patients compared to healthy controls [
20,
21]. Previous studies suggested that miR-34a regulated fat metabolism and insulin secretion by targeting
SIRT1 and acyl-CoA synthetase long-chain family member 1 (
ACSL1) [
15,
22,
23].
SIRT1 is a key metabolic sensor and regulator in cells and directly deacetylates and modulates some important metabolic regulators, including
PGC-1α (peroxisome proliferator-activated receptor γ coactivator 1-α), peroxisome proliferator-activated receptors (
PPARs),
p53, and forkhead box protein O1 (
FOXO1) which change the expression of transcriptional programs that modulate cholesterol, lipid and energy homeostasis [
24]. Moreover, researchers demonstrated that miR-34a was involved in insulin hyposecretion, insulin resistance, and cell injury in a mammalian T2D model [
25]. Therefore, we hypothesized that miR-34a might play an important role in promoting glucose intolerance in human and animals.
Researches on the dysregulation of miR-34a in glucose metabolism have been widely reported in rodents and human [
26,
27], but little is known in the teleosts yet, especially in
M. amblycephala. Hence, we used next-generation sequencing technologies to identify the potential regulatory networks of miR-34a in
M. amblycephala. In this study, we conducted an in vivo experiment by injecting the inhibitor of miR-34a into
M. amblycephala, and then constructed three poly(A)
+ libraries from livers in consideration of the suggestion that the main target organ of miR-34a was liver tissue [
28]. After sequencing and bioinformatics analysis, a number of differentially expressed genes (DEGs) including the target genes of miR-34a would be identified. These results may provide new insights into the regulatory mechanism of miR-34a in high-glucose metabolism in
M. amblycephala.
3. Discussion and Conclusions
M. amblycephala is a widely cultured freshwater fish in China, but the molecular mechanism of nutritional utilization of dietary carbohydrates is still unclear [
32]. Previous studies had demonstrated that altered dietary and metabolic conditions were affected by gene regulation in the cell nucleus [
24]. We had previously performed a next-generation sequencing study between normal starch diet and high starch diet treated fishes, and identified hundreds of DEMs that responded to HSD treatment in intestine, liver, and brain in
M. amblycephala, respectively, suggesting that miRNAs might play crucial roles in glucose metabolism [
9]. Among the DEMs, we found that the miR-34a was significantly upregulated in HSD group. miR-34a was a key regulator in the glucose metabolism [
17,
18,
19,
20,
21], and miR-34a upregulated expression had been identified in T2D patients compared to health controls [
21]. A plausible molecular mechanism for the role and function of miR-34a in metabolism was the loop of miR-34a,
SIRT1 and
p53 [
33]. But how miR-34a affected the course of glucose intolerance responses in
M. amblycephala was still unknown.
So we conducted an in vivo experiment with intraperitoneally injection of miR-34a inhibitor into M. amblycephala, and identified the DEGs related to miR-34a inhibition. The qRT-PCR analysis showed that miR-34a was significant inhibited at 12, 24 and 48 h post the antagomiR34a treatments, with dysregulation of 2212 DEGs. GO and KEGG pathway analysis showed that the DEGs were enriched in the signaling pathways, including PPAR pathway, insulin signaling pathway, JAK/STAT signal pathway, Type I diabetes mellitus and Type II diabetes mellitus, which were associated with glucose/lipid metabolic pathways and biosynthetic processes. All these results implied that the inhibition of miR-34a could regulate a series of genes, which might be crucial for glucose metabolism in M. amblycephala.
Previous studies have demonstrated that miR-34a could reduce the expression level of
SIRT1 and prevent the activation of
PGC-1α,
PPARα,
p53 and
FOXO1 to alter the expression of transcriptional progresses and supervise lipid/glucose, cholesterol and energy homeostasis [
34,
35,
36,
37,
38,
39,
40,
41]. But the expression levels of these genes were not significantly differentially expressed in our study. We found that one of the PPARs family members,
PPARb, was upregulated in antagomiR34a treated samples by qRT-PCR (
Figure 8).
PPARb was a nuclear hormone receptor which governs a variety of biological processes and may be involved in the development of several diabetes and obesity diseases [
42,
43]. For example, Sanderson et al., proved that
PPARb/
δ deletion could downregulate the pathways associated with lipoprotetin metabolism and various pathways related to glucose utilization [
44]. Consistent with these results, we found
PPARb was upregulated in samples injected with miR-34a inhibitors, implying that the deletion of miR-34a could upregulate
PPARb and the pathways associated with the glucose metabolism in
M. amblycephala. These might provide a potential novel role of miR-34a in the regulation of glucose metabolism in
M. amblycephala.
In addition to the genes involved in the putative regulatory loop of miR-34a and
SIRT1 (
Figure 9) [
45], other regulators responded to insulin and glucose homeostasis were found dysregulated in the analysis of RNA-seq data. For example,
NSIG1, the negative regulator of SREBPs, was downregulated in antagomiR34a-12h sample. Previous study showed that the overexpression of
INSIG1 could significantly inhibit
SREBP-1c expression and thereby blocking the expression of downstream genes related to insulin and lipid metabolic pathways (
Figure 9) [
46]. Consistent with this, the downregulated
INSIG1 was detected in antagomiR34a-12h sample, which might increase the expression of SREBPs and the downstream genes. We also found that
PLCB (phosphatidylinositol phospholipase C, β),
GADD45A (Growth arrest and DNA-damage-inducible protein GADD45 α), and
FABP (fatty acid-binding protein) and
ACSL1 were downregulated after inhibiting miR-34a in
M. amblycephala (
Table S3).
ACSL1 is one of the long chain acyl-CoA synthetases in lipid metabolism and also been implicated in the cellular uptake of fatty acids. The overexpression of
ACSL1 showed increased acyl-CoA synthetase activity and fatty acid uptake [
47]. In this study, the
ACSL1 was found downregulated in antagomiR34a-12h sample comparing with control. These results implied that the inhibition of miR-34a might play potential novel roles in glucose homeostasis in
M. amblycephala by directly or indirectly regulating these key genes.
The DEGs in response to miR-34a inhibition in
M. amblycephala livers was associated with JAK/STAT and PPAR signaling pathways. Some not significantly enriched pathways, including insulin-signaling pathway, Type I diabetes mellitus and Type II diabetes mellitus, were also found in the result of KEGG analysis. These pathways were related to DEGs including upregulated
PPP1C (serine/threonine-protein phosphatase PP1 catalytic subunit) and
IL6RA (interleukin 6 receptor α, downregulated) and downregulated
CNTF (ciliary neurotrophic factor). Elevated
IL6 had been reported in diabetes mellitus type 2 [
48]. We checked the expression level of
IL6 and found that
IL6 was upregulated in the antagomiR34a-treated sample (
Figure 5). All these results suggested that inhibited miR-34a might regulate the glucose metabolism by altering pathways related to glucose utilization.
To avoid the incomplete results of RNA-seq from being less sequences samples, we carried out an additional qRT-PCR analysis to detect the expression profiles of glucose metabolism related genes. The results showed that the profiles of 7 selected key genes were all as expected and most of these genes were involved in the AMPK signaling pathway (
Figure 8 and
Figure 9).
AMPK is an energy sensor that regulates cellular metabolism which could stimulate glucose uptake to produce energy when activated by a deficit in nutrient status [
49]. Evidence has shown that gluconeogenesis in the liver is regulated by multiple enzymes such as
PEPCK and
G6Pase (
Figure 9) [
50], and the activation of
AMPK could suppress the transcription of
G6Pase in hepatoma cells [
51]. Andreelli et al., suggested that there was glucose intolerant and fasting hyperglycemia in
AMPK α2 liver-specific knockout mice, which presumably due to the increased
PEPCK and
G6Pase activity [
52]. In our study,
AMPK was found to be upregulated in the miR-34a inhibitor-injected samples and the expression level of G6Pase was decreased by qRT-PCR analysis (
Figure 8). Another gene
PPARb was involved in the glucose metabolism by activating a program that increased the coupling of glycolysis to glucose oxidation in muscle via the cooperation with AMPK signaling pathway to activate
Ldhb gene transcription [
53]. As expected, the relative expression level of
PPARb was found to be increased in the miR-34a-inhibited group by qRT-PCR analysis, which revealed that the knock-down of miR-34a might upregulate
PPARb expression, which then subsequently interacted with
AMPK to regulate the glucose metabolism in the liver tissue in
M. amblycephala. In addition, the expression level of 3/7 selected AMPK signaling pathway related genes showed a progressively accumulation along with handling time post injection of miR-34a inhibitor (
Figure 8). These results implied that miR-34a could interact with AMPK signaling pathway to regulate the glucose embolism in
M. amblycephala.All the results in this study showed that the miR-34a was an important regulator in the glucose metabolism by activating or inactivating the downstream genes involved in the glycometabolism. The regulation of the expression level of miR-34a or its targets genes might provide a novel regulatory role in glucose metabolism in M. amblycephala.