Abnormalities of liver associated with adipocyte dicer deficiency

To be more convincing that adipocyte dicer affected systemic metabolism, we went on to characterize liver, which is an important organ influencing systemic metabolism.

Interestingly, the result of H&E counter staining exhibited that white dots appeared in phase contract microscope illustrating lipid overfilling in livers from iADicer KO mice treated with tamoxifen, suggesting that adipocyte dicer deficiency possibly result in lipid overload in liver (Figure 4A). To confirm this result and better understand the source of lipids in liver from iADicer KO mice, RT-qPCR was performed to detect the genes involved in lipid metabolism. Fat-specific protein 27 (FSP27), a lipid droplet-associated protein that promotes lipid droplet growth and TG storage was induced from iADicer KO mice compared to that from from control mice (Figure 3B), supporting ectopic lipid development in liver with adipocyte dicer deficiency. Moreover, genes involved in DNL pathways such as PAPRγ1/2, FAS, and CD36 also significantly increased in liver from iADicer KO mice compared to that from control mice (Figure 3B). In addition, immunoblot results of PAPRγ1/2 and FAS further confirmed that DNL pathway was activated in liver consecutively to adipocyte dicer deficiency (Figure 3C). These results indicated that adipocyte dicer deficiency resulted in ectopic lipids in liver associated with upregulation of DNL pathway.

Since the accumulation of lipids is proposed to be crucial in the activation of hepatic stellate cells (HSCs) and the development of fibrosis in liver³⁷¹, we went on to investigate whether the ectopic lipids in liver resulted from adipocyte dicer deficiency was associated with fibrosis in the liver. Then, the result of semi-quantification of sirius red stained area suggested that fibrosis was indeed increased in liver from iADicer KO mice compared to control mice (Figure 3D). Furthermore, the results of gene markers involved in extracellular matrix (ECM) depositions had a trend to be increased by adipocyte dicer deficiency (Figure 3E). Moreover, genes involved in inflammatory pathways such as MCP1 and TGF β receptor 1 (TGFβ r1) were significantly increased in mice from the group of iADicer KO mice (Figure 3E).

Since MCP1 is an important chemokine that triggers infiltrations of immune cells, in particular macrophages, it suggested that increased infiltrations of macrophages might lead to the activation of TGF β pathway and accumulation of ECM and thereby fibrosis in liver by dicer deficiency in adipocytes.

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Figure 4: Adipocyte dicer deficiency promoted lipid accumulation and fibrosis in liver.

(A) The result of H&E staining suggested that adipocyte dicer deficiency resulted in lipid accumulation in liver.

(B) Increased mRNA levels of genes involved in DNL pathway in liver as a result of dicer deficiency in adipocytes.

(C) Increased levels of PPARγ and FAS in liver due to adipocyte dicer deficiency by immunobot. (D) Sirius red staining of liver exhibited increased fibrosis as a result of adipocyte dicer deficiency. (E) mRNA levels of genes involved in fibrosis and inflammation in liver. Data represent the mean ± SEM, *P<0.05, **P<0.01. N=4-8.

94 F. Regulation of dicer level in adipocytes

According to our previous data that adipocyte dicer deficiency reduced the lipid storage capability of adipocytes as well as induced abnormalities in systemic metabolism, like ectopic lipid overload and fibrosis in liver. Therefore, adipocyte dicer might have a positive role in the prevention of age-related complications, and adipocyte dicer might be proposed as a target to delay the onset of age-related complications. So, to know how this protein is regulated in adipocytes will be helpful to develop pharmaceutical compounds targeting it. Since adipocytes are sensitive to nutrient availability, we then wondered whether nutrient availability would affect dicer in adipocytes.

Interestingly, 24h fasting significantly increased dicer mRNA level in perigonadal adipose tissue in wildtype mice (Figure 5A). Then, in vitro, overnight deprivation of glucose in the culture media markedly increased dicer protein level in 3T3-F442A adipocytes (Figure 5B). Additionally, fetal bovine serum (FBS) contents in the culture media negatively regulated dicer protein level in 3T3-F442A adipocytes (Figure 5C). Therefore, negative energy state can positively regulate dicer level in adipose tissue and adipocytes.

Since nutrient deprivation is known to activate the lipolysis process in adipocytes by β-adrenergic signaling pathway⁵⁷. Therefore, we went on to see if dicer upregulation under nutrient deprivation is downstream of activation of β-adrenergic signaling pathway, by stimulation of adipocytes with 10^-6 isoproterenol overnight. However, immunoblot result indicated that isoproterenol did not exhibit a similar upregulation effect on dicer as starvation (Figure 5D). Therefore, activation of β-adrenergic signaling pathway might not affect dicer level in adipocytes.

To continue to explore the underlying mechanisms how nutrient availability regulates dicer in adipocytes, we went on to see whether classical cellular nutrient sensor AMP-activated protein kinase (AMPK) is involved. Under nutrient deprivation, AMPK can be activated by the increased ratio of AMP/ATP, ultimately turning up ATP-generating catabolic processes³⁷². In line with the data that fasting or glucose/serum deprivation upregulated dicer, treatment of 1mM 5-Aminoimidazole-4-carboxamide-1-β-^D-ribofuranoside (AICAR), the activator of AMPK, possibly induced dicer expression in 3T3-F442A adipocytes (N=2)

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(Figure 5E). In contrast, mild inhibition of AMPK with 20µM Compound C (CC) seemed to reduce the basal level of dicer while not affecting the effect of serum deprivation on dicer level (N=2) (Figure 5F). But strong inhibition of AMPK with 50µM CC exhibited no obvious effect on dicer expression in adipocyts (N=2) (Figure 5F).

In sum, AMPK might positively regulate dicer expression in adipocytes. However, there might be other unknown factors participating in the regulation of adipocyte dicer expression.

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Figure 5: Regulation of dicer level in adipose tissue and in in vitro adipocytes.

(A) 24h fasting significantly increased mRNA level of dicer in perigonadal adipose tissue in wildtype mice (N=5).

(B) Overnight glucose deprivation significantly increased dicer protein level in 3T3-F442A adipocytes (N=3). (C) Fetal bovine serum contents negatively affected dicer protein levels in 3T3-F442A adipocytes (N=3). (D) Overnight stimulation of 10^-6 isoproterenol did not increase dicer protein expression as serum starvation (N=3).

(E) Overnight treatment of 1mM AICAR might increase dicer protein level in 3T3-F442A adipocytes (N=2). (F) Overnight treatment of various concentrations of AMPK inhibitor compound C differentially affected dicer protein level in 3T3-F442A adipocytes (N=2). Data represent the mean ± SEM, *P<0.05.

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