Scientists have implicated an RNA splicing regulator protein in the mechanism that drives metabolic phenotypes associated with obesity. An international team led by researchers at the Joslin Diabetes Center in Boston has found that the splicing factor SFRS10 is downregulated in both the liver and muscle of obese individuals. Their studies in mice showed that lack of sfrs10 alters splicing of lipid metabolism regulatory protein LPIN1, which lead to lipogenesis.
Mary Elizabeth Patti, M.D., and colleagues, suggest modulating splicing factors and alternative splicing may represent a potential therapeutic approach against obesity-associated tissue lipid accumulation and the resulting metabolic consequences. They report their findings in Cell Metabolism in a paper titled “Expression of the Splicing Factor Gene SFRS10 Is Reduced in Human Obesity and Contributes to Enhanced Lipogenesis.”
Obesity consistently leads to abnormal lipid metabolism and can be associated with other metabolic complications including insulin resistance, type 2 diabetes, and increased cardiovascular disease risk, the researchers note. To try and discover mechanisms associated with these metabolic consequences of obesity, the researchers looked at gene-expression signatures in the liver and muscle of human obese subjects.
Studies in two independent cohorts of obese patients demonstrated that the top-ranking downregulated pathways in both muscle and liver tissues were related to RNA processing and splicing: In particular, 46 of 199 RNA splicing genes were downregulated in the liver, and 41 were downregulated in muscle. Downregulation of RNA processing and splicing genes was also observed in a mouse model of diet-induced obesity.
Dr. Patti’s team chose SFRS10 as a representative splicing factor for further analysis as it was most consistently altered in both human and animal models. They first analyzed the effects of experimentally reducing SFRS10 expression in HepG2 cells using an siRNA that led to a 50–70% reduction in levels SFRS10 mRNA and protein expression.
The resulting reduction in SFRS10 was associated with a 1.5–2.5-fold increase in lipogenic genes, including SREBP1c, FASN, ACC1 and DGAT2, combined with a 1.6-fold increase in lipogenesis, and a 1.4-fold increase in the cellular accumulation of triglycerides (TAG). Equivalent changes were observed in C2C12 myotubes. Conversely, forcing the overexpression of SFRS10 in Hepa1c hepatoma cells resulted in a significant decrease in the expression of lipogenic genes.
Moving on to evaluate SFRS10 in vivo, the authors generated experimental mice that were heterozygous for sfrs10 (homozygocity was lethal) and exhibited 30% lower levels of the gene’s mRNA than wild-type animals. This level of reduction is equivalent to that observed in obese humans, they note. In agreement with the in vitro data, sfrs10 heterozygous mice showed up to a 4.6-fold increase in the hepatic expression of lipogenic and TAG synthesis genes in the postprandial state.
Significantly, while liver TAG accumulation remained unchanged, plasma TAG levels were 52% higher in the heterozygous mice and there was a 3-fold increase in plasma levels of the TAG-enriched VLDL fraction, which indicated a hepatic origin for higher plasma TAG. “Together, these data indicate that decreased expression of sfrs10 is sufficient to increase hepatic lipogenic gene expression, increase VLDL secretion, and induce hypertriglyceridemia in a mouse model in vivo,” the authors state.
The next stage was to evaluate which lipogenesis-related genes with alternatively spliced isoforms SFRS10 might be acting on. One candidate, LPIN1, has been shown to regulate lipid metabolism and exists in two major alternatively spliced isoforms, α and β, and a third variant, γ, which was recently identified in the brain.
The β isoform is generated by the inclusion of exon 6 and is associated with increased expression of lipogenic genes. Notably, the team remarks, an alternatively spliced exon 6 of LPIN1 contains a particular sequence motif that binds to SFRS10, and these sequences are highly conserved in both the human and mouse LPIN1 genes.
To determine if alterations in SFRS10 expression could modulate splicing of LPIN1, the team employed a minigene construct containing the alternative exon 6 of the human LPIN1 gene. Cotransfection of the minigene construct with a plasmid expressing SFRS10 caused exon 6 skipping. Conversely, SFRS10 siRNA increased the inclusion of exon 6.
The effects of SFRS10 knockdown on LPIN1 splicing were evident in HepG2 cells. Treatment with an SFRS10 siRNA didn’t change total LPIN1 expression but did lead to an increase in the β isoform and a parallel decrease in the α isoform. Similar changes in the relative amounts of the α and β LIPIN1 isoforms were also seen as a result of SFRS10 knockdown in C2C12 mytotypes. “This effect was specific to SFRS10, since knockdown of the constitutive splicing factor SF3A1 did not alter LPIN1 splicing,” the authors stress.
Similar changes in LPIN1 isoform ratios were also observed in the livers of Sfrs10 heterozygous mice, wild-type mice fed a high fat diet and, indeed, the livers of obese humans: There was no change in total LPIN1 expression, but there was an increase in levels of the LPIN1 β isoform, relative to the α isoform.
To whether the effects of SFRS10 knockdown on lipogenic gene expression are mediated by increases in the LPIN1 β isoform, the researchers developed distinct siRNA oligonucleotides directed against either total LPIN1 or specifically against the LPIN1 β-specific exon 6. Isolated knockdown of either total or the β-specific isoform of LPIN1 had no significant effects on FASN expression.
However, co-transfection with when the LPIN1 β-specific siRNA and the SFRS10 siRNA abolished FRS10-mediated increases in genes regulating fatty acid synthesis and TAG synthesis. Importantly, they add, LPIN1 β knockdown also prevented the SFRS10 siRNA-induced increase in lipogenesis and TAG accumulation, and prevented increases in lysophosphatidic acid (LPA), an intermediate in the TAG synthesis pathway.
The authors admit that their studies focused on just one RNA splicing factor among a number that were found to be downregulated in obese humans and mice. Nevertheless, they state, the results demonstrate that altered expression and splicing function of genes such as SFRS10 may modulate metabolic pathways critical for obesity and related metabolic phenotypes.
“These findings have several implications,” they conclude. “First, genes and molecules regulating mRNA processing should be investigated as potential candidates in obesity and insulin-resistant states. Second, alternatively spliced isoforms of known metabolic genes should be identified and characterized, as their alternative splicing may serve as an important regulatory step. Moreover, identification of pathways regulating alternative splicing in obesity may have implications for other chronic diseases linking with insulin resistance.”