Obesity and genetics regulate microRNAs in islets, liver and
adipose of diabetic mice
Enpeng Zhao
1
, Mark P. Keller
1
, Mary E. Rabaglia
1
, Angie T. Oler
1
, Donnie S. Stapleton
1
,
Kathryn L. Schueler
1
, Elias Chaibub Neto
3
, Jee Young Moon
3
, Ping Wang
2
, I-Ming Wang
4
,
Pek Lum
4
, Irena Ivanovska
4
, Danielle Greenawalt
4
, John Tsang
4
, YounJeong Choi
2
, Robert
Kleinhanz
4
, Jin Shang
5
, Yun-Ping Zhou
5
, Andrew D. Howard
5
, Bei B. Zhang
5
, Christina
Kendziorski
2
, Nancy A. Thornberry
5
, Brian S. Yandell
3
, Eric E. Schadt
6
, and Alan D. Attie
1,@
1
Biochemistry Department, University of Wisconsin, Madison, WI, 53706
2
Biostatistics and Medical Informatics, University of Wisconsin, Madison, WI, 53706
3
Statistics Department, University of Wisconsin, Madison, WI, 53706
4
Rosetta Inpharmatics, Seattle, WA, 98109
5
Merck Research Laboratories, Rahway, NJ, 07065
6
Sage Bionetworks, Seattle, WA, 98109
Abstract
Type 2 diabetes results from severe insulin resistance coupled with a failure of β-cells to compensate
by secreting sufficient insulin. Multiple genetic loci are involved in the development of diabetes,
although the effect of each gene on diabetes susceptibility is thought to be small. MicroRNAs
(miRNA) are non-coding 19–22 nucleotide RNA molecules that potentially regulate the expression
of thousands of genes. To understand the relationship between miRNA regulation and obesity-
induced diabetes, we quantitatively profiled ~220 miRNAs in pancreatic islets, adipose tissue, and
liver from diabetes-resistant (B6) and diabetes-susceptible (BTBR) mice. More than half of the
miRNAs profiled were expressed in all 3 tissues, with many miRNAs in each tissue showing
significant changes in response to genetic obesity. Further, several miRNAs in each tissue were
differentially responsive to obesity in B6 versus BTBR mice, suggesting that they may be involved
in the pathogenesis of diabetes. In liver, there were ~40 miRNAs that were down-regulated in
response to obesity in B6, but not BTBR mice, indicating that genetic differences between the mouse
strains play a critical role in miRNA regulation. In order to elucidate the genetic architecture of
hepatic miRNA expression, we measured the expression of miRNAs in genetically obese F2 mice.
Approximately 10% of the miRNAs measured showed significant linkage (miR-eQTLs), identifying
loci that control miRNA abundance. Understanding the influence that obesity and genetics exert on
the regulation of miRNA expression will reveal the role miRNAs play in the context of obesity-
induced type 2 diabetes.
Introduction
MicroRNAs (miRNAs) are endogenously expressed single-stranded non-coding RNAs of 19–
22 nucleotides in length. Approximately 500 miRNAs are listed in the current mouse miRNA
registry (microRNA.sanger.ac.uk). MiRNAs regulate gene expression by destabilizing target
mRNAs through multiple rounds of RNA cleavage (Hutvagner and Zamore 2002), or by
@
Corresponding author, adattie@wisc.edu, 608-262-1372 (voice), 608-263-9609 (fax).
NIH Public Access
Author Manuscript
Mamm Genome. Author manuscript; available in PMC 2010 June 1.
Published in final edited form as:
Mamm Genome. 2009 August ; 20(8): 476–485. doi:10.1007/s00335-009-9217-2.
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repressing the transcription of its target gene by binding to its complementary sequence, usually
at the 3’UTR (Filipowicz et al. 2008; He and Hannon 2004).
Over-expression of miRNAs in human cells has been shown to down-regulate the expression
level of hundreds of putative mRNA targets (Lim et al. 2005). MiRNAs are transcribed as long
RNA precursor molecules (pri-miRNAs) that contain a stem-loop structure of ~70 nucleotides
(Lee et al. 2002). Pri-miRNAs are processed in the nucleus by the RNase III enzyme Drosha
and its partner, DGCR8/Pasha, to generate a 60–70 nucleotide hairpin-structured pre-miRNA
(Denli et al. 2004; Gregory et al. 2004; Han et al. 2004). Pre-miRNAs are transported from the
nucleus into the cytoplasm by the nuclear membrane protein, Exportin-5 (Yi et al. 2003). In
the cytoplasm, the RNase III enzyme, Dicer, cleaves the pre-miRNA hairpin to yield short
miRNA duplexes (Hutvagner et al. 2001). The miRNA duplexes are subsequently unwound
to liberate single-stranded mature miRNAs by the RNA-induced Silencing Complex
(Khvorova et al. 2003; Schwarz et al. 2003).
MiRNAs have been shown to be involved in multiple biological processes, including glucose
homeostasis and lipid metabolism (Krutzfeldt and Stoffel 2006; Tang et al. 2008; Zhang and
Farwell 2008). For example, over-expression of miR-375 was shown to inhibit insulin secretion
from the mouse insulinoma cell line, Min6, by directly targeting myotrophin (Poy et al.
2004), which is an actin-binding protein (Bhattacharya et al. 2006). Actin is known to play an
important role in insulin secretion (Wang and Thurmond 2009). Further, in the rat insulinoma
cell line, Ins-1E, miR-375 over-expression resulted in decreased insulin gene expression, by
targeting the PI3K-pathway gene, phosphoinositide-dependent protein kinase-1 (El Ouaamari
et al. 2008). Recently, miR-34a over-expression was shown to decrease glucose-stimulated
insulin secretion and mediate FFA-induced apoptosis in Min6 cells by targeting Vamp2 and
Bcl2, respectively (Lovis et al. 2008). Over-expression of miR-9 in Ins-1E cells results in
decreased expression of the transcription factor, Onecut-2, leading to increased expression of
Granuphilin/Slp4 and increased insulin secretion (Plaisance et al. 2006).
In addition to playing important roles in pancreatic islets, miRNA-dependent regulation has
been reported in liver and adipose tissue in various model systems. MiR-122a is abundantly
expressed in mouse liver (Chang et al. 2004). Injection of anti-sense oligonucleotides against
miR-122a leads to a significant reduction in hepatic steatosis and plasma cholesterol (Esau et
al. 2006). Similar findings have been reported in primate liver (Elmen et al. 2008), suggesting
that miR-122a plays an important role in lipid metabolism across multiple species. MiRNAs
103 and 143 have been reported to increase adipogenesis in 3T3L1 adipocytes by affecting
several key lipid metabolism genes, including Pparg, Fabp4 and adiponectin (Xie et al.
2009). Taken together, these studies clearly demonstrate that miRNAs are critically involved
in important metabolic processes in multiple tissues. To more fully understand miRNA-
dependent regulation in our model of obesity-induced type 2 diabetes, we set out to
quantitatively profile miRNA expression in pancreatic islets, liver, and adipose tissue.
Our laboratory has modeled the genetics of obesity-induced type 2 diabetes in two mouse
strains, diabetes-resistant C57BL/6 (B6) mice and diabetes-susceptible BTBR T
+
tf/J (BTBR)
mice. When made morbidly obese by the leptin mutation (Lep
ob/ob
), B6-ob/ob mice experience
moderate and only transient hyperglycemia, due to a large expansion of β-cell mass, resulting
in a 20–50 fold increase in plasma insulin levels (Clee et al. 2005; Keller et al. 2008). In contrast,
BTBR-ob/ob mice experience severe hyperglycemia due to a failure to increase their
circulating insulin levels. An in vivo measure of cellular replication showed that B6-ob/ob mice
experience a ~3-fold increase in islet cell proliferation, whereas BTBR-ob/ob mice do not
increase islet cellular replication in response to obesity (Keller et al. 2008).
Zhao et al. Page 2
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