Assessment of Hepatic Insulin Signaling in a Murine Model for Alström Syndrome

Ayla Kanber, Boston University

The Jackson Laboratory Summer Student Program

August 11, 2015

Ayla Kanber Student Gayle Bouchard-Collin, M.S. Mentor Jürgen K. Naggert, Ph.D. Principal Investigator

CONFIDENTIAL MATERIAL: DO NOT COPY OR DISTRIBUTE WITHOUT SPONSOR’S PERMISSION.

Assessment of Hepatic Insulin Signaling in a Murine Model for Alström Syndrome

Ayla Kanber

The Jackson Laboratory Summer Student Program

Abstract

Alström Syndrome is a metabolic disease that results from mutations in the

ALMS1 gene. One of the several organs the disease affects is the liver. Often,

the disease causes liver hepatosteatosis that often progresses to hepatosteatitis.

It is not uncommon that patients who suffer with Alström Syndrome die from liver

failure (Marshall et al., 2011). The liver disease in Alström Syndrome is clinically

similar to obesity-related Non–Alcoholic Fatty Liver Disease (NAFLD) and can

serve as a model for this common disease. The purpose of this study was to

examine the insulin-signaling pathway in the liver of pre-obese mice that have a

mutation in the ALMS1 gene. ALMS1Gt/GT mice develop insulin resistance and

form excess lipid deposits in the liver (Collin et al., 2005). Western analysis

revealed that at 6 weeks of age, the AKT pathway is not disturbed in Alms1GT/GT

livers as phosphorylation of AKT is successful before the onset of

hyperinsulinemia. Altogether, these results show that hepatic insulin resistance is

not a primary defect of in Alström Syndrome.

Introduction

Alström Syndrome is a recessive, progressive and fatal ciliopathy that is

characterized by childhood obesity, hyperinsulinemia, type 2 diabetes, steatosis,

dilated cardiomyopathy, and neurosensory deficits such as cone-rod dystrophy

and hearing loss (Marshall et al., 2011). The disease is caused by mutations in

the ALMS1 gene, which encodes the ciliary protein ALMS1. Alström Syndrome is

a rare disease affecting at least 1018 patients worldwide (Marshall, Personal

comm.), however the individual metabolic phenotypes of obesity, type 2 diabetes

and fatty liver disease can affect up to 30% of the population in westernized

countries (Bhala et al., 2013; Nguyen et al., 2011). The progression of NAFLD to

hepatosteatitis is one of the major causes of morbidity and mortality in Alström

Syndrome (Marshall et al., 2011).

When normal cells detect glucose, the insulin-signaling pathway is

stimulated through a cascade of phosphorylations. When the protein kinase AKT

is phosphorylated, it induces the translocation of glucose transporter 4 (GLUT4)

to the plasma membrane and the glucose is metabolized properly. Previously,

insulin stimulated Alms1GT/GT mice have been shown to undergo proper

activation of AKT in adipose tissue, a downstream target of insulin signaling.

Before the onset of obesity and hyperinsulinemia, insulin signaling in Alms1GT/GT

adipose tissue appears to be normal. However, mutant mice have reduced

translocation of GLUT4 to the plasma membrane (Favaretto et al.,

2014). Alms1GT/GT mice progressively accumulate lipid in their hepatocytes

(Favaretto et al., 2014), which in other models has been attributed to liver insulin

resistance. We propose that hepatic insulin resistance may be caused by ALMS1

gene mutations that cause a defect in the insulin-signaling pathway.

In the latter stages of hepatic disease in Alms1 mutant mice, livers

develop collagen deposits that result in extensive fibrosis. In particular, on the B6

genetic background, Alms1 disruption causes hepatosteatitis and severe liver

fibrosis. This increased fibrosis may be due to aberrant TGFβ signaling, which

further increases SMAD activation that precedes collagen deposition in the liver

(Gressner et al., 2006). Recently, it has been shown that miR-21 deficiency can

modify body weight and liver disease in ALMS1 male mice (Collin et al,

unpublished). Previously, miR-21 has been shown to inhibit TGF-β1 expression

in bone barrow mesenchymal stem cells (Wu et al., 2015). Whether miR21

pathway molecules interact with TGFβ signaling molecules to protect against

steatosis and fibrosis is currently unknown.

This study aimed to elucidate the role of ALMS1 in the liver and to

understand how mutations in Alms1 result in hepatic steatosis, a common

molecular disturbance also observed in the obese population. To determine

whether the hepatic insulin-signaling pathway may be perturbed in Alström

Syndrome, we examined livers of pre-obese mice for deficits in the AKT pathway.

Materials and Methods

Alms1Gt(XH152)Byg(Alms1GT/GT)mice were used to recapitulate clinical

features of Alström Syndrome patients. These mice are on a C57BL/6Ei

background. Six week-old, six month-old, and one year-old mice were fasted in

the early morning for 4 hours. They were then given intraperitoneal injections of

either saline or insulin (0.5U/kg). Thirty minutes later, whole liver samples were

collected and frozen in liquid nitrogen. Proteins were extracted from the liver

samples for Western blot analysis to detect pAKT Ser473 and AKT expression.

β-actin expression was used as a loading control for the analysis.

DNA isolation

To isolate DNA, tail tips were heated at 95°C for 30 minutes in 50 mM

NaOH and neutralized in 1M Tris (pH 8.0). Samples were centrifuged for 6

minutes at 3.6K.

Genotyping

A forward primer specific to Alms1 intron 13 upstream of the gene trap

(GT-F) and a reverse primer specific to the gene-trap cassette (GT-R) were

designed to detect the mutant Alms1 allele. A forward primer specific to Alms1

intron 13 upstream of gene trap (WT-F) and a reverse primer specific to Alms1

intron 13 downstream of the gene trap (WT-R) were used to detect the wild-type

allele. The PCR conditions were as follows: initial denaturation at 94°Cfor 2

minutes, followed by 45 cycles of 94°C for 20 seconds, 54°C for 30 seconds and

72°C for 30 seconds and a final extension for 72°C for 7 minutes (Collin et al.,

2005).

Western Analysis

Mice were euthanized by CO2 asphyxiation followed by cervical

dislocation. Then livers were removed and frozen in liquid nitrogen to preserve

the integrity of the proteins. The livers were homogenized in RIPA buffer (3ml

RIPA per gram of tissue) using a PowerGen 125 homogenizer (Fisher Sci). The

livers were incubated at 4°C for 30 minutes then centrifuged at 12.5K for 20

minutes at 4°C. Supernatants were removed and protein concentrations were

determined using Direct Detect (Millipore). Proteins were separated on a 10%

Mini Protean TGX gel (Biorad). For each gel lane, 5 ul of 4X loading buffer

NuPage LDS (Biorad) and 2 ul 10X sample reducing agent were used for a final

20 µl sample. Prior to loading, samples were denatured at 70°C for 10 minutes.

The gel was run at 200V for 35 minutes. Proteins were transferred onto

nitrocellulose membranes using the Transblot Turbo system (Biorad). After the

transfer, the membrane was blocked in Blotto A for one hour then incubated in

primary antibody (1:1000 in Blotto A) overnight at 4°C. Subsequently, the

membrane was washed (TBS-T) 3 times for 20 minutes each time. The

membrane was incubated in secondary antibody (1:1000 in Blotto A) for an hour

at room temperature then washed 3 times in TBS-T for 30 minutes. Chemiluscent

reaction were performed using equal parts of each solution for ECL Western

blotting Detection reagents (Amersham) were used (350 ul of each solution for

one blot). The film was exposed to membranes in the dark for varying times and

processed by X (see dark room for name of machine).

Immunohistochemistry

Wax slides holding 4µM liver specimens were deparaffinized with Xylene

and other alcohol solutions. Blocking solution (1:50 PBS-Triton (0.3%) to horse

serum) was used on each section for 30 minutes. Primary antibody (1:100 to

blocking solution) was added to liver specimens and kept in a humidifying

chamber overnight at 4°C. Slides were washed in 1X PBS three times for 10

minutes on an orbital shaker. Secondary antibody (1:200 PBS-Triton(0.3%)to

blocking solution) was used on each section and kept in a humidifying chamber

in the dark at room temperature for 1 hour. Slides were washed in 1X PBS 3

times for 10 minutes each time on a shaker with a covered top. One drop of

Vectashield with DAPI (Vector) was used on each section and then slides were

covered with a slip and examined under a fluorescent scope (Leica).

Results and Discussion

Figure 1 shows that at 6 months of age, Alms1Gt/GT livers (a) have an

accumulation of lipid deposits compared to wild type control mice(b). A time

course progression of liver disease in Alström Syndrome mice is shown in Figure

2. By 9 months, the liver begins to express collagen fibers (Dark Red;Sirius red

stain) while continuing to retain lipid deposits (Fig2A). At 12 and 20 months of

age, the liver disease continues to cause lipid deposit retention and fibrosis. By

21-24 months old, the liver progresses from hepatosteatosis to hepatosteatitis.

This progression is marked by collagen fibers that are produced to help repair

liver damage, as shown in Figure 2d-e.

Figure 1. Histology showing a comparison of a 6 month-old Alms1Gt/GT and wild type liver.

Figure 2. Histology showing the progression of liver disease in Alms1Gt/GT mice harboring a gene trap mutation in the ALMS1 gene. Figure 2a: arrow points to collagen fibers indicative of fibrosis. Figure 2d: arrow points to (blue) lymphocyte infiltration that indicates the advancement to hepatosteatitis.

IBA1 is a macrophage marker used in immunohistochemistry. In the liver,

macrophages are called Kupffer cells. Kupffer cells are activated for repair when

the liver is damaged. As shown in Figure 3b, mutant livers at 8 months old are

highly activated with Kupffer cells. This shows that the liver damage at this time

point is high in comparison to the control at 8 months old (Figure 3a).

A

B

A

Bc

C

D

E

Figure 3. Immunohistochemistry of macrophage marker IBA1 for a control at 8 months old and an Alms1Gt/GT mutant at 8 months old.

GFAP is a marker that activates stellate cells in the liver. Similar to the

activation of Kupffer cells, hepatic stellate cells are activated when liver damage

is detected. As shown in Figure 4, by 8 months of age, hepatic stellate cells

become activated in Alms1Gt/GT livers (Figure 4a).

Figure 4. Immunohistochemistry of hepatic stellate cell marker GFAP in a littermate control and Alms1Gt/GT mutant at 8 months of age.

A B

A B

Figure 5 shows the Western blot results after injecting fasted 6 week-old

Alms1Gt/GT mutant and wild type mice with either saline or insulin

intraperitoneally. Using anti-pAKT ser473 (Cell Signaling), we observed that the

saline-injected mice express less phosphorylated AKT than the insulin-injected

mice in both control and mutant animals (Figure 5). These results indicate that

wild-type controls and Alms1Gt/GT mutants showed a similar response to insulin

Therefore, at six weeks of age, no insulin resistance in the livers of the

Alms1Gt/GT mutants. AKT expression was relatively equal between saline-

injected mice and insulin-injected mice. Alms1Gt/GT mutants undergo proper

phosphorylation of AKT, which means that at this early age, the insulin-signaling

pathway is not defective. The loading control, β-actin, showed relatively equal

expression between saline-injected mice and insulin-injected mice.

Figure 5. Western blot results of insulin-injected and saline-injected mice at 6 weeks old.

Figure 6. Relative pAKT ser473 expression to β-actin expression values for 6 week-old wild type and Alms1Gt/GT mice that were either insulin- or saline-injected i.p.

Figure 6 shows the relative pAKT ser473 expression to β-actin expression

ratios. The values for both wild type and mutant insulin-injected mice are higher

than the values for both wild type and mutant saline-injected mice. T-tests were

conducted and p-values less than 0.05 are considered significant. The p-value

for wild type, saline-injected mice and wild type, insulin-injected mice is 0.028

and significant. The p-value for Alms1Gt/GT, saline-injected mice and Alms1Gt/GT

insulin-injected mice is also 0.028 and significant. Alternatively, the p-value for

wild type, saline-injected mice and Alms1Gt/GT, saline-injected mice is 0.56 and

insignificant. Similarly, the p-value for wild type, insulin-injected mice and

Alms1Gt/GT, insulin-injected mice is 0.22 and insignificant.

One possibility is that the ALMS1 gene does not interfere with the insulin-

signaling pathway but instead causes liver insulin resistance by some other

mechanism. Another scenario is that the insulin receptors remain in the

endosome recycling pathway and cannot reach the cell surface.

Conclusion In conclusion, the western blot analysis of pAKT ser-473 expression in 6

week-old wild type and Alms1Gt/GT mice shows that the insulin-signaling pathway

in Alms1Gt/GT mice is undergoes proper AKT phosphorylation. At this age, insulin-

signaling pathway is functional, suggesting that the liver of 6 week-old Alms1Gt/GT

mice is not insulin resistant. Thus, hepatic insulin resistance does not appear to

be a primary defect of Alström Syndrome.

It would be meaningful to uncover the mechanisms in which the livers of

Alms1Gt/GT mice become insulin resistant. Although pre-obese Alms1 mutant

mice respond to insulin, it remains possible that there may be signaling defects

downstream to AKT. Future investigation of insulin-signaling molecules

downstream of AKT, such as AS160 or Glut2 will be necessary to unravel the

basis of hepatic insulin resistance in AS. Ultimately, new therapeutic strategies

may benefit both Alström Syndrome patients as well as patients afflicted with

NAFLD.

Acknowledgements

I would like to especially thank my mentor, Gayle Collin, for teaching and

helping me throughout the course of this program. Thank you to Dr. Jürgen K.

Naggert and the Jackson Laboratory Summer Student Program for giving me the

opportunity to conduct this research. I would also like to thank all of the members

in the Naggert/Nishina lab for welcoming and supporting me in the lab. This

Summer Student Fellowship was supported by the Beverly Coleman

Endowment.

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