Anastasia's CV Poster PDF

Defining a novel pathway to lipodystrophy

Anastasia Pimenova, Marie-Elodie Cattin, Esther Mak-Washburn, Patrick G. Burgon University of Ottawa Heart Institute, Ottawa, ON, Canada

Introduction Lipodystrophies are associated with adipose tissue abnormalities and metabolic defects such as insulin resistance. Mutations in A-type lamin, a ubiquitous nuclear protein, are responsible for such diseases. We recently discovered a new A-type lamin interacting protein (MLIP) (Ahmady et al. JBC, 2011). MLIP is ubiquitously expressed; its biological function remains elusive but it may be of high relevance for cardiac function and metabolism due to abundant expression in the heart. MLIP has no paralogous homologue, suggesting no functional redundancy. To elucidate MLIP’s function, we generated both heterozygous (MLIP+/-) and homozygous (MLIP-/-) null 129Sv mice. The main goal of this project is to investigate whether MLIP deletion impacts metabolism in mice.

First author’s emails: [email protected], [email protected]

MLIP+/+ MLIP+/- MLIP-/-

Initial observations of MLIP-/- mice

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Figure 2. Body mass (g) of MLIP +/+, +/- and -/- mice measured over a span of 18 weeks .

Since we observed such a visible difference in body mass between the three genotypes the question was raised – is metabolism in null mice altered as a result of MLIP absence? To answer this question, the following experiments were performed on MLIP-/- mice: 1. Glucose and free fatty acid (FFA) uptake analysis 2. Response of mice to insulin 3. Measurement of Glut4 expression in the heart

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• Representative FDG uptake images at 60 min post-injection

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Figure 3. Micro-PET image of FDG uptake in 12 week old MLIP+/+, +/- and -/- mice.

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Free fatty acid and glucose uptake

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As seen in Figures 3-6, both FFA and glucose uptake was decreased in the heart of MLIP -/- and +/- mice. The decrease in glucose uptake was not restricted to the heart – it was also observed to be lower in brown adipose tissue (Figure 6) and the brain (results not shown).

Figure 5. FTHA uptake in left ventricle of MLIP +/+ and +/- mice. Measured with microPET. Mean±SD,

Figure 6. FDG uptake in MLIP+/+, +/- and -/- mice in brown adipose tissue. MicroPET imaged. Mean±SD.

An initial tracking of body weight was done over a span of 18 weeks. MLIP -/- mice appeared to be leaner than their wild type counterparts.

Figure 4. FDG uptake in MLIP+/+, +/- and -/- mice in the left ventricle. MicroPET imaged . Mean±SD,

14-[18F]fluoro-6-thia-heptadecanoate (FTHA) was used as a radiolabeled free-fatty acid analogue to see how FFA was taken up in the left ventricle of MLIP+/+ and MLIP +/- mice, measured by microPET (Figure 5). 2-[18F]fluoro-2-deoxy-D-glucose (FDG) was used as a radioactive glucose analogue and micro-PET imaged in mice (Figures 3, 4, 6).

Hypersensitivity to insulin



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We next investigated the response to insulin in MLIP +/- and -/- mice. FDG uptake was analyzed before and after insulin injection in the mice. As shown by Figure 7, the glucose uptake in MLIP -/- and MLIP +/- was slightly lower than the MLIP +/+ mouse at base level (as in Figure 3). After insulin injection however, the glucose uptake was significantly higher, suggesting that MLIP +/- and -/- were hyper responsive to insulin. If there is insulin hypersensitivity, how is Glut4 expression in the null versus wild type heart?

Figure 7. FDG uptake in MLIP +/+, +/- and -/- hearts following insulin injection. Imaged with microPET.

Glut4 expression in the heart

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To investigate whether the lower glucose uptake in the heart was due to Glut 4 expression, a Western blot was done on MLIP +/+, +/- and -/- 12 week old mice heart lysates. As shown, Glut4 levels were higher in +/- and -/- hearts compared to +/+.

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Figure 8. Western blot of Glut 4 in MLIP +/+, +/- and -/- heart lysates. Tubulin is loading control.

High fat diet

Conclusions

After doing the Glut 4 experiment, a few questions were raised: • Why is glucose uptake decreased if Glut4 expression and insulin

sensitivity are increased in MLIP +/- and -/- hearts? • Is Glut4 retained in the cytoplasm of MLIP +/- and -/- mouse tissues? • How do MLIP -/- mice respond to metabolic stress? To answer the last question, MLIP +/+ and -/- mice were put on a high fat diet.

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Figure 9. A graph of male MLIP +/+ and -/- mice body mass (g) undergoing a chow and HFD for 31 weeks.

MLIP +/+ and -/- male and female mice groups (n = 3/4) were concurrently fed chow and HFD food over a span of 31 weeks. The HFD contained 60% fat by calorie intake. Males: T- test results gave a p<0.05 for +/+ HFD versus chow diet. Male MLIP-/- mice under HFD gained weight with the same rate as the +/+ HFD mice during the first 4 weeks of diet. After 4 weeks, the -/- mice became “resistant” to HFD as compared with the +/+ HFD mice. (Figure 9) Females: In contrast to males, the female MLIP -/- mice were sensitive to HFD like the +/+ mice. (Figure 10)

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Figure 10. A graph of female MLIP +/+ and -/- mice body mass (g) undergoing a chow and HFD for 31 weeks.

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Figure 11. A graph of male and female MLIP +/+ and -/- mice blood glucose levels (mmol/L) before and after 30 weeks of chow and HFD

Blood glucose of male and female mice was taken before the HFD and after 30 weeks (Figure 11).

Generation of MLIP knockout mice

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Figure 1. A. Targeting vector leading, after Cre-recombinase mediated recombination, to the removal of Mlip promoter and exon 1. Arrows indicate the primers used for mice genotyping. B. Mlip genotyping by PCR of Wild-type (+/+), heterozygous (+/-) and homozygous (-/-) MLIP null mice. C. Western blot confirming that MLIP protein is reduced by 50% in MLIP +/- hearts and absent in -/- hearts, without major changes in Lamin A/C and Lamin B1 expression.

Our results showed that the deletion of MLIP leads to a decrease in glucose and fatty acid uptake in the mouse heart at basal conditions. After insulin injection, however, null mice exhibited insulin hypersensitivity. Glut4 was expressed at a higher level in null mice, making the decreased glucose uptake result puzzling. When subjected to a HFD, male null mice showed resistance to weight gain, unlike the females which gained weight similarly to the wild type female mice. We are doing an ongoing HFD study with a larger sample size than shown here. In parallel, the response of null mice to a high glucose diet will be investigated. Further experiments are ongoing to investigate in detail the metabolic consequences of the deletion of MLIP and elucidate their mechanisms. More generally, our results suggest that disturbances of MLIP:lamin interactions could account for part of the phenotype of A-type lamin associated lipodystrophy.


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