TITLE: Can light be used to treat obesity and diabetes?
AUTHORS: Rédoane Daoudi

[DESCRIPTION]


This document is highly theoretical lab method paper. It may be used to perform
thermogenesis from several cells in a living organism instead of only brown
adipocytes. We perform thermogenesis from white adipocytes. The purpose is to
uptake the blood glucose and lipids to produce heat and subsequent weight loss
and to diminish blood glucose levels for people who suffering from diabetes and
obesity. In fact thermogenesis consumes energy (i.e blood glucose and blood
lipids). So we now describe a novel and original lab method based on
thermogenin (called thermogenin-like system, UCP1-like system or TLS) that is
involved in heat production and glucose/lipids capture by white adipocytes.




[STEPS]
SECTION: In vitro assays
1.  Use a cell line: white adipose cells in DMEM medium.



SECTION: In vitro assays
2. 
Transfect cells by electroporation and with the following DNA constructs:


1) the light-driven
inward H+ pump PoXeR targeted to the inner mitochondrial membrane: 


2) firefly luciferase 


3) luciferin-regenerating
enzyme (LRE) 


4) cysteine racemase 


5) thioesterase



SECTION: In vitro assays
3. 

Measure oxygen consumption, ATP
levels and glucose uptake to estimate the thermogenic capacity of white adipose
cells and subsequent the glucose and lipids uptake.



SECTION: In vivo transfections
4. 
The first step of this model is the in vivo transduction of adipose tissue with
adeno-associated viral (AAV) vectors. The following transgenic DNA constructs
are used in vivo: 


1) the light-driven inward H+ pump PoXeR targeted to the inner
mitochondrial membrane: 


2) firefly luciferase 


3) luciferin-regenerating enzyme (LRE) 


4) cysteine racemase 


5) thioesterase



SECTION: In vivo transfections
5. 

PoXeR is a natural light-driven inward proton pump found in Parvularcula oceani, a
deep-ocean marine bacterium. This pump controls the unusual directionality
opposite to normal proton pumps and may lower the proton motive force when
expressed in the inner mitochondrial membrane, producing heat similarly to
thermogenin. A study shows that PoXeR can be expressed in mouse neural cells
and it is functional in these cells. Another study shows that it is possible to
construct photoenergetic mitochondria in cultured mammalian cells expressing
another light-driven proton pump derived from Haloterrigena turkmenica. Taken
together, these results suggest that PoXeR may be a good candidate for the TLS
in vivo in mammals.



SECTION: In vivo transfections
6. 

The TLS may be used in normal cells to promote uncoupling-induced weight loss without significant
adverse effects. It may be possible to express PoXeR (and other transgenes of
the TLS) under the control of a weak promoter to reduce the functionality of
the system and subsequent weight loss. It may be also possible to express PoXeR
(and other transgenes) under the control of a strong promoter to enhance weight
loss without significant side effects. In addition, it is interesting to obtain
a spatial control of the system in order to restrict transgenes expression to
the adipose tissue. For this reason, we propose to use the following transgenic
DNA constructs in vivo: 


[Adiponectin promoter]-PoXeR targeted to the inner mitochondrial membrane 

[Adiponectin promoter]-firefly luciferase 


[Adiponectin promoter]-LRE 


[Adiponectin promoter]-cysteine racemase 


[Adiponectin promoter]-thioesterase



SECTION: Injections
7. 

Because the bioluminescence of firefly luciferase decreases over time, it is necessary either to continually bring the substrate (i.e firefly luciferin) by continuous infusion and/or to
use a system that regenerates the firefly luciferin from oxyluciferin, the
product of the reaction. A study shows that oxyluciferin is enzymatically
regenerated into firefly luciferin by LRE in vitro. 

This system can be improvable by using cysteine racemase and thioesterase. Finally, it is possible to
concentrate the substrate firefly luciferin in adipose tissue in vivo by using
a suitable linker (firefly luciferin prodrug). Then, mice are injected with a
firefly luciferin or a firefly luciferin prodrug so that the active firefly
luciferin is mainly released in adipose tissue.



SECTION: Injections
8.  Now, the TLS needs to be sensitized
to the light. In fact, all trans retinal is required for light-induced PoXeR
activation in vivo. 

All trans retinal may be not naturally present in adipose tissue, thus it is necessary to administer it
by continuous infusion, it is not enzymatically regenerated in contrast to
firefly luciferin. Because all trans retinal is hydrophobic it can be
encapsulated in liposomes to reach the adipose tissue in vivo. Hydrophobic
compounds have affinity to the phospholipid bilayer of liposomes.

Finally, few hours after all trans retinal injection, mice are injected with a firefly luciferin or a firefly
luciferin prodrug.  



SECTION: Summary
9. 

Firstly perform in vitroassays on white adipocytes cells by transfecting them with the previous
enzymes. Evaluate oxygen consumption, ATP levels and glucose uptake with
specific kits. If the in vitro assays are conclusive, continue with
transduction of adipose tissue in vivo in mice.



SECTION: Summary
10. 

Then, use the previous transgenic DNA
constructs for the in vivo transduction of adipose tissue with AAV
vectors. In fact, a study indicates that AAV-mediated genetic engineering of
white and brown adipose tissue is possible in adult mice.



SECTION: Summary
11. 

Then, use a continuous infusion of all trans retinal in mice. Few hours after all trans retinal injection, use a continuous infusion of firefly luciferin or firefly luciferin prodrug.



SECTION: Summary
12. 

Finally, estimate the weight loss, blood glucose levels or measure body temperature of mice using infrared imaging. Indeed, a study shows that it is possible to measure brown adipose
tissue thermogenesis using infrared imaging in mice.