Maravilhas

2. Expression of myo-inositol cotransporters in
nervous tissues during the development of
experimental diabetes
Advisors: Dr. Luiz Roberto G. Britto (USP)
Dr. Nilberto Robson Falcão do Nascimento (UECE)
Vanessa Ximenes Farias
DOUTORADO EM FISIOLOGIA – UFRJ/USP/UFMG - UECE
- MODALIDADE TURMA FORA DE SEDE -

4. Myo-inositol – Background
 Inositols: Are a group of non-toxic small polar molecules, whose
empirical formula is C6H12O6, which have a wide range of biological
functions (Michell, 2008)
(Crouze & Soulage, 2013)

5.  Essential nutrient for cell growth in culture; (Eagle et al., 1957)
 Organic osmolyte; (Handler & Kwon, 1996)
 It is a precursor of phosphoinositolglycan and phosphoinositides , signal
transduction molecules, which are potential mediators of some of the
actions of insulin.
(Larner et al., 1988)
Myo-inositol – Background

6. Myo-inositol - Maintenance of their
intracellular concentrations
(Croze & Soulage, 2013)

7. Na+ / Myo-inositol Transporter (SMIT-1)
 Originally cloned from MDCK cells; (Kwon et al., 1992)
 Molecular weight ~ 79,5 KDa;
 pH-dependent uptake;
 2 Na+ / myo-inositol; (Hager et al., 1995)
 Expression and activity regulated by several factors, osmotic stress
being the most well documented.

8.  Widely distributed in the CNS :
cortex
hippocampus,
cerebellum
diencephalon,
pineal
choroid plexus. (Isaacks et al. 1997; Lubrich et al. 2000)
 Expressed in peripheral nerves and kidney, especially in the renal
medulla.
(Chau et al., 2005; Lahjouji et al., 2007)
Na+ / Myo-inositol Transporter (SMIT-1)

9. Na+ / Myo-inositol Transporter (SMIT-1)

10.  43% similar to SMIT-1;
 Molecular weight ~ 74KDa;
 Expressed :
brain,
skeletal muscle,
intestine,
liver;
kidney, especially in the renal cortex.
(Aouameur et al., 2007; Lahjouji et al., 2007;Lin et al.,
2009)
Na+ / Myo-inositol Transporter (SMIT-2)

11.  Symporter hydrogen / myo-inositol;
 Molecular weight :75 - 90 kDa, (Uldry et al. 2001)
 Activated by pH decrease;
 Expressed:
Brain
(Uldry et al., 2001; Di Daniel et al.,
2009)
Hippocampus
Cerebellum
Cortex
Hypothalamus
Brainstem
H+ / Myo-inositol Transporter
(HMIT)

12. Myo-inositol and Diabetic Neuropathy
Diabetic neuropathy:
Metabolic;
Neurotrophic;
Vascular
defects.
Demyelination,
Loss of axons
↓Conduction
velocity

13. ↓ myo-inositol content in nerves → 4 weeks after
induction of diabetes.
(Coppey et al., 2001) (Stevens et al., 2000)

14. •Myoinositol and some of its derivatives have
been used both in the clinical and experimental
set as a therapy to prevent diabetes
complications, such as diabetic neuropathy.
(Farias et al., 2011)
Nerve
Conduction
Velocity

15. AIMS
To investigate the expression of myo-inositol co-
transporters in central and peripheral nervous system
structures and their relative importance in the development of
diabetic neuropathy.

16. Materials and Methods
• Experimental groups
1. Euglycemic rats
2. 4 weeks diabetic rats;
3. 8 weeks diabetic rats;
4. 12 weeks diabetic rats;
 Diabetes induction:
Streptozotocin
60 mg/Kg, or vehicle, i.p.
Blood glucose >
200mg/dl
Diabetics

17. Materials and Methods
Euglycemic rats
or
Diabetic rats
4, 8 or 12 weeks after
diabetes induction
Sciatic nerve;
Dorsal root ganglia
Cerebellum
Hipocampus
Brain
Striatum
sacrifice
“Western Blotting” Myo-inositol cotransporters protein
expression pattern
RT-PCR
Myoinositol cotransporters
gene expression pattern

18. RESULTS

19. Groups n
Glycemia on the day of
diagnosis (mg/dl)
Glycemia on the day
of sacrifice (mg/dl)
Euglycemics 6 100 ± 3,7 99,5 ± 3,6
Diabetic – 4 weeks 6 335,3 ± 29,6 414,3 ± 15,2*
Diabetic – 8 weeks 6 341,2 ± 31,8 514,5 ± 21,2**
Diabetic – 12 weeks 6 373,5 ± 31,7 548,5 ± 28,9**
Glucose level
* p<0,05 e ** p< 0,01 vs Glycemia on the day of diagnosis

20. Screening of tissues expressing the
Myo-inositol cotransporters:
MM NEG HIPO CEREB DRG NE SCIA LIV INT KID MUSC
Products of PCR subjected to eletroforesis in agarose gel.

21. Relative gene expression of myo-inositol
cotransporters in tissues of the PNS
SCIATIC NERVE
* p <0.05 vs. euglycemic group (0)

22. Relative gene expression of myo-inositol
cotransporters in tissues of the PNS
DORSAL ROOT GANGLIA
* p <0.05, ** p<0,01 vs. euglycemic group (0)

23. BRAIN
Relative gene expression of myo-inositol
cotransporters in tissues of the CNS
* p <0.05 vs. euglycemic group (0)

24. HIPPOCAMPUS
Relative gene expression of myo-inositol
cotransporters in tissues of the CNS
* p <0.05, ** p<0,01 vs. euglycemic group (0)

25. STRIATUM
Relative gene expression of myo-inositol
cotransporters in tissues of the CNS
* p <0.05 vs. euglycemic group (0)

26. CEREBELLUM
Relative gene expression of myo-inositol
cotransporters in tissues of the CNS
* p <0.05 vs. euglycemic group (0)

27. SMIT-1 SMIT-2 HMIT
Weeks after
diabetes induction
Weeks after
diabetes induction
Weeks after
diabetes induction
TISSUES 4w 8w 12w 4w 8w 12w 4w 8w 12w
Sciatic nerve - - - x x x ↑ - -
DRG ↓ - - - - ↑ - ↑ ↑
Striatum - ↓ - - - - - ↓ -
Brain ↓ - - - - - ↓ ↓ -
Cerebellum ↓ ↓ - - - - - ↓ -
Hippocampus ↓ ↓ - ↓ - ↑ ↓ ↓ ↓
Relative gene expression of myo-inositol
cotransporters in tissues of the CNS and PNS

28. Myo-inositol cotransporters protein expression in
sciatic nerve
SMIT- 1
* p <0.05, ** p<0,01 vs. euglycemic group (0)

29. Myo-inositol cotransporters protein expression in
sciatic nerve
* p <0.05, ** p<0,01 vs. euglycemic group (0)
HMIT

30.  In conclusion, we observed that occurs downregulation of SMIT-1,
SMIT-2 and HMIT in most tissues of the CNS with the time course
of Diabetes Mellitus. In SNP, we observed a decrease in gene
expression of SMIT-1, increased expression of HMIT in sciatic nerve
and DRG, and increased expression of SMIT-2 in the DRG. Protein
expression of SMIT-1 and HMIT gradually decreases throughout the
course of diabetes.

31.  Myo-inositol content quantifying
 Immunohistochemistry → Myo-inositol cotransporters distribution and
expression pattern
 Immunoprecipitation assays→ Level of glycosilation and phosphorilation
of myo-inositol cotransporters
 Compound action potential – sciatic nerve → funcional parameters

32. Thank you !!!