Trans-trigeminal transport of masseter-derived neprilysin to hippocampus.

OBJECTIVE: To show the possible occurrence of exosomal transport of neprilysin from masseter muscle to hippocampus via trigeminal nerve in the living mouse. DESIGN: Mouse C2C12 myotube-derived exosomes were labeled with near-infrared (NIR) dye and injected into the masseter muscle to track their fluorescence from masseter muscle to hippocampus via trigeminal nerve. A plasmid vector encoding green fluorescent protein (GFP)-tagged neprilysin (GFP-neprilysin) was transfected into masseter muscle of C57BL/6 J mice. Expression of mRNA and encoded protein of the transgene was identified in masseter muscle, trigeminal nerve and hippocampus by RT-PCR and Western blot, respectively. RESULTS: Peak of exosomal NIR in masseter muscle at time 0 rapidly reduced at 3 h and 6 h along with the subsequent increases in trigeminal nerve and hippocampus. Expression of GFP-neprilysin mRNA was detected in masseter muscle, but not trigeminal nerve and hippocampus. On the other hand, the corresponding protein of GFP-neprilysin was identified in the three tissues on day 3 after transfection into masseter muscle as a single band on Western blots with anti-GFP and anti-neprilysin antibodies. CONCLUSION: The appearance of GFP-neprilysin protein in trigeminal nerve and hippocampus without a corresponding mRNA expression indicated the protein's origin from the masseter muscle. Concomitant migration of NIR-exosomes from masseter muscle to hippocampus via trigeminal nerve suggested the possible occurrence of exosomal transport of neprilysin.

Genetic dissection reveals diabetes loci proximal to the gimap5 lymphopenia gene.

Congenic DRF.(f/f) rats are protected from type 1 diabetes (T1D) by 34 Mb of F344 DNA introgressed proximal to the gimap5 lymphopenia gene. To dissect the genetic factor(s) that confer protection from T1D in the DRF.(f/f) rat line, DRF.(f/f) rats were crossed to inbred BBDR or DR.(lyp/lyp) rats to generate congenic sublines that were genotyped and monitored for T1D, and positional candidate genes were sequenced. All (100%) DR.(lyp/lyp) rats developed T1D by 83 days of age. Reduction of the DRF.(f/f) F344 DNA fragment by 26 Mb (42.52-68.51 Mb) retained complete T1D protection. Further dissection revealed that a 2 Mb interval of F344 DNA (67.41-70.17 Mb) (region 1) resulted in 47% protection and significantly delayed onset (P < 0.001 compared with DR.(lyp/lyp)). Retaining <1 Mb of F344 DNA at the distal end (76.49-76.83 Mb) (region 2) resulted in 28% protection and also delayed onset (P < 0.001 compared with DR.(lyp/lyp)). Comparative analysis of diabetes frequency in the DRF.(f/f) congenic sublines further refined the RNO4 region 1 interval to approximately 670 kb and region 2 to the 340 kb proximal to gimap5. All congenic DRF.(f/f) sublines were prone to low-grade pancreatic mononuclear cell infiltration around ducts and vessels, but <20% of islets in nondiabetic rats showed islet infiltration. Coding sequence analysis revealed TCR Vbeta 8E, 12, and 13 as candidate genes in region 1 and znf467 and atp6v0e2 as candidate genes in region 2. Our results show that spontaneous T1D is controlled by at least two genetic loci 7 Mb apart on rat chromosome 4.

Increased lipid oxidation heralds diabetes onset in DR.lyp/lyp rats.

AIM: The BB rat model of type 1 diabetes exhibits altered body weight gain and body temperature regulation prior to hyperglycemia onset, implying the existence of as yet unidentified biomarkers of autoimmune processes that destroy pancreatic beta cells. To investigate this hypothesis, we compared the metabolic profile of diabetes-resistant DR.lyp/+ rats and their diabetes-prone, congenic DR.lyp/lyp littermates in the days leading up to diabetes onset. METHODS: Except for the Gimap5 mutation on chromosome 4, congenic DR.lyp/lyp rats are genetically identical to DR.lyp/+ littermates. They invariably develop hyperglycemia at 46-81 days of age, whereas DR.lyp/+ rats do not develop diabetes. In addition to daily food intake and body weight, indirect calorimetry was performed continuously on male DR.lyp/lyp and DR.lyp/+ rats (n=6/group) for 6-18 days to measure locomotor activity, VO (2), VCO (2) and RQ. RESULTS: DR.lyp/lyp rats exhibited a progressive decrease of RQ compared to DR.lyp/+ rats 0.005+/-0.001 units/day (p<0.005). Limiting the analysis to the six days prior to diabetes onset revealed a larger decrease of 0.007+/-0.002 units/day (p<0.001) in DR.lyp/lyp animals, whereas RQ of the DR.lyp/+ rats remained unchanged. This metabolic change occurred prior to hyperglycemia onset and was not associated with changes of any other parameter. CONCLUSIONS: Diabetes onset in DR.lyp/lyp rats is heralded by a progressive shift towards lipid oxidation relative to carbohydrate metabolism.