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In addition 201 the intrinsic and extrinsic apoptotic pathways, apoptosis due to prolonged endoplasmic reticulum ER stress 1213 has been reported in various diseases, including Alzheimer and Parkinson diseases dde Evidence from the Akita 1516 and NOD. Subsequently, the ceramides trigger mitochondrial apoptotic processes and cell death ensues. The sources for the material used were as follows: The Akita and wild type WT cells were generated as follows.
The medium was exchanged every 2 days, and the cells were split, as required, once a week. The cells were harvested at various times 2—16 h and prepared for various analyses described below. Incidence of apoptosis was assessed under a fluorescence microscope Nikon Eclipse TE using a fluorescein isothiocyanate filter.
DAPI staining was used to determine the total number of cells in a field. A minimum of three fields per slide was used to calculate the percentage of apoptotic cells. Fluorescence in cells was monitored at an excitation wavelength of nm. Although these analyses yield quantitative results, they were hampered by the tendency of these cells to clump together, causing the fluorescence peaks to be fe.
Cells were plated in a 6-well plate with the leei and cultured up to semiconfluence. After 20 min, the coverslips were rinsed eli PBS, mounted on slides, and the cells were immediately examined using a confocal laser-scanning microscope Zeiss with a nm argon laser and a nm diode laser.
The targeted factors and the primary antibody concentrations were as follows: The secondary antibody concentration was 1: Immunoreactive bands were visualized by ECL. Cytosol fraction was prepared from Akita and WT cells and harvested, and protein concentration was determined using Coomassie reagent. Sphingomyelins are formed by reaction of a ceramide with CDP-choline, and similar to GPC lipids, they contain a phosphocholine as the polar headgroup.
The prominent ions in the total ion current spectrum are those of the even mass PC molecular species, and these mask the odd mass sphingomyelin signals Lipid extracts were prepared as above in the presence of a Sphingomyelins content in the samples was determined based on standard curves generated using commercially available brain and egg sphingomyelins with a known percentage of each fatty acid constituent and The plasmid developed by Kim and Spiegelman 49 expresses truncated ADD1 with a tyrosine to alanine mutation at amino acid The mixture was incubated for 20 min at room temperature and used to transfect WT and Akita cells cultured in a 6-well plate.
The mixture was then incubated at room temperature for 10—15 min to allow formation of transfection complexes. The complexes were then gently added to the cells.
The cells were harvested at 8 h for various experimental protocols, each done 3—5 times.
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CNSMase message. Dmitochondrial membrane potential. The abdomen was isolated, and pancreata were isolated as described The vial was then vigorously shaken for 90 s, followed by washing three times of the pancreas with Krebs-Ringer buffer containing 1 m m CaCl 2 50 kei.
The cells were washed further with incomplete RPMI 75 ml. Typical islet yields ranged from 25 to 50 from the Akita mice and from WT mice. The islets were used to prepare total RNA for message and for immunostaining analyses, as described below.
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The mixture was spun down quickly to settle the islets at one surface of the agarose, which was then allowed to solidify. 01185 sections were incubated overnight with primary antibodies 1: The ed value for each analysis is indicted in the figure legends. As part of the unfolded protein response, various ER factors are induced to alleviate ER stress.
One such marker of unfolded protein response activation is pPERK As shown in Fig. Basal expression of ER stress factors and apoptotic factors. Tubulin was used as a loading control. These analyses were done three separate times. We reported that ER stress-induced ceramide generation via NSMase-catalyzed sphingomyelin hydrolysis was a critical contributor to INS-1 cell apoptosis 41 If this pathway is activated by ER stress, this should be reflected by reduced abundances of sphingomyelin molecular species in the Akita cells.
The spectra in Fig. Re spectra were acquired by monitoring constant neutral loss of 59, as described 52df WT Fig. As reflected by the decreases in the intensity of ions representing them, the relative abundances of the sphingomyelin 1015 species are decreased in the Akita cells in comparison with WT cells.
Normalization of individual sphingomyelin molecular species to lipid phosphorus revealed the These findings indicate that hydrolysis of sphingomyelins is ramped up in the Akita cells and suggest the possibility of consequential triggering of mitochondrial abnormalities.
Ctotal sphingomyelin pool. This reagent concentrates in the mitochondria of healthy cells, but the mitochondria of cells undergoing apoptosis become compromised and accumulate less of the reagent, and this is reflected by a decrease in the fluorescence signal and the appearance of a second peak that is left of the original. The spectra leo in Fig. This resulted in broader peaks in the spectra and an unexpectedly higher M1 value, even in the WT cells.
We therefore established a second protocol in which mitochondria-associated DiOC 6 3 staining green was monitored in live cells by confocal microscopy. As seen in Fig.
Further, Hoechst staining blue reveals that the nuclei in Akita cells are irregular in shape and size, as compared with the nuclei 101855 WT cells, suggesting that the cell death process is under way in the Akita cells. Aflow cytometry. These analyses were done four separate times. BDiOC 6 3 staining. Cells were loaded with the blue nuclear Hoechst; left panels and green mitochondrial DiOC 6 3 DIOC ; middle panels stain and examined 101885 confocal microscopy.
The merged images are shown in the right panels. In view of the above findings, we 20001 examined if the Akita cells are more susceptible to apoptosis. Exposure to thapsigargin induced activation of caspase-3 in both wild type and Akita with peak activation achieved earlier in the Akita cells than in the wild type cells.
This is reflected by a greater -fold increase, relative to basal, in Akita cell apoptosis at 8 h WT, 1. Aapoptosis quantitation. Bcaspase-3 activation. Cell lysates were prepared and processed for full-length and activated caspase-3 Casp-3 and aCasp-3respectively immunoblotting analyses. Cactivated caspase-3 densitometry.
Because induction of ER stress led to mitochondrial abnormalities in the INS-1 cells, we examined whether a similar affect was evident in the Akita cells. Compared with basal conditions Fig. These outcomes were accelerated in the Akita cells and ve that activation of the mitochondrial apoptotic pathway contributes to ER stress-induced apoptosis of these cells also. Also, as shown in Fig. Cytosol was prepared 1018 control or thapsigargin-treated xe.
The increase in NSMase message occurred earlier and reached a higher -fold increase in the Akita cells, relative to WT cells. These analyses were done 3—5 separate times.
Also, as seen in Fig. It has been reported that although the DN form itself is unable to activate transcription, it interferes with the processing of endogenous SREBP-1 and its binding to the SRE element The cells were harvested under basal conditions or following an 8-h exposure to thapsigargin. As illustrated in Fig. The mice were sacrificed within a week of weaning 4—5 weeks of age.
Islets were isolated from WT and Akita mice at 4 weeks of age. The insets in B are magnifications of islet 201 nuclei.
The exposure times for each were the same in WT and AK sections. It is therefore important to determine the underlying mechanisms that contribute to this process. The ceramides, generated from NSMase-catalyzed hydrolysis of sphingomyelins, promote mitochondrial abnormalities and amplify the apoptosis outcome.
We therefore chose to address this issue in the Akita mouse model. The greater propensity of the Akita cells to develop ER stress under basal conditions is also revealed by the nearly 2-fold higher basal splicing of XBP1 mRNA in the Akita cells, relative to isogenic WT counterparts Following its release into the cytosol, the proapoptotic cytochrome c forms the apoptosome complex with apoptosis protease-activating factor-1 to induce caspases and subsequent apoptosis To further establish this possibility, the effects of accelerating ER stress in the Akita cells were assessed.
However, these outcomes were accelerated in the Akita cells. Thapsigargin also induced NSMase in both WT and Akita cells, and this occurred earlier and the -fold increases in message were greater in the Akita cells. In fact, the higher basal apoptosis was also significantly decreased in these cells. The transcription factor ATF6 plays a key role in the unfolded protein response, and ER stress leads to its cleavage by site 1 and site 2 proteases that are also activated by ER stress These proteases process SREBPs 65 into active mature forms that enter the nucleus and transactivate target genes This coincided with higher pAkt levels, which is consistent with previous reports that phosphorylation of Akt stimulates the synthesis and nuclear localization of mSREBP-1 55— These mice develop mild hyperglycemia rapidly after birth, although they continue to live for over 8 months.
These mice contain a spontaneous dominant mutation in the insulin 2 gene on a C57BL6 mouse background that results in the replacement of cysteine with tyrosine at position 96, causing disruption of a disulfide bridge required for proper insulin folding. This causes proinsulin to accumulate in the ER and leads to the initiation of the unfolded protein response.