Figure 1-Total RNA isolated from mouse heart (Lane 2), liver (Lane 3), spleen (Lane 4) and hind limb (Lane 5). Lane 1 is the 100 bp ladder.
Figure 2-Expression Analysis of GFAT1, GFAT1Alt and Actin in mouse tissues by RT-PCR. A. Lane 1. 100 bp ladder. Lane 2, 3 and 4. GFAT1 in heart, liver and spleen respectively. B. Lane 1. 100 bp ladder. Lane 2 and 5. GFAT1Alt in heart and hind limb respectively. Lane 3 and 4. Non-expression of GFAT1Alt in liver and spleen respectively. Lane 6, 7, 8 and 9. Actin in heart, liver, spleen and hind limb respectively. Lane 10. A doublet from hind limb, representing the variant, GFAT1Alt (top) and GFAT1 (bottom), obtained using primers mGFAT1-5' and mGFAT1-3'.
Figure 3-Expression of the doublet in hind limb. A doublet representing the variant GFAT1Alt (top) and GFAT1 (bottom), was obtained using the primers mGFAT1-5' AND altGFAT1-3'.
Figure 4-Sequence Alignment of the derived GFAT1 and GFAT1Alt sequences with the sequences from GenBank. The 48 bp insert is between 246th-295th nucleotides. The sequence between these nucleotides in GFAT1, which does not have the insert, is shown with Ns (GFAT1, GFAT1 doublet hind limb reverse and GFAT1 liver forward and reverse). Note that for GFAT1 from liver, both sequences obtained with the forward (between 93rd-181st nucleotides) and reverse primers (between 242nd-396th nucleotides) are shown in the same sequences.
Materials and Methods
2 sets of primers, mGFAT1 forward (5’-TGAAAACAGACACAGAAACCATTGCC-3’) and mGFAT1 reverse (5’-CTGCTGCAACATCATCATCTTCCA -3’); mGFAT1 forward and altGFAT1 reverse (5’-TCTTTGCCTCGTTCTGCCTGTGAT-3’) were used to amplify of 416 bp fragments of GFAT1 gene and of 306 bp fragments of GFAT1Alt gene respectively. A doublet of 464 bp, with 48 bp insert (Fig 3. top, GFAT1Alt) and of 416 bp (Fig 3. bottom, GFAT1), was observed only in hind limb using the first set of primers (mGFAT1 forward and mGFAT1 reverse) in RT-PCR. 50 ul of each primer was dried for 1 hour, diluted with 50 ul of distilled water. The volume of distilled water to add was calculated in order to get a 10 pmol of final concentration for each primer.
Total RNA Extraction
Total RNA was extracted from four tissues using RNAqueous (Ambion, Austin, TX) according to the manufacturer’s specifications. Briefly, the liver, heart, spleen and hind limb were removed from a mouse (strain BALB/C) anesthetized by carbon dioxide asphyxiation. The organs were homogenized in 12 mL of cold lysis/binding buffer. The debris from each homogenized tissue was removed by centrifugation for 2-3 minutes, at 4000 rpm. To 1mL of homogenized tissue in lysis buffer, 1 mL 64% ethanol was added. (For hind limb, 2 sets of sample were prepared: sample 1 was prepared by adding 900 ul 64% ethanol to 900 ul of the homogenized tissue; sample 2 was prepared by adding 400 ul lysis buffer, 900 ul ethanol to 500 ul of the homogenized tissue). 600 ul was applied to a spin column supplied with the kit, and centrifuged for 2 minutes at 12,000X g or until all of the homogenate passed through the column. This step was repeated 2 additional times after discarding the flow through between spins. Subsequently, the column was washed once with 700 ul solution 1, centrifuged for 2 minute at 12,000X g and the flow through removed. The column was then washed twice with 500 ul solution 2/3 and centrifuged for 2 minutes at 12,000X g after removing the flow through from the previous wash. Finally, the column was centrifuged at 12,000X g for 2 minutes to remove any residual buffer. The RNA was eluted by adding 60 ul of preheated (70°C) distilled water to the column, incubated at 70°C for 10 minutes, then centrifuged at 12,000X g for 1 minute. This elution step was then repeated in order to increase the RNA yield. (For hind limb, 40 ul of water was used; in order to obtain a more concentrated RNA).
Total RNA was Dnase treated using DNA-free (Ambion, Austin, TX) according to the manufacturer’s specifications. Briefly, a common stock of 188 ng/ul was prepared for each tissue. 10.6 ul of RNA was used for each tissue, to which were added 1.06 ul 10 X Dnase 1 buffer, and 1 ul of Dnase I. These samples were incubated at 37°C for 30 minutes. To inactivate the Dnase I, 1.26 ul of resuspended inactivating solution were added to the samples and incubated at room temperature for 2 minutes. The Dnase I and inactivating solution were then pelleted by centrifuging at 12,000X g for 1 minute. (For hind limb, 12.5 ul of RNA was taken directly, without diluting with water, to which 1.25 ul 10 X Dnase 1 buffer and 1 ul Dnase 1 were added. After the incubation 1.47 ul of resupended Dnase inactivation agent was added).
A two-step RT-PCR was used. First strand synthesis was completed by adding RNase free water to 1 micro gram of RNA to a total volume of 9.4 ul. The mixtures were incubated at 70°C for 10 minutes, cooled on ice and centrifuged for 1 minute at 12,000X g. Next, a master mix of 5 X was prepared, by adding the following solutions: 4 ul 5x RT first strand buffer (20 ul), 4 ul 2.5 mM dNTP’s (20 ul), 2 ul 0.1M DTT (10 ul), and 0.6 ul Qt primer (3 ul). 10.6 ul was taken for each tube and added to Dnased RNA. The tubes were mixed well, then 1 ul superscript II RT was added. The mixtures were incubated at room temperature for 5 minutes, followed by a three step cycling process in a Perkin Elmer 2400 thermal cycler, 42°C for 1 hour, 50°C for 10 minutes, and finally 70°C for 15 minutes. After the cycling was complete 1.5 units of Rnase H was added to the products of the first strand synthesis and incubated at 37°C for 20 minutes. These first strand synthesis products were then used as templates for the subsequent PCR amplification.
1 micro liter of the each first strand synthesis product was used for the PCR reaction. 5 ul of 10X PCR buffer, 4 ul of 2.5mM dNTP’s, 1.5 ul magnesium chloride (50mM), 0.25 ul of Taq polymerase and 36.25 ul of distilled water were added to this template (heart, liver, spleen, hind limb cDNA respectively). Next, the GFAT1 specific primers (mGFAT1 forward and mGFAT1 reverse), the GFAT1Alt specific primers (mGFAT1 forward and ALTGF reverse) and finally, actin specific primers (Actin 1 and Actin 2) were used to add to the reaction mixtures (one set for each reaction). There were 12 PCR reactions in total: 4 reactions for the expression of GFAT1 in each 4 tissues, 4 reactions for the expression of GFAT1Alt in each 4 tissues and finally 4 reactions for the expression of actin which was used as a control. Again, a Perkin Elmer 2400 thermal cycler was used for the PCR amplification. The mixtures were first heated to 94°C for 5 minutes, then cycled 50 times at 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute. A final extension was performed at 72 °C for 7 minutes.
The PCR products from heart, liver and spleen were purified by QIAquick Purification Kit following the protocol provided by the kit. Additionally, the “Rapid Gel Extraction Protocol” was used after running the doublet (obtained by using the first primer set) from hind limb on the gel for 1 hour and cutting separately the 2 different bands under UV light. (The top one representing GFAT1Alt and the bottom one representing GFAT1).
DNA sequencing was performed using a modified version of the Sanger Sequencing method. A master mix of 17 X was prepared, because there were 16 reactions. 50 fmoles of purified PCR products were mixed with 4 ul of 10X cycling buffer, 0.2 ul of 33PdATP and distilled water to a total volume of 30 ul. 6 ul of this master mix was then added to four tubes, each containing 2 ul of ddGTP, ddATP, ddTTP, or ddCTP respectively. Next, a drop of mineral oil was added to each tube in order to prevent evaporation during the sequencing reaction in the PCR machine. The sequencing reactions were conducted for 35 cycles by denaturing at 94°C for 30 seconds, annealing at 58°C for 30 seconds, and elongating at 72°C for 1 minute. Following the sequencing reactions, 4 ul of stop solution was added to each tube and they were then heated to 94°C for 3 minutes to denature the products before running them on a sequencing gel.
RNA extraction from heart, liver, spleen and hind limb yielded both 28S and 18S ribosomal subunits. There was some DNA contamination, as evidence by the presence of a bright band at the top of each sample loaded on the gel (Fig1 ).
The primer pair mGFAT1-5’/mGFAT1-3’ was designed for use in RT-PCR. Using this primer pair and the other tissues; heart, liver, spleen and hind limb respectively, the gel electrophoresis revealed that GFAT1 appears as a single band in heart, liver and spleen (Fig 2A Lane 2, 3 and 4 respectively) but it migrated as a doublet in hind limb muscle (Fig 2B Lane 10). The GFAT1Alt mRNA in mouse is expressed both in the heart and in hind limb, using the primer pair mGFAT1-5’/ALTGF-3’ (Fig 2B Lane 2 and 5) and is undetectable in liver and spleen (Fig 2B Lane 3 and 4). The PCR product from hind limb, which yielded a doublet, could be clearly seen after running the sample on the 0.8% agarose gel for 1 hour (Fig 3).
Sequencing by the specific primers confirms that the bands amplified correspond to GFAT1 and GFAT1Alt genes (Figure 4). The change in the sequence, for GFAT1Alt gene, can be clearly seen starting from the 247th nucleotide. Each sequence was compared with the previously published corresponding cDNA in GenBank and the purified PCR products used as templates for sequencing, showed high homology with the previously published sequence of the genes. As the reverse primers were used both for GFAT1 and GFAT1Alt, on sequencing, all sequencing results were complimented and reversed on Mac Vector, before aligning with the corresponding published sequence.
The muscle-specific variant of GFAT1 mRNA differs from the GFAT1 mRNA only by the presence of a 48 bp insert. Thus, the variant is most likely a result of alternative splicing of GFAT1 in muscle. Kinetic studies of GFAT activity from partially purified tissue extracts showed that GFAT from mouse and rat skeletal muscle differed from liver, fibroblasts and many other cell lines (14) in that the apparent Km of the enzyme for F-6-P was higher in skeletal muscle than in other tissues. The approximate twofold increase in the apparent Km for F-6-P observed with GFAT1Alt versus GFAT1 suggests that the selective expression of the former in muscle may contribute to these findings. Glucose is absorbed into muscle and fat cells through glucose transporters at the cell surface. The number of transporters at the cell surface is controlled by insulin. In type II diabetes, insulin resistance is a central problem. The number of glucose transporters recruited to the cell surface in response to insulin is deficient in these people. This could result from reduced movement of glucose transporters. On the other hand, there is increasing evidence that accumulation of products of the HSP may contribute to insulin resistance (2,3,7,8,11,12). This can be shown in subjects who are genetically
predisposed to type II diabetes. Increased glucose flux via GFAT increases the intracellular concentrations of UDP-GlcNAc (7,8) which in turn modulates the activity of of UDP-GlcNAc:polypeptide beta-N-acetylglucosaminyl transferase (15). The modification of serine and threonine residues of susceptible proteins by a single O-linked GlcNAc had been demonstrated. Numerous cytosolic and nuclear proteins can be modified in this manner, including numerous transcriptional regulators such as, Sp1 (16), and this may result in altered gene expression. The markedly increased susceptibility of GFAT1Alt versus GFAT1 to feedback inhibition by UDP-GlcNAc suggests that GFAT1Alt may protect against the development of insulin resistance, under conditions, which stimulate glucose flux via the HSP in muscle.
I would like to thank Sabrina Volpi and Rocco Coli, for their endless patience and useful suggestions and guidance throughout this project, without whose support this project could not have been completed. In addition, I would like to thank Dr. Berish Rubin for the use of his laboratory, providing me the mouse as well as for giving me the opportunity to perform this individual experiment.
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