Increased Renal Methylglyoxal Formation with Down-Regulation of PGC-1α-FBPase Pathway in Cystathionine γ-Lyase Knockout Mice

We have previously reported that hydrogen sulfide (H2S), a gasotransmitter and vasodilator has cytoprotective properties against methylglyoxal (MG), a reactive glucose metabolite associated with diabetes and hypertension. Recently, H2S was shown to up-regulate peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α, a key gluconeogenic regulator that enhances the gene expression of the rate-limiting gluconeogenic enzyme, fructose-1,6-bisphosphatase (FBPase). Thus, we sought to determine whether MG levels and gluconeogenic enzymes are altered in kidneys of 6–22 week-old cystathionine γ-lyase knockout (CSE-/-; H2S-producing enzyme) male mice. MG levels were determined by HPLC. Plasma glucose levels were measured by an assay kit. Q-PCR was used to measure mRNA levels of PGC-1α and FBPase-1 and -2. Coupled-enzymatic assays were used to determine FBPase activity, or triosephosphate levels. Experimental controls were either age-matched wild type mice or untreated rat A-10 cells. Interestingly, we observed a significant decrease in plasma glucose levels along with a significant increase in plasma MG levels in all three age groups (6–8, 14–16, and 20–22 week-old) of the CSE-/- mice. Indeed, renal MG and triosephosphates were increased, whereas renal FBPase activity, along with its mRNA levels, were decreased in the CSE-/- mice. The decreased FBPase activity was accompanied by lower levels of its product, fructose-6-phosphate, and higher levels of its substrate, fructose-1,6-bisphosphate in renal extracts from the CSE-/- mice. In agreement, PGC-1α mRNA levels were also significantly down-regulated in 6-22 week-old CSE-/- mice. Furthermore, FBPase-1 and -2 mRNA levels were reduced in aorta tissues from CSE-/- mice. Administration of NaHS, a H2S donor, increased the gene expression of PGC-1α and FBPase-1 and -2 in cultured rat A-10 cells. In conclusion, overproduction of MG in CSE-/- mice is due to a H2S-mediated down-regulation of the PGC-1α-FBPase pathway, further suggesting the important role of H2S in the regulation of glucose metabolism and MG generation

Plasma glucose levels in 6-22 week-old CSE^-/- mice.

<p>Plasma glucose levels were measured in 6-8 (<i>n</i> = 5–7), 14-16 (<i>n</i> = 4–5), and 20-22 (<i>n</i> = 6–7) week-old CSE^-/- and CSE^+/+ mice after starving for 16 h. *<i>P</i><0.05 and **<i>P</i><0.01 vs. corresponding age-matched CSE^+/+ mice; ^#<i>P</i><0.05 vs. 6–8 week-old CSE^-/- mice; ^†<i>P</i><0.05 vs. 6–8 week-old CSE^+/+ mice<b>.</b></p

Real-time PCR primer sequences for gene targets in mice.

<p>Real-time PCR primer sequences for gene targets in mice.</p

mRNA levels of FBPase-1 and -2 in the aorta of 14-16 week-old CSE^-/- mice.

<p>FBPase-1 <b>(Panel A)</b> and -2 <b>(Panel B)</b> mRNA expression levels were measured in aortic extracts from 14-16 week-old CSE^–/– mice (<i>n</i> = 3–4). The values from CSE^-/- are presented as a percentage of the mean of age-matched CSE^+/+ mice. *<i>P</i><0.05 and **<i>P</i><0.01 vs. 14-16 week-old CSE^+/+ mice.</p

Fructose-6-phosphate, fructose-1,6-bisphosphate, and dihydroxyacetone phosphate and glyceraldehyde 3-phosphate levels in renal tissues of CSE^–/– mice.

<p><b>Panel A:</b> Fructose-6-phosphate (F-6-P) levels were measured in kidneys of 6-8 (<i>n</i> = 6), 14–16 (<i>n</i> = 6), and 20–22 (<i>n</i> = 6-8) week-old mice. <b>Panel B:</b> Fructose-1,6-bisphosphate (F-1,6-P) levels in kidneys were analyzed in 6-22 (<i>n</i> = 5) week-old mice. <b>Panel C:</b> The triosephosphates, dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (GA3P), were measured in kidneys of 6-22 (<i>n</i> = 5) week-old mice. <b>Panel D:</b> Renal phosphofructokinase (PFK) activity was measured in 6-8 (<i>n</i> = 4), 14–16 (<i>n</i> = 4–5), 20-22 (<i>n</i> = 4-5) week-old mice. The values from CSE^-/- are presented as a percentage of the mean of age-matched CSE^+/+ mice. *<i>P</i><0.05, **<i>P</i><0.01, and ***<i>P</i><0.001 vs. corresponding age groups of CSE^+/+ mice.</p

mRNA levels of proliferator-activated receptor-γ coactivator-1α, fructose-1,6-bisphosphatase-1 and -2, and estrogen-related receptor-α in NaHS-treated rat A-10 cells.

<p>Rat A-10 cells were treated with NaHS at different concentrations for 24 h to determine mRNA levels of proliferator-activated receptor-γ coactivator (PGC)-1α <b>(Panel A)</b>, fructose-1,6-bisphosphatase (FBPase)-1 <b>(Panel B)</b>, FBPase-2 <b>(Panel C)</b>, and estrogen-related receptor-α (ERRα) <b>(Panel D)</b>. <i>n</i> = 5 for each group in <b>Panels A</b>, <b>B</b>, <b>C</b>, and <b>D</b>. The mRNA values obtained from NaHS-treated A-10 cells are presented as a percentage of the mean of control cells. *<i>P</i><0.05 and **<i>P</i><0.01 vs. control group.</p

Methylglyoxal and H₂S levels in plasma and renal tissues in CSE^–/– mice.

<p><b>Panel A:</b> Methylglyoxal (MG) levels in the plasma where measured in 6-8 (<i>n</i> = 7), 14-16 (<i>n</i> = 4), and 20-22 (<i>n</i> = 7) week-old CSE^-/- and CSE^+/+ mice. <b>Panel B:</b> H₂S levels in plasma in 6-8 (<i>n</i> = 4-6), 14-16 (<i>n</i> = 4–6), and 20-24 (<i>n</i> = 3–5) week-old CSE^-/- and CSE^+/+ mice. <b>Panel C:</b> MG levels in renal tissues of 6-8 (<i>n</i> = 5), 14-16 (<i>n</i> = 6), and 20-22 (<i>n</i> = 7) week-old mice. <b>Panel D:</b> Renal H₂S levels in 6-8 (<i>n</i> = 3–5), 14-16 (<i>n</i> = 4), and 20–22 (<i>n</i> = 3-5) week-old mice. MG and H₂S values from CSE^-/- mice are presented as a percentage of the mean of age-matched CSE^+/+ mice. *<i>P</i><0.05, **<i>P</i><0.01, and ***<i>P</i><0.001 vs. corresponding age groups of CSE^+/+ mice.</p

mRNA levels of peroxisome proliferator-activated receptor-γ coactivator-1α, phosphoenolpyruvate carboxykinase, and estrogen-related receptor-α in renal tissues of CSE^–/– mice.

<p><b>Panel A:</b> Peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α mRNA levels in kidneys of CSE^–/– mice in 6-8 (<i>n</i> = 5), 14–16 (<i>n</i> = 6), and 20-22 (<i>n</i> = 5) week-old mice. <b>Panel B:</b> Phosphoenolpyruvate carboxykinase (PEPCK) mRNA in 6-8 (<i>n</i> = 5), 14-16 (<i>n</i> = 6), and 20-22 (<i>n</i> = 5) week-old mice. <b>Panel C:</b> Estrogen-related receptor-α (ERRα) mRNA in 6-8 (<i>n</i> = 5), 14-16 (<i>n</i> = 6), and 20-22 (<i>n</i> = 5) week-old mice. The mRNA values from CSE^–/– mice are presented as a percentage of the mean of age-matched CSE^+/+ mice. *<i>P</i><0.05 and **<i>P</i><0.01 vs. corresponding age groups of CSE^+/+ mice.</p

Total fructose-1,6-bisphosphatase activity and fructose-1,6-bisphosphatase-1 and -2 mRNA levels in renal tissues of CSE^–/– mice.

<p><b>Panel A:</b> Total fructose-1,6-bisphosphatase (FBPase) activity in kidneys of 6-8 (<i>n</i> = 6), 14–16 (<i>n</i> = 6), and 20-22 (<i>n</i> = 4) week-old mice. <b>Panel B:</b> Real-time PCR results of fructose-1,6-bisphosphatase (FBPase)-1 levels in renal tissues of CSE^–/– mice ages 6-8 (<i>n</i> = 5), 14–16 (<i>n</i> = 7), and 20-22 (<i>n</i> = 5) weeks. <b>Panel C:</b> Real-time PCR results of FBPase-2 mRNA levels in kidneys of 6-8 (<i>n</i> = 4), 14–16 (<i>n</i> = 4), and 20-22 (<i>n</i> = 5) week-old mice. FBPase activity, FBPase-1 and -2 mRNA values in CSE^–/– mice are presented as a percentage of the mean of age-matched CSE^+/+ mice. *<i>P</i><0.05, **<i>P</i><0.01, and ***<i>P</i><0.001 vs. corresponding age groups of CSE^+/+ mice.</p