Knockdown of both mitochondrial isocitrate dehydrogenase enzymes in pancreatic beta cells inhibits insulin secretion

Abstract

Background: There are three isocitrate dehydrogenases (IDHs) in the pancreatic insulin cell; IDH1 (cytosolic) and IDH2 (mitochondrial) use NADP(H). IDH3 is mitochondrial, uses NAD(H) and was believed to be the IDH that supports the citric acid cycle.

Methods: With shRNAs targeting mRNAs for these enzymes we generated cell lines from INS-1 832/13 cells with severe (80%-90%) knockdown of the mitochondrial IDHs separately and together in the same cell line.

Results: With knockdown of both mitochondrial IDH's mRNA, enzyme activity and protein level, (but not with knockdown of only one mitochondrial IDH) glucose- and BCH (an allosteric activator of glutamate dehydrogenase)-plus-glutamine-stimulated insulin release were inhibited. Cellular levels of citrate, α-ketoglutarate, malate and ATP were altered in patterns consistent with blockage at the mitochondrial IDH reactions. We were able to generate only 50% knockdown of Idh1 mRNA in multiple cell lines (without inhibition of insulin release) possibly because greater knockdown of IDH1 was not compatible with cell line survival.

Conclusions: The mitochondrial IDHs are redundant for insulin secretion. When both enzymes are severely knocked down, their low activities (possibly assisted by transport of IDH products and other metabolic intermediates from the cytosol into mitochondria) are sufficient for cell growth, but inadequate for insulin secretion when the requirement for intermediates is certainly more rapid. The results also indicate that IDH2 can support the citric acid cycle.

General significance: As almost all mammalian cells possess substantial amounts of all three IDH enzymes, the biological principles suggested by these results are probably extrapolatable to many tissues.

Keywords: Cytosolic isocitrate dehydrogenase; INS-1 832/13 cell line; Insulin secretion; Mitochondrial isocitrate dehydrogenase; Stable knockdown of isocitrate dehydrogenase; shRNA.

Figure 1. Pathways of precursors for substrates and products of the three isocitrate dehydrogenase reactions in the pancreatic beta cell

Abbreviations: GluDH, glutamate dehydrogenase; IDH, isocitrate dehydrogenase.

Figure 2. Knockdown of Idh2 and Idh3a mRNA levels individually or both mRNAs in the same cell line with stable transfection of shRNA in INS-1 832/13-derived cell lines without knockdown of untargeted Idh mRNAs

mRNA levels are expressed relative to the HygC, a nontargeting vector control. Results are the mean ± SE of four or more cell preparations. All values < 25% of the HygC control were significant to the p < 0.001 level.

Figure 3. Moderate knockdown of cytosolic NADP isocitrate dehydrogenase (Idh1) mRNA with stable transfection of shRNA in INS-1 832/13 cells does not lower its enzyme activity or glucose- or BCH-plus-glutamine-stimulated insulin release

A. mRNA measurements are the average of two measurements. mRNA levels were measured by qRT-PCR and were normalized to glutamate dehydrogenase mRNA. B. Enzyme activity of IDH1 and, as controls, two other NADP requiring enzymes, malic enzyme and glucose-6-phosphate dehydrogenase. Activities are expressed as a percent of the control INS-1 832/13 equal to 100% and are the mean ± SE of measurements on 3 or 4 cell preparations. C. Insulin release measurements were the averages of average values from eight separate experiments with cells incubated with no insulin secretagogue, 11.1 mM glucose or BCH (10 mM)-plus-glutamine (10 mM). There were four replicate incubations per condition per each experiment. Results are expressed as the mean ± SE.

Figure 4. Severe single and double lowering of IDH2 and IDH3A proteins using stable knockdown with shRNA in INS-1 832/13-derived cell lines

Immunoblots of IDH2 and IDH3A protein and the mitochondrial glycerol phosphate dehydrogenase (GPD2) as a protein loading control in cell lines. INS-1 832/13 is the parent cell line. HygC is the nontargeting vector control cell line. Blots are representative of 4 repetitions with IDH3A and 2 repetitions with IDH2.

Figure 5. Severe lowering of IDH2 and/or IDH3 enzyme activity with shRNA stable knockdown of Idh2 and/or Idh3a mRNAs in INS-1 832/13 derived cell lines

Activity is presented relative to that of the cell line containing a nontargeting vector HygC as a control. The average enzyme activities of IDH1, IDH2 and IDH3 in the HygC control cell line were, respectively, 32, 125 and 24 nmol NAD(P)H/min/mg cytosol protein (IDH1) or mitochondrial protein (IDH2 and IDH3). On a total cell basis, calculated from the total activity in each compartment, IDH1 activity accounts for 69% of the activity in these control cells and IDH2 and IDH3 account for 26% and 5%, respectively. Results are the mean ± SE of three or four separate cell preparations. Values < 20% were significant to the p < 0.001 level vs the HygC control.

Figure 6. Low level of glucose-stimulated α-ketoglutarate and/or malate and increased citrate in INS-1 832/13-derived cell lines with knocked down mitochondrial NAD-IDH (IDH3) and/or mitochondrial NADP-IDH (IDH2)

Cells were incubated with 11.1 mM glucose or glutamine (10 mM)-plus-BCH (10 mM) for 30 min. Results are the mean ± SE from five separate experiments. ^ap < 0.001, ^bp < 0.01 and ^cp < 0.05 vs HygC control same condition.

Figure 7. Lower ATP levels in the glucose-stimulated cell line Idh2-5981/Idh3a-251 with IDH2 and IDH3 enzyme activities knocked down

Cells were incubated 30 min with or without the presence of an insulin secretagogue as in Figure 6. Results are expressed as the mean ± SE of five experiments. ^ap < 0.006 vs HygC, glucose.

Figure 8. Inhibition of glucose- or glutamine-plus-BCH-stimulated insulin release in the cell line Idh2-598/Idh3a-251 with both mitochondrial IDH enzymes knocked down

Cells were incubated with no insulin secretagogue, 11.1 mM glucose or BCH (10 mM)-plus-glutamine (10 mM) for 1 h. Results are the mean ± SE of 3–9 experiments calculated from average values of quadruplicate incubations for each condition per experiment. ^ap < 0.05 and ^bp < 0.01 vs HygC control same condition.