Silencing of cytosolic or mitochondrial isoforms of malic enzyme has no effect on glucose-stimulated insulin secretion from rodent islets - PubMed (original) (raw)

. 2008 Oct 24;283(43):28909-17.

doi: 10.1074/jbc.M804665200. Epub 2008 Aug 28.

Mette V Jensen, Hans E Hohmeier, Shawn C Burgess, Yun-Ping Zhou, Su Qian, Douglas MacNeil, Andrew Howard, Nancy Thornberry, Olga Ilkayeva, Danhong Lu, A Dean Sherry, Christopher B Newgard

Affiliations

Silencing of cytosolic or mitochondrial isoforms of malic enzyme has no effect on glucose-stimulated insulin secretion from rodent islets

Sarah M Ronnebaum et al. J Biol Chem. 2008.

Abstract

We have previously demonstrated a role for pyruvate cycling in glucose-stimulated insulin secretion (GSIS). Some of the possible pyruvate cycling pathways are completed by conversion of malate to pyruvate by malic enzyme. Using INS-1-derived 832/13 cells, it has recently been shown by other laboratories that NADP-dependent cytosolic malic enzyme (MEc), but not NAD-dependent mitochondrial malic enzyme (MEm), regulates GSIS. In the current study, we show that small interfering RNA-mediated suppression of either MEm or MEc results in decreased GSIS in both 832/13 cells and a new and more glucose- and incretin-responsive INS-1-derived cell line, 832/3. The effect of MEm to suppress GSIS in these cell lines was linked to a substantial decrease in cell growth, whereas MEc suppression resulted in decreased NADPH, shown previously to be correlated with GSIS. However, adenovirus-mediated delivery of small interfering RNAs specific to MEc and MEm to isolated rat islets, while leading to effective suppression of the targets transcripts, had no effect on GSIS. Furthermore, islets isolated from MEc-null MOD1(-/-) mice exhibit normal glucose- and potassium-stimulated insulin secretion. These results indicate that pyruvate-malate cycling does not control GSIS in primary rodent islets.

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Figures

FIGURE 1.

FIGURE 1.

Characterization of INS-1-derived 832/13 and 832/3 cell lines. Glucose-stimulated insulin secretion was measured in 832/13 (A) and 832/3 (B) cells at 3 and 15 m

m

glucose in the presence or absence of 50 n

m

GLP-1, 50 n

m

PACAP, or 50 n

m

GIP. The data represent the means ± S.E. of three independent measurements and are representative of three other independent experiments.

FIGURE 2.

FIGURE 2.

Suppression of MEm expression in 832/13 cells impairs GSIS and cell proliferation. A, 832/13 cells were transfected with an siRNA with no known target (siControl) or two independent siRNAs targeting MEm, and the levels of MEm RNA were measured via RT-PCR. B, following 72 h of culture, GSIS was measured in 832/13 cells transfected with MEm or siControl siRNA duplexes and normalized to total protein content. C, [methyl-3H]thymidine incorporation was measured in 832/13 cells transfected with MEm or siControl siRNA duplexes, and the data were normalized to total protein content. For all three panels, the results represent the means ± S.E. of three independent experiments. *, p < 0.05; **, p < 0.005.

FIGURE 3.

FIGURE 3.

Adenovirus-mediated suppression of MEm expression in rat islets does not impair GSIS. A, MEm siRNA siMEm-2 was cloned into a recombinant adenovirus and used to transduce isolated rat islets. MEm mRNA was measured by RT-PCR. B, GSIS was measured in isolated rat islets treated with Ad-siControl or Ad-siMEm adenoviruses and normalized to islet number. For both panels, the results represent the means ± S.E. of three independent experiments. *, p < 0.05; **, p < 0.005.

FIGURE 4.

FIGURE 4.

Suppression of MEc expression impairs GSIS in 832/13 and 832/3 cells. An siRNA targeting MEc (siMEc) or siControl was transfected into 832/13 cells and 832/3 cells, respectively. Following 72 h of culture, MEc mRNA was measured via RT-PCR (832/13 (A) and 832/3 cells (C)), and GSIS was measured and normalized to total cellular protein (832/13 (B) and 832/3 cells (D)). The results represent the means ± S.E. of three independent experiments. *, p < 0.05; **, p < 0.005.

FIGURE 5.

FIGURE 5.

Suppression of MEc expression does not impair GSIS in rat islets. An adenovirus containing siMEc (Ad-siMEc) or a control adenovirus containing an siRNA with no known target (Ad-siControl) was used to transduce isolated rat islets. MEc mRNA was measured (A), and insulin secretion at 2.8 and 16.7 m

m

glucose was normalized to islet number (B). The results represent the means ± S.E. of three independent experiments. *,p < 0.05; **, p < 0.005.

FIGURE 6.

FIGURE 6.

Metabolic effects of suppression of MEc expression in 832/13 and 832/3 cells. 832/13 or 832/3 cells were transfected with siMEc or siControl siRNA duplexes. [U-14C]Glucose oxidation to CO2 was measured at 2.5 and 12 m

m

glucose in 832/13 cells (A) and 832/3 cells (B), and the data were normalized to total cellular protein. C and D, pyruvate (C) and other organic acids (D) were measured at 2.5 and 12 m

m

glucose by gas chromatography/mass spectrometry in 832/13 cells and normalized to total cellular protein. E, NADPH was measured at 2.5 and 12 m

m

glucose in 832/13 cells and normalized to total cellular protein. F, [U-14C]glucose incorporation into lipids was measured at 2.5 and 12 m

m

glucose and normalized to total cellular protein. The results in all of the panels represent the means ± S.E. of three independent experiments. *, p < 0.05.

FIGURE 7.

FIGURE 7.

Suppression of MEc in 832/13 cells has no effect on pyruvate cycling. 832/13 cells were transfected with siMEc or siControl siRNA duplexes. Following 72 h of culture, the cells were incubated in 2.5 or 12 m

m

[U-13C]glucose for 3 h. Cell extracts were collected, and pyruvate cycling was measured via NMR-based mass isotopomer analysis. The results represent the means ± S.E. of five independent experiments.

FIGURE 8.

FIGURE 8.

Expression of MEc in mouse and rat islets and lack of effect of MEc deletion on GSIS in mouse islets. Islets were isolated from Wistar rats and from wild type (WT) and MOD1–/– mice.A, RNA was harvested, and MEc expression was measured in islets from Wistar rats and wild type mice by RT-PCR. The data are expressed relative to MEc expression in mouse islets, using an identical internal standard (cyclophilin) for both the rat and mouse RNA samples, and mouse and rat-specific MEc primers with similar binding affinities. B, insulin secretion was measured in wild type and MOD1–/– mouse islets in response to 2 m

m

glucose, 16 m

m

glucose, and 16 m

m

glucose plus 30 m

m

KCl. The results in all of the panels represent the means ± S.E. of three independent experiments.

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