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Abstract

We studied a mouse doubly homozygous for mutations in the genes encoding malic enzyme (EC 1.1.1.40) and cytosolic glycerol phosphate dehydrogenase (EC 1.1.1.8) (cGPD). This mouse, which we call the mmgg mouse and which is the product of intercrosses between the Mod-1 mouse and the BALB/cHeA mouse, lacks activity of both enzymes. Like both parental strains the mmgg mouse is completely normal in appearance. cGPD is one of the two enzymes that catalyze the reactions of the glycerol phosphate shuttle. The activity of the other enzyme of the glycerol phosphate shuttle, mitochondrial glycerol phosphate dehydrogenase (EC 1.1.99.5) (mGPD), is abundant in tissues, such as brain, skeletal muscle and the pancreatic islet, suggesting that the glycerol phosphate shuttle is important in these tissues which rapidly metabolize glucose. Cytosolic malic enzyme activity is important for shuttles which transport NADPH equivalents from mitochondria to the cytosol. The major finding of the study was a highly abnormal metabolite pattern in tissues of the mmgg mouse suggesting a block in the glycerol phosphate shuttle due to cGPD deficiency. The metabolite pattern did not suggest that malic enzyme deficiency caused an abnormality. Tissue levels of glycerol phosphate (low) and dihydroxyacetone phosphate (high) were only abnormal in skeletal muscle. Glycolytic intermediates, situated at or before the triose phosphates in the pathway, such as fructose bisphosphate and glyceraldehyde phosphate were increased depending on the tissue. Taken together with previous extensive data on the mouse deficient only in cGPD, this suggests a block in glycolysis at the step catalyzed by glyceraldehyde phosphate dehydrogenase caused by an abnormally low NAD/NADH ratio resulting from a nonfunctional glycerol phosphate shuttle. Consistent with this idea the lactate/pyruvate ratio was high in skeletal muscle signifying a low cytosolic NAD/NADH ratio. The mmgg mouse was normal in all other factors studied including blood glucose and serum insulin levels, pancreatic islet mass, insulin release from isolated pancreatic islets, as well as the activities of five metabolic enzymes, including mGPD, in liver, kidney, skeletal muscle and pancreatic islets. cGPD enzyme activity was undetectable in pancreatic islets, 0.5% of normal in liver, and 2.1% of normal in kidney and skeletal muscle. Malic enzyme activity was undetectable in these same tissues.

Silencing of Cytosolic or Mitochondrial Isoforms of Malic Enzyme Has No Effect on Glucose-stimulated Insulin Secretion from Rodent Islets

“…Real time PCR studies with mouse islet cDNA reported herein demonstrate that MEc is clearly expressed in mouse islets, although at significantly lower levels than in rat islets. Importantly, and consistent with the findings described above, we demonstrate that islets from MEcnull MOD1 Ϫ/Ϫ mouse are indistinguishable from wild type islets in terms of GSIS, which is also consistent with normal glucose homeostasis in the mmgg mouse, which lacks both MEc and glycerol phosphate dehydrogenase (36,37).…”

“…8B). Furthermore, physiologic features of MEc Ϫ/Ϫ mice have been described by multiple groups (35)(36)(37). In particular, the mmgg mouse, which lacks both MEc and glycerol phosphate dehydrogenase, has normal blood glucose and serum insulin levels, and islets isolated from these animals demonstrate normal GSIS.…”

Chronic Reduction of the Cytosolic or Mitochondrial NAD(P)-malic Enzyme Does Not Affect Insulin Secretion in a Rat Insulinoma Cell Line

“…These results are consistent with the finding that normal mouse islets contain little, if any, cytosolic malic enzyme (21,22), whereas mouse, rat, and human islets all contain abundant cytosolic isocitrate dehydrogenase (13,21). Insulin levels and insulin release are normal in the islets of the mouse strain Mod-1, which lacks cytosolic malic enzyme in all tissues (11,13). It is interesting that a recent systems biology paper modeling beta cell metabolism predicted that deletion of the cytosolic malic enzyme would not affect ATP levels or oscillations of cytosolic metabolites and would only modestly affect pyruvate and malate levels (23).…”

“…If confirmed, these results suggest either that isocitrate dehydrogenase is the primary source of cytosolic NADPH in these cells or that multiple routes for NADPH production exist but cannot fully compensate for each other. These results are consistent with the finding that normal mouse islets contain little, if any, cytosolic malic enzyme (21,22), whereas mouse, rat, and human islets all contain abundant cytosolic isocitrate dehydrogenase (13,21). Insulin levels and insulin release are normal in the islets of the mouse strain Mod-1, which lacks cytosolic malic enzyme in all tissues (11,13).…”

Differences between Human and Rodent Pancreatic Islets

“…Pyruvate carboxylase (22), propionyl-CoA carboxylase (22), pyruvate dehydrogenase complex (13), mitochondrial glycerol phosphate dehydrogenase (31), and glutamate dehydrogenase (22) activities were measured in the whole-cell homogenates as described previously. Activities of ATP citrate lyase (24), malic enzyme (14,32,33), NADP isocitrate dehydrogenase (33,34), malate dehydrogenase (35), and aspartate aminotransferase (35) were measured as described previously in a supernatant fraction of cells prepared so as to contain cytosol proteins and mitochondrial matrix proteins but not mitochondrial membrane proteins (the supernatant fraction from centrifuging the frozen-thawed whole-cell homogenate at 20,800 ϫ g for 20 min) (22,26). The concentrations of ingredients of the enzyme reaction mixtures and conditions of the more relevant enzyme assays are briefly described in the supplemental material.…”