J. Antonie Maassen

Mitochondrial diabetes mellitus: a review

We review the relationship between various types of mitochondrial DNA mutations and the prevalence as well as the pathobiochemical and clinical features of mitochondrial diabetes mellitus. An A to G transversion mutation in the tRNA(Leu(UUR)) gene is associated with diabetes in about 1.5% of the diabetic population in different countries and races. Phenotypically this type of mitochondrial diabetes is combined with deafness in more than 60% and is clinically distinguishable with respect to several characteristics from the two idiopathic forms of diabetes. The underlying pathomechanism is probably a delayed insulin secretion due to an impaired mitochondrial ATP production in consequence of the mtDNA defect.

Growth factors can activate ATF2 via a two-step mechanism: phosphorylation of Thr71 through the Ras-MEK-ERK pathway and of Thr69 through RalGDS-Src-p38.

Transcription factor ATF2 regulates gene expression in response to environmental changes. Upon exposure to cellular stresses, the mitogen‐activated proteinkinase (MAPK) cascades including SAPK/JNK and p38 can enhance ATF2s transactivating function through phosphorylation of Thr69 and Thr71. How ever, the mechanism of ATF2 activation by growth factors that are poor activators of JNK and p38 is still elusive. Here, we show that in fibroblasts, insulin, epidermal growth factor (EGF) and serum activate ATF2 via a so far unknown two‐step mechanism involving two distinct Ras effector pathways: the Raf–MEK–ERK pathway induces phosphorylation of ATF2 Thr71, whereas subsequent ATF2 Thr69 phosphorylation requires the Ral–RalGDS–Src–p38 pathway. Cooperation between ERK and p38 was found to be essential for ATF2 activation by these mitogens; the activity of p38 and JNK/SAPK in growth factor‐stimulated fibroblasts is insufficient to phosphorylate ATF2 Thr71 or Thr69 + 71 significantly by themselves, while ERK cannot dual phosphorylate ATF2 Thr69 + 71 efficiently. These results reveal a so far unknown mechanism by which distinct MAPK pathways and Ras effector pathways cooperate to activate a transcription factor.

Heteroplasmy levels of a mitochondrial gene mutation associated with diabetes mellitus decrease in leucocyte DNA upon aging

We showed previously that a mutation in the mitochondrial tRNALeu(UUR) gene at position 3243 associates with maternally inherited diabetes and deafness (MIDD). This mutation shows heteroplasmy in DNA from peripheral blood and other tissues. To examine whether heteroplasmy levels in peripheral blood DNA change upon aging, heteroplasmy levels were determined in DNA samples from peripheral blood, collected recently and 1.5–6 years ago, from 18 individuals carrying the 3243 mutation. It was found that 17 out of 18 carriers showed a decrease upon aging (P = 0.001), the average change being −0.69 ± 0.61% per year. These data indicate a continuous selection against haematopoietic (precursor) cells carrying high levels of the 3243 mutation. Moreover, they imply that heteroplasmy levels may decrease below the detection limit if DNA from peripheral blood is analyzed from elderly individuals. DNA from oral mucosa cells was found to be a good alternative as heteroplasmy levels for the 3243 mutation are on the average 1.7 fold higher than in DNA from peripheral blood.

The Diabetes-associated 3243 Mutation in the Mitochondrial tRNALeu(UUR) Gene Causes Severe Mitochondrial Dysfunction without a Strong Decrease in Protein Synthesis Rate

Cells harboring patient-derived mitochondria with an A-to-G transition at nucleotide position 3243 of their mitochondrial DNA display severe loss of respiration when compared with cells containing the wild-type adenine but otherwise identical mitochondrial DNA sequence. The amount and degree of leucylation of tRNALeu(UUR) were both found to be highly reduced in mutant cells. Despite the low level of leucyl-tRNALeu(UUR), the rate of mitochondrial translation was not seriously affected by this mutation. Therefore, decrease of mitochondrial protein synthesis as such does not appear to be a necessary prerequisite for loss of respiration. Rather, the mitochondrially encoded proteins seem subject to elevated degradation, leading to a severe reduction in their steady state levels. Our results favor a scheme in which the 3243 mutation causes loss of respiration through accelerated protein degradation, leading to a disequilibrium between the levels of mitochondrial and nuclear encoded respiratory chain subunits and thereby a reduction of functional respiratory chain complexes. The possible mechanisms underlying the pathogenesis of mitochondrial diabetes is discussed.

Phosphorylation of PRAS40 on Thr246 by PKB/AKT facilitates efficient phosphorylation of Ser183 by mTORC1

Type 2 diabetes is associated with alterations in protein kinase B (PKB/Akt) and mammalian target of rapamycin complex 1 (mTORC1) signalling. The proline-rich Akt substrate of 40-kDa (PRAS40) is a component of mTORC1, which has a regulatory function at the intersection of the PKB/Akt and mTORC1 signalling pathway. Phosphorylation of PRAS40-Thr246 by PKB/Akt, and PRAS40-Ser183 and PRAS40-Ser221 by mTORC1 results in dissociation from mTORC1, and its binding to 14-3-3 proteins. Although all phosphorylation sites within PRAS40 have been implicated in 14-3-3 binding, substitution of Thr246 by Ala alone is sufficient to abolish 14-3-3 binding under conditions of intact mTORC1 signalling. This suggests that phosphorylation of PRAS40-Thr246 may facilitate efficient phosphorylation of PRAS40 on its mTORC1-dependent sites. In the present study, we investigated the mechanism of PRAS40-Ser183 phosphorylation in response to insulin. Insulin promoted PRAS40-Ser183 phosphorylation after a euglycaemic-hyperinsulinaemic clamp in human skeletal muscle. The insulin-induced PRAS40-Ser183 phosphorylation was further evidenced in vivo in rat skeletal and cardiac muscle, and in vitro in A14 fibroblasts, 3T3L1 adipocytes and L6 myotubes. Inhibition of mTORC1 by rapamycin or amino acid deprivation partially abrogated insulin-mediated PRAS40-Ser183 phosphorylation in cultured cell lines. However, lowering insulin-induced PRAS40-Thr246 phosphorylation using wortmannin or palmitate in cell lines, or by feeding rats a high-fat diet, completely abolished insulin-mediated PRAS40-Ser183 phosphorylation. In addition, replacement of Thr246 by Ala reduced insulin-mediated PRAS40-Ser183 phosphorylation. We conclude that PRAS40-Ser183 is a component of insulin action, and that efficient phosphorylation of PRAS40-Ser183 by mTORC1 requires the phosphorylation of PRAS40-Thr246 by PKB/Akt.

DETECTION OF MITOCHONDRIAL DNA DELETIONS IN HUMAN SKIN FIBROBLASTS OF PATIENTS WITH PEARSON'S SYNDROME BY TWO-COLOR FLUORESCENCE IN SITU HYBRIDIZATION

Pearsons marrow/pancreas syndrome is a disease associated with a large mitochondrial DNA (mtDNA) deletion. The various tissues of a patient contain heteroplasmic populations of wild-type (WT) and deleted mtDNA molecules. The clinical phenotype of Pearsons syndrome is variable and is not correlated with the size and position of the deletion. The histo- and cytological distribution of WT and deleted mtDNA molecules may be factors that correlate with the phenotypical expression of the disease. Here we introduce a new application of two-color FISH to visualize WT and deleted mtDNA simultaneously in a cell population of in vitro cultured skin fibroblasts of two patients with Pearsons syndrome. At the third passage of culturing, fibroblasts showed a remarkable heterogeneity of WT and deleted mtDNA: about 90% of the cells contained almost 100% WT mtDNA, and 10% of the cells contained predominantly deleted mtDNA. At the tenth passage of culturing, fibroblasts showed a reduction of intercellular heteroplasmy from 10% to 1%, while intracellular heteroplasmy was maintained. This new approach enables detailed analysis of distribution patterns of WT and deleted mtDNA molecules at the inter- and intracellular levels in clinical samples, and may contribute to a better understanding of genotype-phenotype relationships in patients with mitochondrial diseases.

In vivo insulin responsiveness for glucose uptake and production at eu- and hyperglycemic levels in normal and diabetic rats

UNLABELLED Whole body glucose uptake (BGU) and hepatic glucose production (HGP) at maximal plasma insulin concentrations (+/- 5000 microU/ml) were determined by eu- (EC) (6 mM) and hyperglycemic (HC) (20 mM) clamps (120 min), combined with [3-3H]glucose infusion, in normal and streptozotocin-treated (65 mg/kg) 3-day diabetic, conscious rats. In normal rats, during EC, BGU was 12.4 +/- 0.4 mg/min and during HC, when urinary glucose loss was 0.54 +/- 0.09 mg/min, BGU was 25.5 +/- 1.6 mg/min. However, throughout the final 60 min of HC, glucose infusion rate (GIR) was not constant but a linear decline in time (r = -0.99) of 17%, P less than 0.0001, was observed indicating a hyperglycemia-induced desensitization process. In diabetic rats, during EC, BGU was 7.7 +/- 0.3 mg/min and during HC, BGU was 15.5 +/- 1.4 mg/min. Throughout the final 60 min of HC, GIR was constant, suggesting that the hyperglycemia-induced desensitization process was already completed. In normal and diabetic rats, HGP was similar: during EC 0.2 +/- 0.5 mg/min and 0.1 +/- 0.5 mg/min, and during HC 0.4 +/- 0.4 mg/min and 0.5 +/- 0.6 mg/min, respectively. In vitro adipocyte and muscle insulin receptor studies showed normal to increased receptor number and increased receptor autophosphorylation in diabetic compared to normal rats. IN CONCLUSION (i) 3-day diabetic rats show, at maximal plasma insulin concentrations, insulin resistance to BGU, but not to HGP. The resistance to BGU is equally present (reduction of 38%) at eu- and hyperglycemic levels as compared to normal rats. (ii) 3-day diabetic rats reveal no defect in adipocyte and muscle insulin receptor function. These data indicate that the diabetes induced insulin resistance for BGU is at the post-receptor level and due to a decreased maximal capacity (Vmax) for glucose uptake, with no change in affinity, or Km.

The nuclear appearance of ERK1/2 and p38 determines the sequential induction of ATF2-Thr71 and ATF2-Thr69 phosphorylation by serum in JNK-deficient cells

Growth factors activate ATF2 via sequential phosphorylation of Thr69 and Thr71, where the ATF2-Thr71-phosphorylation precedes the induction of ATF2-Thr69+71-phosphorylation. Here, we studied the mechanisms contributing to serum-induced two-step ATF2-phosphorylation in JNK1,2-deficient embryonic fibroblasts. Using anion exchange chromatography, ERK1/2 and p38 were identified as ATF2-kinases in vitro. Inhibitor studies as well as nuclear localization experiments show that the sequential nuclear appearance of ERK1/2 and p38 determines the induction of ATF2-Thr71 and ATF2-Thr69+71-phosphorylation in response to serum.

Phosphorus-31 magnetic resonance spectroscopy of skeletal muscle in maternally inherited diabetes and deafness A3243G mitochondrial mutation carriers

To investigate high‐energy phosphate metabolism in striated skeletal muscle of patients with Maternally Inherited Diabetes and Deafness (MIDD) syndrome.

8-Bromo-cAMP induces a proliferative response in an IL-3 dependent leukemic cell line and activates Erk1,2 via a Shc-independent pathway

In a number of cell types, elevation of intracellular cAMP concentrations antagonizes growth factor-induced mitogenesis by abrogating the downstream signaling of RasGTP to extracellular-signal-regulated kinases (Erk 1,2). We studied the effect of elevation of cAMP concentrations on the IL-3-induced mitogenic response in the leukemic cell line AML193. We observed that 8-bromo-cAMP (8-Br-cAMP) had no inhibitory effect on the magnitude of this response. On the contrary. 8-Br-cAMP alone induced a proliferative response in these cells. 8-Br-cAMP activated Erk 1,2 in these cells without involvement of Shc phosphorylation. These findings suggest the presence of a novel cAMP-dependent signaling pathway in AML193 cells, which activates Erk 1,2 via a Shc-independent pathway and leads to the generation of a mitogenic response.