Cuencas Neógeno-Cuaternarias

Based on the findings presented in this Ph.D. thesis and current knowledge one could put forward a hypothesis for the mechanisms by which mitochondrial dysfunction could contribute to insulin resistance and impaired GS activity in subjects with T2DM. Thus, insulin resistance related to mitochondrial dysfunction could be the result of

1) Mutations in mitochondrial and nuclear genes encoding for mitochondrial proteins (328- 330,350) or in factors regulating the coordinated expression of these genes, i.e. PGC-1 (333,350-352).

2) Intrauterine malnutrition or growth retardation (353,354).

3) Metabolic milieu (hyperglycemia, hyperinsulinemia, increased lipid availability,) associated with obesity, IGT and T2DM (52,56-58).

4) Increasing age (303,329,350).

These factors alone or in combination could lead to impaired oxidative phosphorylation and increased ROS production and hence increased oxidative stress (165,350).

There are data to suggest that mutations in mitochondrial DNA and increasing age through decreased mitochondrial ATP synthesis impair pancreatic b-cell function, and hence the capacity to compensate for insulin resistance (303,329,330,350). This may finally lead to b-cell exhaustion as seen in T2DM. To which extent intrauterine malnutrition affect mitochondria in b-cells is unknown.

There are also increasing evidence to support a role for mitochondrial dysfunction and impaired oxidative phosphorylation in skeletal muscle in T2DM (52,56-58,355). This may involve downregulation of several components in the respiratory chain complexes (I-V) including the catalytic b-subunit of ATP synthase (278,327, paper IV). Our data also support a role for altered phosphorylation of ATP synthase b-subunit in this scenario (paper IV). Mitochondrial dysfunction results in increased ROS formation, which in turn leads to increased oxidative stress and hence activation of stress-sensititive kinases such as JNK, p38 MAPK and NF-kB (165). This may impair proximal insulin signaling by inhibitory serine/threonine phosphorylation of IRS-1 and IR (165). In addition, increased oxidative stress leads to increases in cellular stress proteins such as HSP90 and GRP78 (170,171). The activation of p38 MAPK and hence MAPKAP-K2 may be responsible for the observed increase in GRP78 (171). The activation of MAPKAP-K2 may potentially cause increased phosphorylation of GS at site 2 (110). In this way mitochondrial dysfunction is linked to impaired GS activity in muscle in T2DM. The observed increase in HSP90-b may serve to stabilize the function of both PDK1 and Akt in the insulin signaling pathway (172-174,356). In type 2

diabetic muscle this would attenuate the detrimental effects of oxidative stress on insulin signaling and could explain the normal insulin-mediated activation of Akt (214,252, paper I) despite a blunted effect of insulin on proximal signaling components (213,214,229,244,246).

What role the AMPK system plays in this scenario is at present unclear. The finding of normal activity and subunit expression of AMPK in muscle of patients with T2DM (269, paper III), and the normal response to exercise suggest that this system is fully intact (269). Chronic activation of AMPK, whether mediated through training or pharmacologically by AICAR, improves both mitochondrial function and insulin sensitivity (147-155,351). For the reasons outlined above AMPK may therefore serve as a perfect target for the treatment and perhaps even prevention of T2DM. However, AICAR may have detrimental effects on the insulin secretion from b-cells (357). Therefore, at present increased physical activity and weight loss seems to be the treatment of choice to relieve mitochondrial dysfunction and insulin resistance in T2DM and perhaps FDRT2DM _(13,14).

In the near future studies should aim to 1): identify the kinases and hence signaling pathways responsible for the increased phosphorylation of GS at NH2-terminal sites, 2): characterize the role

of O-glycosylation and 14-3-3 proteins in the regulation of GS activity and phosphorylation in normal and diabetic muscle and 3): identify the phosphorylation sites on ATP synthase b-subunit and the kinases responsible for the phosphorylation of these sites. Moreover, to gain further insights into the molecular mechanisms by which the abnormalities associated with mitochondrial dysfunction, increased cellular stress and lipid availability causes insulin resistance and potentially affect the phosphorylation state of GS, and to which extent these abnormalities are inherited or related to environmental factors, it is necessary to study these aspects in glucose-tolerant FDRT2DM

, or even better in non-diabetic co-twins of twins with T2DM (twin approach).

Recent advances in the technology behind functional proteomics will soon enable direct quantitation and identification of huge amounts of proteins from a protein mixture by quantitative MS (358). Furthermore, MS-based proteomic approaches for the identification of glycosylated and serine/ threoinine/tyrosine-phosphorylated proteins have been established (359-361). Applying these novel proteomic appoaches together with the twin approach will help to clarify how genetic, metabolic and environmental factors combine to contribute to the pathogenesis of T2DM.

SUMMARY

The Ph.D. thesis consists of a review and four papers. It is based on experiments carried out during my employment at the Diabetes Research Centre, Odense University Hospital, Denmark, in the period from 1999 to 2002.

Impaired insulin activation of glycogen synthase (GS), a key enzyme in the regulation of glycogen synthesis, plays an important pathophysiological role in the development of insulin resistance in skeletal muscle and hence type 2 diabetes (T2DM). The aim of the Ph.D. study was to elucidate the molecular mechanisms responsible for skeletal muscle insulin resistance in T2DM by two approaches: 1) To investigate the effect of insulin on proximal and distal components of the insulin signaling cascade (IRS-1/PI3K/Akt/GSK-3) and the activation af GS by dephosphorylation of specific serine residues. Moreover, to study other enzymes that may regulate GS activity. 2) To search for alterations in the expression and post-translational modification of proteins in skeletal muscle of patients with T2DM by proteome analysis.

We found that impaired insulin activation of GS in T2DM was not caused by defects in the insulin signaling cascade, but rather due to increased phosphorylation of GS at NH2-terminal sites,

which was not regulated by insulin. We observed no abnormalities in the expression or activity of AMP-activated protein kinase that could explain this defect. Insulin-mediated down-regulation of PP2A protein content was associated with a normal insulin action on glucose storage, glucose and lipid oxidation, but was absent in skeletal muscle of patients with T2DM. Using proteome analysis we identified eight potential markers of skeletal muscle insulin resistance in T2DM. The data suggest a role for increased cellular stress and pertubations in the mitochondrial function including ATP synthesis.

We conclude that future studies of the molecular mechanisms responsible for impaired insulin activation of GS and skeletal muscle insulin resistance should include the potential role of mitochondrial dysfunction and increased cellular stress for the development of T2DM