Subclinical Diabetes.

Type 2 diabetes mellitus (T2DM) is increasing in prevalence worldwide, and those non-diagnosed or misdiagnosed comprise a significant group compared to those diagnosed. Accumulated scientific evidence indicate that the current diagnostic markers (fasting glycemia, 2h glycemia after an oral glucose load and HbA1c) are indeed late diagnostic criteria when considering the incidence of diabetes-related complications and comorbidities, which are also at high risk in some groups among normoglycemic individuals. Additionally, the earlier identification of future risk of diabetes is desirable since it would allow better adherence to preventive actions such as lifestyle intervention, ultimately avoiding complications and minimizing the economic impact/burden on health care expenses. Insulin resistance and hyperhormonemia (insulin, amylin, glucagon) are non-disputable hallmarks of T2DM, which already takes place among these normoglycemic, otherwise health subjects, characterizing a state of subclinical diabetes. Insulin resistance and hyperinsulinemia can be computed from fasting plasma insulin as an independent variable in normoglycemia. An overview of the current diagnostic criteria, disease onset, complications, comorbidities and perspectives on lifestyle interventions are presented. A proposal for early detection of subclinical diabetes from routine evaluation of fasting plasma insulin, which is affordable and robust and thus applicable for the general population, is further suggested.

Subclinical Diabetes

ABSTRACT Type 2 diabetes mellitus (T2DM) is increasing in prevalence worldwide, and those non-diagnosed or misdiagnosed comprise a significant group compared to those diagnosed. Accumulated scientific evidence indicate that the current diagnostic markers (fasting glycemia, 2h glycemia after an oral glucose load and HbA1c) are indeed late diagnostic criteria when considering the incidence of diabetes-related complications and comorbidities, which are also at high risk in some groups among normoglycemic individuals. Additionally, the earlier identification of future risk of diabetes is desirable since it would allow better adherence to preventive actions such as lifestyle intervention, ultimately avoiding complications and minimizing the economic impact/burden on health care expenses. Insulin resistance and hyperhormonemia (insulin, amylin, glucagon) are non-disputable hallmarks of T2DM, which already takes place among these normoglycemic, otherwise health subjects, characterizing a state of subclinical diabetes. Insulin resistance and hyperinsulinemia can be computed from fasting plasma insulin as an independent variable in normoglycemia. An overview of the current diagnostic criteria, disease onset, complications, comorbidities and perspectives on lifestyle interventions are presented. A proposal for early detection of subclinical diabetes from routine evaluation of fasting plasma insulin, which is affordable and robust and thus applicable for the general population, is further suggested.

Amylin induces hypoglycemia in mice.

Amylin is a 37-aminoacid pancreatic protein that exerts control over several metabolic events such as glycemia and lacticemia. Amylin has long been shown to induce increases in arterial plasma glucose. We decided to investigate whether amylin plays additional roles in the glucose metabolism. We evaluated glucose homeostasis using whole blood from the tail tip of fasting, conscious, unrestrained normal and streptozotocyn-induced diabetic mice following subcutaneous administration of mouse amylin. Subcutaneous injection of 1 µg mouse amylin caused a transient decrease in whole blood glucose in both normal and diabetic mice in the absence of insulin. The blood glucose levels were lowest approximately 2 hours after amylin administration, after that they gradually recovered to the levels of the control group. The hypoglycemic effect followed a dose-dependent response ranging from 0.1 to 50 µg / mouse. These results reveal the ability for amylin in the direct control of glycemia at low doses in the absence of insulin.

On the denaturation mechanisms of the ligand binding domain of thyroid hormone receptors.

The ligand binding domain (LBD) of nuclear hormone receptors adopts a very compact, mostly alpha-helical structure that binds specific ligands with very high affinity. We use circular dichroism spectroscopy and high-temperature molecular dynamics simulations to investigate unfolding of the LBDs of thyroid hormone receptors (TRs). A molecular description of the denaturation mechanisms is obtained by molecular dynamics simulations of the TRalpha and TRbeta LBDs in the absence and in the presence of the natural ligand Triac. The simulations show that the thermal unfolding of the LBD starts with the loss of native contacts and secondary structure elements, while the structure remains essentially compact, resembling a molten globule state. This differs from most protein denaturation simulations reported to date and suggests that the folding mechanism may start with the hydrophobic collapse of the TR LBDs. Our results reveal that the stabilities of the LBDs of the TRalpha and TRbeta subtypes are affected to different degrees by the binding of the isoform selective ligand Triac and that ligand binding confers protection against thermal denaturation and unfolding in a subtype specific manner. Our simulations indicate two mechanisms by which the ligand stabilizes the LBD: (1) by enhancing the interactions between H8 and H11, and the interaction of the region between H1 and the Omega-loop with the core of the LBD, and (2) by shielding the hydrophobic H6 from hydration.

Tetramerization of the LexA repressor in solution: implications for gene regulation of the E.coli SOS system at acidic pH.

Structural changes on LexA repressor promoted by acidic pH have been investigated. Intense protein aggregation occurred around pH 4.0 but was not detected at pH values lower than pH 3.5. The center of spectral mass of the Trp increased 400 cm(-1) at pH 2.5 relatively to pH 7.2, an indication that LexA has undergone structural reorganization but not denaturation. The Trp fluorescence polarization of LexA at pH 2.5 indicated that its hydrodynamic volume was larger than its dimer at pH 7.2. 4,4'-Dianilino-1,1'-binaphthyl-5,5'- disulfonic acid (bis-ANS) experiments suggested that the residues in the hydrophobic clefts already present at the LexA structure at neutral pH had higher affinity to it at pH 2.5. A 100 kDa band corresponding to a tetramer was obtained when LexA was subject to pore-limiting native polyacrylamide gel electrophoresis at this pH. The existence of this tetrameric state was also confirmed by small angle X-ray scattering (SAXS) analysis at pH 2.5. 1D 1H NMR experiments suggested that it was composed of a mixture of folded and unfolded regions. Although 14,000-fold less stable than the dimeric LexA, it showed a tetramer-monomer dissociation at pH 2.5 from the hydrostatic pressure and urea curves. Albeit with half of the affinity obtained at pH 7.2 (Kaff of 170 nM), tetrameric LexA remained capable of binding recA operator sequence at pH 2.5. Moreover, different from the absence of binding to the negative control polyGC at neutral pH, LexA bound to this sequence with a Kaff value of 1415 nM at pH 2.5. A binding stoichiometry experiment at both pH 7.2 and pH 2.5 showed a [monomeric LexA]/[recA operator] ratio of 2:1. These results are discussed in relation to the activation of the Escherichia coli SOS regulon in response to environmental conditions resulting in acidic intracellular pH. Furthermore, oligomerization of LexA is proposed to be a possible regulation mechanism of this regulon.

The amino-terminal PrP domain is crucial to modulate prion misfolding and aggregation.

The main hypothesis for prion diseases is that the cellular protein (PrP(C)) can be altered into a misfolded, beta-sheet-rich isoform (PrP(Sc)), which undergoes aggregation and triggers the onset of transmissible spongiform encephalopathies. Here, we investigate the effects of amino-terminal deletion mutations, rPrP(Delta51-90) and rPrP(Delta32-121), on the stability and the packing properties of recombinant murine PrP. The region lacking in rPrP(Delta51-90) is involved physiologically in copper binding and the other construct lacks more amino-terminal residues (from 32 to 121). The pressure stability is dramatically reduced with decreasing N-domain length and the process is not reversible for rPrP(Delta51-90) and rPrP(Delta32-121), whereas it is completely reversible for the wild-type form. Decompression to atmospheric pressure triggers immediate aggregation for the mutants in contrast to a slow aggregation process for the wild-type, as observed by Fourier-transform infrared spectroscopy. The temperature-induced transition leads to aggregation of all rPrPs, but the unfolding temperature is lower for the rPrP amino-terminal deletion mutants. The higher susceptibility to pressure of the amino-terminal deletion mutants can be explained by a change in hydration and cavity distribution. Taken together, our results show that the amino-terminal region has a pivotal role on the development of prion misfolding and aggregation.

Reversible aggregation plays a crucial role on the folding landscape of p53 core domain.

The role of tumor suppressor protein p53 in cell cycle control depends on its flexible and partially unstructured conformation, which makes it crucial to understand its folding landscape. Here we report an intermediate structure of the core domain of the tumor suppressor protein p53 (p53C) during equilibrium and kinetic folding/unfolding transitions induced by guanidinium chloride. This partially folded structure was undetectable when investigated by intrinsic fluorescence. Indeed, the fluorescence data showed a simple two-state transition. On the other hand, analysis of far ultraviolet circular dichroism in 1.0 M guanidinium chloride demonstrated a high content of secondary structure, and the use of an extrinsic fluorescent probe, 4,4'-dianilino-1,1' binaphthyl-5,5'-disulfonic acid, indicated an increase in exposure of the hydrophobic core at 1 M guanidinium chloride. This partially folded conformation of p53C was plagued by aggregation, as suggested by one-dimensional NMR and demonstrated by light-scattering and gel-filtration chromatography. Dissociation by high pressure of these aggregates reveals the reversibility of the process and that the aggregates have water-excluded cavities. Kinetic measurements show that the intermediate formed in a parallel reaction between unfolded and folded structures and that it is under fine energetic control. They are not only crucial to the folding pathway of p53C but may explain as well the vulnerability of p53C to undergo departure of the native to an inactive state, which makes the cell susceptible to malignant transformation.