Diabetes and factors associated with cognitive and functional decline. The screening for CKD among older people across Europe (SCOPE) study.

BACKGROUND: Type 2 diabetes mellitus (DM) in older people is a heterogeneous condition that exhibits differential characteristics in comparison with younger adults. DM increases the risk of disability, is associated with dementia and loss of function, and cognition may often be interrelated and more pronounced in older patients with DM than in those without. AIMS: Our aim was to evaluate the incidence of functional and/or cognitive impairment in older adults with and without DM, and its associated factors in DM participants. METHODS: A 2-year prospective analysis was conducted in a European multicenter prospective cohort (SCOPE study). Older community-dwelling adults (aged ≥ 75 years) underwent a comprehensive geriatric assessment. New functional and/or cognitive decline was explored. RESULTS: Of 1611 participants, 335 (22.0%) had DM at baseline. The percentage of participants scoring at least one ADL impairment and/or cognitive impairment (MMSE < 24) was similar in both groups (9.6%). Factors associated with any new disability in participants with DM in the multivariate analysis were female sex (OR 3.28, 95% CI 1.42-7.56), history of stroke (OR 4.58, 95% CI 1.64-12.7), and greater IADL dependency (OR 1.08 95% CI 1.02-1.15). DISCUSSION: Association between DM and cognitive or functional decline in outpatients of 75 years and older was not found, but factors such as female gender, history of stroke, and IADL dependency could be related. CONCLUSION: Decline in functional and cognitive status of community-dwelling older adults with DM was similar to participants without DM in a short period of 2 years of follow-up, though several clinical factors may increase its risk in this population.

Nelfinavir induces adipocyte insulin resistance through the induction of oxidative stress: differential protective effect of antioxidant agents.

BACKGROUND: Antiretroviral therapy is frequently associated with adverse metabolic effects and lipodystrophy, but the role of HIV protease inhibitors and the mechanisms involved are poorly understood. The HIV protease inhibitor nelfinavir (NFV) impairs insulin signal propagation by inducing similar signalling defects to those induced by exposure to oxidative stress. AIM: We set out to determine if oxidative stress is involved in NFV-induced insulin resistance in 3T3-L1 adipocytes, and whether antioxidant agents with unique modes of action can prevent this effect. RESULTS: Cells exposed to NFV exhibited the following markers of increased oxidative stress: a decrease in both total and low molecular weight reduced thiols, a 20-fold increase in haem oxygenase 1 (HO-1) mRNA, an increase in intracellular reactive oxygen species production (determined by 2',7'-dichlorofluorescein fluorescence), and increased markers of apoptosis. Enhancing cellular thiols with N-acetylcystein prevented the NFV-induced drop in reduced thiols and partially protected against the induction in HO-1, but failed to prevent insulin resistance or cleavage of poly ADP ribose polymerase (PARP), a process indicative of activation of pro-apoptotic caspases. Conversely, the superoxide dismutase-mimetic antioxidant MnTBAP had no effect on cellular thiols in response to NFV, but protected against HO-1 induction and against the impairment in insulin-stimulated Akt/protein kinase B activation and PARP cleavage. CONCLUSIONS: Induction of oxidative stress plays a role in adipocyte insulin resistance and apoptosis induced by NFV through a radical-dependent but thiol-independent mechanism(s). The results may suggest a new mechanism for the adverse effects of NFV on fat cells, and offer potential new intervention approaches.

Agent and cell-type specificity in the induction of insulin resistance by HIV protease inhibitors.

OBJECTIVE: To test agent and cell-type specificity in insulin resistance induced by prolonged exposure to HIV protease inhibitors (HPI), and to assess its relation to the direct, short-term inhibition of insulin-stimulated glucose uptake. METHODS: Following prolonged (18 h) and short (5-10 min) exposure to HPI, insulin-stimulated glucose transport, protein kinase B (PKB) phosphorylation, and GLUT4 translocation were evaluated in 3T3-L1 adipocytes, fibroblasts, L6 myotubes, and L6 cells overexpressing a myc tag on the first exofacial loop of GLUT4 or GLUT1. RESULTS: Prolonged exposure of 3T3-L1 adipocytes to nelfinavir, but not to indinavir or saquinavir, resulted in increased basal lipolysis but decreased insulin-stimulated glucose transport and PKB phosphorylation. In addition, impaired insulin-stimulated glucose uptake and PKB phosphorylation were also observed in the skeletal muscle cell line L6, and in 3T3-L1 fibroblasts. Interestingly, this coincided with increased basal glucose uptake as well as with elevated total-membrane glucose transporter GLUT1 protein content. In contrast to these unique effects of nelfinavir, the mere presence of any of the agents in the 5 min transport assay inhibited insulin-stimulated glucose-uptake activity. This appeared to be caused by direct and specific interaction of the drugs with GLUT4 fully assembled at the plasma membrane, since insulin-stimulated cell-surface exposure of an exofacial myc epitope on GLUT4 was normal. CONCLUSIONS: Independent mechanisms for HPI-induced insulin resistance exist: prolonged exposure to nelfinavir interferes with insulin signaling and alters cellular metabolism of adipocytes and muscle cells, whereas a direct inhibitory effect on insulin-stimulated glucose uptake may occurs through specific interaction of HPI with GLUT4.

Voltage-dependent anion channel 1-based peptides interact with hexokinase to prevent its anti-apoptotic activity.

In brain and tumor cells, the hexokinase isoforms, HK-I and HK-II, bind to the voltage-dependent anion channel (VDAC) in the outer mitochondrial membrane. The VDAC domains interacting with these anti-apoptotic proteins were recently defined using site-directed mutagenesis. Now, we demonstrate that synthetic peptides corresponding to the VDAC1 N-terminal region and selected sequences bound specifically, in a concentration- and time-dependent manner, to immobilized HK-I, as revealed by real time surface plasmon resonance technology. The same VDAC1-based peptides also detached HK bound to brain or tumor-derived mitochondria. Moreover, expression of the VDAC1-based peptides in cells overexpressing HK-I or HK-II prevented HK-mediated protection against staurosporine-induced release of cytochrome c and subsequent cell death. One loop-shaped VDAC1-based peptide corresponding to a selected sequence and fused to a cell-penetrating peptide entered the cell and prevented the anti-apoptotic effects of HK-I and HK-II. This peptide detached mitochondrial-bound HK better than did the same peptide in its linear form. Both cell-expressed and exogenously added cell-penetrating peptide detached mitochondrial-bound HK-I-GFP. These results point to HK-I and HK-II as promoting tumor cell survival through binding to VDAC1, thereby inhibiting cytochrome c release and apoptotic cell death. Moreover, VDAC1-based peptides interfering with HK-mediated anti-apoptotic activity may potentiate the efficacy of conventional chemotherapeutic agents.

Postreceptoral adipocyte insulin resistance induced by nelfinavir is caused by insensitivity of PKB/Akt to phosphatidylinositol-3,4,5-trisphosphate.

Adipocyte insulin resistance can be caused by proximal insulin signaling defects but also from postreceptor mechanisms, which in large are poorly characterized. Adipocytes exposed for 18 h to the HIV protease inhibitor nelfinavir manifest insulin resistance characterized by normal insulin-stimulated tyrosine phosphorylation of the insulin receptor and insulin receptor substrate proteins, preserved in vitro phosphatidylinositol 3-kinase (PI 3-kinase) assay activity but impaired activation of PKB/Akt and stimulation of glucose uptake. Here we aimed to assess whether impaired PKB/Akt activation is indeed rate limiting for insulin signaling propagation in response to nelfinavir and the mechanism for defective PKB/Akt activation. Nelfinavir treatment of 3T3-L1 adipocytes impaired the insulin-stimulated translocation and membrane fusion of myc-glucose transporter (GLUT)-4-green fluorescent protein (GFP) reporter. Phosphorylation of PKB/Akt substrates including glycogen synthase kinase-3 and AS160 decreased in response to nelfinavir, and this remained true, even in cells with forced generation of phosphatidylinositol-3,4,5-trisphohphate (PIP(3)) by a membrane-targeted active PI 3-kinase, confirming that impaired PKB/Akt activation was rate limiting for insulin signal propagation. Cells expressing a GFP-tagged pleckstrin homology domain of general receptors for phosphoinositides 1, which binds PIP(3), revealed intact PIP(3)-mediated plasma membrane translocation of this reporter in nelfinavir-treated cells. However, expression of a membrane-targeted catalytic subunit of PI 3-kinase failed to induce myc-GLUT4-GFP translocation in the absence of insulin, as it did in control cells. Conversely, a membrane-targeted and constitutively active PKB/Akt mutant was normally phosphorylated on S473 and T308, confirming intact PKB/Akt kinases activity, and induced myc-GLUT4-GFP translocation. Collectively, nelfinavir uncovers a postreceptor mechanism for insulin resistance, caused by interference with the sensing of PIP(3) by PKB/Akt, leading to impaired GLUT4 translocation and membrane fusion.

The VDAC1 N-terminus is essential both for apoptosis and the protective effect of anti-apoptotic proteins.

The release of mitochondrial-intermembrane-space pro-apoptotic proteins, such as cytochrome c, is a key step in initiating apoptosis. Our study addresses two major questions in apoptosis: how are mitochondrial pro-apoptotic proteins released and how is this process regulated? Accumulating evidence indicates that the voltage-dependent anion channel (VDAC) plays a central role in mitochondria-mediated apoptosis. Here, we demonstrate that the N-terminal domain of VDAC1 controls the release of cytochrome c, apoptosis and the regulation of apoptosis by anti-apoptotic proteins such as hexokinase and Bcl2. Cells expressing N-terminal truncated VDAC1 do not release cytochrome c and are resistant to apoptosis, induced by various stimuli. Employing a variety of experimental approaches, we show that hexokinase and Bcl2 confer protection against apoptosis through interaction with the VDAC1 N-terminal region. We also demonstrate that apoptosis induction is associated with VDAC oligomerization. These results show VDAC1 to be a component of the apoptosis machinery and offer new insight into the mechanism of cytochrome c release and how anti-apoptotic proteins regulate apoptosis and promote tumor cell survival.

Regulation of adipocyte lipolysis by degradation of the perilipin protein: nelfinavir enhances lysosome-mediated perilipin proteolysis.

A decrease in the lipid droplet-associated protein perilipin may constitute a mechanism for enhanced adipocyte lipolysis under nonstimulated (basal) conditions, and increased basal lipolysis has been linked to whole body metabolic dysregulation. Here we investigated whether the lipolytic actions of the human immunodeficiency virus protease inhibitor, nelfinavir, are mediated by decreased perilipin protein content and studied the mechanisms by which it occurs. Time course analysis revealed that the decrease in perilipin protein content preceded the increase in lipolysis. A causative relationship was suggested by demonstrating that nelfinavir potently increased lipolysis in adipocytes derived from mouse embryonal fibroblasts expressing perilipin but not in mouse embryonal fibroblast adipocytes devoid of perilipin and that adenoviral mediated overexpression of perilipin in 3T3-L1 adipocytes blocked the lipolytic actions of nelfinavir. Nelfinavir did not alter mRNA content of perilipin but rather decreased perilipin proteins t((1/2)) from >70 to 12 h. Protein degradation of perilipin in both control and nelfinavir-treated adipocytes could be prevented by inhibiting lysosomal proteolysis using leupeptin or NH(4)Cl but not by the proteasome inhibitor MG-132. We propose that proteolysis of perilipin involving the lysosomal protein degradation machinery may constitute a novel mechanism for enhancing adipocyte lipolysis.