Teresa L. Z. Jones

Diabetic neuropathies: Update on definitions, diagnostic criteria, estimation of severity, and treatments

Preceding the joint meeting of the 19th annual Diabetic Neuropathy Study Group of the European Association for the Study of Diabetes (NEURODIAB) and the 8th International Symposium on Diabetic Neuropathy in Toronto, Canada, 13–18 October 2009, expert panels were convened to provide updates on classification, definitions, diagnostic criteria, and treatments of diabetic peripheral neuropathies (DPNs), autonomic neuropathy, painful DPNs, and structural alterations in DPNs.

Crystal Structure of the Human Acyl Protein Thioesterase I from a Single X-Ray Data Set to 1.5 Å

BACKGROUND Many proteins undergo posttranslational modifications involving covalent attachment of lipid groups. Among them is palmitoylation, a dynamic, reversible process that affects trimeric G proteins and Ras and constitutes a regulatory mechanism for signal transduction pathways. Recently, an acylhydrolase previously identified as lysophospholipase has been shown to function as an acyl protein thioesterase, which catalyzes depalmitoylation of Galpha proteins as well as Ras. Its amino acid sequence suggested that the protein is evolutionarily related to neutral lipases and other thioesterases, but direct structural information was not available. RESULTS We have solved the crystal structure of the human putative Galpha-regulatory protein acyl thioesterase (hAPT1) with a single data set collected from a crystal containing the wild-type protein. The phases were calculated to 1.8 A resolution based on anomalous scattering from Br(-) ions introduced in the cryoprotectant solution in which the crystal was soaked for 20 s. The model was refined against data extending to a resolution of 1.5 A to an R factor of 18.6%. The enzyme is a member of the ubiquitous alpha/beta hydrolase family, which includes other acylhydrolases such as the palmitoyl protein thioesterase (PPT1). CONCLUSIONS The human APT1 is closely related to a previously described carboxylesterase from Pseudomonas fluorescens. The active site contains a catalytic triad of Ser-114, His-203, and Asp-169. Like carboxylesterase, hAPT1 appears to be dimeric, although the mutual disposition of molecules in the two dimers differs. Unlike carboxylesterase, the substrate binding pocket and the active site of hAPT1 are occluded by the dimer interface, suggesting that the enzyme must dissociate upon interaction with substrate.

The G protein connection: molecular basis of membrane association

Two distinct types of lipid modification, myristoylation and isoprenylation, are critical for membrane association of heterotrimeric G proteins. Elucidation of the molecular basis for G protein membrane association has important implications for understanding G protein structure and function, and is relevant to potential therapeutic approaches to AIDS and cancer.

Nitric oxide modulates beta(2)-adrenergic receptor palmitoylation and signaling.

To determine whether nitric oxide (NO) modulates the β-adrenergic signaling pathway, we treated cells expressing β2-adrenergic receptors (β2AR) with the NO donors, 3-morpholinosydnonimine (SIN-1) and 1,2,3,4-oxatriazolium,5-amino-3-(3-chloro-2-methylphenyl)chloride and determined the intracellular production of cAMP after exposure to β-adrenergic receptor agonists, cholera toxin and forskolin. NO significantly decreased the potency of the β-adrenergic agonist, isoproterenol, to stimulate cAMP production without affecting the stimulatory action of forskolin and cholera toxin, which directly activate adenylyl cyclase and Gs, respectively. Treatment with the NO donor increased the guanyl nucleotide-sensitive high affinity constant for the agonist, isoproterenol, thus suggesting that it reduced functional coupling between the receptor and Gs. Stimulation of endogenous NO production by lipopolysaccharide in RAW 264.7 macrophages also caused a significant increase in the EC50 for isoproterenol-stimulated cAMP production. SIN-1 treatment also led to a reduction in both basal and isoproterenol-stimulated incorporation of [3H]palmitate into the β2AR. Signaling through the nonpalmitoylated, Gly341β2AR mutant was unchanged by SIN-1 treatment. Given the link between β2AR palmitoylation and its responsiveness to agonist, these results suggest that the primary action of NO was depalmitoylation of the β2AR resulting in decreased signaling through the β2AR.

Regulation of meiotic prophase arrest in mouse oocytes by GPR3, a constitutive activator of the Gs G protein

The arrest of meiotic prophase in mouse oocytes within antral follicles requires the G protein Gs and an orphan member of the G protein–coupled receptor family, GPR3. To determine whether GPR3 activates Gs, the localization of Gαs in follicle-enclosed oocytes from Gpr3 +/+ and Gpr3 −/− mice was compared by using immunofluorescence and GαsGFP. GPR3 decreased the ratio of Gαs in the oocyte plasma membrane versus the cytoplasm and also decreased the amount of Gαs in the oocyte. Both of these properties indicate that GPR3 activates Gs. The follicle cells around the oocyte are also necessary to keep the oocyte in prophase, suggesting that they might activate GPR3. However, GPR3-dependent Gs activity was similar in follicle-enclosed and follicle-free oocytes. Thus, the maintenance of prophase arrest depends on the constitutive activity of GPR3 in the oocyte, and the follicle cell signal acts by a means other than increasing GPR3 activity.

Hsp90 Interactions and Acylation Target the G Protein Gα12 but Not Gα13 to Lipid Rafts

The heterotrimeric G proteins, G12and G13, are closely related in their sequences, signaling partners, and cellular effects such as oncogenic transformation and cytoskeletal reorganization. Yet G12 and G13can act through different pathways, bind different proteins, and show opposing actions on some effectors. We investigated the compartmentalization of G12 and G13 at the membrane because other G proteins reside in lipid rafts, membrane microdomains enriched in cholesterol and sphingolipids. Lipid rafts were isolated after cold, nonionic detergent extraction of cells and gradient centrifugation. Gα12 was in the lipid raft fractions, whereas Gα13 was not associated with lipid rafts. Mutation of Cys-11 on Gα12, which prevents its palmitoylation, partially shifted Gα12 from the lipid rafts. Geldanamycin treatment, which specifically inhibits Hsp90, caused a partial loss of wild-type Gα12 and a complete loss of the Cys-11 mutant from the lipid rafts and the appearance of a higher molecular weight form of Gα12 in the soluble fractions. These results indicate that acylation and Hsp90 interactions localized Gα12 to lipid rafts. Hsp90 may act as both a scaffold and chaperone to maintain a functional Gα12 only in discrete membrane domains and thereby explain some of the nonoverlapping functions of G12 and G13 and control of these potent cell regulators.

Retinal Vascular Caliber as a Biomarker for Diabetes Microvascular Complications

Globally, the number of people with diabetes is expected to reach 552 million in 2030 (1). Both the macrovascular (coronary artery disease, peripheral artery disease, and stroke) and microvascular (retinopathy, nephropathy, and neuropathy) complications of diabetes are major causes of morbidity and mortality (2). In theory, the long preclinical phase should provide a window to apply interventions that preempt progression to clinical disease, but to date no strategy achieves this aim for diabetes complications. Intensive control of hyperglycemia and blood pressure implemented with the imperfect means available today delays the appearance and reduces the severity of most complications, but does not prevent their development (3). Drugs such as fenofibrate have reduced the risk of diabetic retinopathy (DR) progression in type 2 diabetic patients, and inhibitors of the renin-angiotensin system (e.g., enalapril, losartan) have reduced the risk of progression in type 1 diabetic patients, but have not reduced the risk of DR incidence. Thus, new approaches are required to derive further benefit from current interventions at an earlier stage of diabetes and to support the development of new interventions. One such approach is to identify biomarkers that will enable systematic patient surveillance and the identification of high-risk patients and also surrogate end points that will accelerate the discovery of new interventions. The retinal vessels are early and prevalent targets of diabetic damage. They can be seen, measured, and tested by noninvasive means (4). Hence, investigators have attempted over the decades to identify early changes in the retinal vessels in diabetes and determine whether they could inform about the development of retinopathy and other complications. New measuring techniques have steadily come along, and others are in development. It seems timely to assess which concepts have emerged and which needs must be met toward fulfilling the expectation that retinal vessels may be able …

Prevalence of diabetic peripheral neuropathy and relation to glycemic control therapies at baseline in the BARI 2D cohort

Abstract  We evaluated the associations between glycemic therapies and prevalence of diabetic peripheral neuropathy (DPN) at baseline among participants in the Bypass Angioplasty Revascularization Investigation 2 Diabetes (BARI 2D) trial on medical and revascularization therapies for coronary artery disease (CAD) and on insulin‐sensitizing vs. insulin‐providing treatments for diabetes. A total of 2,368 patients with type 2 diabetes and CAD was evaluated. DPN was defined as clinical examination score >2 using the Michigan Neuropathy Screening Instrument (MNSI). DPN odds ratios across different groups of glycemic therapy were evaluated by multiple logistic regression adjusted for multiple covariates including age, sex, hemoglobin A1c (HbA1c), and diabetes duration. Fifty‐one percent of BARI 2D subjects with valid baseline characteristics and MNSI scores had DPN. After adjusting for all variables, use of insulin was significantly associated with DPN (OR = 1.57, 95% CI: 1.15–2.13). Patients on sulfonylurea (SU) or combination of SU/metformin (Met)/thiazolidinediones (TZD) had marginally higher rates of DPN than the Met/TZD group. This cross‐sectional study in a cohort of patients with type 2 diabetes and CAD showed association of insulin use with higher DPN prevalence, independent of disease duration, glycemic control, and other characteristics. The causality between a glycemic control strategy and DPN cannot be evaluated in this cross‐sectional study, but continued assessment of DPN and randomized therapies in BARI 2D trial may provide further explanations on the development of DPN.

Impact of glycemic control strategies on the progression of diabetic peripheral neuropathy in the Bypass Angioplasty Revascularization Investigation 2 Diabetes (BARI 2D) Cohort.

OBJECTIVE The Bypass Angioplasty Revascularization Investigation 2 Diabetes (BARI 2D) trial demonstrated similar long-term clinical effectiveness of insulin-sensitizing (IS) versus insulin-providing (IP) treatments for type 2 diabetes on cardiovascular outcomes in a cohort with documented coronary artery disease. We evaluated the effects of randomized glycemic control strategy (IS vs. IP) on the prevalence and incidence of diabetic peripheral neuropathy (DPN). RESEARCH DESIGN AND METHODS DPN (defined as Michigan Neuropathy Screening Instrument [MNSI] clinical examination score >2) was assessed at baseline and yearly for 4 years. DPN prevalence and incidence were compared by intention-to-treat modeling by logistic generalized estimating equation models for prevalence and Kaplan-Meier estimates and Cox regression models for incidence rates. RESULTS Results are reported for 2,159 BARI 2D participants (70% males) with valid baseline and at least one follow-up MNSI score (mean age 62 ± 9 years, mean HbA1c 7.7 ± 1.6%, diabetes duration 10 ± 9 years). There were no differences in the prevalence of DPN between the IS and the IP groups throughout the 4 years of follow-up. In 1,075 BARI 2D participants with no DPN at baseline, the 4-year cumulative incidence rate of DPN was significantly lower in the IS (66%) than in the IP (72%) strategy group (P = 0.02), which remained significant after adjusting for the in-trial HbA1c (P = 0.04). In subgroup analyses, IS strategy had a greater benefit in men (hazard ratio 0.75 [99% CI 0.58–0.99], P < 0.01). CONCLUSIONS Among patients with type 2 diabetes followed for up to 4 years during BARI 2D, a glycemic control therapy with IS significantly reduced the incidence of DPN compared with IP therapy and may add further benefit for men.

RGS16 inhibits signalling through the Gα13–Rho axis

Gα13 stimulates the guanine nucleotide exchange factors (GEFs) for Rho, such as p115Rho-GEF. Activated Rho induces numerous cellular responses, including actin polymerization, serum response element (SRE)-dependent gene transcription and transformation. p115Rho-GEF contains a Regulator of G protein Signalling domain (RGS box) that confers GTPase activating protein (GAP) activity towards Gα12 and Gα13 (ref. 3). In contrast, classical RGS proteins (such as RGS16 and RGS4) exhibit RGS domain-dependent GAP activity on Gαi and Gαq, but not Gα12 or Gα13 (ref 4). Here, we show that RGS16 inhibits Gα13-mediated, RhoA-dependent reversal of stellation and SRE activation. The RGS16 amino terminus binds Gα13 directly, resulting in translocation of Gα13 to detergent-resistant membranes (DRMs) and reduced p115Rho-GEF binding. RGS4 does not bind Gα13 or attenuate Gα13-dependent responses, and neither RGS16 nor RGS4 affects Gα12-mediated signalling. These results elucidate a new mechanism whereby a classical RGS protein regulates Gα13-mediated signal transduction independently of the RGS box.