LRRK2 negatively regulates glucose tolerance via regulation of membrane translocation of GLUT4 in adipocytes.

Epidemiological studies have shown that abnormalities of glucose metabolism are involved in leucine-rich repeat kinase 2 (LRRK2)-associated Parkinson's disease (PD). However, the physiological significance of this association is unclear. In the present study, we investigated the effect of LRRK2 on high-fat diet (HFD)-induced glucose intolerance using Lrrk2-knockout (KO) mice. We found for the first time that HFD-fed KO mice display improved glucose tolerance compared with their wild-type (WT) counterparts. In addition, high serum insulin and leptin, as well as low serum adiponectin resulting from HFD in WT mice were improved in KO mice. Using western blotting, we found that Lrrk2 is highly expressed in adipose tissues compared with other insulin-related tissues that are thought to be important in glucose tolerance, including skeletal muscle, liver, and pancreas. Lrrk2 expression and phosphorylation of its kinase substrates Rab8a and Rab10 were significantly elevated after HFD treatment in WT mice. In cell culture experiments, treatment with a LRRK2 kinase inhibitor stimulated insulin-dependent membrane translocation of glucose transporter 4 (Glut4) and glucose uptake in mouse 3T3-L1 adipocytes. We conclude that increased LRRK2 kinase activity in adipose tissue exacerbates glucose tolerance by suppressing Rab8- and Rab10-mediated GLUT4 membrane translocation.

Klotho Supplementation Reverses Renal Dysfunction and Interstitial Fibrosis in Remnant Kidney.

INTRODUCTION: While recent investigations show that klotho exerts renoprotective actions, it has not been fully addressed whether klotho protein supplementation reverses renal damage. METHODS: The impacts of subcutaneous klotho supplementation on rats with subtotal nephrectomy were examined. Animals were divided into 3 groups: group 1 (short remnant [SR]): remnant kidney for 4 weeks, group 2 (long remnant [LR]): remnant kidney for 12 weeks, and group 3 (klotho supplementation [KL]): klotho protein (20 µg/kg/day) supplementation on the remnant kidney. Blood pressure, blood and urine compositions with conventional methods such as enzyme-linked immunosorbent assay and radioimmunoassay, kidney histology, and renal expressions of various genes were analyzed. In vitro studies were also performed to support in vivo findings. RESULTS: Klotho protein supplementation decreased albuminuria (-43%), systolic blood pressure (-16%), fibroblast growth factor (FGF) 23 (-51%) and serum phosphate levels (-19%), renal angiotensin II concentration (-43%), fibrosis index (-70%), renal expressions of collagen I (-55%), and transforming growth factor ß (-59%) (p < 0.05 for all). Klotho supplementation enhanced fractional excretion of phosphate (+45%), glomerular filtration rate (+76%), renal expressions of klotho (+148%), superoxide dismutase (+124%), and bone morphogenetic protein (BMP) 7 (+174%) (p < 0.05 for all). CONCLUSION: Our data indicated that klotho protein supplementation inactivated renal renin-angiotensin system, reducing blood pressure and albuminuria in remnant kidney. Furthermore, exogenous klotho protein supplementation elevated endogenous klotho expression to increase phosphate excretion with resultant reductions in FGF23 and serum phosphate. Finally, klotho supplementation reversed renal dysfunction and fibrosis in association with improved BMP7 in remnant kidney.

Nrf2 Expression Is Decreased in LRRK2 Transgenic Mouse Brain and LRRK2 Overexpressing SH-SY5Y Cells.

Mutations in leucine rich-repeat kinase 2 (LRRK2) cause autosomal-dominant, late-onset Parkinson's disease (PD). Accumulating evidence indicates that PD-associated LRRK2 mutations induce neuronal cell death by increasing cellular reactive oxygen species levels. However, the mechanism of increased oxidative stress associated with LRRK2 kinase activity remains unclear. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that protects cells from oxidative stress by inducing the expression of antioxidant genes. In the present, it was found that decreased expression of Nrf2 and mRNA expression of its target genes in Lrrk2-transgenic mouse brain and LRRK2 overexpressing SH-SY5Y cells. Furthermore, knockdown of glycogen synthase kinase-3ß (GSK-3ß) recovered Nrf2 expression and mRNA expression of its target genes in LRRK2 overexpressing SH-SY5Y cells. We concluded that since Nrf2 is transcriptional factor for antioxidative responses, therefore, reduction of Nrf2 expression by LRRK2 may be part of a mechanism that LRRK2-induces vulnerability to oxidative stress in neuronal cells.

Air contamination of therapeutic drug monitoring assay reagents results in falsely high plasma ammonia levels.

BACKGROUND: Accumulating evidence shows that contamination of blood samples by atmospheric ammonia affects blood ammonia test levels; however, reports on the effect of ammonia contamination of assay reagents are limited. Here, we aimed to clarify the effect of ammonia contamination of assay reagents, particularly the therapeutic drug monitoring (TDM) reagents, on the detection levels of blood ammonia using enzymatic assays. METHODS: Ammonia gas was measured in the refrigerator compartment of the automatic analyser and the reaction tank water, probe wash water and drain outlets connected to the automatic analyser. At different time points following the closure of the cold storage, ammonia levels in quality control plasma samples were measured using three commercial assay reagents to evaluate the effect of air contamination. The distribution of evaporated ammonia in the reagent was measured using the CicaLiquid NH3 assay kit containing the assay reagent most affected by air contamination. RESULTS: It was confirmed that ammonia gas was generated in the cold storage of the automatic analyser. More than half of the reagents detected >0.25 ppm ammonia, and the highest concentration was detected in the TDM reagent. The ammonia levels obtained using all three reagents increased significantly after 3 h of air contamination. The effect was resolved by measuring a 'dummy' sample or mixing the reagents by inversion. CONCLUSIONS: We demonstrated that air contamination by TDM reagents placed in cold storage could result in significantly falsely high ammonia measurements. Preventing this effect would improve the accuracy of ammonia measurements.

In vitro study on effect of bardoxolone methyl on cisplatin-induced cellular senescence in human proximal tubular cells.

Bardoxolone methyl [methyl-2-cyano-3, 12-dioxooleana-1, 9(11)dien-28-oate (CDDO-Me)], an activator of the nuclear factor erythroid-derived 2-related factor2 pathway, is a potential therapeutic candidate for the treatment of kidney diseases. However, its effect against cellular senescence remains unclear. This study aimed to investigate whether CDDO-Me protects cells against cisplatin-induced cellular senescence using an in vitro model. The human renal proximal tubular epithelial cell line HK-2 was treated with cisplatin for 6 h, followed by treatment with or without CDDO-Me (0.1 or 0.2 µmol/L). Senescence markers were analyzed using western blotting and real-time PCR. Apoptosis was evaluated through TUNEL staining. Cisplatin induced changes in the levels of markers specific for proliferation, cell cycle, and senescence in a time- and dose-dependent manner. Furthermore, IL-6 and IL-8 levels in the culture medium increased markedly. These data suggested that cellular senescence-like alterations occurred in HK-2 cells exposed to cisplatin. CDDO-Me treatment reversed the cisplatin-mediated alterations in the levels of cellular senescence markers. The antioxidant enzymes, HO1, NQO1, GPX1, and CAT were upregulated by CDDO-Me treatment. Furthermore, CDDO-Me treatment induced apoptosis in cisplatin-exposed HK-2 cells. Pretreatment with Ac-DEVD-CHO, the caspase inhibitor, suppressed the reversal effect of CDDO-Me against cisplatin-induced cellular senescence-like alterations. This study showed that CDDO-Me attenuated cisplatin-induced premature senescence of HK-2 cells. This beneficial effect may be related to Nrf2 activation. Our findings also showed that CDDO-Me induced apoptosis in cisplatin-treated HK-2 cells, potentially protecting the kidneys from cellular senescence. CDDO-Me appears to be a candidate treatment for acute kidney injury.

LRRK2 Inhibition Ameliorates Dexamethasone-Induced Glucose Intolerance via Prevents Impairment in GLUT4 Membrane Translocation in Adipocytes.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are associated with Parkinson's disease. LRRK2 is a large protein with multiple functional domains, including a guanosine 5'-triphosphate (GTP)-binding domain and a protein kinase domain. Recent studies indicated that the members of the Rab GTPase family, Rab8a and Rab10, which are involved in the membrane transport of the glucose transporter type 4 (GLUT4) during insulin-dependent glucose uptake, are phosphorylated by LRRK2. However, the physiological role of LRRK2 in the regulation of glucose metabolism is largely unknown. In the present study, we investigated the role of LRRK2 using dexamethasone (DEX)-induced glucose intolerance in mice. LRRK2 knockout (KO) mice exhibited suppressed glucose intolerance, even after treatment with DEX. The phosphorylation of LRRK2, Rab8a and Rab10 was increased in the adipose tissues of DEX-treated wild-type mice. In addition, inhibition of the LRRK2 kinase activity prevented the DEX-induced inhibition of GLUT4 membrane translocation and glucose uptake in cultured 3T3-L1 adipocytes. These results suggest that LRRK2 plays an important role in glucose metabolism in adipose tissues.

Oxidative stress increases megalin expression in the renal proximal tubules during the normoalbuminuric stage of diabetes mellitus.

Megalin, an endocytic receptor expressed in proximal tubule cells, plays a critical role in renal tubular protein reabsorption and is associated with the albuminuria observed in diabetic nephropathy. We have previously reported increased oxidant production in the renal cortex during the normoalbuminuric stage of diabetes mellitus (DM); however, the relationship between oxidative stress and renal megalin expression during the normoalbuminuric stage of DM remains unclear. In the present study, we evaluated whether oxidative stress affects megalin expression in the normoalbuminuric stage of DM in a streptozotocin-induced diabetic rat model and in immortalized human proximal tubular cells (HK-2). We demonstrated that increased expression of renal megalin accompanies oxidative stress during the early stage of DM, before albuminuria development. Telmisartan treatment prevented the diabetes-induced elevation in megalin level, possibly through an oxidative stress-dependent mechanism. In HK-2 cells, hydrogen peroxide significantly increased megalin levels in a dose- and time-dependent manner; however, the elevation in megalin expression was decreased following prolonged exposure to severe oxidative stress induced by 0.4 mmol/l hydrogen peroxide. High-glucose treatment also significantly increased megalin expression in HK-2 cells. Concurrent administration of the antioxidant N-acetyl-cysteine blocked the effects of high glucose on megalin expression. Furthermore, the hydrogen peroxide-induced increase in megalin expression was blocked by treatment with phosphatidylinositol 3-kinase and Akt inhibitors. Increase of phosphorylated Akt expression was also seen in the renal cortex of diabetic rats. Taken together, our results indicate that mild oxidative stress increases renal megalin expression through the phosphatidylinositol 3-kinase-Akt pathway in the normoalbuminuric stage of DM.

Heat-shock protein 72 promotes platelet aggregation induced by various platelet activators in rats.

Increase of thrombus in the coronary arteries is positively correlated with the level of heat-shock protein 72 (HSP72) in the blood of patients with acute myocardial infarction (AMI). Platelet aggregation participates in thrombus formation on ruptured plaque in AMI. In this study, we aimed to clarify the role of HSP72 in thrombus formation by evaluating the effects of HSP72 on platelet aggregation. Platelet aggregation activities were measured in platelet-rich plasma obtained from male Sprague-Dawley rats with or without the platelet activators, such as adenosine diphosphate (ADP), collagen, thrombin receptor-activating peptide-6 (TRAP-6), ristocetin, and arachidonic acid. Changes in aggregation were estimated by the co-addition of recombinant HSP72 and anti-HSP72 antibodies. Our results showed that addition of HSP72 increased platelet aggregation in the presence of low concentrations of ADP, collagen, TRAP-6, ristocetin, and arachidonic acid. Increased platelet aggregation stimulated by ADP and HSP72 was reduced by the co-addition of anti-HSP72 antibodies. Thus, these findings suggested that HSP72 was released extracellularly in response to stress, promoting thrombus formation and AMI. Additionally, treatment with anti-HSP72 antibodies may control platelet aggregation induced by extracellular HSP72.

Aminophylline increases respiratory muscle activity during hypercapnia in humans.

BACKGROUND: Theophylline is an old drug traditionally used as a bronchodilator, although it was recently shown to possess anti-inflammatory properties, enhance the actions of corticosteroid actions, and stimulate the respiratory neuronal network. Theophylline has been recognized as an important drug for not only asthma but also corticosteroid-insensitive chronic obstructive pulmonary disease (COPD). To clarify the role of theophylline in hypercapnic ventilatory responses in humans, we analyzed the effects of aminophylline administered at the usual clinical therapeutic doses on ventilation and augmentation of respiratory muscle contractility in room air and under 3 conditions of hypercapnia. STUDY DESIGN: We performed electromyography (EMG) of the parasternal intercostal muscle (PARA) and transversus abdominis muscle (TA) in 7 healthy subjects and recorded both ventilatory parameters and EMG data in room air and under 3 conditions of hypercapnia before (control) and during aminophylline administration. RESULTS: Before aminophylline administration (control), hypercapnic stimulation elicited ventilatory augmentation in a hypercapnia intensity-dependent manner. Ventilatory parameters (tidal volume, frequency of respiration, and minute ventilation) showed significant increases from lower PaCO2 levels during aminophylline administration when compared with the corresponding values before aminophylline administration. EMG activity of both PARA and TA increased significantly at each level of hypercapnia, and those augmentations were shown from lower PaCO2 levels during aminophylline administration. CONCLUSION: Aminophylline administered at the usual clinical therapeutic dose increases ventilation and EMG activity of both inspiratory and expiratory muscles during hypercapnia in healthy humans.

Is there a relation between triglyceride concentrations in very low density lipoprotein and the index of insulin resistance in nondiabetic subjects?

BACKGROUND: Serum very low density lipoprotein (VLDL) levels increase during the early stages of insulin resistance; therefore, determination of VLDL levels would be useful for evaluating the progression of metabolic syndrome and diabetes mellitus. The aim of this study was to clarify the clinical utility of triglyceride in VLDL (VLDL-TG) level, determined using a homogeneous assay kit (Shino-test Corporation, Tokyo, Japan), as an index of insulin resistance. METHODS: We enrolled 74 subjects in this study (diabetic subjects, n = 42; nondiabetic subjects, n = 32). The levels of VLDL-TG, remnant-like lipoprotein particle cholesterol, preheparin lipoprotein lipase mass, and other biochemical markers were determined. RESULTS: VLDL-TG levels were significantly higher in the diabetic group (1.04 ± 0.84 mmol/l vs. 0.64 ± 0.42 mmol/l, P < 0.01) than in the nondiabetic group. In the nondiabetic group, VLDL-TG was significantly correlated with the homeostasis model assessment of insulin resistance (HOMA-IR), the index for insulin resistance (r = 0.513, P = 0.003). VLDL-TG levels, but not TG levels, were higher in the highest quartile (HOMA-IR) of the nondiabetic group. CONCLUSION: VLDL-TG level was a useful early marker for insulin resistance, especially in nondiabetic subjects. The homogeneous VLDL-TG assay is a simple, low-cost method for determining insulin resistance.

Semiquantitative analysis of apolipoprotein A-I modified by advanced glycation end products in diabetes mellitus.

BACKGROUND: Apolipoprotein A-I (Apo A-I), the major component of high-density lipoprotein (HDL), is modified by reactive α-oxoaldehydes, such as methylglyoxal (MG) and glycolaldehyde (GA), and these modifications affect the function of Apo A-I. GA- and MG-modified Apo A-I serum levels were semiquantitatively evaluated in diabetic patients to elucidate the association of each protein with diabetes and to determine its appropriateness as a serum marker of diabetes. METHODS: We enrolled 44 subjects in this study (diabetic subjects, n = 24; nondiabetic subjects, n = 20). GA- and MG-modified Apo A-I levels in serum were determined by sandwich enzyme-linked immunosorbent assay (ELISA) by using anti-GA or anti-MG antibody and anti-Apo A-I antibody. RESULTS: The GA-modified Apo A-I levels did not significantly differ between the diabetic and nondiabetic subjects (1.00 ± 0.38 vs. 0.96 ± 0.22). However, the MG-modified Apo A-I levels in the diabetic subjects were significantly higher than those in the nondiabetic subjects (1.33 ± 0.52 vs. 0.90 ± 0.20). In addition, MG-modified Apo A-I levels correlated with the glycated hemoglobin (HbA1c) levels, HDL-cholesterol levels, and the homeostasis model assessments of insulin resistance, which are indicators of insulin resistance. CONCLUSION: The MG-modified Apo A-I level may be an indicator of diabetic dyslipidemia and insulin resistance.

Phosphoglucomutase activity as a novel biomarker in patients with acute myocardial infarction.

BACKGROUND: Phosphoglucomutase (PGM), a key enzyme in cellular glucose utilization and energy homeostasis, has been reported to show a relationship with oxidative stress. However, the clinical importance of PGM activity has not been investigated in patients with ischemic heart disease (IHD). The aim of the present pilot study was to clarify whether PGM activity has potential as a cardiovascular risk predictor in patients with IHD. METHODS AND RESULTS: The levels of serum PGM activity in 237 patients with IHD (63 patients with acute myocardial infarction (AMI) and 174 patients with stable effort angina pectoris (EAP)) were evaluated. PGM activity was compared with levels of various myocardial, thrombosis, and inflammatory biomarkers on admission. PGM activity in the AMI group was significantly increased relative to that in the EAP group on admission (AMI, 55.5 µmol·min(-1)·L(-1) (U/L); EAP, 14.4 U/L (P<0.001)), and was observed to increase in parallel with well-established myocardial markers (P<0.001). Moreover, PGM activity and the lipid, thrombosis, and inflammatory biomarkers in the AMI group were higher than those in the EAP group. CONCLUSIONS: PGM activity increased with levels of myocardial, thrombosis, and inflammatory biomarkers in patients with AMI, and might be useful in diagnostic applications during the acute phase in patients with AMI.

With a novel evaluation method comparison of 2 homogeneous assay kits for high density lipoprotein cholesterol.

BACKGROUND: 2 homogeneous assay kits, Determiner L HDL-C from Kyowa Medex, Co., Ltd. (Tokyo, Japan) (KWM) and Cholestest NHDL from Daiichi Pure Chemical, Co., Ltd. (Tokyo, Japan) (DIC), for HDL-C are widely used in laboratory tests worldwide. Meanwhile, it was reported that free cholesterol (FC) in HDL is not measured with one of these kits. We devised a novel evaluation method and analyzed the 2 kits in detail. METHODS: Using HPLC, the residual amounts of cholesterol in reaction mixtures were compared before and after reaction with homogeneous reagents. Healthy sera were reacted with homogeneous reagents or with heated, non-enzyme reagents from each kit. The FC and total cholesterol in each lipoprotein were continuously measured with HPLC. RESULTS: With KWM, the HDL-C was measured >95%.With DIC, all FC in sera was eliminated in the first reaction, and FC in HDL was not measured. Moreover, the peak X that was assumed to be a part of HDL was separated with DIC, and the cholesterol in the peak X was not measured. CONCLUSIONS: 2 HDL-C assay kits were compared using a novel evaluation method. KWM was good overall. DIC was found to have 2 problems: 1) FC in HDL was not measured, and 2) the cholesterol in peak X that was assumed to be a part of HDL was not measured.