Inhibitor treatment of peripheral mononuclear cells from Parkinson's disease patients further validates LRRK2 dephosphorylation as a pharmacodynamic biomarker.

Activating mutations in leucine-rich repeat kinase 2 (LRRK2) are strongly associated with increased risk of Parkinson's disease (PD). Thus, LRRK2 kinase inhibitors are in development as potential Parkinson's disease therapeutics. A reduction in the constitutive levels of phosphorylation on leucine-rich repeat kinase 2 (LRRK2) is currently used to measure target engagement of LRRK2 kinase inhibitors in cell and animal models. We aimed to determine if reduced phosphorylation of LRRK2 following inhibitor treatment is also a valid measure of target engagement in peripheral mononuclear cells from Parkinson's disease patients. Peripheral mononuclear cells from idiopathic Parkinson's disease patients and controls were treated ex vivo with two structurally distinct inhibitors of LRRK2, at four different doses, and immunoblotting was used to assess the reduction in LRRK2 phosphorylation at Ser910, Ser935, Ser955 and Ser973. Both inhibitors showed no acute toxicity in primary cells and both inhibitors reduced the constitutive phosphorylation of LRRK2 at all measured residues equally in both control and Parkinson's disease groups. Measuring the reduction in LRRK2 phosphorylation resulting from LRRK2 kinase inhibition, is thus a valid measure of acute peripheral target engagement in Parkinson's disease patients. This is important if LRRK2 kinase inhibitors are to be used in a clinical setting.

Inflammation is genetically implicated in Parkinson's disease.

Inflammation has long been associated with the pathogenesis of Parkinson's disease (PD) but the extent to which it is a cause or consequence is sill debated. Over the past decade a number of genes have been implicated in PD. Relatively rare missense mutations in genes such as LRRK2, Parkin, SNCA and PINK1 are causative for familial PD whereas more common variation in genes, including LRRK2, SNCA and GBA, comprise risk factors for sporadic PD. Determining how the function of these genes and the proteins they encode are altered in PD has become a priority, as results will likely provide much needed insights into contributing causes. Accumulating evidence indicates that many of these genes function in pathways that regulate aspects of immunity, particularly inflammation, suggesting close associations between PD and immune homeostasis.

Contraction-induced skeletal muscle FAT/CD36 trafficking and FA uptake is AMPK independent.

The aim of this study was to investigate the molecular mechanisms regulating FA translocase CD36 (FAT/CD36) translocation and FA uptake in skeletal muscle during contractions. In one model, wild-type (WT) and AMP-dependent protein kinase kinase dead (AMPK KD) mice were exercised or extensor digitorum longus (EDL) and soleus (SOL) muscles were contracted, ex vivo. In separate studies, FAT/CD36 translocation and FA uptake in response to muscle contractions were investigated in the perfused rat hindlimb. Exercise induced a similar increase in skeletal muscle cell surface membrane FAT/CD36 content in WT (+34%) and AMPK KD (+37%) mice. In contrast, 5-aminoimidazole-4-carboxamide ribonucleoside only induced an increase in cell surface FAT/CD36 content in WT (+29%) mice. Furthermore, in the perfused rat hindlimb, muscle contraction induced a rapid (1 min, +15%) and sustained (10 min, +24%) FAT/CD36 relocation to cell surface membranes. The increase in cell surface FAT/CD36 protein content with muscle contractions was associated with increased FA uptake, both in EDL and SOL muscle from WT and AMPK KD mice and in the perfused rat hindlimb. This suggests that AMPK is not essential in regulation of FAT/CD36 translocation and FA uptake in skeletal muscle during contractions. However, AMPK could be important in regulation of FAT/CD36 distribution in other physiological situations.

AMPK-dependent hormonal regulation of whole-body energy metabolism.

AMP-dependent protein kinase (AMPK) is an evolutionarily conserved serine/threonine protein kinase central to the regulation of energy balance at both the cellular and whole-body levels. In its classical role as an intracellular metabolic stress-sensing kinase, AMPK switches on fatty acid oxidation and glucose uptake in muscle, while switching off hepatic gluconeogenesis. AMPK also has a broader role in metabolism through the control of appetite. Regulation of AMPK activity at the whole-body level is coordinated by a growing number of hormones and cytokines secreted from adipose tissue, skeletal muscle, pancreas and the gut including leptin, adiponectin, insulin, interluekin-6, resistin, TNF-alpha and ghrelin. Understanding how these secreted signalling proteins regulate AMPK activity to control fatty acid oxidation, glucose uptake, gluconeogenesis and appetite may yield therapeutic treatments for metabolic disorders such as diabetes, insulin resistance and obesity.

Direct demonstration of lipid sequestration as a mechanism by which rosiglitazone prevents fatty-acid-induced insulin resistance in the rat: comparison with metformin.

AIMS/HYPOTHESIS: Thiazolidinediones can enhance clearance of whole-body non-esterified fatty acids and protect against the insulin resistance that develops during an acute lipid load. The present study used [(3)H]-R-bromopalmitate to compare the effects of the thiazolidinedione, rosiglitazone, and the biguanide, metformin, on insulin action and the tissue-specific fate of non-esterified fatty acids in rats during lipid infusion. METHODS: Normal rats were treated with rosiglitazone or metformin for 7 days. Triglyceride/heparin (to elevate non-esterified fatty acids) or glycerol (control) were then infused for 5 h, with a hyperinsulinaemic clamp being performed between the 3rd and 5th hours. RESULTS: Rosiglitazone and metformin prevented fatty-acid-induced insulin resistance (reduced clamp glucose infusion rate). Both drugs improved insulin-mediated suppression of hepatic glucose output but only rosiglitazone enhanced systemic non-esterified fatty acid clearance (plateau plasma non-esterified fatty acids reduced by 40%). Despite this decrease in plateau plasma non-esterified fatty acids, rosiglitazone increased fatty acid uptake (two-fold) into adipose tissue and reduced fatty acid uptake into liver (by 40%) and muscle (by 30%), as well as reducing liver long-chain fatty acyl CoA accumulation (by 30%). Both rosiglitazone and metformin increased liver AMP-activated protein kinase activity, a possible mediator of the protective effects on insulin action, but in contrast to rosiglitazone, metformin had no significant effect on non-esterified fatty acid kinetics or relative tissue fatty acid uptake. CONCLUSIONS/INTERPRETATION: These results directly demonstrate the "lipid steal" mechanism, by which thiazolidinediones help prevent fatty-acid-induced insulin resistance. The contrasting mechanisms of action of rosiglitazone and metformin could be beneficial when both drugs are used in combination to treat insulin resistance.