PNPLA3 variant M148 causes resistance to starvation-mediated lipid droplet autophagy in human hepatocytes.

The mechanism of how patatin-like phospholipase domain-containing protein 3 (PNPLA3) variant M148 is associated with increased risk of development of hepatic steatosis is still debated. Here, we propose a novel role of PNPLA3 as a key player during autophagosome formation in the process of lipophagy. A human hepatocyte cell line, HepG2 cells, expressing recombinant I148 or 148M, was used to study lipophagy under energy deprived conditions, and lipid droplet morphology was investigated using florescence microscopy, image analysis and biochemical assays. Autophagic flux was studied using the golden-standard of LC3-II turnover in combination with the well characterized GFP-RFP-LC3 vector. To discriminate between, perturbed autophagic initiation and lysosome functionality, lysosomes were characterized by Lysotracker staining and LAMP1 protein levels as well as activity and activation of cathepsin B. For validation, human liver biopsies genotyped for I148 and 148M were analyzed for the presence of LC3-II and PNPLA3 on lipid droplets. We show that the M148-PNPLA3 variant is associated with lipid droplets that are resistant to starvation-mediated degradation. M148 expressing hepatocytes reveal decreased autophagic flux and reduced lipophagy. Both I148-PNPLA3 and M148-PNPLA3 colocalize and interact with LC3-II, but the M148-PNPLA3 variant has lower ability to bind LC3-II. Together, our data indicate that PNPLA3 might play an essential role in lipophagy in hepatocytes and furthermore that the M148-PNPLA3 variant appears to display a loss in this activity, leading to decreased lipophagy.

A six months exercise intervention influences the genome-wide DNA methylation pattern in human adipose tissue.

Epigenetic mechanisms are implicated in gene regulation and the development of different diseases. The epigenome differs between cell types and has until now only been characterized for a few human tissues. Environmental factors potentially alter the epigenome. Here we describe the genome-wide pattern of DNA methylation in human adipose tissue from 23 healthy men, with a previous low level of physical activity, before and after a six months exercise intervention. We also investigate the differences in adipose tissue DNA methylation between 31 individuals with or without a family history of type 2 diabetes. DNA methylation was analyzed using Infinium HumanMethylation450 BeadChip, an array containing 485,577 probes covering 99% RefSeq genes. Global DNA methylation changed and 17,975 individual CpG sites in 7,663 unique genes showed altered levels of DNA methylation after the exercise intervention (q<0.05). Differential mRNA expression was present in 1/3 of gene regions with altered DNA methylation, including RALBP1, HDAC4 and NCOR2 (q<0.05). Using a luciferase assay, we could show that increased DNA methylation in vitro of the RALBP1 promoter suppressed the transcriptional activity (p = 0.03). Moreover, 18 obesity and 21 type 2 diabetes candidate genes had CpG sites with differences in adipose tissue DNA methylation in response to exercise (q<0.05), including TCF7L2 (6 CpG sites) and KCNQ1 (10 CpG sites). A simultaneous change in mRNA expression was seen for 6 of those genes. To understand if genes that exhibit differential DNA methylation and mRNA expression in human adipose tissue in vivo affect adipocyte metabolism, we silenced Hdac4 and Ncor2 respectively in 3T3-L1 adipocytes, which resulted in increased lipogenesis both in the basal and insulin stimulated state. In conclusion, exercise induces genome-wide changes in DNA methylation in human adipose tissue, potentially affecting adipocyte metabolism.

ApoA-I Milano stimulates lipolysis in adipose cells independently of cAMP/PKA activation.

ApoA-I, the main protein component of HDL, is suggested to be involved in metabolic homeostasis. We examined the effects of Milano, a naturally occurring ApoA-I variant, about which little mechanistic information is available. Remarkably, high-fat-fed mice treated with Milano displayed a rapid weight loss greater than ApoA-I WT treated mice, and a significantly reduced adipose tissue mass, without an inflammatory response. Further, lipolysis in adipose cells isolated from mice treated with either WT or Milano was increased. In primary rat adipose cells, Milano stimulated cholesterol efflux and increased glycerol release, independently of ß-adrenergic stimulation and phosphorylation of hormone sensitive lipase (Ser563) and perilipin (Ser522). Stimulation with Milano had a significantly greater effect on glycerol release compared with WT but similar effect on cholesterol efflux. Pharmacological inhibition or siRNA silencing of ABCA1 did not diminish Milano-stimulated lipolysis, although binding to the cell surface was decreased, as analyzed by fluorescence microscopy. Interestingly, methyl-ß-cyclodextrin, a well-described cholesterol acceptor, dose-dependently stimulated lipolysis. Together, these results suggest that decreased fat mass and increased lipolysis following Milano treatment in vivo is partly explained by a novel mechanism at the adipose cell level comprising stimulation of lipolysis independently of the canonical cAMP/protein kinase A signaling pathway.

Single injections of apoA-I acutely improve in vivo glucose tolerance in insulin-resistant mice.

AIMS/HYPOTHESIS: Apolipoprotein A-I (apoA-I), the main protein constituent of HDL, has a central role in the reverse cholesterol-transport pathway, which together with the anti-inflammatory properties of apoA-I/HDL provide cardioprotection. Recent findings of direct stimulation of glucose uptake in muscle by apoA-I/HDL suggest that altered apoA-I and HDL functionality may be a contributing factor to the development of diabetes. We have studied the in vivo effects of short treatments with human apoA-I in a high-fat diet fed mouse model. In addition to native apoA-I, we investigated the effects of the cardioprotective Milano variant (Arg173Cys). METHODS: Male C57Bl6 mice on a high-fat diet for 2 weeks that received a single injection of human apoA-I proteins (wild-type and Milano) were analysed for blood glucose and insulin levels during a 3 h incubation followed by glucose tolerance tests. Incorporation of injected human apoA-I protein into HDLs was analysed by native gel electrophoresis. RESULTS: ApoA-I treatment significantly improved insulin secretion and blood glucose clearance in the glucose tolerance test, with an efficiency exceeding that of lean control animals, and led to decreased basal glucose during the 3 h incubation. Notably, the two apoA-I variants triggered insulin secretion and glucose clearance to the same extent. CONCLUSIONS/INTERPRETATION: ApoA-I treatment leads to insulin- and non-insulin-dependent effects on glucose homeostasis. The experimental model of short-term (2 weeks) feeding of a high-fat diet to C57Bl6 mice provides a suitable and time-efficient system to unravel the resulting tissue-specific mechanisms of acute apoA-I treatment that lead to improved glucose homeostasis.

Adiponutrin: a multimeric plasma protein.

The interest in adiponutrin stems from adiponutrin variant I148M, which is strongly associated to non-alcoholic fatty liver disease. Adiponutrin has to date been considered to be solely an intracellular protein, with a role in lipid metabolism in liver and adipose tissue. However, a physiologically relevant role for adiponutrin has not been found. The aim of this study was to investigate the presence of adiponutrin in human plasma, a new facet of adiponutrin research. We demonstrate that adiponutrin is present in plasma as disulfide-bond dependent multimers, estimated to circulate at a concentration of 1.25-4 nM. Experiments reveal that adiponutrin is released from HepG2 cells in the presence of oleate. The presence of adiponutrin in plasma makes it accessible for clinical investigations and use as a potential biomarker for metabolic disease.

The AMPK-related kinase SIK2 is regulated by cAMP via phosphorylation at Ser358 in adipocytes.

SIK2 (salt-inducible kinase 2) is a member of the AMPK (AMP-activated protein kinase) family of kinases and is highly expressed in adipocytes. We investigated the regulation of SIK2 in adipocytes in response to cellular stimuli with relevance for adipocyte function and/or AMPK signalling. None of the treatments, including insulin, cAMP inducers or AICAR (5-amino-4-imidazolecarboxamide riboside), affected SIK2 activity towards peptide or protein substrates in vitro. However, stimulation with the cAMP-elevating agent forskolin and the ß-adrenergic receptor agonist CL 316,243 resulted in a PKA (protein kinase A)-dependent phosphorylation and 14-3-3 binding of SIK2. Phosphopeptide mapping of SIK2 revealed several sites phosphorylated in response to cAMP induction, including Ser(358). Site-directed mutagenesis demonstrated that phosphorylation of Ser(358), but not the previously reported PKA site Ser(587), was required for 14-3-3 binding. Immunocytochemistry illustrated that the localization of exogenously expressed SIK2 in HEK (human embryonic kidney)-293 cells was exclusively cytosolic and remained unchanged after cAMP elevation. Fractionation of adipocytes, however, revealed a significant increase of wild-type, but not Ser358Ala, HA (haemagglutinin)-SIK2 in the cytosol and a concomitant decrease in a particulate fraction after CL 316,243 treatment. This supports a phosphorylation-dependent relocalization in adipocytes. We hypothesize that regulation of SIK2 by cAMP could play a role for the critical effects of this second messenger on lipid metabolism in adipocytes.

Visualization of lipid directed dynamics of perilipin 1 in human primary adipocytes.

Perilipin 1 is a lipid droplet coating protein known to regulate lipid metabolism in adipocytes by serving as a physical barrier as well as a recruitment site for lipases to the lipid droplet. Phosphorylation of perilipin 1 by protein kinase A rapidly initiates lipolysis, but the detailed mechanism on how perilipin 1 controls lipolysis is unknown. Here, we identify specific lipid binding properties of perilipin 1 that regulate the dynamics of lipolysis in human primary adipocytes. Cellular imaging combined with biochemical and biophysical analyses demonstrate that perilipin 1 specifically binds to cholesteryl esters, and that their dynamic properties direct segregation of perilipin 1 into topologically distinct micro domains on the lipid droplet. Together, our data points to a simple unifying mechanism that lipid assembly and segregation control lipolysis in human primary adipocytes.

Parathyroid hormone induces adipocyte lipolysis via PKA-mediated phosphorylation of hormone-sensitive lipase.

Parathyroid hormone (PTH) is secreted from the parathyroid glands in response to low plasma calcium levels. Besides its classical actions on bone and kidney, PTH may have other important effects, including metabolic effects, as suggested for instance by increased prevalence of insulin resistance and type 2 diabetes in patients with primary hyperparathyroidism. Moreover, secondary hyperparathyroidism may contribute to the metabolic derangements that characterize states of vitamin D deficiency. PTH has been shown to induce adipose tissue lipolysis, but the details of the lipolytic action of PTH have not been described. Here we used primary mouse adipocytes to show that intact PTH (1-84) as well as the N-terminal fragment (1-37) acutely stimulated lipolysis in a dose-dependent manner, whereas the C-terminal fragment (38-84) was without lipolytic effect. The lipolytic action of PTH was paralleled by phosphorylation of known protein kinase A (PKA) substrates, i.e. hormone-sensitive lipase (HSL) and perilipin. The phosphorylation of HSL in response to PTH occurred at the known PKA sites S563 and S660, but not at the non-PKA site S565. PTH-induced lipolysis, as well as phosphorylation of HSL at S563 and S660, was blocked by both the PKA-inhibitor H89 and the adenylate cyclase inhibitor MDL-12330A, whereas inhibitors of extracellular-regulated kinase (ERK), protein kinase B (PKB), AMP-activated protein kinase (AMPK) and Ca(2+)/calmodulin-dependent protein kinase (CaMK) had little or no effect. Inhibition of phosphodiesterase 4 (PDE4) strongly potentiated the lipolytic action of PTH, whereas inhibition of PDE3 had no effect. Our results show that the lipolytic action of PTH is mediated by the PKA signaling pathway with no or minor contribution of other signaling pathways and, furthermore, that the lipolytic action of PTH is limited by simultaneous activation of PDE4. Knowledge of the signaling pathways involved in the lipolytic action of PTH is important for our understanding of how metabolic derangements develop in states of hyperparathyroidism, including vitamin D deficiency.

Berry intake changes hepatic gene expression and DNA methylation patterns associated with high-fat diet.

The liver is a critical organ for regulation of energy homeostasis and fatty liver disease is closely associated with obesity and insulin resistance. We have previously found that lingonberries, blackcurrants and bilberries prevent, whereas açai berries exacerbate, the development of hepatic steatosis and obesity in the high-fat (HF)-fed C57BL/6J mouse model. In this follow-up study, we investigated the mechanisms behind these effects. Genome-wide hepatic gene expression profiling indicates that the protective effects of lingonberries and bilberries are accounted for by several-fold downregulation of genes involved in acute-phase and inflammatory pathways (e.g. Saa1, Cxcl1, Lcn2). In contrast, açai-fed mice exhibit marked upregulation of genes associated with steatosis (e.g. Cfd, Cidea, Crat) and lipid and cholesterol biosynthesis, which is in line with the exacerbation of HF-induced hepatic steatosis in these mice. In silico transcription factor analysis together with immunoblot analysis identified NF-κB, STAT3 and mTOR as upstream regulators involved in mediating the observed transcriptional effects. To gain further insight into mechanisms involved in the gene expression changes, the HELP-tagging assay was used to identify differentially methylated CpG sites. Compared to the HF control group, lingonberries induced genome-wide hypermethylation and specific hypermethylation of Ncor2, encoding the corepressor NCoR/SMRT implicated in the regulation of pathways of metabolic homeostasis and inflammation. We conclude that the beneficial metabolic effects of lingonberries and bilberries are associated with downregulation of inflammatory pathways, whereas for blackcurrants, exerting similar metabolic effects, different mechanisms of action appear to dominate. NF-κB, STAT3 and mTOR are potential targets of the health-promoting effects of berries.

Perilipin 1 binds to aquaporin 7 in human adipocytes and controls its mobility via protein kinase A mediated phosphorylation.

Accumulating evidence suggests that dysregulated glycerol metabolism contributes to the pathophysiology of obesity and type 2 diabetes. Glycerol efflux from adipocytes is regulated by the aquaglyceroporin AQP7, which is translocated upon hormone stimulation. Here, we propose a molecular mechanism where the AQP7 mobility in adipocytes is dependent on perilipin 1 and protein kinase A. Biochemical analyses combined with ex vivo studies in human primary adipocytes, demonstrate that perilipin 1 binds to AQP7, and that catecholamine activated protein kinase A phosphorylates the N-terminus of AQP7, thereby reducing complex formation. Together, these findings are indicative of how glycerol release is controlled in adipocytes, and may pave the way for the future design of drugs against human metabolic pathologies.

cAMP-elevation mediated by ß-adrenergic stimulation inhibits salt-inducible kinase (SIK) 3 activity in adipocytes.

Salt-inducible kinase (SIK) 3 is a virtually unstudied, ubiquitously expressed serine/threonine kinase, belonging to the AMP-activated protein kinase (AMPK)-related family of kinases, all of which are regulated by LKB1 phosphorylation of a threonine residue in their activation (T)-loops. Findings in adrenal cells have revealed a role for cAMP in the regulation of SIK1, and recent findings suggest that insulin can regulate an SIK isoform in Drosophila. As cAMP has important functions in adipocytes, mainly in the regulation of lipolysis, we have evaluated a potential role for cAMP, as well as for insulin, in the regulation of SIK3 in these cells. We establish that raised cAMP levels in response to forskolin and the ß-adrenergic receptor agonist CL 316,243 induce a phosphorylation of SIK3 in HEK293 cells and primary adipocytes. This phosphorylation coincides with increased 14-3-3 binding to SIK3 in these cell types. Our findings also show that cAMP-elevation results in reduced SIK3 activity in adipocytes. Phosphopeptide mapping and site-directed mutagenesis reveal that the cAMP-mediated regulation of SIK3 appears to depend on three residues, T469, S551 and S674, that all contribute to some extent to the cAMP-induced phosphorylation and 14-3-3-binding. As the cAMP-induced regulation can be reversed with the protein kinase A (PKA) inhibitor H89, and a role for other candidate kinases, including PKB and RSK, could be excluded, we believe that PKA is the kinase responsible for SIK3 regulation in response to elevated cAMP levels. Our findings of cAMP-mediated regulation of SIK3 suggest that SIK3 may mediate some of the effects of this important second messenger in adipocytes.

Expression and regulation of cyclic nucleotide phosphodiesterases in human and rat pancreatic islets.

As shown by transgenic mouse models and by using phosphodiesterase 3 (PDE3) inhibitors, PDE3B has an important role in the regulation of insulin secretion in pancreatic ß-cells. However, very little is known about the regulation of the enzyme. Here, we show that PDE3B is activated in response to high glucose, insulin and cAMP elevation in rat pancreatic islets and INS-1 (832/13) cells. Activation by glucose was not affected by the presence of diazoxide. PDE3B activation was coupled to an increase as well as a decrease in total phosphorylation of the enzyme. In addition to PDE3B, several other PDEs were detected in human pancreatic islets: PDE1, PDE3, PDE4C, PDE7A, PDE8A and PDE10A. We conclude that PDE3B is activated in response to agents relevant for ß-cell function and that activation is linked to increased as well as decreased phosphorylation of the enzyme. Moreover, we conclude that several PDEs are present in human pancreatic islets.