A-769662 inhibits adipocyte glucose uptake in an AMPK-independent manner.

Activation of AMP-activated protein kinase (AMPK) is considered a valid strategy for the treatment of type 2 diabetes. However, despite the importance of adipose tissue for whole-body energy homeostasis, the effect of AMPK activation in adipocytes has only been studied to a limited extent and mainly with the AMP-mimetic 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AICAR), which has limited specificity. The aim of this study was to evaluate the effect of the allosteric AMPK activators A-769662 and 991 on glucose uptake in adipocytes. For this purpose, primary rat or human adipocytes, and cultured 3T3-L1 adipocytes, were treated with either of the allosteric activators, or AICAR, and basal and insulin-stimulated glucose uptake was assessed. Additionally, the effect of AMPK activators on insulin-stimulated phosphorylation of Akt and Akt substrate of 160 kDa was assessed. Furthermore, primary adipocytes from ADaM site binding drug-resistant AMPKß1 S108A knock-in mice were employed to investigate the specificity of the drugs for the observed effects. Our results show that insulin-stimulated adipocyte glucose uptake was significantly reduced by A-769662 but not 991, yet neither activator had any clear effects on basal or insulin-stimulated Akt/AS160 signaling. The use of AMPKß1 S108A mutant-expressing adipocytes revealed that the observed inhibition of glucose uptake by A-769662 is most likely AMPK-independent, a finding which is supported by the rapid inhibitory effect A-769662 exerts on glucose uptake in 3T3-L1 adipocytes. These data suggest that AMPK activation per se does not inhibit glucose uptake in adipocytes and that the effects of AICAR and A-769662 are AMPK-independent.

Investigation of the specificity and mechanism of action of the ULK1/AMPK inhibitor SBI-0206965.

SBI-0206965, originally identified as an inhibitor of the autophagy initiator kinase ULK1, has recently been reported as a more potent and selective AMP-activated protein kinase (AMPK) inhibitor relative to the widely used, but promiscuous inhibitor Compound C/Dorsomorphin. Here, we studied the effects of SBI-0206965 on AMPK signalling and metabolic readouts in multiple cell types, including hepatocytes, skeletal muscle cells and adipocytes. We observed SBI-0206965 dose dependently attenuated AMPK activator (991)-stimulated ACC phosphorylation and inhibition of lipogenesis in hepatocytes. SBI-0206965 (≥25 µM) modestly inhibited AMPK signalling in C2C12 myotubes, but also inhibited insulin signalling, insulin-mediated/AMPK-independent glucose uptake, and AICA-riboside uptake. We performed an extended screen of SBI-0206965 against a panel of 140 human protein kinases in vitro, which showed SBI-0206965 inhibits several kinases, including members of AMPK-related kinases (NUAK1, MARK3/4), equally or more potently than AMPK or ULK1. This screen, together with molecular modelling, revealed that most SBI-0206965-sensitive kinases contain a large gatekeeper residue with a preference for methionine at this position. We observed that mutation of the gatekeeper methionine to a smaller side chain amino acid (threonine) rendered AMPK and ULK1 resistant to SBI-0206965 inhibition. These results demonstrate that although SBI-0206965 has utility for delineating AMPK or ULK1 signalling and cellular functions, the compound potently inhibits several other kinases and critical cellular functions such as glucose and nucleoside uptake. Our study demonstrates a role for the gatekeeper residue as a determinant of the inhibitor sensitivity and inhibitor-resistant mutant forms could be exploited as potential controls to probe specific cellular effects of SBI-0206965.

Inhibition of AMPK activity in response to insulin in adipocytes: involvement of AMPK pS485, PDEs, and cellular energy levels.

Insulin resistance in obesity and type 2 diabetes has been shown to be associated with decreased de novo fatty acid (FA) synthesis in adipose tissue. It is known that insulin can acutely stimulate FA synthesis in adipocytes; however, the mechanisms underlying this effect are unclear. The rate-limiting step in FA synthesis is catalyzed by acetyl-CoA carboxylase (ACC), known to be regulated through inhibitory phosphorylation at S79 by the AMP-activated protein kinase (AMPK). Previous results from our laboratory showed an inhibition of AMPK activity by insulin, which was accompanied by PKB-dependent phosphorylation of AMPK at S485. However, whether the S485 phosphorylation is required for insulin-induced inhibition of AMPK or other mechanisms underlie the reduced kinase activity is not known. To investigate this, primary rat adipocytes were transduced with a recombinant adenovirus encoding AMPK-WT or a nonphosphorylatable AMPK S485A mutant. AMPK activity measurements by Western blot analysis and in vitro kinase assay revealed that WT and S485A AMPK were inhibited to a similar degree by insulin, indicating that AMPK S485 phosphorylation is not required for insulin-induced AMPK inhibition. Further analysis suggested an involvement of decreased AMP-to-ATP ratios in the insulin-induced inhibition of AMPK activity, whereas a possible contribution of phosphodiesterases was excluded. Furthermore, we show that insulin-induced AMPK S485 phosphorylation also occurs in human adipocytes, suggesting it to be of an importance yet to be revealed. Altogether, this study increases our understanding of how insulin regulates AMPK activity, and with that, FA synthesis, in adipose tissue.

Persistent whole day meal effects of three dipeptidyl peptidase-4 inhibitors on glycaemia and hormonal responses in metformin-treated type 2 diabetes.

AIM: Dipeptidyl peptidase-4 (DPP-4) inhibition has effects on both fasting and postprandial glucose. However, the extent of this effect over the whole day and whether different DPP-4 inhibitors have the same effects have not been established. We therefore explored the whole day effects of three different DPP-4 inhibitors versus placebo on glucose, islet and incretin hormones after ingestion of breakfast, lunch and dinner in subjects with metformin-treated and well-controlled type 2 diabetes. METHODS: The study was single-centre and crossover designed, involving 24 subjects [12 men, 12 women, mean age 63 years, body mass index 31.0 kg/m2 , glycated haemoglobin 44.7 mmol/mol (6.2%)], who underwent four test days in random order. Each whole day test included ingestion of standardized breakfast (525 kcal), lunch (780 kcal) and dinner (560 kcal) after intake of sitagliptin (100 mg) or vildagliptin (50 mg twice), or saxagliptin (5 mg) or placebo. RESULTS: Compared with placebo, DPP-4 inhibition reduced glucose levels, increased beta-cell function (insulin secretory rate in relation to glucose), suppressed glucagon, increased intact glucagon-like-peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) but suppressed total GLP-1 and GIP after all three meals. The effects were sustained throughout the daytime period with similar changes after each meal and did not differ between the DPP-4 inhibitors. CONCLUSIONS: DPP-4 inhibition has persistent daytime effects on glucose, islet and incretin hormones with no difference between three different DPP-4 inhibitors.

Salt-inducible kinase 2 regulates TFEB and is required for autophagic flux in adipocytes.

Dysregulation of autophagy has been observed in obesity and type 2 diabetes. Salt-inducible kinase 2 (SIK2), a member of the AMPK-related kinase family, is downregulated in adipocytes from obese or insulin resistant individuals and was previously demonstrated to regulate autophagy in cancer and normal cell lines. The aim of this study was thus to investigate a potential role of SIK2 in the regulation of adipocyte autophagy. To do so, SIK2 siRNA silencing or SIKs pharmacological inhibition of SIK2 was employed in murine differentiated 3T3-L1 adipocytes and autophagic flux was monitored. Our data indicate that SIK2 is required for both autophagic flux and expression of TFEB, the transcription factor that regulates autophagy, in adipocytes. The effect of SIK2 on autophagic flux occurs before the regulation of TFEB protein levels, suggesting different mechanisms whereby SIK2 stimulates autophagy. This study broadens the current knowledge on autophagy regulation and SIK2 function in adipocytes.

AMPK activation by A-769662 and 991 does not affect catecholamine-induced lipolysis in human adipocytes.

Activation of AMP-activated protein kinase (AMPK) is considered an attractive strategy for the treatment of type 2 diabetes. Favorable metabolic effects of AMPK activation are mainly observed in skeletal muscle and liver tissue, whereas the effects in human adipose tissue are only poorly understood. Previous studies, which largely employed the AMPK activator 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AICAR), suggest an antilipolytic role of AMPK in adipocytes. The aim of this work was to reinvestigate the role of AMPK in the regulation of lipolysis, using the novel allosteric small-molecule AMPK activators A-769662 and 991, with a focus on human adipocytes. For this purpose, human primary subcutaneous adipocytes were treated with A-769662, 991, or AICAR, as a control, before being stimulated with isoproterenol. AMPK activity status, glycerol release, and the phosphorylation of hormone-sensitive lipase (HSL), a key regulator of lipolysis, were then monitored. Our results show that both A-769662 and 991 activated AMPK to a level that was similar to, or greater than, that induced by AICAR. In contrast to AICAR, which as expected was antilipolytic, neither A-769662 nor 991 affected lipolysis in human adipocytes, although 991 treatment led to altered HSL phosphorylation. Furthermore, we suggest that HSL Ser660 is an important regulator of lipolytic activity in human adipocytes. These data suggest that the antilipolytic effect observed with AICAR in previous studies is, at least to some extent, AMPK independent.

Effect of single-dose DPP-4 inhibitor sitagliptin on ß-cell function and incretin hormone secretion after meal ingestion in healthy volunteers and drug-naïve, well-controlled type 2 diabetes subjects.

To explore the effects of a single dose of the DPP-4 inhibitor sitagliptin on glucose-standardized insulin secretion and ß-cell glucose sensitivity after meal ingestion, 12 healthy and 12 drug-naïve, well-controlled type 2 diabetes (T2D) subjects (mean HbA1c 43 mmol/mol, 6.2%) received sitagliptin (100 mg) or placebo before a meal (525 kcal). ß-cell function was measured as the insulin secretory rate at a standardized glucose concentration and the ß-cell glucose sensitivity (the slope between glucose and insulin secretory rate). Incretin levels were also monitored. Sitagliptin increased standardized insulin secretion, in both healthy and T2D subjects, compared to placebo, but without increasing ß-cell glucose sensitivity. Sitagliptin also increased active glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) and reduced total (reflecting the secretion) GIP, but not total GLP-1 levels. We conclude that a single dose of DPP-4 inhibition induces dissociated effects on different aspects of ß-cell function and incretin hormones after meal ingestion in both healthy and well-controlled T2D subjects.

GFAT1 phosphorylation by AMPK promotes VEGF-induced angiogenesis.

Activation of AMP-activated protein kinase (AMPK) in endothelial cells regulates energy homeostasis, stress protection and angiogenesis, but the underlying mechanisms are incompletely understood. Using a label-free phosphoproteomic analysis, we identified glutamine:fructose-6-phosphate amidotransferase 1 (GFAT1) as an AMPK substrate. GFAT1 is the rate-limiting enzyme in the hexosamine biosynthesis pathway (HBP) and as such controls the modification of proteins by O-linked ß-N-acetylglucosamine (O-GlcNAc). In the present study, we tested the hypothesis that AMPK controls O-GlcNAc levels and function of endothelial cells via GFAT1 phosphorylation using biochemical, pharmacological, genetic and in vitro angiogenesis approaches. Activation of AMPK in primary human endothelial cells by 5-aminoimidazole-4-carboxamide riboside (AICAR) or by vascular endothelial growth factor (VEGF) led to GFAT1 phosphorylation at serine 243. This effect was not seen when AMPK was down-regulated by siRNA. Upon AMPK activation, diminished GFAT activity and reduced O-GlcNAc levels were observed in endothelial cells containing wild-type (WT)-GFAT1 but not in cells expressing non-phosphorylatable S243A-GFAT1. Pharmacological inhibition or siRNA-mediated down-regulation of GFAT1 potentiated VEGF-induced sprouting, indicating that GFAT1 acts as a negative regulator of angiogenesis. In cells expressing S243A-GFAT1, VEGF-induced sprouting was reduced, suggesting that VEGF relieves the inhibitory action of GFAT1/HBP on angiogenesis via AMPK-mediated GFAT1 phosphorylation. Activation of GFAT1/HBP by high glucose led to impairment of vascular sprouting, whereas GFAT1 inhibition improved sprouting even if glucose level was high. Our findings provide novel mechanistic insights into the role of HBP in angiogenesis. They suggest that targeting AMPK in endothelium might help to ameliorate hyperglycaemia-induced vascular dysfunction associated with metabolic disorders.

Salt-inducible kinase 2 and -3 are downregulated in adipose tissue from obese or insulin-resistant individuals: implications for insulin signalling and glucose uptake in human adipocytes.

AIMS/HYPOTHESIS: Salt-inducible kinases (SIKs) are related to the metabolic regulator AMP-activated protein kinase (AMPK). SIK2 is abundant in adipose tissue. The aims of this study were to investigate the expression of SIKs in relation to human obesity and insulin resistance, and to evaluate whether changes in the expression of SIKs might play a causal role in the development of disturbed glucose uptake in human adipocytes. METHODS: SIK mRNA and protein was determined in human adipose tissue or adipocytes, and correlated to clinical variables. SIK2 and SIK3 expression and phosphorylation were analysed in adipocytes treated with TNF-α. Glucose uptake, GLUT protein levels and localisation, phosphorylation of protein kinase B (PKB/Akt) and the SIK substrate histone deacetylase 4 (HDAC4) were analysed after the SIKs had been silenced using small interfering RNA (siRNA) or inhibited using a pan-SIK-inhibitor (HG-9-91-01). RESULTS: We demonstrate that SIK2 and SIK3 mRNA are downregulated in adipose tissue from obese individuals and that the expression is regulated by weight change. SIK2 is also negatively associated with in vivo insulin resistance (HOMA-IR), independently of BMI and age. Moreover, SIK2 protein levels and specific kinase activity display a negative correlation to BMI in human adipocytes. Furthermore, SIK2 and SIK3 are downregulated by TNF-α in adipocytes. Silencing or inhibiting SIK1-3 in adipocytes results in reduced phosphorylation of HDAC4 and PKB/Akt, less GLUT4 at the plasma membrane, and lower basal and insulin-stimulated glucose uptake in adipocytes. CONCLUSION/INTERPRETATION: This is the first study to describe the expression and function of SIKs in human adipocytes. Our data suggest that SIKs might be protective in the development of obesity-induced insulin resistance, with implications for future treatment strategies.

SIK2 regulates CRTCs, HDAC4 and glucose uptake in adipocytes.

Salt-inducible kinase 2 (SIK2) is an AMP-activated protein kinase (AMPK) related kinase abundantly expressed in adipose tissue. Our aim was to identify molecular targets and functions of SIK2 in adipocytes, and to address the role of PKA-mediated phosphorylation of SIK2 on Ser358. Modulation of SIK2 in adipocytes resulted in altered phosphorylation of CREB-regulated transcription co-activator 2 (CRTC2), CRTC3 and class IIa histone deacetylase 4 (HDAC4). Furthermore, CRTC2, CRTC3, HDAC4 and protein phosphatase 2A (PP2A) interacted with SIK2, and the binding of CRTCs and PP2A to wild-type but not Ser358Ala SIK2, was reduced by cAMP elevation. Silencing of SIK2 resulted in reduced GLUT4 (also known as SLC2A4) protein levels, whereas cells treated with CRTC2 or HDAC4 siRNA displayed increased levels of GLUT4. Overexpression or pharmacological inhibition of SIK2 resulted in increased and decreased glucose uptake, respectively. We also describe a SIK2­CRTC2­HDAC4 pathway and its regulation in human adipocytes, strengthening the physiological relevance of our findings. Collectively, we demonstrate that SIK2 acts directly on CRTC2, CRTC3 and HDAC4, and that the cAMP­PKA pathway reduces the interaction of SIK2 with CRTCs and PP2A. Downstream, SIK2 increases GLUT4 levels and glucose uptake in adipocytes.

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.

Cocaine- and amphetamine-regulated transcript (CART) protects beta cells against glucotoxicity and increases cell proliferation.

Cocaine- and amphetamine-regulated transcript (CART) is an islet peptide that promotes glucose-stimulated insulin secretion in beta cells via cAMP/PKA-dependent pathways. In addition, CART is a regulator of neuronal survival. In this study, we examined the effect of exogenous CART 55-102 on beta cell viability and dissected its signaling mechanisms. Evaluation of DNA fragmentation and chromatin condensation revealed that CART 55-102 reduced glucotoxicity-induced apoptosis in both INS-1 (832/13) cells and isolated rat islets. Glucotoxicity in INS-1 (832/13) cells also caused a 50% reduction of endogenous CART protein. We show that CART increased proliferation in INS-1 (832/13) cells, an effect that was blocked by PKA, PKB, and MEK1 inhibitors. In addition, CART induced phosphorylation of CREB, IRS, PKB, FoxO1, p44/42 MAPK, and p90RSK in INS-1 (832/13) cells and isolated rat islets, all key mediators of cell survival and proliferation. Thus, we demonstrate that CART 55-102 protects beta cells against glucotoxicity and promotes proliferation. Taken together our data point to the potential use of CART in therapeutic interventions targeted at enhancing functional beta cell mass and long-term insulin secretion in T2D.

Survival of pancreatic beta cells is partly controlled by a TCF7L2-p53-p53INP1-dependent pathway.

The transcription factor T-cell factor 7-like 2 (TCF7L2) confers type 2 diabetes risk mainly through impaired insulin secretion, perturbed incretin effect and reduced beta-cell survival. The aim of this study was to identify the molecular mechanism through which TCF7L2 influences beta-cell survival. TCF7L2 target genes in INS-1 cells were identified using Chromatin Immunoprecipitation. Validation of targets was obtained by: siRNA silencing, real-time quantitative polymerase chain reaction, electrophoretic mobility shift assay, luciferase reporter assays and western blot. Apoptosis rate was measured by DNA degradation and caspase-3 content. Islet viability was estimated by measuring metabolic rate. TCF7L2 binds to 3646 gene promoters in INS-1 cells in high or low glucose, including Tp53, Pten, Uggt1, Adamts9 and Fto. SiRNA-mediated reduction in TCF7L2 activity resulted in increased apoptosis and increased expression of Tp53, which resulted in elevated p53 protein activity and an increased expression of the p53 target gene Tp53inp1 (encoding p53-induced-nuclear-protein 1). Reversing the increase in p53INP1 protein expression, seen after Tcf7l2 silencing, protected INS-1 cells from Tcf7l2 depletion-induced apoptosis. This result was replicated in primary rat islets. The risk T-allele of rs7903146 is associated with increased TCF7L2 mRNA expression and transcriptional activity. On the other hand, in vitro silencing of TCF7L2 lead to increased apoptosis. One possibility is that the risk T-allele increases expression of an inhibitory TCF7L2 isoform with lower transcriptional activity. These results identify the p53-p53INP1 pathway as a molecular mechanism through which TCF7L2 may affect beta-cell survival and established a molecular link between Tcf7l2 and two type 2 diabetes-associated genes, Tp53inp1 and Adamts9.

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.

Metabolic control by AMPK in white adipose tissue.

White adipose tissue (WAT) plays an important role in the integration of whole-body metabolism by storing fat and mobilizing triacylglycerol when needed. The released free fatty acids can then be oxidized by other tissues to provide ATP. AMP-activated protein kinase (AMPK) is a key regulator of metabolic pathways, and can be targeted by a new generation of direct, small-molecule activators. AMPK activation in WAT inhibits insulin-stimulated lipogenesis and in some situations also inhibits insulin-stimulated glucose uptake, but AMPK-induced inhibition of ß-adrenergic agonist-stimulated lipolysis might need to be re-evaluated in vivo. The lack of dramatic effects of AMPK activation on basal metabolism in WAT could be advantageous when treating type 2 diabetes with pharmacological pan-AMPK activators.

Mechanism of TNFα-induced downregulation of salt-inducible kinase 2 in adipocytes.

Salt-inducible kinase 2 (SIK2) is highly expressed in white adipocytes, but downregulated in individuals with obesity and insulin resistance. These conditions are often associated with a low-grade inflammation in adipose tissue. We and others have previously shown that SIK2 is downregulated by tumor necrosis factor α (TNFα), however, involvement of other pro-inflammatory cytokines, or the mechanisms underlying TNFα-induced SIK2 downregulation, remain to be elucidated. In this study we have shown that TNFα downregulates SIK2 protein expression not only in 3T3L1- but also in human in vitro differentiated adipocytes. Furthermore, monocyte chemoattractant protein-1 and interleukin (IL)-1ß, but not IL-6, might also contribute to SIK2 downregulation during inflammation. We observed that TNFα-induced SIK2 downregulation occurred also in the presence of pharmacological inhibitors against several kinases involved in inflammation, namely c-Jun N-terminal kinase, mitogen activated protein kinase kinase 1, p38 mitogen activated protein kinase or inhibitor of nuclear factor kappa-B kinase (IKK). However, IKK may be involved in SIK2 regulation as we detected an increase of SIK2 when inhibiting IKK in the absence of TNFα. Increased knowledge about inflammation-induced downregulation of SIK2 could ultimately be used to develop strategies for the reinstalment of SIK2 expression in insulin resistance.

Characterization of choroid plexus in the preterm rabbit pup following subcutaneous administration of recombinant human IGF-1/IGFBP-3.

Insulin-like growth factor-1 (IGF-1) is essential for normal brain development and regulates essential processes of vascular maturation and stabilization. Importantly, preterm birth is associated with reduced serum levels of IGF-1 as compared to in utero levels. Using a preterm rabbit pup model, we investigated the uptake of systemic recombinant human (rh) IGF-1 in complex with its main binding protein IGF-binding protein 3 (BP-3) to the brain parenchyma via the choroid plexus. Five hours after subcutaneous administration, labeled rhIGF-1/rhIGFBP-3 displayed a widespread presence in the choroid plexus of the lateral and third ventricle, however, to a less degree in the fourth, as well as in the perivascular and subarachnoid space. We found a time-dependent uptake of IGF-1 in cerebrospinal fluid, decreasing with postnatal age, and a translocation of IGF-1 through the choroid plexus. The impact of systemic rhIGF-1/rhIGFBP-3 on IGF-1 receptor activation in the choroid plexus decreased with postnatal age, correlating with IGF-1 uptake in cerebrospinal fluid. In addition, choroid plexus gene expression was observed to increase with postnatal age. Moreover, using choroid plexus in vitro cell cultures, gene expression and protein synthesis were further investigated upon rhIGF-1/rhIGFBP-3 stimulation as compared to rhIGF-1 alone, and found not to be differently altered. Here, we characterize the uptake of systemic rhIGF-1/rhIGFBP-3 to the preterm brain, and show that the interaction between systemic rhIGF-1/rhIGFBP-3 and choroid plexus varies over time.

Salt-inducible kinases are required for glucose uptake and insulin signaling in human adipocytes.

OBJECTIVE: Salt-inducible kinase 2 (SIK2) is abundantly expressed in adipocytes and downregulated in adipose tissue from individuals with obesity or insulin resistance. The main aims of this work were to investigate the involvement of SIKs in the regulation of glucose uptake in primary mature human adipocytes and to identify mechanisms underlying this regulation. METHODS: Primary mature adipocytes were isolated from human, rat, or mouse adipose tissue and treated with pan-SIK inhibitors. Adipocytes isolated from wild type, ob/ob, and SIK2 knockout mice were also used. Glucose uptake was examined by glucose tracer assay. The insulin signaling pathway was monitored by Western blotting, co-immunoprecipitation, and total internal reflection fluorescence microscopy. RESULTS: This study demonstrates that SIK2 is downregulated in obese ob/ob mice and that SIK activity is required for intact glucose uptake in primary human and mouse adipocytes. The underlying mechanism involves direct effects on the insulin signaling pathway, likely at the level of phosphatidylinositol (3,4,5)-trisphosphate (PIP3) generation or breakdown. Moreover, lack of SIK2 alone is sufficient to attenuate glucose uptake in mouse adipocytes. CONCLUSIONS: SIK2 is required for insulin action in human adipocytes, and the mechanism includes direct effects on the insulin signaling pathway.

AMPKß isoform expression patterns in various adipocyte models and in relation to body mass index.

AMP-activated protein kinase (AMPK) activation is considered a useful strategy for the treatment of type 2 diabetes (T2D). It is unclear whether the expression and/or activity of AMPK in adipocytes is dysregulated in obesity. Also, the expression/activity pattern of AMPKß isoforms, which are targets for AMPK activators, in adipocytes remains elusive. In this study we show that the two AMPKß isoforms make roughly equal contributions to AMPK activity in primary human and mouse adipocytes, whereas in cultured 3T3-L1 adipocytes of mouse origin and in primary rat adipocytes, ß1-associated activity clearly dominates. Additionally, we found that obesity is not associated with changes in AMPK subunit expression or kinase activity in adipocytes isolated from subcutaneous adipose tissue from individuals with various BMI.