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.

MAFB as a novel regulator of human adipose tissue inflammation.

AIMS/HYPOTHESIS: Dysregulated expression of metabolic and inflammatory genes is a prominent consequence of obesity causing insulin resistance and type 2 diabetes. Finding causative factors is essential to understanding progression of these pathologies and discovering new therapeutic targets. The transcription factor V-maf musculoaponeurotic fibrosarcoma oncogene homologue B (MAFB) is highly expressed in human white adipose tissue (WAT). However, its role in the regulation of WAT function is elusive. We aimed to characterise MAFB expression and function in human WAT in the context of obesity and insulin resistance. METHODS: MAFB mRNA expression was evaluated in human WAT from seven cohorts with large inter-individual variation in BMI and metabolic features. Insulin-induced adipocyte lipogenesis and lipolysis were measured and correlated with MAFB expression. MAFB regulation during adipogenesis and the effects of MAFB suppression in human adipocytes was investigated. MAFB regulation by TNF-α was examined in human primary adipocytes and THP-1 monocytes/macrophages. RESULTS: MAFB expression in human adipocytes is upregulated during adipogenesis, increases with BMI in WAT, correlates with adverse metabolic features and is decreased after weight loss. MAFB downregulation decreases proinflammatory gene expression in adipocytes and interferes with TNF-α effects. Interestingly, MAFB is differentially regulated by TNF-α in adipocytes (suppressed) and THP-1 cells (upregulated). Further, MAFB is primarily expressed in WAT macrophages/monocytes and its expression correlates with macrophage and inflammatory markers. CONCLUSIONS/INTERPRETATION: Our findings indicate that MAFB is a regulator and a marker of adipose tissue inflammation, a process that subsequently causes insulin resistance.

Liver X receptor (LXR) regulates human adipocyte lipolysis.

The Liver X receptor (LXR) is an important regulator of carbohydrate and lipid metabolism in humans and mice. We have recently shown that activation of LXR regulates cellular fuel utilization in adipocytes. In contrast, the role of LXR in human adipocyte lipolysis, the major function of human white fat cells, is not clear. In the present study, we stimulated in vitro differentiated human and murine adipocytes with the LXR agonist GW3965 and observed an increase in basal lipolysis. Microarray analysis of human adipocyte mRNA following LXR activation revealed an altered gene expression of several lipolysis-regulating proteins, which was also confirmed by quantitative real-time PCR. We show that expression and intracellular localization of perilipin1 (PLIN1) and hormone-sensitive lipase (HSL) are affected by GW3965. Although LXR activation does not influence phosphorylation status of HSL, HSL activity is required for the lipolytic effect of GW3965. This effect is abolished by PLIN1 knockdown. In addition, we demonstrate that upon activation, LXR binds to the proximal regions of the PLIN1 and HSL promoters. By selective knock-down of either LXR isoform, we show that LXRα is the major isoform mediating the lipolysis-related effects of LXR. In conclusion, the present study demonstrates that activation of LXRα up-regulates basal human adipocyte lipolysis. This is at least partially mediated through LXR binding to the PLIN1 promoter and down-regulation of PLIN1 expression.

Insulin induces Thr484 phosphorylation and stabilization of SIK2 in adipocytes.

AIMS/HYPOTHESIS: Salt-inducible kinase 2 (SIK2) is downregulated in adipose tissue from obese or insulin-resistant individuals and inhibition of SIK isoforms results in reduced glucose uptake and insulin signalling in adipocytes. However, the regulation of SIK2 itself in response to insulin in adipocytes has not been studied in detail. The aim of our work was to investigate effects of insulin on various aspects of SIK2 function in adipocytes. METHODS: Primary adipocytes were isolated from human subcutaneous and rat epididymal adipose tissue. Insulin-induced phosphorylation of SIK2 and HDAC4 was analyzed using phosphospecific antibodies and changes in the catalytic activity of SIK2 with in vitro kinase assay. SIK2 protein levels were analyzed in primary adipocytes treated with the proteasome inhibitor MG132. RESULTS: We have identified a novel regulatory pathway of SIK2 in adipocytes, which involves insulin-induced phosphorylation at Thr484. This phosphorylation is impaired in individuals with a reduced insulin action. Insulin stimulation does not affect SIK2 catalytic activity or cellular activity towards HDAC4, but is associated with increased SIK2 protein levels in adipocytes. CONCLUSION/INTERPRETATION: Our data suggest that downregulation of SIK2 in the adipose tissue of insulin-resistant individuals can partially be caused by impaired insulin signalling, which might result in defects in SIK2 expression and function.