Salt-inducible Kinase 3 Signaling Is Important for the Gluconeogenic Programs in Mouse Hepatocytes

Salt-inducible kinases (SIKs), members of the 5′-AMP-activated protein kinase (AMPK) family, are proposed to be important suppressors of gluconeogenic programs in the liver via the phosphorylation-dependent inactivation of the CREB-specific coactivator CRTC2. Although a dramatic phenotype for glucose metabolism has been found in SIK3-KO mice, additional complex phenotypes, dysregulation of bile acids, cholesterol, and fat homeostasis can render it difficult to discuss the hepatic functions of SIK3. The aim of this study was to examine the cell autonomous actions of SIK3 in hepatocytes. To eliminate systemic effects, we prepared primary hepatocytes and screened the small compounds suppressing SIK3 signaling cascades. SIK3-KO primary hepatocytes produced glucose more quickly after treatment with the cAMP agonist forskolin than the WT hepatocytes, which was accompanied by enhanced gluconeogenic gene expression and CRTC2 dephosphorylation. Reporter-based screening identified pterosin B as a SIK3 signaling-specific inhibitor. Pterosin B suppressed SIK3 downstream cascades by up-regulating the phosphorylation levels in the SIK3 C-terminal regulatory domain. When pterosin B promoted glucose production by up-regulating gluconeogenic gene expression in mouse hepatoma AML-12 cells, it decreased the glycogen content and stimulated an association between the glycogen phosphorylase kinase gamma subunit (PHKG2) and SIK3. PHKG2 phosphorylated the peptides with sequences of the C-terminal domain of SIK3. Here we found that the levels of active AMPK were higher both in the SIK3-KO hepatocytes and in pterosin B-treated AML-12 cells than in their controls. These results suggest that SIK3, rather than SIK1, SIK2, or AMPKs, acts as the predominant suppressor in gluconeogenic gene expression in the hepatocytes.     

Fsp27/CIDEC is a CREB target gene induced during early fasting in liver and regulated by FA oxidation rate

FSP27 [cell death-inducing DFFA-like effector c (CIDEC) in humans] is a protein associated with lipid droplets that downregulates the fatty acid oxidation (FAO) rate when it is overexpressed. However, little is known about its physiological role in liver. Here, we show that fasting regulates liver expression of Fsp27 in a time-dependent manner. Thus, during the initial stages of fasting, a maximal induction of 800-fold was achieved, whereas during the later phase of fasting, Fsp27 expression decreased. The early response to fasting can be explained by a canonical PKA-CREB-CRTC2 signaling pathway because: i) CIDEC expression was induced by forskolin, ii) Fsp27 promoter activity was increased by CREB, and iii) Fsp27 expression was upregulated in the liver of Sirt1 knockout animals. Interestingly, pharmacological (etomoxir) or genetic (Hmgcs2 interference) inhibition of the FAO rate increases the in vivo expression of Fsp27 during fasting. Similarly, CIDEC expression was upregulated in HepG2 cells by either etomoxir or HMGCS2 interference. Our data indicate that there is a kinetic mechanism of autoregulation between short- and long-term fasting, by which free FAs delivered to the liver during early fasting are accumulated/exported by FSP27/CIDEC, whereas over longer periods of fasting, they are degraded in the mitochondria through the carnitine palmitoyl transferase system.     

Analysis of microRNA expression profile induced by AICAR in mouse hepatocytes

AMP-activated protein kinase (AMPK) has been proposed to act as a key energy sensor mediating the metabolism of glucose and lipids, and pharmacological activation of AMPK may provide a new strategy for the management of type 2 diabetes. MicroRNAs (miRNAs) are a group of endogenous noncoding RNA that play important roles in many biological processes including energy metabolism. Whether miRNAs mediate AMPK action in regulating metabolic process is not clear. In this study, 0.5 mM 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) was added to increase activation of AMPK in 8 week old C57BL/6 mice primary hepatocytes. MiRNA microarray was performed to compare the miRNA expression profiles of hepatocytes treated with or without AICAR. We discovered that 41 miRNAs were significantly altered in AICAR-treated sample (fold change: > 2) compared with untreated control sample. Among them, 19 miRNAs were upregulated. MiRNA targets were predicted by TargetScan. Further bioinformatic analysis indicated that these predicted targets might be mainly involved in pathways of cellular metabolism and tumor pathogenesis. FUNDO analysis suggested that these predicted targets were enriched in cancer, diabetes mellitus, hypertension, obesity and heart failure (P < 0.01). A series of miRNAs could be regulated by the activation of AMPK and might mediate the action of AMPK during metabolic processes and tumor pathogenesis. Predicted target genes discovered in this study and pathway analysis provide new insights into hepatic metabolism and tumor pathogenesis regulated by AMPK signaling and clues to the possible molecular mechanism underlying the effect of AMPK.     

The contribution of the Drosophila model to lipid droplet research

Intracellular lipid droplets have long been misconceived as evolutionarily conserved but functionally frugal components of cellular metabolism. An ever-growing repertoire of functions has elevated lipid droplets to fully-fledged cellular organelles. Insights into the multifariousness of these organelles have been obtained from a range of model systems now employed for lipid droplet research including the fruit fly, Drosophila melanogaster. This review summarizes the progress in fly lipid droplet research along four main avenues: the role of lipid droplets in fat storage homeostasis, the control of lipid droplet structure, the lipid droplet surface as a dynamic protein-association platform, and lipid droplets as mobile organelles. Moreover, the research potential of the fruit fly model is discussed with respect to the prevailing general questions in lipid droplet biology.