Effects of fatty acids on translocation of the γ- and ε-subspecies of protein kinase C (PKC) in living cells were investigated using their proteins fused with green fluorescent protein (GFP). γ-PKC–GFP and ε-PKC–GFP predominated in the cytoplasm, but only a small amount of γ-PKC–GFP was found in the nucleus. Except at a high concentration of linoleic acid, all the fatty acids examined induced the translocation of γ-PKC–GFP from the cytoplasm to the plasma membrane within 30 s with a return to the cytoplasm in 3 min, but they had no effect on γ-PKC–GFP in the nucleus. Arachidonic and linoleic acids induced slow translocation of ε-PKC–GFP from the cytoplasm to the perinuclear region, whereas the other fatty acids (except for palmitic acid) induced rapid translocation to the plasma membrane. The target site of the slower translocation of ε-PKC–GFP by arachidonic acid was identified as the Golgi network. The critical concentration of fatty acid that induced translocation varied among the 11 fatty acids tested. In general, a higher concentration was required to induce the translocation of ε-PKC–GFP than that of γ-PKC–GFP, the exceptions being tridecanoic acid, linoleic acid, and arachidonic acid. Furthermore, arachidonic acid and the diacylglycerol analogue (DiC8) had synergistic effects on the translocation of γ-PKC–GFP. Simultaneous application of arachidonic acid (25 μM) and DiC8 (10 μM) elicited a slow, irreversible translocation of γ-PKC– GFP from the cytoplasm to the plasma membrane after rapid, reversible translocation, but a single application of arachidonic acid or DiC8 at the same concentration induced no translocation.These findings confirm the involvement of fatty acids in the translocation of γ- and ε-PKC, and they also indicate that each subspecies has a specific targeting mechanism that depends on the extracellular signals and that a combination of intracellular activators alters the target site of PKCs.

No abstract

Prostaglandin (PG) F 2␣ suppresses adipocyte differentiation by inhibiting the function of peroxisome proliferatoractivated receptor ␥. However, PGF 2␣ synthase (PGFS) in adipocytes remains to be identified. Here, we studied the expression of members of the aldo-keto reductase (AKR) 1B family acting as PGFS during adipogenesis of mouse 3T3-L1 cells. AKR1B3 mRNA was expressed in preadipocytes, and its level increased about 4-fold at day 1 after initiation of adipocyte differentiation, and then quickly decreased the following day to a level lower than that in the preadipocytes. In contrast, the mRNA levels of Akr1b8 and 1b10 were clearly lower than that level of Akr1b3 in preadipocytes and remained unchanged during adipogenesis. The transient increase in Akr1b3 during adipogenesis was also observed by Western blot analysis. The mRNA for the FP receptor, which is selective for PGF 2␣ , was also expressed in preadipocytes. Its level increased about 2-fold within 1 h after the initiation of adipocyte differentiation and was maintained at almost the same level throughout adipocyte differentiation. The small interfering RNA for Akr1b3, but not for Akr1b8 or 1b10, suppressed PGF 2␣ production and enhanced the expression of adipogenic genes such as peroxisome proliferator-activated receptor ␥, fatty acid-binding protein 4 (aP2), and stearoylCoA desaturase. Moreover, an FP receptor agonist, Fluprostenol, suppressed the expression of those adipogenic genes in 3T3-L1 cells; whereas an FP receptor antagonist, AL-8810, efficiently inhibited the suppression of adipogenesis caused by the endogenous PGF 2␣ . These results indicate that AKR1B3 acts as the PGFS in adipocytes and that AKR1B3-produced PGF 2␣ suppressed adipocyte differentiation by acting through FP receptors.

Background: Gut dysbiosis associated with the use of proton-pump inhibitors (PPIs) has been found to lead to the occurrence of infectious and inflammatory adverse events. A longitudinal observational cohort study has demonstrated the heightened risk of death associated with PPI use. Summary: We evaluated meta-analyses to determine the association between PPI use and infectious and inflammatory diseases. Meta-analyses showed that PPI use is a potential risk for the development of enteric infections caused by Clostridium difficile, as well as small intestinal bacterial overgrowth, spontaneous bacterial peritonitis, community-acquired pneumonia, hepatic encephalopathy, and adverse outcomes in inflammatory bowel disease. We also examined changes in the composition and function of the gut microbiota with the use of PPIs. PPI use significantly increased the presence of Streptococcaceae and Enterococcaceae, which are risk factors for C. difficile infection, and decreased that of Faecalibacterium, a commensal anti-inflammatory microorganism. Key Message: High-throughput, microbial 16S rRNA gene sequencing has allowed us to investigate the association between the gut microbiome and PPI use. Future prospective comparison studies are necessary to confirm this association, and to develop new strategies to prevent complications of PPI use

The molecular mechanisms by which arachidonic acid (AA) and ceramide elicit translocation of protein kinase C (PKC) were investigated. Ceramide translocated ⑀PKC from the cytoplasm to the Golgi complex, but with a mechanism distinct from that utilized by AA. Using fluorescence recovery after photobleaching, we showed that, upon treatment with AA, ⑀PKC was tightly associated with the Golgi complex; ceramide elicited an accumulation of ⑀PKC which was exchangeable with the cytoplasm. Stimulation with ceramide after AA converted the AA-induced Golgi complex staining to one elicited by ceramide alone; AA had no effect on the ceramide-stimulated localization. Using point mutants and deletions of ⑀PKC, we determined that the ⑀C1B domain was responsible for the ceramide-and AA-induced translocation. Switch chimeras, containing the C1B from ⑀PKC in the context of ␦PKC (␦(⑀C1B)) and vice versa (⑀(␦C1B)), were generated and tested for their translocation in response to ceramide and AA. ␦(⑀C1B) translocated upon treatment with both ceramide and AA; ⑀(␦C1B) responded only to ceramide. Thus, through the C1B domain, AA and ceramide induce different patterns of ⑀PKC translocation and the C1B domain defines the subtype specific sensitivity of PKCs to lipid second messengers.The PKC 1 family of serine/threonine protein kinase contains at least 10 subtypes. They are divided into three subgroups based on structural differences and requirement for activators (1-4). The conventional PKCs (cPKC; ␣, ␤I, ␤II, and ␥) are Ca 2ϩ -dependent and activated by diacylglycerol or phorbol esters. The novel PKCs (nPKC; ␦, ⑀, , and ) are activated by diacylglycerol (DG) or phorbol esters, but are Ca 2ϩ -independent (5-7). The atypical PKCs (aPKC; and /) are insensitive to DG/phorbol ester, and are Ca 2ϩ -independent (8 -10). All PKCs possess an amino-terminal regulatory domain and a catalytic domain in the carboxyl terminus. The regulatory domain of the PKCs contains a variable region 1 (V1), a pseudosubstrate motif (PS), and a conserved region 1 (C1). The V1 of ⑀PKC has been reported to be a selective inhibitor of ⑀PKC translocation (11, 12). In the resting state, the PS is bound in the active site of the catalytic domain, keeping the enzyme inactive by blocking the catalytic site. The binding of activators to the regulatory domain causes a conformational change which releases the PS from the active site and activates the enzyme (13). DG and phorbol ester binding have been localized to the C1 domain (2,8,14,15). Additionally, the C1 domain mediates protein-protein interactions: that of ⑀PKC binds actin (16 -18).The C1 domain of cPKCs and nPKCs have two cysteine-rich loops (C1A and C1B), each consisting of ϳ50-amino acids including six cysteine and two histidine residues arranged in a zinc finger motif. The C1B of cPKCs and nPKCs showed strong phorbol esters binding, but all C1A except for ␥PKC showed very weak affinity for phorbol esters (19). GFP-tagged C1A-C1B or C1A translocated to the plasma membrane in response to receptor or phorbol esters s...