Tong-Jin Zhao

Ghrelin O-acyltransferase (GOAT) is essential for growth hormone-mediated survival of calorie-restricted mice

Ghrelin O-acyltransferase (GOAT) attaches octanoate to proghrelin, which is processed to ghrelin, an octanoylated peptide hormone that stimulates release of growth hormone (GH) from pituitary cells. Elimination of the gene encoding ghrelin or its receptor produces only mild phenotypes in mice. Thus, the essential function of ghrelin is obscure. Here, we eliminate the Goat gene in mice, thereby eliminating all octanoylated ghrelin from blood. On normal or high fat diets, Goat−/− mice grew and maintained the same weights as wild-type (WT) littermates. When subjected to 60% calorie restriction, WT and Goat−/− mice both lost 30% of body weight and 75% of body fat within 4 days. In both lines, fasting blood glucose initially declined equally. After 4 days, glucose stabilized in WT mice at 58–76 mg/dL. In Goat−/− mice, glucose continued to decline, reaching 12–36 mg/dL on day 7. At this point, WT mice showed normal physical activity, whereas Goat−/− mice were moribund. GH rose progressively in calorie-restricted WT mice and less in Goat−/− mice. Infusion of either ghrelin or GH normalized blood glucose in Goat−/− mice and prevented death. Thus, an essential function of ghrelin in mice is elevation of GH levels during severe calorie restriction, thereby preserving blood glucose and preventing death.

Inhibition of ghrelin O-acyltransferase (GOAT) by octanoylated pentapeptides

The discovery of ghrelin O-acyltransferase (GOAT) opens the way to the design of drugs that block the attachment of an octanoyl group to the appetite-stimulating peptide hormone ghrelin, potentially preventing obesity. Here, we develop a biochemical assay that uses membranes from insect cells infected with baculovirus encoding mouse GOAT. The GOAT-containing membranes transferred the [3H]octanoyl group from [3H]octanoyl CoA to recombinant proghrelin in vitro. Transfer depended on the serine at residue 3 of proghrelin, which is the known site of acylation. GOAT also transferred [3H]octanoyl to a pentapeptide containing only the N-terminal five amino acids of proghrelin. GOAT activity could be inhibited by an octanoylated ghrelin pentapeptide, and its potency was enhanced 45-fold when the octanoylated serine-3 was replaced by octanoylated diaminopropionic acid. The data suggest that GOAT is subjected to end-product inhibition and this inhibition is better achieved with substrates having the octanoyl group attached through an amide linkage rather than the corresponding ester. These insights may facilitate the future design of useful inhibitors of GOAT.

Downregulation of AMP-activated protein kinase by Cidea-mediated ubiquitination and degradation in brown adipose tissue

We previously showed that Cidea−/− mice are resistant to diet‐induced obesity through the upregulation of energy expenditure. The AMP‐activated protein kinase (AMPK), consisting of catalytic α subunit and regulatory subunits β and γ, has a pivotal function in energy homoeostasis. We show here that AMPK protein levels and enzymatic activity were significantly increased in the brown adipose tissue of Cidea−/− mice. We also found that Cidea is colocalized with AMPK in the endoplasmic reticulum and forms a complex with AMPK in vivo through specific interaction with the β subunit of AMPK, but not with the α or γ subunit. When co‐expressed with Cidea, the stability of AMPK‐β subunit was dramatically reduced due to increased ubiquitination‐mediated degradation, which depends on a physical interaction between Cidea and AMPK. Furthermore, AMPK stability and enzymatic activity were increased in Cidea−/− adipocytes differentiated from mouse embryonic fibroblasts or preadipocytes. Our data strongly suggest that AMPK can be regulated by Cidea‐mediated ubiquitin‐dependent proteosome degradation, and provide a molecular explanation for the increased energy expenditure and lean phenotype in Cidea‐null mice.

Regulating the Drought-responsive Element (DRE)-mediated Signaling Pathway by Synergic Functions of Trans-active and Trans-inactive DRE Binding Factors in Brassica napus

DREB1/C-repeat binding factor (CBF) is a plant-specific family of transcription factors and plays a crucial role in freeze tolerance. In the present work, two groups of drought-responsive element binding factor (DREB)-like genes were isolated from Brassica napus, named Group I and Group II. The two groups of genes were both induced by low temperature, but the expression of Group I preceded that of Group II. The Group I DREBs could specifically bind with the DRE cis-acting element and activate the expression of downstream genes, but Group II factors were trans-inactive although they still had the ability to bind with DRE, which was confirmed by electrophoretic mobility shift assay. Fluorescence quenching assays indicated that the DRE binding ability of the two groups was similar. Co-expression of Group II could depress the trans-activation activity of Group I DREB in a concentration-dependent manner. These results strongly suggested that the trans-active Group I DREBs were expressed at the early stage of cold stress to open the DRE-mediated signaling pathway in cold stress, whereas the trans-inactive Group II DREBs were expressed at the later stage to close the signal pathway in a competitive manner. The results herein provide a new insight into the regulation mechanisms of the DRE-mediated signaling pathway in response to cold stress.

Induced Ablation of Ghrelin Cells in Adult Mice Does Not Decrease Food Intake, Body Weight, or Response to High-Fat Diet

Injection of the peptide hormone ghrelin stimulates food intake in mice and humans. However, mice born without ghrelin demonstrate no significant loss of appetite. This paradox suggests either that compensation develops in mice born without ghrelin or that ghrelin is not essential for appetite control. To distinguish these possibilities, we generated transgenic mice (Ghrl-DTR) that express the diphtheria toxin receptor in ghrelin-secreting cells. Injection of diphtheria toxin in adulthood ablated ghrelin cells and reduced plasma ghrelin by 80%-95%. Ghrelin cell-ablated mice exhibited no loss of appetite or body weight and no resistance to a high-fat diet. To stimulate food intake in mice by ghrelin injection, we had to raise plasma levels many-fold above normal. Like germline ghrelin-deficient mice, the ghrelin cell-ablated mice developed profound hypoglycemia when subjected to prolonged calorie restriction, confirming that ghrelin acts to maintain blood glucose under famine conditions.

The conserved Ala37 in the ERF/AP2 domain is essential for binding with the DRE element and the GCC box

Four AP2/EREBP genes encoding putative ethylene‐responsive element binding factor (ERF)/AP2 domains were cloned from Brassica napus, and these genes could be induced by low temperature, ethylene, drought, high salinity, abscisic acid and jasmonate treatments. These four genes, named BnDREBIII‐1 to BnDREBIII‐4, were highly homologous and the 37th amino acid was the only difference among their ERF/AP2 domains. BnDREBIII‐1 was demonstrated to be able to bind to both dehydration‐responsive element and the GCC box and transactivate the expression of downstream genes, while BnDREBIII‐4 could bind neither. Further results suggested that Ala37 might play a crucial role in the DNA binding or the stability of the ERF/AP2 domain.

Profound Hypoglycemia in Starved, Ghrelin-deficient Mice Is Caused by Decreased Gluconeogenesis and Reversed by Lactate or Fatty Acids

Background: Calorie-restricted, fat-depleted Goat−/− mice develop profound hypoglycemia resulting from lack of ghrelin-mediated growth hormone release. Results: Hypoglycemia is caused by decreased gluconeogenesis and reversed by gluconeogenic precursors (lactate and alanine) or fatty acids. Conclusion: In absence of fatty acids, growth hormone maintains gluconeogenic precursors, allowing survival. Significance: Maintenance of blood glucose by ghrelin-growth hormone axis is crucial for evolutionary adaptation to starvation. When mice are subjected to 7-day calorie restriction (40% of normal food intake), body fat disappears, but blood glucose is maintained as long as the animals produce ghrelin, an octanoylated peptide that stimulates growth hormone secretion. Mice can be rendered ghrelin-deficient by knock-out of the gene encoding either ghrelin O-acyltransferase, which attaches the required octanoate, or ghrelin itself. Calorie-restricted, fat-depleted ghrelin O-acyltransferase or ghrelin knock-out mice fail to show the normal increase in growth hormone and become profoundly hypoglycemic when fasted for 18–23 h. Glucose production in Goat−/− mice was reduced by 60% when compared with similarly treated WT mice. Plasma lactate and pyruvate were also low. Injection of lactate, pyruvate, alanine, or a fatty acid restored blood glucose in Goat−/− mice. Thus, when body fat is reduced by calorie restriction, ghrelin stimulates growth hormone secretion, which allows maintenance of glucose production, even when food intake is eliminated. In humans with anorexia nervosa or kwashiorkor, ghrelin and growth hormone are known to be elevated, just as they are in fat-depleted mice. We suggest that these two hormones prolong survival in starved humans as they do in mice.

The Generation of the Oxidized Form of Creatine Kinase Is a Negative Regulation on Muscle Creatine Kinase

Muscle creatine kinase (CK) is a crucial enzyme in energy metabolism, and it exists in two forms, the reduced form (R-CK) and the oxidized form (O-CK). In contrast with R-CK, O-CK contained an intrachain disulfide bond in each subunit. Here we explored the properties of O-CK and its regulatory role on muscle CK. The intrachain disulfide bond in O-CK was demonstrated to be formed between Cys74 and Cys146 by site-directed mutagenesis. Biophysical analysis indicated that O-CK showed decreased catalytic activity and that it might be structurally unstable. Further assays through guanidine hydrochloride denaturation and proteolysis by trypsin and protease K revealed that the tertiary structure of O-CK was more easily disturbed than that of R-CK. Surprisingly, O-CK, unlike R-CK, cannot interact with the M-line protein myomesin through biosensor assay, indicating that O-CK might have no role in muscle contraction. Through in vitro ubiquitination assay, CK was demonstrated to be a specific substrate of muscle ring finger protein 1 (MURF-1). O-CK can be rapidly ubiquitinated by MURF-1, while R-CK can hardly be ubiquitinated, implying that CK might be degraded by the ATP-ubiquitin-proteasome pathway through the generation of O-CK. The results above were further confirmed by molecular modeling of the structure of O-CK. Therefore, it can be concluded that the generation of O-CK was a negative regulation of R-CK and that O-CK might play essential roles in the molecular turnover of MM-CK.

Catalysis of Creatine Kinase Refolding by Protein Disulfide Isomerase Involves Disulfide Cross-link and Dimer to Tetramer Switch

Protein disulfide isomerase (PDI) functions as an isomerase to catalyze thiol:disulfide exchange, as a chaperone to assist protein folding, and as a subunit of prolyl-4-hydroxylase and microsomal triglyceride transfer protein. At a lower concentration of 0.2 μm, PDI facilitated the aggregation of unfolded rabbit muscle creatine kinase (CK) and exhibited anti-chaperone activity, which was shown to be mainly due to the hydrophobic interactions between PDI and CK and was independent of the cross-linking of disulfide bonds. At concentrations above 1 μm, PDI acted as a protector against aggregation but an inhibitor of reactivation during CK refolding. The inhibition effect of PDI on CK reactivation was further characterized as due to the formation of PDI-CK complexes through intermolecular disulfide bonds, a process involving Cys-36 and Cys-295 of PDI. Two disulfide-linked complexes containing both PDI and CK were obtained, and the large, soluble aggregates around 400 kDa were composed of 1 molecule of tetrameric PDI and 2 molecules of inactive intermediate dimeric CK, whereas the smaller one, around 200 kDa, was formed by 1 dimeric PDI and 1 dimeric CK. To our knowledge this is the first study revealing that PDI could switch its conformation from dimer to tetramer in its functions as a foldase. According to the observations in this research and our previous study of the folding pathways of CK, a working model was proposed for the molecular mechanism of CK refolding catalyzed by PDI.

Slow skeletal muscle myosin-binding protein-C (MyBPC1) mediates recruitment of muscle-type creatine kinase (CK) to myosin

Muscle contraction requires high energy fluxes, which are supplied by MM-CK (muscle-type creatine kinase) which couples to the myofibril. However, little is known about the detailed molecular mechanisms of how MM-CK participates in and is regulated during muscle contraction. In the present study, MM-CK is found to physically interact with the slow skeletal muscle-type MyBPC1 (myosin-binding protein C1). The interaction between MyBPC1 and MM-CK depended on the creatine concentration in a dose-dependent manner, but not on ATP, ADP or phosphocreatine. The MyBPC1-CK interaction favoured acidic conditions, and the two molecules dissociated at above pH 7.5. Domain-mapping experiments indicated that MM-CK binds to the C-terminal domains of MyBPC1, which is also the binding site of myosin. The functional coupling of myosin, MyBPC1 and MM-CK is further corroborated using an ATPase activity assay in which ATP expenditure accelerates upon the association of the three proteins, and the apparent K(m) value of myosin is therefore reduced. The results of the present study suggest that MyBPC1 acts as an adaptor to connect the ATP consumer (myosin) and the regenerator (MM-CK) for efficient energy metabolism and homoeostasis.