Anti-diabetic activity of Chiliadenus iphionoides.

ETHNOPHARMACOLOGICAL RELEVANCE: Chiliadenus iphionoides (Boiss. & Blanche) Brullo (Asteraceae), a small aromatic shrub found throughout Israel, is used traditionally in the treatment of diabetes mellitus. In this study, Chiliadenus iphionoides anti-diabetic activity was characterized using cellular and animal models. MATERIALS AND METHODS: Pancreatic ß cells, adipocytes, and skeletal myotubes were treated with an ethanolic extract of Chiliadenus iphionoides to study the extract's effects on insulin secretion and glucose uptake. The sand rat (Psammomys obesus) was used to study Chiliadenus iphionoides acute and long term effects in vivo. An oral starch tolerance test was performed as well as a 30 day feeding study. RESULTS: Chiliadenus iphionoides extract increased insulin secretion in ß cells as well as glucose uptake in adipocytes and skeletal myotubes. The extract also displayed hypoglycemic activity in the diabetic sand rat. CONCLUSIONS: Chiliadenus iphionoides exhibits considerable anti-diabetic activity, although the mechanism of action remains to be determined.

Fatty liver is associated with impaired activity of PPARγ-coactivator 1α (PGC1α) and mitochondrial biogenesis in mice.

Accumulating evidence indicates that mitochondria have a key role in non-alcoholic fatty liver disease (NAFLD). C57BL/6J mice were fed a choline-deficient, ethionine-supplemented (CDE) diet. Histological studies demonstrated accumulation of fat vacuoles in up to 90% of hepatocytes in mice fed the CDE diet for 14 days. In addition, a decrease in mitochondrial levels, together with an increase in superoxide radicals' levels were observed, indicating elevation of oxidative stress in hepatocytes. ATP levels were decreased in livers from CDE-fed mice after overnight fasting. This was accompanied by a compensative and significant increase in peroxisome-proliferator-activated receptor-γ coactivator 1α (PGC1α) mRNA levels in comparison to control livers. However, there was a reduction in PGC1α protein levels in CDE-treated mice. Moreover, the expression of mitochondrial biogenesis genes nuclear respiratory factor 1 (NRF-1), mitochondrial transcription factor A (TFAM), mitochondrial transcription factor B1 (TFB1M) and mitochondrial transcription factor B2 (TFB2M), which are all regulated by PGC1α activity, remained unchanged in fasted CDE-treated mice. These results indicate impaired activity of PGC1α. The impaired activity was further confirmed by chromatin immunoprecipitation analysis, which demonstrated decreased interaction of PGC1α with promoters containing NRF-1 and NRF-2 response elements in mice fed the CDE diet. A decrease in PGC1α ability to activate the expression of the gluconeogenic gene phosphoenol-pyruvate carboxykinase was also observed. This study demonstrates, for the first time, that attenuated mitochondrial biogenesis in steatotic livers is associated with impaired biological activity of PGC1α.

The configuration of the alpha and beta subunit domains in single-chain bovine LH analogs influences the secretion and steroidogenic response.

The gonadotrophins LH, FSH and human (h) CG are non-covalent heterodimers composed of a common alpha and the hormone-unique beta subunit. LH regulates the production of androgens and progestins in the follicle, and the levels of these steroids are critical for the ovarian function. Structural features of the gonadotrophins involved in the steroidogenic response of the ovary are not completely understood. As an approach to address how the topology of the ligand affects steroidogenesis we exploited the single-chain (SC) gonadotrophin methodology because manipulating the relative position of the tethered subunit domains in SC hCG analogs enabled to change in the conformation, secretion, receptor binding and adenylyl cyclase activity. We genetically engineered a SC bovine LH analog with a linker derived from the CTP domain of the hCGbeta subunit, NH2-alpha-CTP-LHbeta-COOH (denoted as alphaCTPLHbeta; AB configuration) and evaluated the secretion form transfected CHO cells and steroidogenesis in follicular derived cells in comparison to the variant NH2-LHbeta-CTP-alpha-COOH (LHbetaCTPalpha; BA configuration). The secretion of the analogs from CHO cells was quantitative, and that of alphaCTPLHbeta was more efficient than that of LHbetaCTPalpha The experiments suggested that both variants were N- and O- glycosylated, though the posttranslational modifications are likely to be non-identical in the AB and BA analogs. The analogs stimulated progesterone secretion by immortalized rat granulosa cells that express the rat LH receptor but the EC50 of alphaCTPLHbeta (AB orientation) was higher by 20 fold, as compared to LHbetaCTPalpha (BA). In primary cultures of bovine theca cells, alphaCTPLHbeta stimulated progesterone release with a reduced sensitivity (by at least 50 folds) and smaller magnitude over the basal levels (about 3 folds) relative to LHbetaCTPalpha. In contrast, the accumulation of androstenedione in the media of the same primary cultures appeared to be nearly identical. As a result, the androstenedione/progesterone ratio for the alphaCTPLHbeta analog was significantly increased relative to LHbetaCTPalpha (2-3 folds). This unequal response suggests a distinct regulation of progesterone and androstenedione biosynthesis. Our data demonstrate major differences in steroid balance following stimulation of the receptor with structural LH analogs and provide further insight into gonadotrophin regulation of ovarian steroid production.

The steroidogenic effect of single-chain bovine LH analogs in cultured bovine follicular cells.

Single-chain gonadotropin analogs had been constructed for the purpose of structure-function studies and analog design. Incorporation of a spacer derived from the carboxyl terminal peptide (CTP) of the choriogonadotropin (CG) beta subunit between the tethered subunit domains of the human gonadotropins is beneficial for the secretion of the single-chain variants without compromising biocactivity. Although the CGbeta subunit containing the CTP domain is expressed only in primates and equids, a CTP-like sequence exists in the untranslated region of the LHbeta gene of several mammalian species, including the bovine species. The CTP encrypted in the bovine LHbeta DNA (designated as 'boCTP') and the CTP derived from the human CGbeta subunit (denoted as 'huCTP') served as a linker sequence in the design of bovine single-chain luteinizing hormone (LH) analogs. The purpose of the present study was to evaluate steroidogenesis in cultured bovine theca cells following stimulation with these single-chain analogs. The concentration of the LHbetaboCTPalpha and LHbetahuCTPalpha analogs in the conditioned media of the expressing CHO cells was three- to six-fold higher than that of the "linkerless" LHbetaalpha and LHbeta111alpha variants. The four analogs induced androstenedione and progesterone secretion from the primary theca cells in a dose-dependent manner, but differences in the steroidogenic response were observed. The LHbetaboCTPalpha analog (10 ng/ml) effectively induced androstenedione and progesterone secretion over unstimulated levels (4.0- and 4.4-fold increase for androstenedione and progesterone, respectively). The response to the pituitary bovine LH standard (10 ng/ml) was less pronounced for both steroids (two- to three-fold increase over basal levels). The activities of LHbetahuCTPalpha, LHbetaalpha and LHbeta111alpha were comparable and sightly reduced relative to the LHbetaboCTPalpha activity. The data suggested that LHbetaboCTPalpha was ranked as the most potent and this was even more prominent when analogs were used at a lower dose (1 ng/ml). These data suggest that the design, including the huCTP or boCTP linker, is favorable for the production of single-chain bovine LH analogs. Furthermore, spacing of the tethered subunit domains with the cryptic boCTP sequence that originated from the bovine LHbeta gene appears advantageous for the purpose of stimulating steroid production in the species-specific bioassay. Thus, an effective strategy to produce bioactive single-chain LH analogs in non-primate, non-equid species would be the mutatation of the LHbeta genes with the aim of expressing the cryptic CTP sequence as a spacer derived from the DNA of the same organism.