Relationship between weight change and glycaemic control in patients with type 2 diabetes receiving once-weekly dulaglutide treatment.

AIM: To assess the relationship between weight change and glycated haemoglobin (HbA1c) change in dulaglutide-treated patients by analysing data from six head-to-head phase III AWARD clinical trials. METHODS: At 26 weeks, the relationship between weight and HbA1c was analysed in each trial rather than by pooling data because of differences in design and background therapy. The effect of baseline characteristics was also evaluated with regard to weight and HbA1c response. RESULTS: Across the studies, 87-97% and 83-95% of patients treated with dulaglutide 1.5 and 0.75 mg, respectively, had reductions in HbA1c levels, while 57-88% and 43-84% of patients treated with dulaglutide 1.5 and 0.75 mg, respectively, experienced weight loss. The majority (55-83%) of patients receiving dulaglutide 1.5 mg experienced weight loss and HbA1c reductions, while 41-79% of patients in the dulaglutide 0.75 mg arm lost weight and had reductions in HbA1c level. A weak and inconsistent correlation was observed between the changes in weight and HbA1c (range from -0.223 to 0.267) in patients treated with dulaglutide. The baseline characteristics of gender, age, duration of diabetes, HbA1c, body weight and BMI were not related to different combinations of weight and HbA1c responses. CONCLUSIONS: Dulaglutide is an effective treatment option across the type 2 diabetes treatment spectrum. Dulaglutide showed dose-dependent effects on both weight loss and HbA1c reduction. These effects had a weak correlation and appeared to be independent.

Inosine-5'-monophosphate dehydrogenase: regulation of expression and role in cellular proliferation and T lymphocyte activation.

Guanine nucleotide synthesis is essential for the maintenance of normal cell growth and function, as well as for cellular transformation and immune responses. The expression of two genes encoding human inosine-5'-monophosphate dehyrogenase (IMPDH) type I and type II results in the translation of catalytically indistinguishable enzymes that control the rate-limiting step in the de novo synthesis of guanine nucleotides. Cellular IMPDH activity is increased more than 10-fold in activated peripheral blood T lymphocytes and is attributable to the increased expression of both the type I and type II enzymes. In contrast, abrogation of cellular IMPDH activity by selective inhibitors prevents T lymphocyte activation and establishes a requirement for elevated IMPDH activity in T lymphocytic responses. In order to assess the molecular mechanisms governing the expression of the IMPDH type I and type II genes in resting and activated peripheral blood T lymphocytes, we have cloned the human IMPDH type I and type II genes and characterized their genomic organization and their respective 5'-flanking regions. Both genes contain 14 highly conserved exons that vary in size from 49 to 207 base pairs. However, the intron structures are completely divergent, resulting in disparities in gene length (18 kilobases for type I and 5.8 kilobases for type II). In addition, the 5'-regulatory sequences are highly divergent; expression of the IMPDH type I gene is controlled by three distinct promoters in a tissue specific manner while the type II gene is regulated by a single promoter and closely flanked in the 5' region by a gene of unknown function. The conservation of the IMPDH type I and type II coding sequence in the presence of highly divergent 5'-regulatory sequences points to a multifactorial control of enzyme expression and suggests that tissue-specific and/or developmentally specific regulation of expression may be important. Delineation of these regulatory mechanisms will aid in the elucidation of the signaling events that ultimately lead to the synthesis of guanine nucleotides required for cellular entry into S phase and the initiation of DNA replication.

Prevention of calcium-induced membrane structural alterations in erythrocyte membranes by flunarizine.

The calcium antagonist flunarizine is shown to be able to prevent particle aggregation, membrane aggregation and blebbing resulting from elevated calcium concentrations. The anti-ischemic effects of flunarizine may therefore result in part from its ability to directly interfere with calcium-membrane interactions and thus prevent the lethal membrane reorganizations which occur after a period of ischemia during intracellular calcium overload.

Liquid chromatographic determination of nicarbazin in feeds.

A new liquid chromatographic method has been developed for determination of nicarbazin in feeds. Approximately 40 g feed is extracted with 200 mL acetonitrile-water (80 + 20, v/v). An aliquot of the extract is filtered and assayed using a reversed-phase isocratic method that measures the 4,4'-dinitrocarbanilide moiety of nicarbazin at a wavelength of 340 nm. For medicated feeds, the method uses a standard linear range of 5 to 100 microg/mL. For lower levels, a linear range of 50 to 150 ng/mL can be used. The method has a limit of detection of 250 ng/g and a limit of quantitation of 500 ng/g in a 40 g feed sample. Recovery was 99.1%, with a range of 95.2 to 101.8%. In the typical U.S. dosing range of 27 to 113.5 g/ton, the precision of the method based on one analyst, one day, and 2 weighings ranged from 2.8% (113.5 g/ton) to 4.7% (27 g/ton).

Characterization of the human inosine-5'-monophosphate dehydrogenase type II gene.

Inosine-5'-monophosphate dehydrogenase (IMPDH) activity and mRNA levels are induced up to 15-fold upon mitogenic or antigenic stimulation of human peripheral blood T lymphocytes. This increase in IMPDH activity is required for cellular proliferation and has been associated with malignant transformation. We have cloned the human IMPDH type II gene and show that it contains 14 exons and is approximately 5.8 kilobases in length. Exons vary in size from 49 to 207 base pairs and introns from 73 to 1065 base pairs. The transcription start site was mapped to a position 50 nucleotides upstream of the translation initiation site. The 5'-flanking region consisting of 463 base pairs upstream of the translation initiation site confers induced transcription and differential regulation upon a chloramphenicol acetyltransferase reporter gene when transfected into Jurkat T cells and human peripheral blood T lymphocytes, respectively. DNase I footprinting analysis using Jurkat T cell nuclear extract identified four protected regions in the promoter which coincide with consensus transcription factor binding sites for the nuclear factors AP2, ATF, CREB, Egr-1, Nm23, and Sp1. These findings suggest that several of these nuclear factors may play a critical role in the regulation of IMPDH type II gene expression during T lymphocyte activation.

Tissue-specific regulation of the ecto-5'-nucleotidase promoter. Role of the camp response element site in mediating repression by the upstream regulatory region.

We have isolated the 5' region of the ecto-5'-nucleotidase (low K(m) 5'-NT) gene and established that a 969-base pair (bp) fragment confers cell-specific expression of a CAT reporter gene that correlates with the expression of endogenous ecto-5'-NT mRNA and enzymatic activity. A 768-bp upstream negative regulatory region has been identified that conferred lymphocyte-specific negative regulation in a heterologous system with a 244-bp deoxycytidine kinase core promoter. DNase I footprinting identified several protected areas including Sp1, Sp1/AP-2, and cAMP response element (CRE) binding sites within the 201-bp core promoter region and Sp1, NRE-2a, TCF-1/LEF-1, and Sp1/NF-AT binding sites in the upstream regulatory region. Whereas the CRE site was essential in mediating the negative activity of the upstream regulatory region in Jurkat but not in HeLa cells, mutation of the Sp1/AP-2 site decreased promoter activity in both cell lines. Electrophoretic mobility shift assay analysis of proteins binding to the CRE site identified both ATF-1 and ATF-2 in Jurkat cells. Finally, phorbol 12-myristate 13-acetate increased the activity of both the core and the 969-bp promoter fragments, and this increase was abrogated by mutations at the CRE site. In summary, we have identified a tissue-specific regulatory region 5' of the ecto-5'-NT core promoter that requires the presence of a functional CRE site within the basal promoter for its suppressive activity.

Regulation of inosine-5'-monophosphate dehydrogenase type II gene expression in human T cells. Role for a novel 5' palindromic octamer sequence.

Expression of the gene encoding human inosine- 5'-monophosphate dehydrogenase (IMPDH) type II, an enzyme catalyzing the rate-limiting step in the generation of guanine nucleotides, is increased more than 10-fold in activated peripheral blood T lymphocytes and is required for T cell activation. We have examined the 5'-regulatory sequences that are important for the transcriptional regulation of this gene in T cells. DNase I mapping of genomic DNA identified a hypersensitive element near the transcription initiation site. Fine mapping by in vivo footprinting demonstrated five transcription factor binding sites that are occupied in both resting and activated peripheral blood T lymphocytes; these are tandem CRE motifs, a Sp1 site, an overlapping Egr-1/Sp1 site, and a novel palindromic octamer sequence (POS). The tandem CRE and POS sites are of major functional importance as judged by mutational and electrophoretic mobility shift analyses. These data provide evidence that expression of the human IMPDH type II gene is predominantly regulated by the nuclear factors ATF-2 and an as yet unidentified POS-binding protein. Additional major protein-DNA interactions do not occur within the promoter region after T lymphocyte activation, indicating a requirement for additional protein-protein interactions and/or post-translational modifications of pre-bound transcription factors to account for the observed increase in IMPDH type II gene expression.

Chemical evolution of the citric acid cycle: sunlight photolysis of alpha-ketoglutaric acid.

Sunlight photolysis of alpha-ketoglutaric acid produces succinic acid as a major product. Other higher molecular weight products are identified by GC-MS analysis. These results provide further support for the important role of succinic acid in chemical evolution.