Role of maternal Oct-4 during oogenesis and early embryogenesis in the mouse

Bibliography: p. 167-179

Genome-wide operon prediction in Staphylococcus aureus

Identification of operon structure is critical to understanding gene regulation and function, and pathogenesis, and for identifying targets towards the development of new antibiotics in bacteria. Recently, the complete genome sequences of a large number of important human bacterial pathogens have become available for computational analysis, including the major human Gram-positive pathogen Staphylococcus aureus. By annotating the predicted operon structure of the S.aureus genome, we hope to facilitate the exploration of the unique biology of this organism as well as the comparative genomics across a broad range of bacteria. We have integrated several operon prediction methods and developed a consensus approach to score the likelihood of each adjacent gene pair to be co-transcribed. Gene pairs were separated into distinct operons when scores were equal to or below an empirical threshold. Using this approach, we have generated a S.aureus genome map with scores annotated at the intersections of every adjacent gene pair. This approach predicted about 864 monocistronic transcripts and 533 polycistronic operons from the protein-encoding genes in the S.aureus strain Mu50 genome. When compared with a set of experimentally determined S.aureus operons from literature sources, this method successfully predicted at least 91% of gene pairs. At the transcription unit level, this approach correctly identified at least 92% of complete operons in this dataset. This consensus approach has enabled us to predict operons with high accuracy from a genome where limited experimental evidence for operon structure is available

Potentiation of insulin-mediated glucose lowering without elevated hypoglycemia risk by a small molecule insulin receptor modulator.

Insulin resistance is the key feature of type 2 diabetes and is manifested as attenuated insulin receptor (IR) signaling in response to same levels of insulin binding. Several small molecule IR activators have been identified and reported to exhibit insulin sensitization properties. One of these molecules, TLK19781 (Cmpd1), was investigated to examine its IR sensitizing action in vivo. Our data demonstrate that Cmpd1, at doses that produced minimal efficacy in the absence of insulin, potentiated insulin action during an OGTT in non-diabetic mice and enhanced insulin-mediated glucose lowering in diabetic mice. Interestingly, different from insulin alone, Cmpd1 combined with insulin showed enhanced efficacy and duration of action without increased hypoglycemia. To explore the mechanism underlying the apparent glucose dependent efficacy, tissue insulin signaling was compared in healthy and diabetic mice. Cmpd1 enhanced insulin's effects on IR phosphorylation in both healthy and diabetic mice. In contrast, the compound potentiated insulin's effects on Akt phosphorylation in diabetic but not in non-diabetic mice. These differential effects on signaling corresponding to glucose levels could be part of the mechanism for reduced hypoglycemia risk. The in vivo efficacy of Cmpd1 is specific and dependent on IR expression. Results from these studies support the idea of targeting IR for insulin sensitization, which carries low hypoglycemia risk by standalone treatment and could improve the effectiveness of insulin therapies

Combination of cmpd1 with insulin in normoglycemic mice extends glucose lowering duration of action but does not increase hypoglycemia risk.

<p>(A) Glucose lowering in normoglycemic C57BL/6 mice. Cmpd1 (10 or 30 mg/kg) and insulin (1 or 3 U/kg; only 3 U/kg data are shown) were dosed at time 0. Glucose was measured hourly for 4 hours. ** p<0.01 and *** p<0.001 vs. vehicle; # p<0.05 and ## p<0.01 vs. insulin alone. The data are means ± SEM with n = 8 in each group. Individual animal glucose values and their means of 3 U/kg insulin alone (B), 3 U/kg insulin plus 10 mg/kg Cmpd1 (C) or 3 U/kg insulin plus 30 mg/kg Cmpd1 (D) are shown. No significant increase of hypoglycemia was detected in the combination groups.</p

Increased bile acid synthesis and impaired bile acid transport in human obesity

Context: Alterations in bile acid (BA) synthesis and transport have the potential to affect multiple metabolic pathways in the pathophysiology of obesity. Objective: The objective of the study was to investigate the effects of obesity on serum fluctuations of BAs and markers of BA synthesis. Design: We measured BA fluctuations in 11 nonobese and 32 obese subjects and BA transporter expression in liver specimens from 42 individuals and specimens of duodenum, jejunum, ileum, colon, and pancreas from nine individuals. Main Outcome Measures: We analyzed serum BAs and markers of BA synthesis after overnight fasting, during a hyperinsulinemic-euglycemic clamp, or a mixed-meal tolerance test and the association of BA transporter expression with body mass index. Results: BA synthesis markers were 2-fold higher (P < .01) and preferentially 12α-hydroxylated (P < .05) in obese subjects, and both measures were correlated with clamp-derived insulin sensitivity (r = -0.62, P < .0001, and r = -0.39, P = .01, respectively). Insulin infusion acutely reduced serum BAs in nonobese subjects, but this effect was blunted in obese subjects (δBAs -44.2% vs -4.2%, P < .05). The rise in serum BAs postprandially was also relatively blunted in obese subjects (δBAs +402% vs +133%, P < .01). Liver expression of the Na+-taurocholate cotransporting polypeptide and the bile salt export pump were negatively correlated with body mass index (r = -0.37, P = .02, and r = -0.48, P = .001, respectively). Conclusions: Obesity is associated with increased BA synthesis, preferential 12α-hydroxylation, and impaired serum BA fluctuations. The findings reveal new pathophysiological aspects of BA action in obesity that may lend themselves to therapeutic targeting in metabolic disease

Increased bile acid synthesis and deconjugation after Biliopancreatic diversion

Biliopancreatic diversion (BPD) improves insulin sensitivity and decreases serum cholesterol out of proportion with weight loss. Mechanisms of these effects are unknown. One set of proposed contributors to metabolic improvements after bariatric surgeries is bile acids (BAs). We investigated the early and late effects of BPD on plasma BA levels, composition, and markers of BA synthesis in 15 patients with type 2 diabetes (T2D). We compared these to the early and late effects of Roux-en-Y gastric bypass (RYGB) in 22 patients with T2D and 16 with normal glucose tolerance. Seven weeks after BPD, insulin sensitivity had doubled and serum cholesterol had halved. At this time, BA synthesis markers and total plasma BAs, particularly unconjugated BAs, had markedly risen; this effect could not be entirely explained by low FGF19. In contrast, after RYGB, insulin sensitivity improved gradually with weight loss and cholesterol levels declined marginally; BA synthesis markers were decreased at an early time point (2 weeks) after surgery and returned to the normal range 1 year later. These findings indicate that BA synthesis contributes to the decreased serum cholesterol after BPD. Moreover, they suggest a potential role for altered enterohepatic circulation of BAs in improving insulin sensitivity and cholesterol metabolism after BPD

Cmpd1 potentiates insulin-mediated glycemic control.

<p>(A) Glucose lowering in STZ-diabetic mice. Cmpd1 (30 mg/kg) and insulin (0.3 or 1 U/kg) were dosed at time 0. Glucose was measured hourly for 4 hours. *p < 0.05 vs. insulin 1 U/kg. (B) Glucose AUC (0–4 h) calculated according to data in (A). *p < 0.05 and **p<0.01 vs. comparators as indicated. (C) Glucose lowering in db/db mice were conducted similarly as in (A). Cmpd1 (30 mg/kg) and insulin (0.6 or 2 U/kg) were dosed and AUC (0–4 h) calculated accordingly. **p<0.01 and ***p<0.001 vs. comparators as indicated. (D) Glucose tolerance test in normoglycemic mice. Cmpd1 (30 mg/kg) was dosed i.p.at—30 minutes and 5 g /kg glucose (p.o.) and insulin (0.5 U/kg; i.p.) were administered at time 0. Glucose was measured at specified time points up to 120 min. *p < 0.05 and **p<0.01 vs. insulin. The data are means ± SEM with n = 8 in each group.</p

Cmpd1 has differential effects on insulin signaling in diabetic and normoglycemic tissues.

<p>Mice were fasted for 4 hours and received Cmpd1 i.p. and 1 U/kg insulin administration at time 0. 30 min post injection, liver and gastrocnemius muscle were collected to examine insulin signaling. Tissue lysate phospho-protein analysis was conducted using phosphor-IR (Y1150/1151) from Cell Signaling Technology and phospho-Akt (Ser473) Assay kit from Meso Scale Technology. Fold changes of compound treated vs. basal untreated samples were calculated for pIR normoglycemic mice (A), pIR diabetic mice (B), pAkt normoglycemic mice (C) and pAkt diabetic mice (D). Basal liver pIR and pAkt levels in STZ-diabetic mice were ~3 fold higher than that in normoglycemic mice but overall magnitude of stimulation by insulin or Cmpd1 insulin combination are comparable in the two models. Basal muscle pIR and pAkt levels are similar in the two models. Results shown are means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 vs. comparators as indicated; n = 4 per condition.</p

Increased Bile Acid Synthesis and Impaired Bile Acid Transport in Human Obesity

Context: Alterations in bile acid (BA) synthesis and transport have the potential to affect multiple metabolic pathways in the pathophysiology of obesity. Objective: The objective of the study was to investigate the effects of obesity on serum fluctuations of BAs and markers of BA synthesis. Design: We measured BA fluctuations in 11 nonobese and 32 obese subjects and BA transporter expression in liver specimens from 42 individuals and specimens of duodenum, jejunum, ileum, colon, and pancreas from nine individuals. Main Outcome Measures: We analyzed serum BAs and markers of BA synthesis after overnight fasting, during a hyperinsulinemic-euglycemic clamp, or a mixed-meal tolerance test and the association of BA transporter expression with body mass index. Results: BA synthesis markers were 2-fold higher (P < .01) and preferentially 12α-hydroxylated (P < .05) in obese subjects, and both measures were correlated with clamp-derived insulin sensitivity (r = -0.62, P < .0001, and r = -0.39, P = .01, respectively). Insulin infusion acutely reduced serum BAs in nonobese subjects, but this effect was blunted in obese subjects (δBAs -44.2% vs -4.2%, P < .05). The rise in serum BAs postprandially was also relatively blunted in obese subjects (δBAs +402% vs +133%, P < .01). Liver expression of the Na+-taurocholate cotransporting polypeptide and the bile salt export pump were negatively correlated with body mass index (r = -0.37, P = .02, and r = -0.48, P = .001, respectively). Conclusions: Obesity is associated with increased BA synthesis, preferential 12α-hydroxylation, and impaired serum BA fluctuations. The findings reveal new pathophysiological aspects of BA action in obesity that may lend themselves to therapeutic targeting in metabolic disease