Stacy Branch

Glut-1 expression and its response to hypoglycemia in the embryonic mouse heart

The embryonic heart depends on glucose during early organogenesis. Glut-1 functions in constitutive glucose uptake in adult tissues and is the predominant glucose transporter in embryonic and fetal tissues. This study focuses on Glut-1 expression in the heart during normal organogenesis using immunohistochemistry for Glut-1 distribution, Western analysis for Glut-1 protein levels, and reverse transcriptase polymerase chain reaction for Glut-1 mRNA levels. The role of Glut in glucose uptake response to hypoglycemia in the embryonic heart is evaluated using the Glut inhibitor cytochalasin B. Cardiac Glut-1 expression is also evaluated after in vitro hypoglycemic exposure. Glut-1 levels are highest on gestational days 9–10, intermediate on gestational day 10.5, and lowest on gestational days 11.5–13.5 in the normal embryonic heart. Cardiac Glut-1 mRNA levels similarly decline between gestational days 9.5 and gd 13.5. Cytochalasin B produces a dose-dependent decrease in glucose uptake in hearts exposed to hypoglycemia for 30 min or 6 h, implicating Glut in this response. Glut-1 protein expression is unchanged after 2 or 6 h but increased after 12 and 24 h of hypoglycemia in the gestational day 9.5 heart. Thus, Glut-1 expression is prominent in the embryonic heart and is correlated with changes in cardiac glucose requirements during normal organogenesis. Glut activity increases in response to acute hypoglycemia and the expression of Glut-1 increases in response to prolonged hypoglycemia. These results support the importance of Glut-1 during normal cardiogenesis and in response to hypoglycemia in the embryonic heart.

Hexokinase I expression and activity in embryonic mouse heart during early and late organogenesis.

Abstract Hexokinase (HK) catalyzes the first step in glucose metabolism, that is, the conversion of glucose to glucose-6-phosphate (G6P). Four HK isoforms have been identified, of which HK-I is predominant in embryonic and fetal tissues. HK-I has been studied in preimplantation embryos and in fetal stages, but little is known about its activity or expression in the early postimplantation embryo. We evaluated HK-I expression, HK-I activity, and glycolytic metabolism in the embryonic mouse heart during early [gestational day (gd) 9.5] and late (gd 13.5) organogenesis. Immunohistochemistry demonstrated that HK-I is localized mainly in the heart at both stages, with stronger expression on gd 13.5. Densitometry after SDS-PAGE/western analysis confirmed higher immunodetectable HK-I protein levels in hearts on gd 13.5 vs gd 9.5. By contrast, RT-PCR demonstrated higher HK-I mRNA expression on gd 9.5 vs gd 13.5. Similarly, cardiac HK-I activity (conversion of glucose to G6P) and glycolysis (conversion of glucose to lactate) were higher on gd 9.5 than on gd 13.5. These results suggest a complex regulation of HK-I expression and activity in the embryonic heart during organogenesis, involving a change in the intrinsic activity of the enzyme with development. HK-I appears to play an important role in glucose metabolism during this critical stage of cardiogenesis.

Expression of glucose-regulate proteins (GRP78 and GRP94) in hearts and fore-limb buds of mouse embryos exposed to hypoglycemia in vitro

Hypoglycemia, the classic inducer of glucose-related protein (GRP) synthesis, is dysmorphogenic in rodent embryos and detrimentally affects the heart. This study compares GRP induction in a target vs non-target tissue by evaluating GRP expression in hearts and fore-limb buds of mouse embryos following exposure to hypoglycemia in vitro. Gestational day 9.5 embryos were exposed to 2, 6, and 24 h of either mild (80 mg/dl glucose) or severe (40 mg/dl glucose) hypoglycemia using the method of whole-embryo culture. GRP78 increased in a dose- and time-dependent fashion in embryonic hearts exposed to either 40 mg/dl or 80 mg/dl glucose, whereas GRP94 levels increased in hearts only after 24 h of hypoglycemia. In contrast to the heart, GRP induction in fore-limb buds occurred only with GRP78 following the most severe level and duration of hypoglycemia. RT-PCR analysis demonstrated an elevation in GRP78 and GRP94 message levels in embryonic hearts following severe hypoglycemia. However, mRNA levels did not increase in response to mild hypoglycemia. Overall, these data demonstrate the preferential induction of GRPs in the heart as compared to fore-limb buds in mouse embryos exposed to hypoglycemia. Increases in GRP protein levels may be a more reliable biomarker of stress than message levels. However, both tissues and methods should be examined for enhanced biomarker sensitivity.

Differentially expressed genes associated with 5-Aza2′-deoxycytidine-induced hindlimb defects in the Swiss Webster Mouse

5‐Aza‐2′‐deoxycytidine (d‐AZA) inhibits methylation of DNA, a process that serves as an epigenetic regulator of gene expression. We have shown that d‐AZA causes temporally related defects in mice. Gestational day (GD) 10 treatment induced severe long‐bone defects of the hindlimb but not the forelimb. Exposure of younger embryos (GD 8 or 9) does not induce similar defects in forelimbs. This limb‐dependent response suggests that methylation alterations in genes specific for fore‐ or hindlimbs may contribute to the observed pattern of defects. Subtraction hybridization (SH) studies were conducted to identify differential expression of DNA subsequent to the administration of d‐AZA to mice on GD 10. Hindlimb buds collected from both treated and untreated embryos at 4, 12, and 24 hours post‐treatment were used. A clone isolated from the untreated sample (down‐regulation in treated tissue) was identified as a member of the murine B1 family of repetitive sequences. The two other clones isolated from the treated tissue (up‐regulation) were homologous to avian myogenic regulatory protein mRNA and activin receptor type II gene. Both species are active during embryogenesis. These findings suggest that the isolated clones may have roles in abnormal embryonic development when inappropriately expressed.