BACE (beta-secretase) modulates the processing of APLP2 in vivo.

BACE is an aspartyl protease that cleaves the amyloid precursor protein (APP) at the beta-secretase cleavage site and is involved in Alzheimer's disease. The aim of our study was to determine whether BACE affects the processing of the APP homolog APLP2. To this end, we developed BACE knockout mice with a targeted insertion of the gene for beta-galactosidase. BACE appeared to be exclusively expressed in neurons as determined by differential staining. BACE was expressed in specific areas in the cortex, hippocampus, cerebellum, pons, and spinal cord. APP processing was altered in the BACE knockouts with Abeta levels decreasing. The levels of APLP2 proteolytic products were decreased in BACE KO mice, but increased in BACE transgenic mice. Overexpression of BACE in cultured cells led to increased APLP2 processing. Our results strongly suggest that BACE is a neuronal protein that modulates the processing of both APP and APLP2.

Expression of chick Tbx-2, Tbx-3, and Tbx-5 genes during early heart development: evidence for BMP2 induction of Tbx2.

Expression patterns of Tbx2, -3, and -5 genes were analyzed during chick embryonic heart development. Transcripts of these three cTbx genes were detected in overlapping patterns in the early cardiac crescent. cTbx2 and cTbx3 expression patterns closely overlapped with that of bmp2. cTbx5 expression diverged from cTbx2 and bmp2 during the elaboration and folding of the heart tube. In comparison, cTbx2 expression overlapped significantly with that of bmp2 and bmp4 during all stages of heart development and during later embryonic stages, suggestive of a specialized role for Tbx2 in septation. Coexpression of cTbx 2 and cTbx3 genes with bmp2 transcripts raised the possibility that these cTbx genes might be downstream bmp2 targets. Application of bmp2 selectively induced cTbx2 and cTbx3 expression in noncardiogenic embryonic tissue, and the bmp antagonist Noggin down-regulated cTbx2 gene activity. Moreover, the appearance of murine mTbx2 was blocked in bmp2 null mouse embryos. cTbx2 and to a lesser extent cTbx3 gene activity appears to be directed by BMPs during early cardiogenesis.

Metabolic response to various beta-adrenoceptor agonists in beta3-adrenoceptor knockout mice: evidence for a new beta-adrenergic receptor in brown adipose tissue.

The beta3-adrenoceptor plays an important role in the adrenergic response of brown and white adipose tissues (BAT and WAT). In this study, in vitro metabolic responses to beta-adrenoceptor stimulation were compared in adipose tissues of beta3-adrenoceptor knockout and wild type mice. The measured parameters were BAT fragment oxygen uptake (MO2) and isolated white adipocyte lipolysis. In BAT of wild type mice (-)-norepinephrine maximally stimulated MO2 4.1+/-0.8 fold. Similar maximal stimulations were obtained with beta1-, beta2- or beta3-adrenoceptor selective agonists (dobutamine 5.1+/-0.3, terbutaline 5.3+/-0.3 and CL 316,243 4.8+/-0.9 fold, respectively); in BAT of beta3-adrenoceptor knockout mice, the beta1- and beta2-responses were fully conserved. In BAT of wild type mice, the beta1/beta2-antagonist and beta3-partial agonist CGP 12177 elicited a maximal MO2 response (4.7+/-0.4 fold). In beta3-adrenoceptor knockout BAT, this response was fully conserved despite an absence of response to CL 316,243. This unexpected result suggests that an atypical beta-adrenoceptor, distinct from the beta1-, beta2- and beta3-subtypes and referred to as a putative beta4-adrenoceptor is present in BAT and that it can mediate in vitro a maximal MO2 stimulation. In isolated white adipocytes of wild type mice, (-)-epinephrine maximally stimulated lipolysis 12.1+/-2.6 fold. Similar maximal stimulations were obtained with beta1-, beta2- or beta3-adrenoceptor selective agonists (TO509 12+/-2, procaterol 11+/-3, CL 316,243 11+/-3 fold, respectively) or with CGP 12177 (7.1+/-1.5 fold). In isolated white adipocytes of beta3-adrenoceptor knockout mice, the lipolytic responses to (-)epinephrine, to the beta1-, beta2-, beta3-adrenoceptor selective agonists and to CGP 12177 were almost or totally depressed, whereas those to ACTH, forskolin and dibutyryl cyclic AMP were conserved.

(-)-CGP 12177 causes cardiostimulation and binds to cardiac putative beta 4-adrenoceptors in both wild-type and beta 3-adrenoceptor knockout mice.

Some blockers of beta1- and beta2-adrenoceptors cause cardiostimulant effects through an atypical beta-adrenoceptor (putative beta4-adrenoceptor) that resembles the beta3-adrenoceptor. It is likely but not proven that the putative beta4-adrenoceptor is genetically distinct from the beta3-adrenoceptor. We therefore investigated whether or not the cardiac atypical beta-adrenoceptor could mediate agonist effects in mice lacking a functional beta3-adrenoceptor gene (beta3 KO). (-)-CGP 12177, a beta1- and beta2-adrenoceptor blocker that causes agonist effects through both beta3-adrenoceptors and cardiac putative beta4-adrenoceptors, caused cardiostimulant effects that were not different in atria from wild-type (WT) mice and beta3 KO mice. The effects of (-)-CGP 12177 were resistant to blockade by (-)-propranolol (200 nM) but were blocked by (-)-bupranolol (1 microM) with an equilibrium dissociation constant of 15 nM in WT and 17 nM in beta3 KO. (-)-[3H]CGP 12177 labeled a similar density of the putative beta4-adrenoceptor in ventricular membranes from the hearts of both WT (Bmax = 52 fmol/mg protein) and beta3 KO (Bmax = 53 fmol/mg protein) mice. The affinity of (-)-[3H]CGP 12177 for the cardiac putative beta4-adrenoceptor was not different between WT (Kd = 46 nM) and beta3 KO (Kd= 40 nM). These results provide definitive evidence that the cardiac putative beta4-adrenoceptor is distinct from the beta3-adrenoceptor.

Targeted gene disruption reveals a leptin-independent role for the mouse beta3-adrenoceptor in the regulation of body composition.

Targeted disruption of mouse beta3-adrenoceptor was generated by homologous recombination, and validated by an acute in vivo study showing a complete lack of effect of the beta3-adrenoceptor agonist CL 316,243 on the metabolic rate of homozygous null (-/-) mice. In brown adipose tissue, beta3-adrenoceptor disruption induced a 66% decrease (P < 0.005) in beta1-adrenoceptor mRNA level, whereas leptin mRNA remained unchanged. Chronic energy balance studies in chow-fed mice showed that in -/- mice, body fat accumulation was favored (+41%, P < 0.01), with a slight increase in food intake (+6%, NS). These effects were accentuated by high fat feeding: -/- mice showed increased total body fat (+56%, P < 0.025) and food intake (+12%, P < 0.01), and a decrease in the fat-free dry mass (-10%, P < 0.05), which reflects a reduction in body protein content. Circulating leptin levels were not different in -/- and control mice regardless of diet. The significant shift to the right in the positive correlation between circulating leptin and percentage of body fat in high fat-fed -/- mice suggests that the threshold of body fat content inducing leptin secretion is higher in -/- than in control mice. Taken together, these studies demonstrate that beta3-adrenoceptor disruption creates conditions which predispose to the development of obesity.

Retinoid signaling is required for the establishment of a ZPA and for the expression of Hoxb-8, a mediator of ZPA formation.

We show that retinoid receptor antagonists applied to the presumptive wing region block the formation of a zone of polarizing activity (ZPA). This suggests a direct relationship between retinoid signaling and the establishment of the ZPA. We provide evidence that the Hox gene, Hoxb-8, is a direct target of retinoid signaling since exogenously applied RA rapidly induces this gene in the absence of protein synthesis and, moreover, retinoid receptor antagonists down-regulate Hoxb-8 expression. In addition, we find that, in the lateral plate mesoderm, the domains of Hoxb-8 expression and of polarizing activity are coextensive. Taken together, these findings support the hypothesis that retinoids are required for the establishment of a ZPA, and that retinoids act, at least in part, through Hoxb-8, a gene associated with ZPA formation (Charité et al., 1994).

Respective degree of expression of beta 1-, beta 2- and beta 3-adrenoceptors in human brown and white adipose tissues.

1. The possible existence of a beta 3-adrenoceptor in human brown and white adipose tissues was investigated by mRNA expression and binding studies. 2. The relative amounts of beta 1-, beta 2- and beta 3-adrenoceptor mRNA, as determined by total RNA Northern blot analysis in newborn brown adipose tissue, were 28, 63 and 9% respectively of the total beta-adrenoceptor mRNA. 3. The beta 1/beta 2-adrenoceptors of human brown adipose tissue plasma membranes were characterized using [3H]-CGP 12177 as a ligand. Their Kd and Bmax values were 1.9 nM and 156 fmol mg-1 of membrane proteins, respectively. The beta 3-adrenoceptor was characterized by use of the new specific radioligand [3H]-SB 206606. The binding of this ligand was stereospecifically displaced by the active R,R- or the inactive S,S-enantiomer of BRL 37344 up to a concentration of about 10 microM. The Kd and Bmax values of the brown adipose tissue membrane beta 3-adrenoceptors were 87 nM and 167 fmol mg-1 of proteins, respectively. A low affinity [3H]-CGP 12177 binding site population was also detected in these membranes. 4. In human omental white adipose tissue, no beta 3-adrenoceptor mRNA could be detected in total RNA Northern blots and the beta 1-and beta 2-adrenoceptor mRNAs represented 9 and 91%, respectively of the total beta-adrenoceptor mRNA, and no specific binding of [3H]-SB 206606 could be measured.

Control of beta 3-adrenergic receptor gene expression in brown adipocytes in culture.

Brown adipose tissue is a mammalian thermogenic tissue. Its ability to dissipate energy as heat is due to a unique mitochondrial protein, uncoupling protein (UCP). Activation and expression of UCP is under control of the sympathetic nervous system acting through beta -adrenergic receptors (AR). In this study we used Siberian hamster brown adipocytes differentiated in vitro to investigate the expression of the fat specific beta 3-AR. Binding studies using the new labelled beta 3 adrenergic ligand [3H]SB 206606 showed a density of beta 3-AR in brown adipocyte plasma membranes comparable to that measured in vivo. beta 3-AR mRNA expression was very high in mature brown adipocytes and was started to be expressed during differentiation before UCP mRNA. Its half-life was approximately 50 min. Treatment of cells with non-specific beta adrenergic agonists, specific beta 3-adrenergic agonists, and dibutyryl cyclic AMP resulted in a marked down regulation of beta 3-AR mRNA level within several hours.

Characterization of a new, highly specific, beta 3-adrenergic receptor radioligand, [3H]SB 206606.

The RR-enantiomer of the beta 3-adrenergic receptor agonist BRL 37344 was tritiated to yield a high specific activity compound, [3H]SB 206606. This new, potentially specific, beta 3-adrenergic receptor ligand was characterized by binding studies using membranes from both Chinese hamster ovary K1 cells transfected with the rat beta 3-adrenergic receptor and rat interscapular brown adipose tissue, where beta 1-, beta 2-, and beta 3-adrenergic receptor subtypes are known to coexist. [3H]SB 206606 was found to bind to a single population of binding sites in both preparations. The Kd values for [3H]SB 206606 binding to membranes from Chinese hamster ovary K1 cells and brown adipose tissue were quite comparable (58 and 38 nM, respectively). At 37 degrees, the time courses of association and dissociation of [3H]SB 206606 with membranes of brown adipose tissue were quite short. At 4 degrees, the T1/2 were found to be 13 and 40 min, respectively. The Ki values for various beta-adrenergic agonists and antagonists in brown adipose tissue membranes were similar to those obtained in Chinese hamster ovary K1 cell membranes with both [3H]SB 206606 and [125I]iodocyanopindolol. The order of binding affinity was BRL 37344 >> (-)-isoproterenol = (-)-norepinephrine > (-)-epinephrine = (+)-isoproterenol. The similarity of the Kd values and of the Ki values for various beta-adrenergic agonists and antagonists in both systems tested indicates that, in a complex membrane system, [3H]SB 206606 binds selectively to the beta 3-adrenergic receptor. The affinity of [3H]SB 206606 is 76 times higher for the beta 3-adrenergic receptor than for the beta 1/beta 2-adrenergic receptors, thus allowing, under controlled conditions, measurement of interactions only with the beta 3-adrenergic receptor in complex membrane systems.

Expression of beta 1- and beta 3-adrenergic-receptor messages and adenylate cyclase beta-adrenergic response in bovine perirenal adipose tissue during its transformation from brown into white fat.

Possible modifications of the beta-adrenergic effector system during the development of bovine perirenal brown adipose tissue (BAT) in utero and its transformation into white-like adipose tissue after birth were studied. The parameters assessed were the level of expression of beta 1-, beta 2- and beta 3-adrenergic receptor (AR) mRNAs and the response of the plasma-membrane adenylate cyclase to (-)-isoprenaline and to the beta 3-agonist BRL 37344. The beta 3-AR mRNA was found to be expressed very early in utero, i.e. before the third month of foetal life. Then it increased dramatically (9-fold) between month 6 of foetal life and birth. A high beta 3-AR mRNA level was maintained after birth up to an age of 3 months. After conversion of BAT into white-like adipose tissue, i.e. in the adult bovine, the beta 3-AR mRNA expression became small or not detectable, and the beta 1-AR mRNA, which was expressed much less than the beta 3-AR mRNA in foetal life, became predominant. A response of the adenylate cyclase to (-)-isoprenaline was observed in foetal life (3.1-fold stimulation). It decreased after birth (1.8-fold stimulation) and then remained constant until adulthood. A response to BRL 37344 was also observed in foetal life (1.8-fold stimulation). It was maintained after birth, but disappeared in the adult. A possible relationship between the beta-AR expression and the adenylate cyclase response to (-)-isoprenaline on the one hand and the uncoupling-protein expression on the other is discussed. The bovine might represent a good model to understand the transition from brown to white fat in the human.

Adipose tissues from various anatomical sites are characterized by different patterns of gene expression and regulation.

We have shown previously the presence of brown adipocytes among white fat pads, and proposed the existence of a spectrum of adipose depots according to the abundance of brown fat cells [Cousin, Cinti, Morroni, Raimbault, Ricquier, Pénicaud and Casteilla (1992) J. Cell Sci. 103, 931-942]. In this study, we tried to characterize this spectrum better. We determined in several adipose depots (i) the richness of pre-adipose cells, as assessed by A2COL6 mRNA levels; (ii) whether a fat pad was characterized by a pattern of mRNA expression; (iii) whether this pattern was close related to abundance of brown adipocytes, and (iv) whether the regulation of this pattern by catecholamines under cold exposure or beta-agonist treatment was similar in the different pads. This was achieved by studying proteins involved in glucose and lipid metabolism such as insulin-sensitive glucose transporter (GLUT4), fatty acid synthase, lipoprotein lipase and fatty acid binding protein aP2, as well as beta 3-adrenergic-receptor expression. Among white adipose depots, the periovarian fat pad was characterized by the highest content of pre-adipocytes and of brown adipocytes, and inguinal fat by the highest lipogenic activity potential. There was no close correlation between beta 3-adrenergic-receptor expression and brown adipocyte content in the tissues, as measured by the degree of uncoupling protein (UCP) gene expression. However, in pads expressing UCP mRNA, mRNA levels of beta 3-adrenergic receptor and other markers were increased in parallel. Under cold exposure or beta 3-agonist treatment, a specific up-regulation of GLUT4 expression was observed in interscapular brown adipose tissue. The regional difference described in this study, could participate in preferential fat-pad growth under physiological conditions as well as in pathological situations.

Expression of the beta 3-adrenergic receptor in human white adipose tissue.

The results of this study suggest that the atypical beta-adrenergic receptor (beta 3 subtype) is expressed in human white adipose tissue. A beta 3-adrenergic receptor mRNA could be detected in human omental fat poly(A)+ RNA by polymerase chain reaction amplification with appropriate primers. The expression of the beta 3-adrenergic receptor in human fat was confirmed by Northern blot analysis; however, the level of its mRNA was lower than those of the beta 1- and beta 2-adrenergic receptors. Two populations of ((-)-4-(3-t-butylamino-2-hydroxypropoxy)-[5,7-3H]benzimidazole-2-one) ([3H]CGP 12177)-binding sites were identified in human omental fat membranes, one with a high affinity (Kd = 1.7 nM) and the other with a low affinity (Kd = 22 nM). The low affinity binding site population, which should represent the beta 3-adrenergic receptor, was predominant (75% of the total binding sites).

Modulation in vivo of beta-adrenergic-receptor subtypes in rat brown adipose tissue by the thermogenic agonist Ro 16-8714.

The number of beta 3-adrenergic receptors (AR) in plasma membranes from interscapular brown adipose tissue (IBAT) was decreased by 62% in lean Zucker rats treated with the thermogenic beta-adrenergic agonist Ro 16-8714 as compared with controls after 72 h of treatment. The loss of beta 3-AR number was preceded by a 93% decrease in the steady-state level of beta 3-AR mRNA at 30 h. Similar results were obtained in obese (fa/fa) Zucker rats. Ro 16-8714 had no effect on the number of beta 1- and beta 2-ARs in IBAT. This is the first report to demonstrate that the beta 3-AR in IBAT can be specifically down-regulated in vivo by exposure to a thermogenic agonist.

Progesterone transcriptionally regulates the beta 2-adrenergic receptor gene in pregnant rat myometrium.

Administration of 5 mg of progesterone to late pregnant rats induced an increase in myometrial beta-adrenergic receptors density detected by 125I-cyanopindolol binding. This increase was significant after 24 h (1.4-fold; p less than 0.05) and reached 1.6-fold (p less than 0.05) after 36 h. The antiprogestin RU 486 or cycloheximide completely suppressed the effect of progesterone injection. Quantification of the beta 1- and beta 2-receptor subtypes revealed that progesterone selectively up-regulated the beta 2-subtype. The increase in beta 2-adrenoreceptors was preceded by an elevation of their mRNA (2.3 kilobases) levels as determined by Northern blot hybridization with a rat heart beta 2-adrenoreceptor cDNA probe. This increase was significant after 18 h of exposure to progesterone (2.1-fold; p less than 0.05) and reached a maximum after 24 h (3.4-fold; p less than 0.01). The rate of beta 2-adrenergic gene transcription evaluated by nuclear run-on transcription assays, increased by 2.5-fold in myometria exposed for 4 h to progesterone. This study indicates that progesterone regulates myometrial beta 2-adrenergic receptor expression by controlling the rate of transcription of the gene.

Radioligand binding studies of the atypical beta 3-adrenergic receptor in rat brown adipose tissue using [3H]CGP 12177.

Two populations of [3H]CGP 12177 binding sites exist in rat interscapular brown adipose tissue (IBAT) plasma membranes. The majority of binding sites are of low affinity with a Kd of 31 nM, a value in close agreement with that for the Kd of [3H]CGP 12177 binding to a cloned rat beta 3-adrenergic receptor (AR) expressed in CHO cells (44 nM). Competition binding studies demonstrate that the Ki values of the cloned rat beta 3-AR and of the low affinity sites in IBAT are 45 and 29 nM, respectively, for BRL 37344 and 1.4 and 1.0 microM, for (-)-propranolol. These findings strongly suggest that the low affinity [3H]CGP 12177 binding site measured in IBAT plasma membranes represents the atypical beta 3-AR in this tissue.

An adipose tissue-specific beta-adrenergic receptor. Molecular cloning and down-regulation in obesity.

Clones encoding an atypical beta-adrenergic receptor were isolated from a rat brown adipose tissue cDNA library. This receptor expressed in Chinese hamster ovary (CHO) cells displays a low affinity for beta-adrenergic antagonists and a high affinity for BRL 37344, an agonist that selectively stimulates lipolysis in adipose tissue. The rank order of potency for agonist-mediated increases in intracellular cAMP in transfected cells correlates with that for agonist-mediated stimulation of lipolysis in brown adipocytes. Northern blot analysis demonstrates that this receptor subtype is expressed only in brown and white adipose tissue where it represents the predominant beta-receptor subtype. The amount of atypical beta-adrenergic receptor present in adipose tissue of obese (fa/fa) Zucker rats is reduced by up to 71% as compared with lean (Fa/Fa) control animals. These findings suggest that a change in the expression of this beta-adrenergic receptor subtype may play a role in obesity.

Changes in beta 1- and beta 2-adrenergic receptor mRNA levels in brown adipose tissue and heart of hypothyroid rats.

The aim of the present work was to study the effect of hypothyroidism on the expression of the beta-adrenergic receptor (beta-AR) in interscapular brown adipose tissue and heart. The total density of plasma membrane beta-AR per tissue is decreased by 44% in hypothyroid rat interscapular brown adipose tissue and by 55% in hypothyroid rat heart compared with euthyroid controls. The effects of hypothyroidism on the density of both beta 1- and beta 2-AR subtypes were also determined in competition displacement experiments. The densities of beta 1- and beta 2-AR per tissue are decreased by 50% and 48% respectively in interscapular brown adipose tissue and by 52% and 54% in the heart. Northern blot analysis of poly(A)+ RNA from hypothyroid rat interscapular brown adipose tissue demonstrated that the levels of beta 1- and beta 2-AR mRNA per tissue are decreased by 73% and 58% respectively, whereas in hypothyroid heart, only the beta 1-AR mRNA is decreased, by 43%. The effect of hypothyroidism on the beta 1-AR mRNA is significantly more marked in the interscapular brown adipose tissue than in the heart. These results indicate that beta-AR mRNA levels are differentially regulated in rat interscapular brown adipose tissue and heart, and suggest that the decrease in beta-AR number in interscapular brown adipose tissue and heart of hypothyroid animals may in part be explained by a decreased steady-state level of beta-AR mRNA.

The novel thermogenic beta-adrenergic agonist Ro 16-8714 increases the interscapular brown-fat beta-receptor-adenylate cyclase and the uncoupling-protein mRNA level in obese (fa/fa) Zucker rats.

In obese (fa/fa) rats both the total (-)-isoprenaline- and NaF-stimulated adenylate cyclase activities of interscapular brown-adipose-tissue (IBAT) plasma membranes were decreased as compared with lean rats by 40 and 38% respectively. An acute treatment with Ro 16-8714 increased (-)-isoprenaline- and NaF-stimulated adenylate cyclase activities by 2.5- and 2.0-fold respectively, and beta-adrenoceptor number 2.8-fold in obese rat IBAT, but it had no effect on these parameters in lean rats. It increased mRNA for mitochondrial uncoupling protein in both lean and obese rats to the same extent.

Fetal glucose utilization in response to maternal starvation and acute hyperketonemia.

The effects of maternal hypoglycemia and/or hyperketonemia on glucose utilization by individual fetal rat tissues have been studied in vivo. To decrease blood glucose and to raise fetal blood ketone body concentrations, 19-day pregnant rats were submitted to 48 or 96 h of starvation. To differentiate between the effects of decreased blood glucose and increased ketone body concentrations, fed pregnant rats were infused for 2 h with DL-beta-hydroxybutyrate. After 96 h of maternal starvation, fetal 2-deoxy-D-glucose (2DG) uptake decreased from 13.6 +/- 0.5 to 8.6 +/- 1.15 micrograms.min-1.g-1. This was mainly due to a decrease in 2DG uptake by fetal hindlimb muscles and heart. By contrast, 2DG uptake in fetal liver and brain was not affected by maternal starvation. Acute hyperketonemia in fed pregnant rats induced a 23% decrease in 2DG uptake by the whole fetus mainly as the result of a lowered 2DG uptake in fetal hindlimb muscles. These data suggest that fetal 2DG uptake does not simply depend on lowered blood glucose level during maternal starvation but that other hormonal, cardiovascular, or metabolic adaptations are implicated. In the rat, most of the fetal tissues including brain are protected against maternal hypoglycemia.

Hyperglycemia and hyperinsulinemia increase glucose utilization in fetal rat tissues.

In vivo measurement of glucose utilization by individual tissues of 19-day rat fetuses have been performed using radioactive 2-deoxy-D-glucose technique. In the basal state, glucose metabolic index was 13.6 +/- 0.5 ng.min-1.mg-1 for the whole fetus, 21 +/- 1 in the hindlimb muscles, 13 +/- 2 in the liver, and 16 +/- 2 in the brain, whereas the fetal heart had the highest value: 62 +/- 5 ng.min-1.mg-1. To raise the fetal glycemia, the basal maternal blood glucose concentration of 0.78 +/- 0.02 g/l was elevated to 1.04 +/- 0.02 g/l by mean of hyperglycemic clamps. The fetal hyperglycemia increased glucose metabolic index by 30-100% over basal values in all the tissues tested except in the brain. To raise fetal insulinemia, maternal euglycemic clamp with supraphysiological insulin concentrations were performed, then a fraction (1%) of exogenous insulin crossed the placenta. Fetal plasma insulin concentrations were thus elevated to 180 +/- 32 and 255 +/- 23 microU/ml. The fetal heart increased significantly its glucose metabolic index in response to the lower insulin level. Glucose metabolic index in hindlimb muscles and liver was increased by 50-100% for the highest insulin level, whereas the brain was unaffected by exogenous insulin. We conclude that glucose metabolic index is stimulated by physiological hyperglycemia in individual fetal tissues and that fetal tissues (heart, liver, and muscle) are sensitive to exogenous insulin.