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Uterine receptivity to embryo implantation depends on appropriate progesterone (P4) and estrogen stimulation. P4 rapidly stimulates production of the morphogen Indian hedgehog (IHH) in murine uterine epithelium as well as downstream molecules in the hedgehog pathway such as Patched homolog 1 (PTCH1) and nuclear receptor subfamily 2, group F, member 2 (NR2F2) in uterine stroma. Studies using IHH-null mice indicate that IHH is obligatory for the normal P4 response in the uterus. To determine whether IHH induction in uterine epithelium is mediated through P4 receptor (PR) in epithelium (E) and/or stroma (S), we produced tissue recombinants using uteri from neonatal PR knockout (ko) mice and wild-type (wt) mice containing PR in S and/or E or lacking PR altogether using a tissue recombinant methodology and assessed their response to P4. In tissue recombinants containing wt-S (wt-S + wt-E and wt-S + ko-E), P4 induced Ihh mRNA expression at 6 h that was 6-fold greater than in oil-treated controls (P < 0.05; n = 6) in both types of tissue recombinants despite the absence of epithelial PR in wt-S + ko-E grafts. Conversely, Ihh mRNA expression was unaffected by P4 in ko-S + ko-E and ko-S + wt-E grafts despite epithelial PR expression in the latter. Nr2f2 and Ptch1 mRNA expression was similar in that it was stimulated by P4 only in recombinants containing stromal PR. These results indicate that stromal PR is both necessary and sufficient for P4 stimulation of epithelial IHH as well as downstream events such as PTCH1 and NR2F2 increases in stroma.

The steroid hormone progesterone, acting via its nuclear receptor, is a major regulator of the process of ovulation. Female mice lacking progesterone receptor (PGR) exhibit an anovulatory phenotype due to failure in follicular rupture. To identify the PGR-regulated pathways that control ovulation, we analyzed global changes in gene expression in the ovaries of wild-type and Pgr-null mice subjected to gonadotropin-induced superovulation. Our analysis uncovered several genes whose expression was reduced in the Pgr-null ovaries compared with the wild-type ovaries immediately preceding ovulation. Interestingly, these genes included three hypoxia-inducible factors (HIFs): HIF-1 alpha, HIF-2 alpha, and HIF-1 beta. These transcription factors form alphabeta-heterodimers, which regulate the transcription of specific cellular genes, thereby mediating adaptive response of the tissue to low-oxygen levels. We observed that the expression of mRNAs and proteins corresponding to HIF-1 alpha, HIF-2 alpha, and HIF-1 beta was induced in a PGR-dependent manner, specifically in the granulosa cells of the preovulatory follicles. Inhibition of the HIF transcriptional activity by echinomycin, a small-molecule inhibitor that suppresses the binding of HIF alphabeta-heterodimers to target genes, blocked ovulation by preventing the rupture of the preovulatory follicles. Echinomycin specifically inhibited the expression of genes that are known regulators of ovulation, such as a disintegrin and metalloproteinase with thrombospondin-like motifs-1 and endothelin-2. Furthermore, echinomycin reduced the expression of vascular endothelial growth factor A, a key factor controlling vascularization/angiogenesis during ovulation. Collectively, these findings unveiled a novel ovarian role for the HIF transcription factors during the ovulatory period in mice.

Skeletal muscle can maintain ATP concentration constant during the transition from rest to exercise, whereas metabolic reaction rates may increase substantially. Among the key regulatory factors of skeletal muscle energy metabolism during exercise, the dynamics of cytosolic and mitochondrial NADH and NAD+ have not been characterized. To quantify these regulatory factors, we have developed a physiologically based computational model of skeletal muscle energy metabolism. This model integrates transport and reaction fluxes in distinct capillary, cytosolic, and mitochondrial domains and investigates the roles of mitochondrial NADH/NAD+ transport (shuttling) activity and muscle glycogen concentration (stores) during moderate intensity exercise (60% maximal O2 consumption). The underlying hypothesis is that the cytosolic redox state (NADH/NAD+) is much more sensitive to a metabolic disturbance in contracting skeletal muscle than the mitochondrial redox state. This hypothesis was tested by simulating the dynamic metabolic responses of skeletal muscle to exercise while altering the transport rate of reducing equivalents (NADH and NAD+) between cytosol and mitochondria and muscle glycogen stores. Simulations with optimal parameter estimates showed good agreement with the available experimental data from muscle biopsies in human subjects. Compared with these simulations, a 20% increase (or approximately 20% decrease) in mitochondrial NADH/NAD+ shuttling activity led to an approximately 70% decrease (or approximately 3-fold increase) in cytosolic redox state and an approximately 35% decrease (or approximately 25% increase) in muscle lactate level. Doubling (or halving) muscle glycogen concentration resulted in an approximately 50% increase (or approximately 35% decrease) in cytosolic redox state and an approximately 30% increase (or approximately 25% decrease) in muscle lactate concentration. In both cases, changes in mitochondrial redox state were minimal. In conclusion, the model simulations of exercise response are consistent with the hypothesis that mitochondrial NADH/NAD+ shuttling activity and muscle glycogen stores affect primarily the cytosolic redox state. Furthermore, muscle lactate production is regulated primarily by the cytosolic redox state.

The synthesis and breakdown of triglycerides in adipose tissue and muscle is a crucial element of energy metabolism because it ensures that adequate fuel is available during starvation. Triglyceride turnover determines the availability of fatty acids for utilization by mammalian tissues, and any dysfunction in this process can lead to alterations in glucose metabolism, insulin resistance and type 2 diabetes. Our understanding of the reactions involved in triglyceride synthesis is currently being reassessed, primarily because of the recently identified role that re-esterification of fatty acids plays in triglyceride deposition and, thus, in controlling fatty-acid availability. Here, we review recent information on triglyceride synthesis and introduce the pathway of glyceroneogenesis as an important and highly regulated source of glyceride-glycerol in adipose tissue.

Control mechanisms of cellular metabolism and energetics in skeletal muscle that may become evident in response to physiological stresses such as reduction in blood flow and oxygen supply to mitochondria can be quantitatively understood using a multi-scale computational model. The analysis of dynamic responses from such a model can provide insights into mechanisms of metabolic regulation that may not be evident from experimental studies. For the purpose, a physiologically-based, multi-scale computational model of skeletal muscle cellular metabolism and energetics was developed to describe dynamic responses of key chemical species and reaction fluxes to muscle ischemia. The model, which incorporates key transport and metabolic processes and subcellular compartmentalization, is based on dynamic mass balances of 30 chemical species in both capillary blood and tissue cells (cytosol and mitochondria) domains. The reaction fluxes in cytosol and mitochondria are expressed in terms of a general phenomenological Michaelis-Menten equation involving the compartmentalized energy controller ratios ATP/ADP and NADH/NAD(+). The unknown transport and reaction parameters in the model are estimated simultaneously by minimizing the differences between available in vivo experimental data on muscle ischemia and corresponding model outputs in coupled with the resting linear flux balance constraints using a robust, nonlinear, constrained-based, reduced gradient optimization algorithm. With the optimal parameter values, the model is able to simulate dynamic responses to reduced blood flow and oxygen supply to mitochondria associated with muscle ischemia of several key metabolite concentrations and metabolic fluxes in the subcellular cytosolic and mitochondrial compartments, some that can be measured and others that can not be measured with the current experimental techniques. The model can be applied to test complex hypotheses involving dynamic regulation of cellular metabolism and energetics in skeletal muscle during physiological stresses such as ischemia, hypoxia, and exercise.

Regulation of lipolysis in adipose tissue is critical to whole body fuel homeostasis and to the development of insulin resistance. Due to the challenging nature of laboratory investigations of regulatory mechanisms in adipose tissue, mathematical models could provide a valuable adjunct to such experimental work. We have developed a computational model to analyze key components of adipose tissue metabolism in vivo in human in the fasting state. The various key components included triglyceride-fatty acid cycling, regulation of lipolytic reactions, and glyceroneogenesis. The model, consisting of spatially lumped blood and cellular compartments, included essential transport processes and biochemical reactions. Concentration dynamics for major substrates were described by mass balance equations. Model equations were solved numerically to simulate dynamic responses to intravenous epinephrine infusion. Model simulations were compared with the corresponding experimental measurements of the arteriovenous difference across the abdominal subcutaneous fat bed in humans. The model can simulate physiological responses arising from the different expression levels of lipases. Key findings of this study are as follows: (1) Distinguishing the active metabolic subdomain ( approximately 3% of total tissue volume) is critical for simulating data. (2) During epinephrine infusion, lipases are differentially activated such that diglyceride breakdown is approximately four times faster than triglyceride breakdown. (3) Glyceroneogenesis contributes more to glycerol-3-phosphate synthesis during epinephrine infusion when pyruvate oxidation is inhibited by a high acetyl-CoA/free-CoA ratio.

Skeletal muscle plays a major role in the regulation of whole-body energy metabolism during physiological stresses such as ischemia, hypoxia, and exercise. Current experimental techniques provide relatively little in vivo data on dynamic responses of metabolite concentrations and metabolic fluxes in skeletal muscle to such physiological stimuli. As a complementary approach to experimental measurements and as a framework for quantitatively analyzing available in vivo data, a physiologically based model of skeletal muscle cellular metabolism and energetics is developed. This model, which incorporates key transport and reaction processes, is based on dynamic mass balances of 30 chemical species in capillary (blood) and tissue (cell) domains. The reaction fluxes in the cellular domain are expressed in terms of a generalized Michaelis?Menten equation involving energy controller ratios ATP/ADP and ATP/ADP and NADH/NAD+ . This formalism introduces a large number of unknown parameters ( approximately 90). Estimating these parameters from in vivo sparse data and evaluating dynamic sensitivities of the model outputs with respect to these parameters is a challenging problem. Parameter estimation is accomplished using an efficient, nonlinear, constraint-based, optimization algorithm that minimizes differences between available experimental data and corresponding model outputs by explicitly utilizing equality constraints on resting fluxes and concentrations. With the estimated parameter values, the model is able to simulate dynamic responses to reduced blood flow (ischemia) of key metabolite concentrations and metabolic fluxes, both measured and nonmeasured. A general parameter sensitivity analysis is carried out to determine and characterize the parameters having the most and least effects on the measured outputs.

The progesterone receptor (PR) plays a critical role during ovulation. Mice lacking the PR gene are anovulatory due to a failure in the rupture of the preovulatory follicles. The pathways that operate downstream of PR to control ovulation are poorly understood. Using gene expression profiling, we identified peroxisome proliferator-activated receptor gamma (PPARgamma) as a target of regulation by PR in the granulosa cells of the preovulatory follicles during the ovulatory process. To investigate the function of PPARgamma during ovulation, we created a conditional knockout mouse in which this gene was deleted via Cre-Lox-mediated excision in granulosa cells. When these mutant mice were subjected to gonadotropin-induced superovulation, the preovulatory follicles failed to rupture and the number of eggs released from the mutant ovaries declined drastically. Gene expression analysis identified endothelin-2, interleukin-6, and cyclic GMP-dependent protein kinase II as novel targets of regulation by PPARgamma in the ovary. Our studies also suggested that cycloxygenase 2-derived metabolites of long-chain fatty acids function as endogenous activating ligands of PPARgamma in the preovulatory follicles. Collectively, these studies revealed that PPARgamma is a key mediator of the biological actions of PR in the granulosa cells and activation of its downstream pathways critically controls ovulation.

A mathematical model of the whole-body metabolism is developed to predict fuel homeostasis during exercise by using hormonal control over cellular metabolic processes. The whole body model is composed of seven tissue compartments: brain, heart, liver, GI (gastrointestinal) tract, skeletal muscle, adipose tissue, and "other tissues". Each tissue compartment is described by dynamic mass balances and major cellular metabolic reactions. The glucagon-insulin controller is incorporated into the whole body model to predict hormonal changes during exercise. Moderate [150 W power output at 60% of peak oxygen consumption (VO(2max))] exercise for 60 min was implemented by increasing ATP utilization rates in heart and skeletal muscle. Arterial epinephrine level was given as an input function, which directly affects heart and skeletal muscle metabolism and indirectly other tissues via glucagon-insulin controller. Model simulations were validated with experimental data from human exercise studies. The exercise induced changes in hormonal signals modulated metabolic flux rates of different tissues in a coordinated way to achieve glucose homeostasis, demonstrating the efficacy of hormonal control over cellular metabolic processes. From experimental measurements of whole body glucose balance and arterial substrate concentrations, this model could predict the dynamic changes of hepatic glycogenolysis and gluconeogenesis, which are not easy to measure experimentally, suggesting the higher contribution of glycogenolysis ( approximately 75%). In addition, it could provide dynamic information on the relative contribution of carbohydrates and lipids for fuel oxidation in skeletal muscle. Model simulations indicate that external fuel supplies from other tissue/organ systems to skeletal muscle become important for prolonged exercise emphasizing the significance of interaction among tissues. In conclusion, this model can be used as a valuable complement to experimental studies due to its ability to predict what is difficult to measure directly, and usefulness to provide information about dynamic behaviors.

The steroid hormone progesterone (P) plays a pivotal role during ovulation. Mice lacking P receptor (Pgr) gene fail to ovulate due to a defect in follicular rupture. The P receptor (PGR)-regulated pathways that modulate ovulation, however, remain poorly understood. To identify these pathways, we performed gene expression profiling using ovaries from mice subjected to gonadotropin-induced superovulation in the presence and in the absence of CDB-2914, a synthetic PGR antagonist. Prominent among the genes that were down-regulated in response to CDB-2914 was endothelin (ET)-2, a potent vasoactive molecule. ET-2 mRNA was transiently induced in mural granulosa cells of the preovulatory follicles immediately preceding ovulation. This induction was absent in the ovaries of PGR null mice, indicating a critical role of this receptor in ET-2 expression. To investigate the functional role of ET-2 during ovulation, we employed selective antagonists of endothelin receptors, ETR-A and ETR-B. Mice treated with an ETR-B antagonist exhibited a dramatic (>85%) decline in the number of released oocytes. Strong expression of ETR-B was observed in the mural and cumulus granulosa cells of the preovulatory follicles as well as in the capillaries lining the inner border of the theca interna. We also identified cGMP-dependent protein kinase II, a previously reported PGR-regulated gene, as a downstream target of ET-2 during ovulation. Collectively, our studies uncovered a unique pathway in which ET-2, produced by PGR in mural granulosa cells, acts in a paracrine or autocrine manner on multiple cell types within the preovulatory follicle to control the final events leading to its rupture.

Overproduction of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) acts as a neurotoxic effector in the central nervous system, resulting in neurodegenerative diseases. From the alcoholic extracts of Perilla frutescens, we have purified an inhibitor of NO production in lipopolysaccharide (LPS)-activated microglia by activity-guided purification. The active compound was identified as luteolin by spectral analysis. Luteolin inhibited the NO production in LPS-activated microglia in a dose-dependent manner (IC50=6.9 microM). Luteolin also suppressed the degradation of I-kappaB-alpha, the expression of protein and mRNA of iNOS in LPS-activated microglia as observed in Western blot analysis and RT-PCR experiments. Luteolin may have beneficial effects in the treatment of neuro-inflammatory diseases through the inhibition of iNOS expression.

A mathematical model of whole-body metabolism is developed to predict glucose homeostasis during exercise by using a hormonal controller over cellular metabolic processes. Model simulations were validated with experimental data from exercise studies in humans. The exercise-induced changes in hormonal signals modulated metabolic flux rates of various tissues in a coordinated way to maintain blood glucose constant. This study demonstrates the efficacy of a multi-tissue controller to accomplish blood glucose homeostasis by integrating the outputs of tissues under hormonal control. In conclusion, this model can be used as a valuable complement to experimental studies due to its ability to predict what is difficult to measure directly and to provide dynamic information about the system.