Kasim A. Mookhtiar

Selective thyroid hormone receptor-β activation: A strategy for reduction of weight, cholesterol, and lipoprotein (a) with reduced cardiovascular liability

Few treatments for obesity exist and, whereas efficacious therapeutics for hyperlipidemia are available, further improvements are desirable. Thyroid hormone receptors (TRs) regulate both body weight and cholesterol levels. However, thyroid hormones also have deleterious effects, particularly on the heart. The TRβ subtype is involved in cholesterol lowering and possibly elevating metabolic rate, whereas TRα appears to be more important for control of heart rate (HR). In the current studies, we examined the effect of TRβ activation on metabolic rate and HR with either TRα1–/– mice or the selective TRβ agonist KB-141 in mice, rats, and monkeys. 3,5,3′-triiodi-l-thyronine (T3) had a greater effect on increasing HR in WT than in TRα–/– mice (ED15 values of 34 and 469 nmol/kg/day, respectively). T3 increased metabolic rate [whole body oxygen consumption (MVO2)] in both WT and TRα–/– mice, but the effect in the TRα1–/– mice at the highest dose was half that of the WT mice. Thus, stimulation of MVO2 is likely due to both TRα and -β. T3 had equivalent potency for cholesterol reduction in WT and TRα–/– mice. KB-141 increased MVO2 with selectivities of 16.5- and 11.2-fold vs. HR in WT and TRα1–/– mice, respectively. KB-141 also increased MVO2 with a 10-fold selectivity and lowered cholesterol with a 27-fold selectivity vs. HR in rats. In primates, KB-141 caused significant cholesterol, lipoprotein (a), and body-weight reduction (up to 7% after 1 wk) with no effect on HR. TRβ-selective agonists may constitute a previously uncharacterized class of drugs to treat obesity, hypercholesterolemia, and elevated lipoprotein (a).

Glucokinase Regulatory Protein May Interact With Glucokinase in the Hepatocyte Nucleus

Glucokinase (GK) plays a central role in the sensing of glucose in pancreatic β-cells and parenchymal cells of the liver. Glucokinase regulatory protein is a physiological inhibitor of GK in the liver. To understand the role of the interaction of these two proteins in glucose sensing, we carried out a series of experiments to localize the protein in the liver cell. The regulatory protein was found to be present mainly in the nucleus of the cell under a variety of conditions that mimicked the glucose status of the fed and fasted state. GK was localized in the nucleus when the cells were exposed to low glucose concentrations. At higher glucose concentrations or in the presence of low concentrations of fructose, GK translocated to the cytoplasm. The effect of fructose was more robust and rapid than the effect of high glucose concentrations. Furthermore, the effect of fructose and high glucose on the translocation of GK from the nucleus could be partially reversed by glucagon. This unusual localization and behavior suggests a role for GK and its regulatory protein in hepatic energy metabolism that may be broader than glucose phosphorylation.

Expression of Human Hepatic Glucokinase in Transgenic Mice Liver Results in Decreased Glucose Levels and Reduced Body Weight

Glucokinase is the predominant hexokinase in pancreatic β-cells and liver parenchymal cells and functions as a critical component of the glucose-sensing apparatus in these glucose-responsive cell types. In the β-cells, the sensing leads to insulin secretion, while the role in hepatocytes is thought to be in hepatic glucose uptake. To determine the physiological response to an increase in hepatic glucokinase expression, transgenic mice expressing the human hepatic glucokinase gene under the control of a liver-specific human apolipoprotein A-I gene enhancer were generated. Transgenic mice had twofold higher total fasting hepatic glucokinase mRNA, which resulted in a modest 20% increase in fasting glucokinase activity. These animals showed lower fasting plasma glucose, insulin, and lactate levels and improved tolerance to glucose. In addition, glucokinase transgenic animals weighed less and had lower BMI than nontransgenic animals. Thus, glucokinase transgenic animals demonstrate that a modest change in hepatic glucokinase activity enhances the metabolism of glucose.

Heterologous expression and characterization of rat liver glucokinase regulatory protein

Glucokinase is a critical component of the physiological glucose sensor found in cell types that are responsive to changes in plasma glucose levels. The acute regulation of glucokinase activity has been shown to occur via a regulatory protein found in liver parenchymal cells (Van Schaftingen E, Detheux M, Da Cunha MV. Faseb J 8:414–419, 1994). The action of this protein is modulated by phosphate esters of fructose. In the presence of fructose-6-phosphate, the protein inhibits glucokinase in an allosteric competitive manner, while fructose-1-phosphate reverses this inhibition. A cDNA potentially encoding the rat liver regulatory protein has been cloned, but its identity is uncertain because of the small amounts of soluble protein obtained by expression in bacteria. We report the heterologous expression of the regulatory protein in Escherichia coli and its purification to homogeneity and high specific activity in a single chromatographic step. The properties of this recombinant protein are very similar to those of the liver protein. Direct demonstration of the binding of the recombinant protein to glucokinase has been obtained in vitro using coprecipitation experiments and in vivo, using the yeast two-hybrid system. These studies establish that the protein encoded by the cDNA is identical to the glucokinase regulatory protein and also validate tools with which to carry out structure-function studies on the interaction of the regulatory protein with glucokinase.