The focus of Dr. Agarwal's research has been on mechanisms of steroid action with emphases on: 1) structure-activity relationships of ligand-steroid receptor interactions, and 2) steroid metabolism. His early work involved interactions of the estrogen receptor with several non-steroidal antiestrogens (triaryl-ethylene and -ethane; TAEs) in order to define the ligand binding site on this receptor. These studies showed that these ligands bound to the same site on the estrogen receptor as estradiol-17ß. The TAEs however have an "accessory" binding site which is responsible for anti-estrogenic activity. Data on binding of ligands to the accessory site could improve anti-estrogen activity for use as anti-tumor agents or fertility regulators.
He carried out additional studies demonstrating that estrogen-receptor complexes needed to be constantly present at nuclear acceptor sites in order to maintain the transcriptional activity of estrogen. Specificity of steroid action in target tissues may be increased by biochemically modifying the steroid. For example, in male genital skin, testosterone is converted by 5a-reductase to dihydrotestosterone which is a more potent androgen than testosterone itself. Some analogs of testosterone, however, are maximally active with no further metabolism. One such synthetic analog of testosterone is 7a- methyl-19nor-testosterone. Using rat liver and a prostate microsomal fraction, he showed that this steroid was not metabolized by 5a-reductase and thus it might be useful for treatment of 5-alpha reductase deficient patients. It is currently being evaluated for its androgenic potency in primates.
His work over the past decade has concentrated on the conversion of cortisol (the main glucocorticoid in humans) to its inactive metabolite, cortisone. This conversion is mediated by 11ß- hydroxysteroid dehydrogenase (11-HSD). Specific defects in this enzyme lead to high levels of cortisol in the kidney and spurious activation of mineralocorticoid receptors by cortisol, an inherited form of hypertension termed apparent mineralocorticoid excess (AME). Dr. Agarwal cloned two isoforms of 11-HSD (types 1 and 2) and expressed them using a number of different systems including Xenopus oocytes, vaccinia virus and transfection of expression plasmids in mammalian cells. He showed that the 11-HSD1 isozyme catalyzes both oxidation of cortisol (and corticosterone) and reduction of cortisone (and 11-dehydrocorticosterone), requires NADP or NADPH as a cofactor, and requires glycosylation for full activity. In contrast, 11-HSD2 catalyzes only oxidation, requires NAD as a cofactor, has a much higher affinity for steroids, and apparently doesn't require glycosylation. Together with his colleagues, he showed that mutations of the 11-HSD2 gene cause AME.
Dr. Agarwal's current focus is to determine if there is an association between polymorphisms in the 11-HSD2 gene and essential hypertension or salt sensitivity and to examine the transcriptional regulation of this gene in placenta and kidney. His most recent work relates to CGL which is an autosomal recessive disorder characterized by extreme lack of body fat since birth, severe insulin resistance, hypertriglyceridemia, hepatic steatosis and early onset of diabetes. Through positional cloning, they identified disease causing mutations in (AGPAT2) gene located on chromosome 9q34, encoding 1-acylglycerol-3-phosphate-O-acyltransferase 2, in the affected subjects from 26 of the 42 pedigrees of various ethnicities. The affected individuals were either homozygous or compound heterozygous for various mutations including, deletions, nonsense, missense, splice-site and those in the 3'-UTR. The AGPAT2 catalyzes the acylation of the lysophosphatidic acid at the sn-2 position to form phosphatidic acid, a key intermediate in the biosynthesis of triacylglycerol (TG) and glycerophospholipids, which are involved in signal transduction.
The high AGPAT2 expression in adipose tissue suggests that the AGPAT2 mutations may cause CGL by inhibiting TG biosynthesis and storage in the adipocytes. It is also of interest to note that only five pedigrees revealed mutations in BSCL2 gene located on chromosome 11q13. The function of BSCL2 remains unknown. These observations suggest that at least two distinct mechanisms may underlie extreme lack of adipose tissue in CGL patients. In addition to these studies they also showed in a pedigree with familial parital lipodystrophy a hyterozygous, R425C, mutation in PPARG gene. It is still unclear how such a mutation could cause regional loss of fat.
More recently we have determined a new genetic loci, a zinc metalloproteinase (ZMPSTE24), in subjects diagnosed with Mandibuloacral Dysplasia. MAD, a rare autosomal recessive disorder, affects multiple tissues including loss of partial adipose tissue leading to insulin resistance and diabetes mellitus. These patients also have progeroid features. Mutations in ZMPSTE24 which is a coax-motif protease, cleaves prelamin A to mature lamin A. Uncleaved prelamin A has been shown to be toxic to the cells.