Elmus G. Beale

Disregulated glyceroneogenesis: PCK1 as a candidate diabetes and obesity gene

Genetics and diet interact to cause type 2 diabetes mellitus and obesity. PCK1 has been implicated as one of many genes associated with type 2 diabetes mellitus. The common assumption is that mutations in PCK1 lead to excessive glucose production through hepatic gluconeogenesis. However, there is an alternative explanation, wherein mutations at the PCK1 locus could selectively affect PCK1 expression in adipose tissue. The result would be changes in glyceroneogenesis that would affect the storage and release of fatty acids. Here, we present the novel hypothesis that a variety of phenotypes could arise from mutations of the various tissue-specific control elements of PCK1. We also suggest specific quantitative metabolic traits that would accompany mutations that selectively affect PCK1 expression in adipose tissue.

Caenorhabditis elegans senses bacterial autoinducers.

ABSTRACT Pseudomonas aeruginosa uses virulence factors controlled by quorum sensing (QS) to kill Caenorhabditis elegans. Here we show that C. elegans is attracted to the acylated homoserine lactones (AHSLs) that mediate QS in P. aeruginosa. Our data also indicate that C. elegans can distinguish AHSLs and may use them to mediate aversive or attractive learning.

FAM20: an evolutionarily conserved family of secreted proteins expressed in hematopoietic cells

BackgroundHematopoiesis is a complex developmental process controlled by a large number of factors that regulate stem cell renewal, lineage commitment and differentiation. Secreted proteins, including the hematopoietic growth factors, play critical roles in these processes and have important biological and clinical significance. We have employed representational difference analysis to identify genes that are differentially expressed during experimentally induced myeloid differentiation in the murine EML hematopoietic stem cell line.ResultsOne identified clone encoded a previously unidentified protein of 541 amino acids that contains an amino terminal signal sequence but no other characterized domains. This protein is a member of family of related proteins that has been named family with sequence similarity 20 (FAM20) with three members (FAM20A, FAM20B and FAM20C) in mammals. Evolutionary comparisons revealed the existence of a single FAM20 gene in the simple vertebrate Ciona intestinalis and the invertebrate worm Caenorhabditis elegans and two genes in two insect species, Drosophila melanogaster and Anopheles gambiae. Six FAM20 family members were identified in the genome of the pufferfish, Fugu rubripes and five members in the zebrafish, Danio rerio. The mouse Fam20a protein was ectopically expressed in a mammalian cell line and found to be a bona fide secreted protein and efficient secretion was dependent on the integrity of the signal sequence. Expression analysis revealed that the Fam20a gene was indeed differentially expressed during hematopoietic differentiation and that the other two family members (Fam20b and Fam20c) were also expressed during hematcpoiesis but that their mRNA levels did not vary significantly. Likewise FAM20A was expressed in more limited set of human tissues than the other two family members.ConclusionsThe FAM20 family represents a new family of secreted proteins with potential functions in regulating differentiation and function of hematopoietic and other tissues. The Fam20a mRNA was only expressed during early stages of hematopoietic development and may play a role in lineage commitment or proliferation. The expansion in gene number in different species suggests that the family has evolved as a result of several gene duplication events that have occurred in both vertebrates and invertebrates.

Adipose Expression of the Phosphoenolpyruvate Carboxykinase Promoter Requires Peroxisome Proliferator-activated Receptor γ and 9-cis-Retinoic Acid Receptor Binding to an Adipocyte-specific Enhancer in Vivo

A putative adipocyte-specific enhancer has been mapped to approximately 1 kilobase pair upstream of the cytosolic phosphoenolpyruvate carboxykinase (PEPCK) gene. In the present study, we used transgenic mice to identify and characterize the 413-base pair (bp) region between −1242 and −828 bp as a bona fide adipocyte-specific enhancer in vivo. This enhancer functioned most efficiently in the context of the PEPCK promoter. The nuclear receptors peroxisome proliferator-activated receptor γ (PPARγ) and 9-cis-retinoic acid receptor (RXR) are required for enhancer function in vivo because: 1) a 3-bp mutation in the PPARγ-/RXR-binding element centered at −992 bp, PCK2, completely abolished transgene expression in adipose tissue; and 2) electrophoretic mobility supershift experiments with specific antibodies indicated that PPARγ and RXR are the only factors in adipocyte nuclear extracts which bind PCK2. In contrast, a second PPARγ/RXR-binding element centered at −446 bp, PCK1, is not involved in adipocyte specificity because inactivation of this site did not affect transgene expression. Moreover, electrophoretic mobility shift experiments indicated that, unlike PCK2, PCK1 is not selective for PPARγ/RXR binding. To characterize the enhancer further, the rat and human PEPCK 5′-flanking DNA sequences were compared by computer and found to have significant similarities in the enhancer region. This high level of conservation suggests that additional transcription factors are probably involved in enhancer function. A putative human PCK2 element was identified by this sequence comparison. The human and rat PCK2 elements bound PPARγ/RXR with the same affinities. This work provides the first in vivo evidence that the binding of PPARγ to its target sequences is absolutely required for adipocyte-specific gene expression.

Glyceroneogenesis comes of age

Glyceroneogenesis is a generally ignored metabolic pathway that occurs in adipose tissues and liver of mammalian species. This short review highlights a series of recent discoveries showing that glyceroneogenesis is important in lipid homeostasis.—Beale, E. G., Hammer, R. E., Antoine, B., Forest, C. Glyceroneogenesis comes of age. FASEB J. 16, 1695–1696 (2002)

A single element in the phosphoenolpyruvate carboxykinase gene mediates thiazolidinedione action specifically in adipocytes

Phosphoenolpyruvate carboxykinase (PEPCK) is the key enzyme in glyceroneogenesis, an important metabolic pathway that functions to restrain the release of non-esterified fatty acids (NEFAs) from adipocytes. The antidiabetic drugs known as thiazolidinediones (TZDs) are thought to achieve some of their benefits by lowering elevated plasma NEFAs. Moreover, peroxisome proliferator activated receptor gamma (PPARgamma) mediates the antidiabetic effects of TZDs, though many TZD responses appear to occur via PPARgamma-independent pathways. PPARgamma is required for adipocyte PEPCK expression, hence PEPCK could be a major target gene for the antidiabetic actions of TZDs. Here we used tissue culture and transfection assays to confirm that the TZD, rosiglitazone, stimulates PEPCK gene transcription specifically in adipocytes. We made the novel observation that this effect was by far the most rapid and robust among several other genes expressed in adipocytes. Adipocytes were transfected with a PEPCK/chloramphenicol acetyltransferase chimeric gene, in which either of the two previously discovered PPARgamma/retinoid X receptor alpha response elements, PCK2 and gAF1/PCK1, had been inactivated by mutagenesis. We demonstrate that PCK2 alone is a bona fide thiazolidinedione response element. We show also that the regulation of PEPCK by PPARs is cell-specific and isotype-specific since rosiglitazone induces PEPCK gene expression selectively in adipocytes, and PPARalpha- and PPARbeta-specific activators are inefficient. Hence, TZDs could lower plasma NEFAs via PPARgamma and PEPCK by enhancing adipocyte glyceroneogenesis.

Cell-specific expression of cytosolic phosphoenolpyruvate carboxykinase in transgenic mice.

The gene encoding cytosolic phosphoenolpyruvate carboxykinase (PEPCK) is expressed in multiple cell types in diverse tissues including liver, kidney, intestine, and white and brown adipose tissues. It can thus be considered a model system for examining the regulation of cell‐specific transcription. The PEPCK gene is transcribed from a single start site, but studies of transgenic mice have revealed that distinct cis‐acting elements (and thus different trans‐acting factors) regulate PEPCK expression in hepatocytes, renal proximal tubule epithelial cells, and adipocytes. Hepatocytes require elements between –457 and +69 bp; renal proximal tubule epithelia require elements between –363 and +69 bp; and adipocytes require elements between –2086 and –888 bp. An additional element downstream of +69 bp is required to either attenuate PEPCK mRNA levels in liver and fat or increase renal PEPCK mRNA. We hypothesize that the transcription factors C/EBP and DBP are the principal tissue‐specific regulators in liver, and that HNF‐1 and perhaps C/EBP are important for kidney‐specific PEPCK expression. We propose that the putative downstream element is involved in regulating PEPCK mRNA turnover in liver and fat. Finally, we suggest that the fat‐specific element is an enhancer that requires a novel adipogenic regulatory factor, ARF6, to function. The long‐term objective will be to fine map the cis‐acting elements and identify the cognate trans‐acting factors that regulate PEPCK in liver, kidney and fat. This information will help elucidate the combinatorial mechanisms that control the cell‐specific expression of this complex gene.— Beale, E. G., Clouthier, D. E., Hammer, R. E. Cell‐specific expression of cytosolic phosphoenolpyruvate carboxykinase in transgenic mice. FASEB J. 6: 3330‐3337; 1992.

A retrospective look at replacing face‐to‐face embryology instruction with online lectures in a human anatomy course

Embryology is integrated into the Clinically Oriented Anatomy course at the Texas Tech University Health Sciences Center School of Medicine. Before 2008, the same instructor presented embryology in 13 face‐to‐face lectures distributed by organ systems throughout the course. For the 2008 and 2009 offerings of the course, a hybrid embryology instruction model with four face‐to‐face classes that supplemented online recorded lectures was used. One instructor delivered the lectures face‐to‐face in 2007 and by online videos in 2008–2009, while a second instructor provided the supplemental face‐to‐face classes in 2008–2009. The same embryology learning objectives and selected examination questions were used for each of the three years. This allowed direct comparison of learning outcomes, as measured by examination performance, for students receiving only face‐to‐face embryology instruction versus the hybrid approach. Comparison of the face‐to‐face lectures to the hybrid approach showed no difference in overall class performance on embryology questions that were used all three years. Moreover, there was no differential effect of the delivery method on the examination scores for bottom quartile students. Students completed an end‐of‐course survey to assess their opinions. They rated the two forms of delivery similarly on a six‐point Likert scale and reported that face‐to‐face lectures have the advantage of allowing them to interact with the instructor, whereas online lectures could be paused, replayed, and viewed at any time. These experiences suggest the need for well‐designed prospective studies to determine whether online lectures can be used to enhance the efficacy of embryology instruction. Anat Sci Educ 7: 234–241.

5'-AMP-activated protein kinase signaling in Caenorhabditis elegans.

5′-AMP-activated protein kinase (AMPK) has been called “the metabolic master switch” because of its central role in regulating fuel homeostasis. AMPK, a heterotrimeric serine/threonine protein kinase composed of α, β, and γ subunits, is activated by upstream kinases and by 5′-AMP in response to various nutritional and stress signals. Downstream effects include regulation of metabolism, protein synthesis, cell growth, and mediation of the actions of a number of hormones, including leptin. However, AMPK research represents a young and growing field; hence, there are many unanswered questions regarding the control and action of AMPK. This review presents evidence for the existence of AMPK signaling pathways in Caenorhabditis elegans, a genetically tractable model organism that has yet to be fully exploited to elucidate AMPK signaling mechanisms.

Insulin Regulates Expression of the Phosphoenolpyruvate Carboxykinase Gene

Publisher Summary This chapter describes insulin regulates expression of the phosphoenolpyruvate carboxykinase (PEPCK) gene. The inhibitory effect of insulin on PEPCK gene transcription is (1) achieved at physiologic concentrations of the hormone, (2) mediated through the insulin receptor, (3) specific, (4) seen in the absence of on-going protein synthesis, (5) readily reversible, (6) dominant over the action of inducing hormones, and (7) probably exerted at the step of transcript initiation. Studies of the regulation of mRNA PEPCK synthesis provided the first evidence of an effect of insulin on gene transcription and would be of interest even if this were the only example of this action of the hormone. In recent years, several additional examples of regulation of specific mRNA metabolism by insulin have been reported and it appears that the regulation of PEPCK synthesis represents a prototype of a general regulatory role of insulin. Considerable progress has been made toward understanding how insulin regulates the rate at which a specific protein is synthesized in a target tissue. There are at present many examples in which insulin affects mRNA metabolism, and in several of these instances, this hormone influences the rate of transcription of specific genes. Based on the rate at which additional examples are appearing, it is obvious that this is an important manifestation of insulin action.