The "thrifty" gene encoding Ahsg/Fetuin-A meets the insulin receptor: Insights into the mechanism of insulin resistance

Ahsg (fetuin-A) is a 55-59 kDa phosphorylated glycoprotein synthesized in the adult predominantly by hepatocytes, from which it enters the circulation. When dysregulated, this glycoprotein operates to influence the clinical sequelae of insulin resistance-type 2 diabetes and cardiovascular disease. The pathological sequelae likely arise from two separable molecular “faces” of Ahsg—one acting at the level of the insulin receptor and a second face influencing ectopic biomineralization in the intima. A detailed understanding of these two functional faces of Ahsg is not yet clear for lack of structural studies. Ahsg has a physiological role in the biomineralization of bone, which when dysregulated can lead to ectopic calcification of soft tissues in the vasculature. Ahsg has a second physiological function in regulating how insulin signals through its receptor, a transmembrane tyrosine kinase. Dysregulation of this “face” of Ahsg results in morbid sequelae such as impaired glucose disposal and fatty liver. Ahsg binds to tandem fibronectin type 3 (Fn3) domains present in the 194 amino acid residue extracellular portion of the β-subunit of the insulin receptor, distant from the high-affinity pocket formed by two complementing α-subunits where insulin binds. Only two proteins are known to bind directly to the insulin receptor ectodomain - insulin and Ahsg - the former turns on the receptor's intrinsic tyrosine kinase (TK) activity, and the latter shuts it down. Recent X-ray crystallographic studies of the ectodomain of the insulin receptor now sharpen our understanding of the receptor's extracellular α-subunit and linked β-subunit. Ahsg genotype and its circulating level have been correlated with body morphometrics (obese versus lean and visceral adiposity) in epidemiological studies enrolling thousands of patients. Epidemiological studies from the clinic reveal high levels of circulating Ahsg in insulin resistance and diabetes. This review endeavors to explain how one protein can mediate diverse pathologies, but specifically addresses its metabolic “face” blunting insulin receptor activity, an action leading to insulin resistance.     

Protein synthesis,polyribosomes, and peptide elongation in early development of Strongylocentrotus purpuratus

The absolute rate of protein synthesis in developing embryos of Strongylocentrotus purpuratus has been measured by lysine incorporation. Protein synthesis rises to about 240 pg hr^?1 embryo^?1 from the two- to eight-cell stage, and then gradually increases to a maximum of over 500 pg hr^?1 embryo^?1 in the blastula. The changes in protein synthesis are accompanied by similar increase in the polyribosomes in the embryo, so that 60–65% of the ribosomes are in polyribosomes by the blastula stage. The data are used to calculate an average peptide elongation rate of 1.8 amino acids ribosome^?1 sec^?1.     

Direct measurement of histone peptide elongation rate in cleaving sea urchin embryos

Regulation of protein synthesis can be exercised at a number of levels. One of the more experimentally difficult levels to approach has been the measurement of peptide elongation rate. This paper presents a new application of the cyanogen bromide (CNBr) cleavage of proteins in a direct measurement of histone peptide elongation rate in cleaving sea urchin embryos (Strongylocentrotus purpuratus). The data indicate an elongation rate (at 15°C) for histones H2B and H1α of 0.69 and 0.80 codons per s, respectively. These values fall within the range of previously published values of average peptide elongation rate for total protein in these cells. This method should be generally applicable to many systems for which the measurement of peptide elongation rate may provide a key to the understanding of the regulation of protein synthesis.     

Two temporal phases for the control of histone gene activity in cleaving sea urchin embryos (S. purpuratus)

This report presents an analysis of histone gene expression in the cleaving embryo of the sea urchin, Strongylocentrotus purpuratus, with emphasis on whether the regulatory site(s) in the pathway of gene expression change as development proceeds. The analysis focuses on the equation, $\text{dP1}\text{dt}\text{= M·f·n·}\text{A}\text{t}$, where $\text{dP1}\text{dt}$ = the absolute rate of histone synthesis; M = the mole quantity of histone messenger RNA; f = the fraction of histone mRNA in polysomes; n = the polysome size; and $\text{A}\text{t}$ = the rate of elongation of nascent histone polypeptide chains. The embryo solves this rate equation differently at different times. Measurements were made (at 15°C) of absolute rates of histone synthesis ($\text{dP1}\text{dt}$). The rate of histone synthesis increases at least 48-fold during the first 6 hr after fertilization from less than 0.5 to 24 pg embryo^?1 hr^?1; in the period from 6 to 12 hr, this rate rises to 182 pg embryo^?1 hr^?1, an additional 7.7-fold rise, resulting in an overall increase of 370-fold between the 1-cell and 200-cell stage. The fraction of newly synthesized (zygotic) histone messenger RNA that partitions into polysomes (f^zygotic) has also been measured during the first 12 hr of development. This fraction increases from 0.2 in the 2-hr embryo to 0.8 in the 6-hr embryo (16-cell stage), increasing slowly thereafter to near unity by 12 hr. The size of histone-synthesizing polysomes (n) does not change substantially over the 12-hr interval, remaining constant at a weighted mean of 5 ribosomes per polysome (range 3 to 7). Utilizing the data on f^zygotic and $\text{dP1}\text{dt}$, the rate of elongation of nascent histone polypeptide chains ($\text{A}\text{t}$) during the first 6 hr of development was estimated; $\text{A}\text{t}$ remains constant at 1.11 codons per second. This calculated value is in fair agreement with a direct measurement of histone peptide elongation rate in the 12-hr embryo. It is proposed that histone gene expression in cleaving sea urchin embryos be divided into two phases, distinguished on the basis of their pivotal translational parameters: Phase I (0–6 hr), during which f is rate determining, and Phase II (6 hr on), during which M is the rate-determining parameter.     

Transforming growth factor type beta can act as a potent competence factor for AKR-2B cells

Transforming growth factor type beta (TGF beta) is a pleiotropic regulator of cell growth with specific high-affinity cell-surface receptors on a large number of cells; its mechanism of action, however, is poorly defined. In this report, we utilized the mouse fibroblast line AKR-2B to explore the question of the temporal requirements during the cell cycle in regard to both the growth inhibitory and the growth stimulatory action of TGF beta. The results indicate that AKR-2B cells are most sensitive to the inhibitory action of TGF beta during early to mid-G1. In addition, TGF beta need be present only briefly (as little as 1 min) in order to exert its inhibitory effect on EGF-induced DNA synthesis. Likewise, the stimulatory effect of TGF beta in the absence of EGF requires only an equally brief exposure to TGF beta. Use of homogeneous 125I-labeled TGF beta in a cell-binding assay demonstrates that TGF beta bound to cell-surface receptors can readily exchange into the culture medium T1/2 = 120 min), helping to rule out the possibility that persistent receptor-bound TGF beta is the source of a continuous stimulus. The data indicate that TGF beta exposure induces a stable state in the cell (T1/2 = 20 h) similar to but distinct from the state of "competence" induced by platelet-derived growth factor (PDGF).     

Transforming growth factor type β can act as a potent competence factor for AKR-2B cells

Transforming growth factor type β (TGFβ) is a pleiotropic regulator of cell growth with specific high-affinity cell-surface receptors on a large number of cells; its mechanism of action, however, is poorly defined. In this report, we utilized the mouse fibroblast line AKR-2B to explore the question of the temporal requirements during the cell cycle in regard to both the growth inhibitory and the growth stimulatory action of TGFβ. The results indicate that AKR-2B cells are most sensitive to the inhibitory action of TGFβ during early to mid-G₁. In addition, TGFβ need be present only briefly (as little as l min) in order to exert its inhibitory effect on EGF-induced DNA synthesis. Likewise, the stimulatory effect of TGFβ in the absence of EGF requires only an equally brief exposure to TGFβ. Use of homogeneous ¹²⁵I-labeled TGFβ in a cell-binding assay demonstrates that TGFβ bound to cell-surface receptors can readily exchange into the culture medium T_1/2 = 120 min), helping to rule out the possibility that persistent receptor-bound TGFβ is the source of a continuous stimulus. The data indicate that TGFβ exposure induces a stable state in the cell (T_1/2 = 20 h) similar to but distinct from the state of “competence” induced by platelet-derived growth factor (PDGF).