Insulin Signaling

From the appearance of insulin in the bloodstream, the insulin signaling pathway begins with the binding of insulin to the extracellular a-subunits of the transmembrane protein insulin receptor (Fig. 1). Insulin receptor consists of two extracellular a-subunits and two transmembrane j8-subunits. This binding of insulin turns the receptor into an

* The invited author (and primary author) of this chapter.

The Nutritional Biochemistry of Chromium(III) John B. Vincent (Editor) ISBN: 0-444-53071-1

© 2007 Elsevier B.V. All rights reserved.

Cellular effects

Fig. 1. Insulin signaling pathway.

autokinase, phosphorylating itself at three tyrosine residues (1158, 1162, and 1663 following the human sequence) of the j8-subunit. This conversion turns the receptor into an active kinase catalytically phosphorylating tyrosine residues of several substrate proteins. Known substrates include the insulin receptor substrate proteins (IRS), Shc, Gab-1, and others. These proteins in turn recruit other proteins inside the cell, which possess phosphotyrosine-binding domains (SH2 and PTB domains), forming signaling centers. These adapter molecules include PI3K (phosphatidylinositol 3-kinase) and Grb2. Association of the p85 subunit of PI3K with IRS-1 or Gab activates the catalytic p110 subunit. PI3K in turn phosphorylates serines and threonine residues of Akt (protein kinase B), activating this kinase. Further propagation along this pathway leads to the major cellular effects associated with insulin action, including glucose uptake and metabolism. Thus, enhancing the binding of insulin to its receptor or the activation of any of the kinases along the cascade could enhance insulin signaling.

Alternatively, preventing the signaling system from being deactivated, for example the inhibition of phosphatase enzymes, could also enhance insulin action. Notably, phosphotyrosine protein phosphatase 1B (PTP1B) has been implicated in the dephos-phorylation of insulin receptor. For reviews, see [1, 2].

LOW-MOLECULAR-WEIGHT CHROMIUM/CHROMODULIN

Two biomolecules are known to bind Cr in vivo: transferrin and low-molecular-weight chromium-binding substance (LMWCr), also termed "chromodulin." Transferrin is responsible for maintaining chromium supplies in the bloodstream and transporting Cr to the tissues [3-6] and possibly from the intestine lining to the bloodstream [7]. The role(s) of LMWCr is less clear.

Low-molecular-weight chromium-binding substance was first reported by the toxicology group of Osamu Wada [8] in 1981. A low-molecular-weight chromium compound was identified by size exclusion chromatography of the cytosol of liver cells of male mice injected with a single dose of potassium chromate. A similar low-molecular-weight compound was found in the feces and urine and 2 hours after injection in the plasma. These researchers suggested that an LMWCr was formed in the liver, which participates in retention and excretion of chromium in the body. The material from the livers of rabbits treated similarly with chromate was partially purified and found apparently to be an anionic organic-chromium complex containing amino acids. This same year, Wu and Wada reported additional studies on LMWCr from urine [9]. Low-molecular-weight chromium-binding substance was found to occur in urine normally, although the amounts were greatly increased after rats were injected with chromate. Normal human and rat urine LMWCr was found not be saturated with chromium. The LMWCr was believed to be similar to that of the liver and other organs of rabbits and dogs and to be involved in removing excess chromium from the body.

Follow-up studies were performed looking at the effects of inhalation exposure of CrCl3. Chromium as LMWCr in the lungs (of rats exposed to the Cr-containing aerosol) slowly decreased while levels in the liver increased. Thus, LMWCr was proposed to be in equilibrium with Cr in the rest of the body; the long half-life of chromium in the lungs was proposed to be the result of low LMWCr levels or a slow rate of synthesis of the LMWCr [10]. Wada and coworkers have also examined the distribution of LMWCr [11]. Low-molecular-weight chromium-binding substance was found in liver, kidney, spleen, intestine, testicle, brain, and blood plasma, with the greatest amount in liver followed by kidney. The organs were obtained from mice 2 hours after injection with potassium dichromate. Supernatants of homogenates of the organs were found to possess more chromium bound to LMWCr when dichromate was added to the homogenate than when the mice were injected with dichromate. The time course of chromium binding to LMWCr after injection of dichromate was also examined [11]. Chromium was found to be associated with liver and kidney LMWCr only 2 minutes after injection and reached a maximum within 1-2 hours after treatment. In these studies, LMWCr was again identified by its elution behavior in size exclusion chromatography, and its Cr-binding ability.

Efforts have continued to isolate and characterize LMWCr. To date LMWCr has been isolated and purified from rabbit liver [12], bovine liver [13], porcine kidney [14], and porcine kidney powder [14] and partially purified from dog [15] and mouse liver [11]. Inclusion of protease inhibitors in buffers during the isolation of bovine liver LMWCr does not affect the amount of oligopeptide isolated [13], suggesting it is not a proteolytic artifact generated during the isolation procedure. The materials from rabbit and dog liver were loaded with Cr by injection of the animal with chromate (or Cr(III) which provides lower yields). For the materials from bovine liver and porcine kidney and kidney powder, chromate was added to the homogenized liver or kidney or suspended kidney powder. Chromium(III) could also be added to the bovine liver homogenate to load LMWCr with Cr, but the loading was not as efficient as when chromate was utilized [13]. A Cr-loading procedure is required so that the material can be followed by its Cr content during the isolation and purification procedures [12, 13]. The isolation procedures are similar involving an ethanol precipitation, anion exchange chromatography, and finally size exclusion chromatography. Thus, LMWCr appears to be a naturally occurring oligopeptide composed of glycine, cysteine, aspartate, and glutamate with the carboxylates comprising more than half of the total amino acid residues (Table 1). The amino acid composition data for the rabbit liver LMWCr (injected chromate) and bovine liver (chromate added to homogenate) are extremely similar, indicating that the type of Cr-loading procedure utilized is probably not critical to the composition of the isolated material. No amino acid sequence data has appeared, despite attempts at sequencing by Edman degradation, nuclear magnetic resonance (NMR), and mass spectrometry. The lack of additional characterization of the organic components of the materials is a matter of concern. Additionally, note that the material from urine has not been isolated and characterized; its assumed identity with the material from liver is based solely on their similar apparent molecular weight from size exclusion and similar chromatographies and their chromium-binding potential. Unfortunately to date, LMWCr has not proven to be antigenic, preventing its presence to be detected using immunological techniques.

The amino acid composition of LMWCr has been used to search the human genome for protein possessing fragments comprised of these amino acids. Two candidate sequences, EDGEECDCGE and DGEECDCGEE, from the beginning of the disintregrin domain of the protein ADAM 19 have been identified [17, 18]. ADAM 19 is a multidomain membrane protein with disintegrin and metalloproteinase domains [19]. The ten-amino acid sequences are conserved in rat, mouse, and human ADAM 19, the only

Table 1

Amino acid composition data for isolated LMWCr's

Table 1

Amino acid composition data for isolated LMWCr's

Source

Glycine

Glutamic acid

Aspartic acid

Cysteine

Ref.

Rabbit liver

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