The culture of primary hepatocytes as spheroids creates an efficient three-dimensional tissue construct for hepatic studies in vitro. Spheroids possess structural polarity and functional bile canaliculi with normal differentiated function. Thus, hepatocyte spheroids have been proposed as the cell source in a variety of diagnostic, discovery, and therapeutic applications, such as a bioartificial liver. Using a novel rocking technique to induce spheroid formation, kinetics of spheroid formation, cell-cell adhesion, gene expression, and biochemical activities of rat hepatocyte spheroids were tested over 14 days of culture. Evidence was provided that the formation of spheroids occurred faster and with fewer nonadherent hepatocytes in rocked suspension culture compared to a traditional rotational system. Hepatocyte spheroids in rocked culture showed stable expression of more than 80% of 242 liver-related genes including those of albumin synthesis, urea cycle, phase I and II metabolic enzymes, and clotting factors. Biochemical activity of rocked spheroid hepatocytes was superior to monolayer culture of hepatocytes on tissue culture plastic and collagen. N ovel systems are needed to facilitate short-term and long-term culture of hepatocytes for diagnostic, discovery, and therapeutic applications. 1 Traditional monolayer culture of primary hepatocytes on tissue culture plastic is problematic and has been associated with a rapid loss of differentiated function. 2 Although single-layer and double-layer surfaces of collagen or other biomatrix materials are associated with improved differentiated functions in vitro, 3 biological surfaces can pose manufacturing hurdles and do not support high cell density culture of primary hepatocytes (exceeding 1 ϫ 10 7 cell/mL). In contrast, spheroids, which are nonadherent multicell aggregates of greater than 40 m diameter, provide a three-dimensional tissue construct which form spontaneously and allow suspension culture of primary hepatocytes at high cell density under oxygenated bioreactor conditions. 4 Spheroid formation allows recapitulation of the cuboidal geometry of primary hepatocytes with relatively stable long-term differentiated function. 5,6 Reports of structural polarity and bile canaliculi formation by primary rat hepatocytes in spheroid aggregates provide further evidence that hepatic spheroids mimic the hepatocellular microanatomy of the liver. 7 Original observations of tissue-like aggregate formation from isolated cells was reported by Moscona in 1961, using fetal liver cells and a rotational technique. 8 The descriptive term "spheroid" was coined years later by Landry in 1985 when multicellular aggregates were formed from isolated rat hepatocytes after 3-5 days of culture on nonadherent plastic surfaces. 9 Later, spheroids
The small heat shock-related protein 20 (HSP20) is present in four isoforms in bovine carotid artery smooth muscles. Three of the isoforms are phosphorylated and one is not. Increases in the phosphorylation of two isoforms of HSP20 (isoform 3, pI 5.9; and 8, pI 5.7) are associated with cyclic nucleotide-dependent relaxation of bovine carotid artery smooth muscles. Increases in the phosphorylation of another isoform (isoform 4, pI 6.0) are associated with phorbol ester-induced contraction of bovine carotid artery smooth muscles. In this investigation we determined that isoforms 3 and 8 are phosphorylated on Ser 16 of the HSP20 molecule during activation of cAMP-dependent signaling pathways. Phosphorylation state-specific antibodies produced against a peptide containing phosphorylated Ser 16 recognized isoforms 3 and 8 but not isoform 4. In human vascular tissue, only isoform 3 is present. Incubation of transiently permeabilized strips of bovine carotid artery smooth muscle with synthetic peptides in which Ser 16 is phosphorylated, inhibits contractile responses to high extracellular KCl and to serotonin. These data suggest that phosphorylation of HSP20 on Ser 16 modulates cAMP-dependent vasorelaxation.A major phosphorylation event that occurs with cyclic nucleotide-dependent relaxation of vascular smooth muscle is an increase in the phosphorylation of two 20-kDa proteins (isoform 3, pI 5.9; and 8, pI 5.7) (1-3). We recently identified these 20-kDa phosphoproteins as different phosphorylated forms of a small heat shock-related protein, HSP20 1 (4). In addition, HSP20 can be phosphorylated in vitro by both cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG) (4). HSP20 is also phosphorylated during endothelialdependent vasorelaxation of isolated segments of bovine carotid artery smooth muscle (5).In a vascular smooth muscle, umbilical artery smooth muscle, that is refractory to cyclic nucleotide-dependent vasorelaxation, there is no significant increase in the phosphorylation of HSP20 in response to activation of PKA or PKG (2). HSP20 is present in umbilical artery smooth muscle and can be phosphorylated by PKA in vitro using homogenates of umbilical smooth muscle (6). Taken together, these data support a role for phosphorylated HSP20 in mediating cyclic nucleotide-dependent vasorelaxation.Histamine and phorbol ester-induced contractions of bovine carotid artery smooth muscle are associated with an increase in the phosphorylation of another 20-kDa protein (isoform 4, pI 6.0) (1). The subsequent activation of cyclic nucleotide-dependent signaling pathways leads to a decrease in the phosphorylation of isoform 4. This 20-kDa protein is immunoreactive with specific polyclonal antibodies raised against HSP20 (4). Thus, increases in the phosphorylation of this isoform of HSP20 are associated with smooth muscle contraction and decreases are associated with activation of cyclic nucleotide-dependent signaling pathways.The purpose of this investigation was to determine the specific site on the HSP20 mo...
OBJECTIVEThe contribution of mitochondrial dysfunction to skeletal muscle insulin resistance remains elusive. Comparative proteomics are being applied to generate new hypotheses in human biology and were applied here to isolated mitochondria to identify novel changes in mitochondrial protein abundance present in insulin-resistant muscle.RESEARCH DESIGN AND METHODSMitochondria were isolated from vastus lateralis muscle from lean and insulin-sensitive individuals and from obese and insulin-resistant individuals who were otherwise healthy. Respiration and reactive oxygen species (ROS) production rates were measured in vitro. Relative abundances of proteins detected by mass spectrometry were determined using a normalized spectral abundance factor method.RESULTSNADH- and FADH2-linked maximal respiration rates were similar between lean and obese individuals. Rates of pyruvate and palmitoyl-dl-carnitine (both including malate) ROS production were significantly higher in obesity. Mitochondria from obese individuals maintained higher (more negative) extramitochondrial ATP free energy at low metabolic flux, suggesting that stronger mitochondrial thermodynamic driving forces may underlie the higher ROS production. Tandem mass spectrometry identified protein abundance differences per mitochondrial mass in insulin resistance, including lower abundance of complex I subunits and enzymes involved in the oxidation of branched-chain amino acids (BCAA) and fatty acids (e.g., carnitine palmitoyltransferase 1B).CONCLUSIONSWe provide data suggesting normal oxidative capacity of mitochondria in insulin-resistant skeletal muscle in parallel with high rates of ROS production. Furthermore, we show specific abundance differences in proteins involved in fat and BCAA oxidation that might contribute to the accumulation of lipid and BCAA frequently associated with the pathogenesis of insulin resistance.
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