Glucose homeostasis is controlled by a glucose sensor in pancreatic fl-cells. Studies on rodent fl-cells have suggested a role for GLUT2 and glucokinase in this control function and in mechanisms leading to diabetes. Little direct evidence exists so far to implicate these two proteins in glucose recognition by human (3-cells. The present in vitro study investigates the role of glucose transport and phosphorylation in fl-cell preparations from nondiabetic human pancreata. Human fl-cells differ from rodent f8-cells in glucose transporter gene expression (predominantly GLUT1 instead of GLUT2), explaining their low K. (3 mmol/liter) and low V m^x (3 mmol/min per liter) for 3-0-methyl glucose transport. The 100-fold lower GLUT2 abundance in human versus rat fl-cells is associated with a 10-fold slower uptake of alloxan, explaining their resistance to this rodent diabetogenic agent. Human and rat f-cells exhibit comparable glucokinase expression with similar flux-generating influence on total glucose utilization. These data underline the importance of glucokinase but not of GLUT2 in the glucose sensor of human fl-cells. (J. Clin. Invest 96:2489-2495

Hepatic stellate cells are considered to be liver-specific pericytes that play a key role in liver fibrosis. Because these cells express desmin and smooth muscle ␣-actin, they were assumed to be of myogenic origin. This hypothesis became doubtful when it was reported that stellate cells also express glial fibrillary acidic protein and neural cell adhesion molecule. In the present study, we show that activated stellate cells express nestin, a class VI intermediate filament protein originally identified as a marker for neural stem cells. Expression of nestin was first studied during spontaneous activation of stellate cells in culture. Immunohistochemistry showed that nestin-positive stellate cells already appeared at day 3, and nearly all the cells became positive for nestin at day 6 and 15. The immunoreaction was present in filaments except in dividing cells. The presence of messenger RNA transcript for nestin was shown by reverse transcription polymerase chain reaction and sequencing of amplified complementary DNA. We then compared the presence of nestin with that of other intermediate filament proteins and smooth muscle ␣-actin. Immunoblotting showed that the relative concentrations of nestin, desmin, and vimentin increased between day 2 and 6 in primary culture. After the initial increase vimentin leveled off, while nestin and desmin showed a tendency to decrease. This pattern was quite different from that of glial fibrillary acidic protein, which kept declining, and smooth muscle ␣-actin, which increased continuously up to day 13 in culture. We then studied the presence of nestin in normal and CCl 4 -injured rat liver. In normal liver, minimal immunoreaction for nestin was observed within the liver parenchyma. During induction of fibrosis by carbon tetrachloride, nestin-positive stellate cells appeared at 6 weeks, which was late in comparison with the induction of desmin and smooth muscle ␣-actin. We conclude that nestin is induced in stellate cells during transition from the quiescent to the activated phenotype; culture activation is a stronger stimulus than in vivo activation by injection of CCl 4 . Taken Hepatic stellate cells exert specific liver functions: storage of large amounts of retinyl esters, synthesis and breakdown of hepatic extracellular matrix, secretion of a variety of cytokines, and control of the diameter of the sinusoids.

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Expression of glucokinase in hepatocytes and pancreatic 13-cells is of major physiologic importance to mammalian glucose homeostasis. Liver glucokinase catalyzes the first committed step in the disposal of glucose, and 18-cell glucokinase catalyzes a rate-limiting step required for glucose-regulated insulin release. The present study reports the expression of glucokinase in rat glucagon-producing a-cells, which are negatively regulated by glucose. Purified rat a-cells express glucokinase mRNA and protein with the same transcript length, nucleotide sequence, and immunoreactivity as the 13-cell isoform. Glucokinase activity accounts for more than 50% of glucose phosphorylation in extracts of a-cells and for more than 90% of glucose utilization in intact cells. The glucagon-producing tumor MSL-G-AN also contained glucokinase mRNA, protein, and enzymatic activity. These data indicate that glucokinase may serve as a metabolic glucose sensor in pancreatic a-cells and, hence, mediate a mechanism for direct regulation of glucagon release by extracellular glucose. Since these cells do not express Glut2, we suggest that glucose sensing does not necessarily require the coexpression of Glut2 and glucokinase.Control of blood glucose levels in mammals is dependent on hormonal and metabolic communication between pancreatic islets of Langerhans and the liver. Acute changes in blood glucose concentration are detected by the endocrine pancreas, which responds by secreting a hormonal mixture that is rich in either glucagon (fasting state) or insulin (during or just after meals). Glucagon stimulates the liver to mobilize glucose from intracellular glycogen stores, while insulin increases postprandial glucose extraction from the portal vein (1). Since glucose itself is the main physiological activator of insulin release (2) and a direct inhibitor of glucagon release (3-5), the system is controlled via short feedback loops that are typical for homeostasis in general.Glucokinase (hexokinase IV) is expressed in liver and islets of Langerhans where it has been proposed to regulate hepatic glucose disposal and pancreatic glucose sensing (6, 7). Unlike the other mammalian hexokinases, which all have high affinity for glucose, the Km of glucokinase is in the millimolar range. Consequently, glycolytic flux becomes proportional to the extracellular glucose concentration in glucokinase-expressing cells as long as glucose uptake is not rate-limiting for its further metabolism (6, 7). Furthermore, the enzyme is not sensitive to feedback inhibition by glucose 6-phosphate (G6P), allowing the liver to sustain high metabolic flux despite elevated intracellular concentration of G6P (7). Glucokinase gene expression level in 13-cells is correlated to cellular glucose sensitivity both in vivo (8, 9) and in vitro (10), suggesting that the enzyme is a constituent of the ,B-cell glucose sensor. Analogous to the situation in 13-cells, it is conceivable that glucokinase expression in pancreatic a-cells is required for glucose to suppress glucagon re...

Activation of hepatic stellate cells (HSC) is a central event in the pathogenesis of liver fibrosis during chronic liver injury. We examined the expression of retinoic acid (RAR) and retinoid X receptors (RXR) during HSC activation and evaluated the influence of natural and synthetic retinoic acids (RA) on the phenotype of culture-activated HSC. The expression of the major RAR/RXR subtypes and isoforms was analyzed by Northern hybridization. Presence of functional receptor proteins was established by gel shift analysis. Retinoic acids, RAR, and RXR selective agonists and an RAR antagonist were used to evaluate the effects of retinoid signalling on matrix synthesis by Northern blotting and immunoprecipitation, and on cell proliferation by BrdU incorporation. The 9-cisRA and synthetic RXR agonists reduced HSC proliferation and synthesis of collagen I and fibronectin. All-trans RA and RAR agonists both reduced the synthesis of collagen I, collagen III, and fibronectin, but showed a different effect on cell proliferation. Synthetic RAR agonists did not affect HSC proliferation, indicating that ATRA inhibits cell growth independent of its interaction with RARs. In contrast, RAR specific antagonists enhance HSC proliferation and demonstrate that RARs control proliferation in a negative way. In conclusion, natural RAs and synthetic RAR or RXR specific ligands exert differential effects on activated HSC. Our observations may explain prior divergent results obtained following retinoid administration to cultured stellate cells or to animals subjected to fibrogenic stimuli. (HEPATOLOGY 2004;39:97-108.)

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Rat pancreatic a-and P-cells are critically dependent on hormonal signals generating cyclic AMP (cAMP) as a synergistic messenger for nutrient-induced hormone release. Several peptides of the glucagon-secretin family have been proposed as physiological ligands for cAMP production in P-cells, but their relative importance for islet function is still unknown. The present study shows expression at the RNA level in p-cells of receptors for glucagon, glucose-dependent insulinotropic polypeptide (GIP), and glucagon-like peptide 1(7-36) amide (GLP-I), while RNA from islet a-cells hybridized only with GIP receptor cDNA. Western blots confirmed that GLP-I receptors were expressed in P-cells and not in a-cells. Receptor activity, measured as cellular cAMP production after exposing islet P-cells for 15 min to a range of peptide concentrations, was already detected using 10 pmol/1 GLP-I and 50 pmol/1 GIP but required 1 nmol/1 glucagon. EC 50 values of GLP-I-and GIP-induced cAMP formation were comparable (0.2 nmol/1) and 45-fold lower than the EC g0 of glucagon (9 nmol/1). Maximal stimulation of cAMP production was comparable for the three peptides. In purified a-cells, 1 nmol/1 GLP-I failed to increase cAMP levels, while 10 pmol/1 to 10 nmol/1 GIP exerted similar stimulatory effects as in P-cells. In conclusion, these data show that stimulation of glucagon,