Effect of starvation on insulin production and insulin binding in Tetrahymena

Tetrahymena pyriformis GL was starved for 24 h and then the immunologically demonstrable insulin content and FITC-insulin binding were measured by flow cytometry and localization was studied by confocal microscopy. The amount of endogeneous insulin as well as FITC insulin binding, was highly significantly elevated. Glucose feeding for 30 min abolished the elevation of FITC-insulin binding. In starved cells, insulin-binding sites disappeared from the surface and FITC-insulin was bound inside the cells, within large food vacuoles. Endogeneous insulin was dispersed in the cytoplasm both in the control and starved cells and food vacuoles did not contain it. The results call attention to the stimulatory effect of starvation on insulin production in Tetrahymena, in parallel with the internal storage of insulin receptors, which points to an autocrine mechanism.     

Evidence of the receptor nature of the binding sites induced in Tetrahymena by insulin treatment. A quantitative cytofluorimetric technique for the study of binding kinetics

Tetrahymena pyriformis GL cells pretreated (imprinted) and not pretreated with insulin showed dissimilar quantitative relations of FITC-insulin binding. Displacement of FITC-insulin by unlabelled insulin was considerably less in the control than in the imprinted series. The curve for saturation of the binding sites with FITC-insulin resembled a true saturation curve. The imprinted cells bound considerably more hormone in a shorter time than the control cells at identical levels of exposure. The dissociation of bound hormone from the imprinted cells increased over the control at 23 degrees C, and to a still greater degree at 4 degrees C. The effect of the pH of the medium on the dissociation of bound FITC-insulin also differed between the imprinted and not imprinted cells. Thus the proposed cytofluorimetric assay of binding kinetics demonstrated the actual conditions of receptor activity, and indicated that the induced insulin binding sites of Tetrahymena behaved similarly to 'classical' receptors.     

The vascular endothelial cell mediates insulin transport into skeletal muscle

The pathways by which insulin exits the vasculature to muscle interstitium have not been characterized. In the present study, we infused FITC-labeled insulin to trace morphologically (using confocal immunohistochemical methods) insulin transport into rat skeletal muscle. We biopsied rectus muscle at 0, 10, 30, and 60 min after beginning a continuous (10 mU x min(-1) x kg(-1)), intravenous FITC-insulin infusion (with euglycemia maintained). The FITC-insulin distribution was compared with that of insulin receptors (IR), IGF-I receptors (IGF-IR), and caveolin-1 (a protein marker for caveolae) in skeletal muscle vasculature. We observed that muscle endothelium stained strongly for FITC-insulin within 10 min, and this persisted to 60 min. Endothelium stained more strongly for FITC-insulin than any other cellular elements in muscle. IR, IGF-IR, and caveolin-1 were also detected immunohistochemically in muscle endothelial cells. We further compared their intracellular distribution with that of FITC-insulin in cultured bovine aortic endothelial cells (bAECs). Considerable colocalization of IR or IGF-IR with FITC-insulin was noted. There was some but less overlap of IR or IGF-IR or FITC-insulin with caveolin-1. Immunoprecipitation of IR coprecipitated caveolin-1, and conversely the precipitation of caveolin-1 brought down IR. Furthermore, insulin increased the tyrosine phosphorylation of caveolin-1, and filipin (which inhibits caveolae formation) blocked insulin uptake. Finally, the ability of insulin, IGF-I, and IGF-I-blocking antibody to diminish insulin transport across bAECs grown on transwell plates suggested that IGF-IR, in addition to IR, can also mediate transendothelial insulin transit. We conclude that in vivo endothelial cells rapidly take up and concentrate insulin relative to plasma and muscle interstitium and that IGF-IR, like IR, may mediate insulin transit through endothelial cells in a process involving caveolae.     

Effects of extremely low concentrations of hormones on the insulin binding of Tetrahymena

FITC-insulin binding to previously hormone-treated Tetrahymena was studied by flow cytometry and confocal microscopy. Hormones produced by Tetrahymena were chosen for study and the hormone concentrations were administered between 10(-6) and 10(-21)M for 30 min. Endorphin, serotonin and insulin significantly reduced the hormone binding however histamine did not influence it at all. Endorphin, serotonin and insulin were significantly effective down to 10(-18)M and the effect of insulin and endorphin suggest a similar mechanism. The results call attention to the efficacy of very low hormone concentrations, which can influence the hormone content (earlier experiments) and receptor binding capacity (present study) of a unicellular organism. This seems to be very important, as in wild (natural) conditions the dilution of signaling materials secreted by a water-living protozoan is very high. In addition, the results point to the selectivity of response, as not all of the hormones that deeply influence other physiological indices (e.g. histamine) have an effect on insulin content or insulin receptors.     

Glucose starvation alters insulin but not IGF-I binding to Chinese hamster ovary (CHO) cells

The pattern of cellular protein glycosylation can be altered in CHO cells by glucose starvation. When wild type CHO cells are deprived of glucose, 125I-insulin binding increases from a B/F of 0.033 +/- 0.004 to 0.063 +/- 0.011, due to an increase in receptor affinity. The already elevated insulin binding to mutant B4-2-1 CHO cells, whose genetic defect causes abnormal glycosylation mimicking the pattern seen in the glucose starved normal cells, is not affected by glucose starvation. In neither cell line is 125I-IGF-I binding affected by glucose starvation. These data support the hypothesis that abnormal glycosylation can alter insulin binding to its receptor. Furthermore, there is a striking difference in the susceptibility of IGF-I and insulin receptors to alterations in glycosylation.     

Gastrointestinal stability and absorption of insulin in suckling pigs

Stability and absorption of orally administered fluorescein-isothiocyanate labeled insulin (FITC-insulin) in the gastrointestinal (GI) tract were investigated in newborn and 3-day-old pigs. The uptake of FITC-insulin by the intestinal epithelial cells was visualized using confocal laser scanning microscopy. Following oral administration, 3 h later 56 and 88% of orally administered fluorescence was found in the GI tract in newborn and 3-day-old piglets, respectively. Chromatographic analysis revealed that 15-37% of fluorescence recovered from the gastric and proximal intestinal contents was eluted in the void volume of a Sephadex G-25 column. It was also observed that oral administration of FITC-insulin at a dose of 100 nmol/kg body weight led to a significant decrease in blood glucose in newborn pigs (P<0. 05) but not in 3-day-old pigs. Microscopic examination showed that FITC-insulin was taken up via the vesicular transport mechanism throughout the whole small intestine but the ileum appeared to be a preferred site for FITC-insulin transport in newborn pigs. In 3-day-old pigs, the uptake of FITC-insulin occurred only in the distal part of the small intestine. These findings suggest that milk-borne insulin may partially survive in the GI lumen and subsequently act on the gastrointestinal tract in suckling piglets, while GI absorption of milk-borne insulin is limited to newborn pigs.     

Caveolin-1 is required for vascular endothelial insulin uptake

As insulin's movement from plasma to muscle interstitium is rate limiting for its metabolic action, defining the regulation of this movement is critical. Here, we address whether caveolin-1 is required for the first step of insulin's transendothelial transport, its uptake by vascular endothelial cells (ECs), and whether IL-6 and TNFα affect insulin uptake or caveolin-1 expression. Uptake of FITC-labeled insulin was measured using confocal microscopy in control bovine aortic ECs (bAECs), in bAECs in which caveolin-1 was either knocked down or overexpressed, in murine ECs from caveolin-1(-/-) mice and in bAECs exposed to inflammatory cytokines. Knockdown of caveolin-1 expression in bAECs using specific caveolin-1 siRNA reduced caveolin-1 mRNA and protein expression by ～ 70%, and reduced FITC-insulin uptake by 67% (P < 0.05 for each). Over-expression of caveolin-1 increased insulin uptake (P < 0.05). Caveolin-1-null mouse aortic ECs did not take up insulin and re-expression of caveolin-1 by transfecting these cells with FLAG-tagged caveolin-1 DNA rescued FITC-insulin uptake. Knockdown of caveolin-1 significantly reduced both insulin receptor protein level and insulin-stimulated Akt1 phosphorylation. Knockdown of caveolin-1 also inhibited insulin-induced caveolin-1 and IGF-1 receptor translocation to the plasma membrane. Compared with controls, IL-6 or TNFα (20 ng/ml for 24 h) inhibited FITC-insulin uptake as well as the expression of caveolin-1 mRNA and protein (P < 0.05 for each). IL-6 or TNFα also significantly reduced plasma membrane-associated caveolin-1. Thus, we conclude that insulin uptake by ECs requires expression of caveolin-1 supporting a role for caveolae mediating insulin uptake. Proinflammatory cytokines may inhibit insulin uptake, at least in part, by inhibiting caveolin-1 expression.     

Insulin down-regulates insulin receptor number and up-regulates insulin receptor affinity in cells expressing a tyrosine kinase-defective insulin receptor

We examined the effect of insulin treatment on HTC cells transfected with large numbers of either normal insulin receptors (HTC-IR) or insulin receptors defective in tyrosine kinase (HTC-IR/M-1030). In both HTC-IR and HTC-IR/M-1030 cells, 20 h of insulin treatment (1 microM) at 37 degrees C resulted in a 65% decrease in the number of binding sites with a reciprocal 6-fold increase in affinity. In contrast, treatment with 10 nM insulin (20 h, 37 degrees C) also increased receptor affinity but had a smaller effect on the number of binding sites. 125I-Insulin binding to soluble receptors from HTC-IR and HTC-IR/M-1030 cells pretreated with insulin showed results similar to those obtained in intact cells. In both HTC-IR and HTC-IR/M-1030 cells, insulin enhanced insulin receptor degradation. In HTC-IR/M-1030 cells a 1-h incubation with insulin did not change receptor number and had only a small effect on receptor affinity; also there was no effect of insulin after a 20-h incubation at 15 degrees C. Inhibiting protein synthesis by pretreatment with cycloheximide (100 microM) did not block either the decrease in receptor number or the increase in receptor affinity. Both HTC-IR and HTC-IR/M-1030 cells exhibited a very slow rate of insulin and insulin receptor internalization and no differences were seen in this parameter when HTC-IR cells were compared to HTC-IR/M-1030 cells. These studies indicate, therefore, that in cells expressing kinase-defective insulin receptors, insulin down-regulates insulin receptor number via enhanced receptor degradation, and up-regulates receptor affinity. These effects were time- and temperature-dependent, but not dependent on new protein synthesis, and suggest that activation of tyrosine kinase may not be a prerequisite for certain mechanisms whereby insulin regulates its receptor.     

Insulin receptor cycling and insulin action in the rat adipocyte

The possibility that the insulin receptor of adipocytes undergoes cycling was examined by a method involving pronase digestion at 12 degrees C, followed by insulin binding studies to determine receptor location and quantity. In the absence of insulin treatment, the amount of internal receptors (i.e. protected from pronase) was 10% of total receptor content. Following a 30-min insulin treatment (0.1 microM) at 37 degrees C, the internal receptor content increased 2-fold (206 +/- 12% of control, 100%). This effect was rapid, and maximum internalization was approached by 5 min of insulin treatment. Warming pronase-digested cells to 37 degrees C allowed the internal receptors to move to the cell surface. This movement was rapid also, and expansion of the internal pool by insulin pretreatment provided a 2.4-fold increase in the reinsertion of cell-surface receptors (238 +/- 28% of nontreated cells, 100%). Insulin-pretreated and nontreated cells had approximately 13 and 6%, respectively, of their original cell-surface receptor content, i.e. their content before pronase digestion. These receptors appeared intact after the cycling process, as judged by affinity labeling and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the receptor and its binding subunit. The ability of the recycled receptor to respond to insulin was examined by studies of glucose incorporation into lipids and the inhibition of isoproterenol-stimulated lipolysis. Cells pretreated with insulin and allowed to recycle (e.g. 13% of normal receptor content) were 2-3-fold more responsive and 7-fold more sensitive to subsequent insulin stimulation than nontreated cells (e.g. 6% of normal receptor content), indicating that the recycled receptors are biologically active and coupled to cellular effector systems.     

A mutant insulin receptor with defective tyrosine kinase displays no biologic activity and does not undergo endocytosis

The cDNAs encoding the normal human insulin receptor (HIRc) and a receptor that had lysine residue 1018 replaced by alanine (A/K1018) were used to transfect Rat 1 fibroblasts. Lysine 1018 is a critical residue in the ATP binding site of the tyrosine kinase domain in the receptor beta-subunit. Untransfected Rat 1 cells express 1700 endogenous insulin receptors. Expressed HIRc receptors had levels of insulin-stimulable autophosphorylation in vitro comparable to normal receptors, whereas A/K1018 receptors had less than 1% of that activity. Stimulation by insulin of HIRc receptors in situ in intact cells led to phosphorylation of beta-subunit tyrosine residues and activation of tyrosine kinase activity that could be preserved and assayed in vitro after receptor purification. In contrast, A/K1018 receptors showed no such activation, either of autophosphorylation or of kinase activity toward histone. Cells expressing HIRc receptors display enhanced sensitivity to insulin of 2-deoxyglucose transport and glycogen synthase activity. This increased sensitivity was proportional to insulin receptor number at low but not at high levels of receptor expression. A/K1018 receptors were unable to mediate these biologic effects and actually inhibited insulin's ability to stimulate glucose transport and glycogen synthase through the endogenous Rat 1 receptors. Expressed HIRc receptors mediated insulin internalization and degradation, whereas A/K1018 receptors mediated little, if any. Endocytotic uptake of the expressed A/K1018 insulin receptors was also markedly depressed compared to normal receptors. Unlike HIRc receptors, A/K1018 receptors also fail to undergo down-regulation after long (24 h) exposures to high (170 nM) concentrations of insulin. We conclude the following. 1) Normal human insulin receptors expressed in Rat 1 fibroblasts display active tyrosine-specific kinase, normal intracellular itinerary after endocytosis, and normal coupling to insulin's biologic effects. 2) A receptor mutated to alter the ATP binding site in the tyrosine kinase domain had little if any tyrosine kinase activity. 3) This loss of kinase activity was accompanied by a nearly complete lack of both endocytosis and biologic activity.     

Receptors for insulin and insulin-like growth factor-I can form hybrid dimers. Characterisation of hybrid receptors in transfected cells.

We have demonstrated the formation of hybrid insulin/insulin-like growth factor-I(IGF-I) receptors in transfected rodent fibroblasts, which overexpress human receptors, by examining reactivity with species- and receptor-specific monoclonal antibodies. In NIH 3T3 and Rat 1 fibroblasts, endogenous IGF-I receptors were unreactive with anti-(human insulin receptor)monoclonal antibodies (47-9, 25-49, 83-14, 83-7, 18-44). However, in transfected cells expressing high levels of insulin receptors, 60-80% of high-affinity IGF-I receptors reacted with these antibodies, as assessed either by inhibition of ligand binding in intact cells or by precipitation of solubilized receptors. Conversely, endogenous insulin receptors in NIH 3T3 cells were unreactive with anti-(IGF-I receptor) antibodies alpha IR-3 and 16-13. However, approx. 50% of high-affinity insulin receptors reacted with these antibodies in cells expressing high levels of human IGF-I receptors. The hybrid receptors in transfected cells bound insulin or IGF-I with high affinity. However, responses to these ligands were asymmetrical, in that binding of IGF-I inhibited subsequent binding of insulin, but prior binding of insulin did not affect the affinity for IGF-I. The existence of hybrid receptors in normal tissues could have important implications for metabolic regulation by insulin and IGF-I.     

Effects of growth and insulin treatment on the levels of insulin receptors and their mRNA in Hep G2 cells

We have studied the variations in the number of insulin receptor and insulin receptor mRNA levels in (Hep G2) cells in response to growth and insulin treatment. The levels of insulin receptors are relatively low in growing cells. After approximately 5 days in culture, if cells are not refed they cease to divide and the number of receptors/cell increases, reaching 4 times the initial values by the 9th day. Refeeding the cells completely prevented both growth arrest and the increase in insulin receptor number. Insulin added daily to cells at 0.33 microM caused receptor down-regulation but did not prevent a 3-fold increase in binding with growth arrest. Pulse-chase studies of metabolically labeled ([35S]methionine) cells showed that the receptor degradation rate (apparent t 1/2, 18-20 h) was comparable in rapidly growing versus growth-arrested cells. The increased receptor level in non-refed cells is not due to generation of a soluble factor by confluent cells, nor is it caused by depletion of insulin, glucose, or insulin-like growth factor I from the culture medium. The levels of insulin receptor mRNA measured on Northern blots increased in growth-arrested cells in parallel to the increase in receptor number. The mRNA value begins to increase from the 3rd day in culture and by the 9th day reaches a level 6.0 times that on the 3rd day. Chronic insulin-induced receptor down-regulation did not alter insulin receptor mRNA levels at any time point studied. These data demonstrate that the increase in insulin receptor number/cell in growth-arrested cells is paralleled by an increase in insulin receptor mRNA content with no change in the receptor degradation rates. This suggests that the increase in the number of insulin receptors is due to enhanced receptor synthesis due to increased receptor mRNA content. Conversely, down-regulation of the insulin receptor does not affect the level of insulin receptor mRNA and thus must be due to increased receptor degradation.     

Discrepancy in hormone binding and information transfer between sister cells of Tetrahymena clones

Tetrahymena cells treated with insulin in mass cultures were separated to single-cell clones or one of the "sister-cells" of dividing Tetrahymena (in single-cell culture) was treated with insulin. In both cases the FITC-insulin binding of sister-cells were compared. The insulin imprinting significantly increased the insulin binding of cells. There was also a significant difference between the imprinted and not imprinted sisters as well as between the not imprinted sisters. This demonstrates the existence of a difference (in hormone binding) between sister-cells and justifies that the information of the first hormone treatment (imprinting) is not equally divided between the sister-cells.     

Mutation of tyrosine residues 1162 and 1163 of the insulin receptor affects hormone and receptor internalization

Insulin internalization and degradation, insulin receptor internalization and recycling, as well as long term receptor down-regulation were comparatively studied in Chinese hamster ovary (CHO) cell lines, either parental or expressing the wild-type human insulin receptor (CHO.R) or a mutated receptor in which the tyrosine residues in positions 1162 and 1163 were replaced by phenylalanines (CHO.Y2). The two transfected cell lines presented very similar binding characteristics, and their pulse labeling with [35S]methionine revealed that the receptors were processed normally. As expected, the mutation of these twin tyrosines resulted in a defective insulin stimulation of both receptor kinase activity and glycogen synthesis. We now present evidence that compared to CHO.R cells, which efficiently internalized and degraded insulin, CHO.Y2 cells exhibited a marked defect in hormone internalization, leading to impaired insulin degradation. Moreover, the mutated receptors were found to be less effective than the wild-type receptors in transducing the hormone signal for receptor internalization, whereas the process of receptor recycling after internalization seemed not to be altered. In parental CHO cells, insulin induced long term receptor down-regulation, but was totally ineffective in both transfected cell lines. These results reveal that the tyrosines 1162 and 1163 in the kinase regulatory domain of the receptor beta-subunit play a pivotal role in insulin and receptor internalization.     

The effect of insulin, serum and dexamethasone on mRNA levels for the insulin receptor in the human lymphoblastoic cell line IM-9

The effect of insulin, serum and dexamethasone on mRNA levels in the insulin receptor in the human lymphoblastoic cell line IM-9 was examined. To this end, mRNA levels were quantitated by Northern blot analysis using a labeled cDNA probe for the insulin receptor. The presence of 0.1 microM dexamethasone in the medium had a strong stimulatory effect on mRNA levels in insulin receptor, suggesting the presence of a glucocorticoid inducible enhancer element near the insulin receptor gene. Also, the nature of the serum had an effect on insulin receptor mRNA levels, as cells maintained in 10% fetal calf serum had insulin receptor mRNA levels that were 40-50% of those found in IM-9 cells maintained in 1% newborn serum. Variations in insulin receptor mRNA levels led in each situation to concordant variations in insulin binding. Insulin levels of up to 1 microM had no effect on hybridizable insulin receptor mRNA levels making an insulin-induced feed-back mechanism on gene expression or mRNA stability unlikely.     

Time dependent effects of dexamethasone on serum insulin level and insulin receptors in rat liver and erythrocytes

The effects of glucocorticoid excess on regulation of insulin receptors were investigated in dexamethasone-treated rats. Glucocorticoid excess was produced by administration of dexamethasone (0.5 mg/100 g b.w.) 30 min, 4, 12, 18, 24, 42 or 70 h before experiments. This treatment caused time-dependent changes of glucose and insulin concentration in blood, as well as in amounts of specific insulin binding and insulin receptors of liver cells and erythrocytes. The time intervals in which dexamethasone produced the increase in insulin concentration were accompanied with decrease in insulin binding to receptors in membranes of liver cells, while significant changes in insulin binding to receptors of erythrocytes were not observed under the same experimental conditions. The effect is maximal 18 and 42 h after dexamethasone treatment that increase insulin blood level by about 85% and 60%, respectively. Receptor analysis revealed that changes in specific binding of insulin could be due to significant changes in amount of binding sites on cell surface rather than to mild alteration in receptor affinity. These findings suggest that besides the changes in insulin level, the alterations in insulin receptor number and affinity may play a major role in the states of altered insulin sensitivity which accompany glucocorticoid excess.     

Acute effects of insulin on surface insulin receptors in isolated hepatocytes. Evidence for a recycling pathway

Isolated rat hepatocytes were incubated for 1 h at 37 degrees C with 10 nM insulin. Following washout of insulin, cells were incubated with [125I] monoiodoinsulin at 15 degrees C to assess surface insulin binding. Preincubation with 10 nM insulin did not cause a decrease in insulin binding. Scatchard analysis confirmed that insulin receptor number remained constant. In the presence of 200 microM chloroquine or 25 microM monensin, surface insulin binding after preincubation with 10 nM insulin fell to 81.1 +/- 1.2% or 39.0 +/- 2.7% of control, respectively. It is suggested that the maintenance of insulin receptor number following acute insulin treatment in vitro is due to an insulin receptor recycling pathway, possibly involving lysosomes and/or the Golgi apparatus.     

The role of cell surface sialic acid in insulin receptor function and insulin action

Removal of cell surface sialic acid from adipocytes with neuraminidase inhibits insulin action. Here, we have examined the effects of mild neuraminidase treatment (5 milliunits/ml, 12 degrees C, 15 min) on insulin receptor structure and function. Neuraminidase treatment sufficient to cause greater than 90% loss of insulin stimulatable lipogenesis had no effect on 125I-insulin binding, tyrosine kinase activity of partially purified insulin receptors, insulin receptor phosphorylation in intact cells, or insulin-induced receptor internalization. However, recycling of the insulin receptor to the plasma membrane was inhibited by 50%. Recycled receptors in neuraminidase-treated cells were unable to mediate insulin action in contrast to recycled receptors from non-neuraminidase-treated cells. Furthermore, when insulin receptors were protected from exposure to neuraminidase, by inducing receptor internalization prior to neuraminidase treatment, the cells were still unable to respond to insulin. Analysis of the alpha and beta subunits of the receptor from neuraminidase-treated cells, affinity-labeled with 125I-insulin, or labeled by autophosphorylation, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis failed to indicate any changes in the holoreceptor or the individual subunits. This suggests there was no detectable release of sialic acid from the receptor. From this data we conclude that loss of sialic acid from nonreceptor glycoconjugates leads to loss of insulin action and inhibition of receptor recycling. The insulin receptor does not appear to be involved in this inhibitory effect. These findings suggest that an uncharacterized plasma membrane glycoprotein is essential in transmitting the "signal" of insulin binding to the cellular effector system.     

Effect of TRB3 on insulin and nutrient-stimulated hepatic p70 S6 kinase activity

Insulin and nutrients activate hepatic p70 S6 kinase (S6K1) to regulate protein synthesis. Paradoxically, activation of S6K1 also leads to the development of insulin resistance. In this study, we investigated the effect of TRB3, which acts as an endogenous inhibitor of Akt, on S6K1 activity in vitro and in vivo. In cultured cells, overexpression of TRB3 completely inhibited insulin-stimulated S6K1 activation by mammalian target of rapamycin, whereas knockdown of endogenous TRB3 increased both basal and insulin-stimulated activity. In C57BL/6 mice, adenoviral overexpression of TRB3 inhibited insulin-stimulated activation of hepatic S6K1. In contrast, overexpression of TRB3 did not inhibit nutrient-stimulated S6K1 activity. We also investigated the effect of starvation, feeding, or insulin treatment on TRB3 levels and S6K1 activity in the liver of C57BL/6 and db/db mice. Both insulin and feeding activate S6K1 in db/db mice, but only insulin activates in the C57BL/6 strain. TRB3 levels were 3.5-fold higher in db/db mice than C57BL/6 mice and were unresponsive to feeding or insulin, whereas both treatments reduced TRB3 in C57BL/6 mice. Akt was activated by insulin alone in the C57BL/6 strain and but not in db/db mice. Both insulin and feeding activated mammalian target of rapamycin similarly in these mice; however, feeding was unable to activate the downstream target S6K1 in C57BL/6 mice. These results suggest that the nutrient excess in the hyperphagic, hyperinsulinemic db/db mouse primes the hepatocyte to respond to nutrients resulting in elevated S6K1 activity. The combination of elevated TRB3 and constitutive S6K1 activity results in decreased insulin signaling via the IRS-1/phosphatidylinositol 3-kinase/Akt pathway.