Insulin differentially regulates protein phosphotyrosine phosphatase activity in rat hepatoma cells.

We have studied the effect of insulin stimulation on phosphotyrosine phosphatase (PTPase) activity in the well-differentiated rat hepatoma cell line Fao. PTPase activity was measured using a 32P-labeled peptide corresponding to the major site of insulin receptor autophosphorylation. Of the PTPase activity in Fao cells, 14% was in the cytosolic fraction, whereas 86% was in the particulate fraction; this latter fraction also had a 4-fold higher specific activity. Purification of the particulate fraction by lectin chromatography resulted in a 50% increase in specific activity, although this glycoprotein-rich fraction contained only 1.5% of the total activity. Both the cytosolic and particulate PTPase fractions were active toward the tyrosyl-phosphorylated insulin receptor in vitro. The activity of the particulate fraction but not the cytosolic fraction was inhibited by addition of a micromolar concentration of a phosphorylated peptide corresponding to residues 1142-1153 of the human insulin receptor sequence. By contrast, addition of the nonphosphorylated peptide even at millimolar concentration was without effect. Both PTPase fractions were inhibited by Zn+ at similar concentrations, whereas the cytosolic PTPase activity was 10-fold more sensitive to vanadate inhibition. Treatment of cells with 100 nM insulin increased PTPase activity in the particulate fraction by 40% and decreased activity in the cytosolic fraction by 35%. These effects occurred within 15 min and were half-maximal at 3-4 nM insulin. When assessed as total activity, the magnitude of the changes in PTPase activity in the particulate and cytosolic fractions could not be explained on the basis of a translocation of PTPases between the two pools.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein tyrosine phosphatase regulation in fibroblasts from patients with an insulin receptor gene mutation

Tyrosine phosphorylation of the insulin receptor is the initial event following receptor binding to insulin, and it induces further tyrosine phosphorylation of various intracellular molecules. This signaling is countered by protein tyrosine phosphatases (PTPases), which reportedly are associated with insulin resistance that can be reduced by regulation of PTPases. Protein tyrosine phosphatase 1B (PTP1B) and leukocyte antigen-related PTPase (LAR) are the PTPases implicated most frequently in insulin resistance and diabetes mellitus. Here, we show that PTP1B and LAR are expressed in human fibroblasts, and we examine the regulation of PTPase activity in fibroblasts from patients with an insulin receptor gene mutation as an in vitro model of insulin resistance. Total PTPase activity was significantly lower in the cytosolic and membrane fractions of fibroblasts with mutations compared with controls (p<0.05). Insulin stimulation of fibroblasts with mutations resulted in a significantly smaller increase in PTP1B activity compared with stimulation of wild-type fibroblasts (p<0.05). This indicates that insulin receptor gene mutations blunt increases in PTPase activity in response to insulin, possibly via a negative feedback mechanism. Our data suggest that the PTPase activity in patients with insulin receptor gene mutation and severe insulin resistance may differ from that in ordinary type 2 diabetes.     

Protein Tyrosine Phosphatase 1B is Impaired in Skeletal Muscle of Diabetic Psammomys Obesus

Protein tyrosine phosphatases (PTPases) have been suggested to modulate the insulin receptor signal transduction pathways.We studied PTPases in Psammomys obesus, an animal model of nutritionally induced insulin resistance. No changes in the protein expression level of src homology PTPase 2 (SHP-2) (muscle, liver) or leukocyte antigen receptor (LAR) (liver) were detected. In contrast, the expression level of PTPase 1B (PTP 1B) in the skeletal muscle, but not in liver, was increased by 83% in the diabetic animals, compared with a diabetes-resistant line. However, PTP 1B– specific activity (activity/protein) significantly decreased (50% to 56%) in skeletal muscle of diabetic animals, compared with both the diabetes-resistant line and diabetes-prone animals. In addition, PTP 1B activity was inversely correlated to serum glucose level (r = –.434, P < .02). These findings suggest that PTP 1B, though overexpressed, is not involved in the susceptibility to insulin resistance in Psammomys obesus and is secondarily attenuated by hyperglycemia or other factors in the diabetic milieu.     

Effects of adenosine receptor antagonism on protein tyrosine phosphatase in rat skeletal muscle

Earlier studies have shown that whole body adenosine receptor antagonism increases skeletal muscle insulin sensitivity in insulin-resistant Zucker rats. To find which steps in the insulin signaling pathway are influenced by adenosine receptors, muscle from lean and obese Zucker rats, treated for 1 week with the adenosine receptor antagonist, 1,3-dipropyl-8-(4-acrylate)-phenylxanthine (BWA1433), were analyzed. All rats were first anesthetized and injected intravenously (i.v.) with 1 IU of insulin. About 3 min later the gastrocnemius was freeze clamped. Insulin receptors were partially purified on wheat germ agglutinin (WGA) columns and insulin receptor kinase activity measured in control and BWA1433-treated lean and obese Zucker rats. Protein tyrosine phosphatase (PTPase) activity was also analyzed in subcellular fractions, including the cytosolic fraction, a high-speed particulate fraction and the insulin receptor fraction eluted from WGA columns. Administration of BWA1433 increased insulin receptor kinase activity in obese but not lean Zucker rats. PTPase activities were higher in the untreated obese rat muscle particulate fractions than in the lean rat particulate fractions. The BWA1433 administration lowered the PTPase activity of the obese rats but not the lean rats. Although the PTPase activity in WGA eluate fractions containing crude insulin receptors were similar in lean and obese animals, BWA1433 administration was found to lower the PTPase activities in the fractions obtained from obese but not from the lean rats. PTPases may be upregulated in muscles from obese rats due to activated adenosine receptors. Adenosine receptor blockade, by reducing PTPase activity, may thereby increase insulin signaling.     

Elevated expression and activity of protein-tyrosine phosphatase 1B in skeletal muscle of insulin-resistant type II diabetic Goto-Kakizaki rats

We investigated the cellular mechanism(s) of insulin resistance associated with non-insulin dependent diabetes mellitus (NIDDM) using skeletal muscles isolated from non-obese, insulin resistant type II diabetic Goto-Kakizaki (GK) rats, a well known genetic rat model for type II diabetic humans. Relative to non-diabetic control rats (WKY), insulin-stimulated insulin receptor (IR) autophosphorylation and insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation were significantly inhibited in GK skeletal muscles. This may be due to increased dephosphorylation by a protein tyrosine phosphatase (PTPase). Therefore, we measured skeletal muscle total PTPase and PTPase 1B activities in the skeletal muscles isolated from control rats (WKY) and diabetic Goto-Kakizaki (GK) rats. PTPase activity was measured using a synthetic phosphopeptide, TRDIY(P)ETDY(P)Y(P)RK, as the substrate. Basal PTPase activity was 2-fold higher (P < 0.001) in skeletal muscle of GK rats when compared to WKY. Insulin infusion inhibited skeletal muscle PTPase activity in both control (26.20% of basal, P < 0.001) and GK (25.35% of basal, P < 0.001) rats. However, PTPase activity in skeletal muscle of insulin-stimulated GK rats was 200% higher than hormone-treated WKY controls (P < 0.001). Immunoprecipitation of PTPase 1B from skeletal muscle lysates and analysis of the enzyme activity in immunoprecipitates indicated that both basal and insulin-stimulated PTPase 1B activities were significantly higher (twofold, P < 0.001) in skeletal muscle of diabetic GK rats when compared to WKY controls. The increase in PTPase 1B activity in diabetic GK rats was associated with an increased expression of the PTPase 1B protein. We concluded that insulin resistance of GK rats is accompanied atleast by an abnormal regulation of PTPase 1B. Elevated PTPase 1B activity through enhanced tyrosine dephosphorylation of the insulin receptor and its substrates, may lead to impaired glucose tolerance and insulin resistance in GK rats.     

Replacement of soybean oil by fish oil increases cytosolic lipases activities in liver and adipose tissue from rats fed a high-carbohydrate diets

Several studies have demonstrated that fish oil consumption improves metabolic syndrome and comorbidities, as insulin resistance, nonalcoholic fatty liver disease, dyslipidaemia and hypertension induced by high-fat diet ingestion. Previously, we demonstrated that administration of a fructose-rich diet to rats induces liver lipid accumulation, accompanied by a decrease in liver cytosolic lipases activities. In this study, the effect of replacement of soybean oil by fish oil in a high-fructose diet (FRUC, 60% fructose) for 8 weeks on lipid metabolism in liver and epididymal adipose tissue from rats was investigated. The interaction between fish oil and FRUC diet increased glucose tolerance and decreased serum levels of triacylglycerol (TAG), VLDL-TAG secretion and lipid droplet volume of hepatocytes. In addition, the fish oil supplementation increased the liver cytosolic lipases activities, independently of the type of carbohydrate ingested. Our results firmly establish the physiological regulation of liver cytosolic lipases to maintain lipid homeostasis in hepatocytes. In epididymal adipose tissue, the replacement of soybean oil by fish oil in FRUC diet did not change the tissue weight and lipoprotein lipase activity; however, there was increased basal and insulin-stimulated de novo lipogenesis and glucose uptake. Increased cytosolic lipases activities were observed, despite the decreased basal and isoproterenol-stimulated glycerol release to the incubation medium. These findings suggest that fish oil increases the glycerokinase activity and glycerol phosphorylation from endogenous TAG hydrolysis. Our findings are the first to show that the fish oil ingestion increases cytosolic lipases activities in liver and adipose tissue from rats treated with high-carbohydrate diets.     

Compartmentalization and in vivo insulin-induced translocation of the insulin-signaling inhibitor Grb14 in rat liver

The molecular adaptor Grb14 binds in vitro to the activated insulin receptor (IR) and inhibits IR signaling. In this study, we have used rat liver subcellular fractionation to analyze in vivo insulin effects on Grb14 compartmentalization and IR phosphorylation and activity. In control rats, Grb14 was recovered mainly in microsomal and cytosolic fractions, but was also detectable at low levels in plasma membrane and Golgi/endosome fractions. Insulin injection led to a rapid and dose-dependent increase in Grb14 content, first in the plasma membrane fraction, and then in the Golgi/endosome fraction, which paralleled the increase in IR beta-subunit tyrosine phosphorylation. Upon sustained in vivo IR tyrosine phosphorylation induced by high-affinity insulin analogs, in vitro IR dephosphorylation by endogenous phosphatases, and in vivo phosphorylation of the IR induced by injection of bisperoxo(1,10 phenanthroline)oxovanadate, a phosphotyrosine phosphatase inhibitor, we observed a striking correlation between IR phosphorylation state and Grb14 content in both the plasma membrane and Golgi/endosome fractions. In addition, coimmunoprecipitation experiments provided evidence that Grb14 was associated with phosphorylated IR beta-subunit in these fractions. Altogether, these data support a model whereby insulin stimulates the recruitment of endogenous Grb14 to the activated IR at the plasma membrane, and induces internalization of the Grb14-IR complex in endosomes. Removal of Grb14 from fractions of insulin-treated rats by KCl treatment led to an increase of in vivo insulin-stimulated IR tyrosine kinase activity, indicating that endogenous Grb14 exerts a negative feedback control on IR catalytic activity. This study thus demonstrates that Grb14 is a physiological regulator of liver insulin signaling.     

Protein kinase C activation patterns are determined by methodological variations. Studies of insulin action in BC3H-1 myocytes and rat adipose tissue

In BC3H-1 myocytes, insulin has been reported to (a) increase diacyglycerol (DAG) production and provoke increases in protein kinase C enzyme activity of crude or DEAE-Sephacel-purified cytosol and membrane fractions in BC3H-1 myocytes (Cooper et al. (1987) J. Biol. Chem. 262, 3633-3739), but (b) decrease cytosolic, and transiently increase membrane, immunoreactive protein kinase C (Acevedo-Duncan et al. (1989) FEBS Lett. 244, 174-176). Presently, we used a Mono-Q column to purify protein kinase C and found that, similar to immunoblot findings, enzyme activity decreased in the cytosol, and increased in the membrane during insulin treatment. Similar differences in protein kinase C activation patterns were observed in rat adipose tissue: insulin stimulated cytosolic protein kinase C enzyme activity as measured after DEAE-Sephacel chromatography, but decreased cytosolic enzyme activity when measured after Mono-Q chromatography or by immunoblotting. We presently evaluated the possibility that insulin-induced increases in endogenous DAG may influence protein kinase C during assay in vitro. Crude cytosol from BC3H-1 myocytes contained 25-35% of total and [3H]glycerol-labelled DAG and insulin increased this DAG. Considerable amounts of [3H]glycerol-labelled DAG were present in insulin-stimulated protein kinase C-containing column fractions following DEAE-Sephacel chromatography of cytosol fractions, whereas lesser amounts were recovered after Mono-Q column chromatography. This difference in recovery of DAG and activation of the enzyme by this endogenous DAG may explain why we were able to discern insulin-induced (presumably translocation 'provoked') decreases in cytosolic protein kinase C in the present Mono-Q column preparations of both BC3H-1 myocytes and rat adipose tissue.     

In vivo 2H2O administration reveals impaired triglyceride storage in adipose tissue of insulin-resistant humans

Indirect evidence suggests that impaired triglyceride storage in the subcutaneous fat depot contributes to the development of insulin resistance via lipotoxicity. We directly tested this hypothesis by measuring, in vivo, TG synthesis, de novo lipogenesis (DNL), adipocyte proliferation, and insulin suppression of lipolysis in subcutaneous adipose tissue of BMI-matched individuals classified as insulin resistant (IR) or insulin sensitive (IS). Nondiabetic, moderately obese subjects with BMI 25–35 kg/m², classified as IR or IS by the modified insulin suppression test, consumed deuterated water (²H₂O) for 4 weeks. Deuterium incorporation into glycerol, palmitate, and DNA indicated TG synthesis, DNL, and adipocyte proliferation, respectively. Net TG synthesis and DNL in adipose cells were significantly lower in IR as compared with IS subjects, whereas adipocyte proliferation did not differ significantly. Plasma FFAs measured during an insulin suppression test were 2.5-fold higher in IR subjects, indicating resistance to insulin suppression of lipolysis. Adipose TG synthesis correlated directly with DNL but not with proliferation. These results provide direct in vivo evidence for impaired TG storage in subcutaneous adipose tissue of IR as compared with IS. Relative inability to store TG in the subcutaneous depot may represent a mechanism contributing to the development of insulin resistance in the setting of obesity.     

Decrease in protein tyrosine phosphatase activities in vanadate-treated obese Zucker (fa/fa) rat liver

The inhibitory action of vanadate towards protein tyrosine phosphatase (PTPase) has been considered as a probable mechanism by which it exerts insulin-like effects. In this study, we have examined thein vivo effects of vanadate on PTPases in the liver of obese Zucker rats, a genetic animal model for obesity and type II diabetes. These animals were characterized by hyperinsulinemia and mild hyperglycemia. The number of insulin receptors were significantly (p<0.01) decreased in liver. After chronic administration of vanadate in obese rats, 80% decrease in the plasma levels of insulin was observed. The insulin receptor numbers were significantly (p<0.01) higher in vanadate-treated obese rats as compared to the untreated ones. The hepatic PTPase activities in cytosolic and particulate fractions, with phosphorylated poly glu:tyr (41) and the insulin receptor peptide (residues 1142–1153) as substrates, increased in obese rats. In vanadate-treated obese rat livers, the PTPase activities in both subcellular fractions with these substrates decreased significantly (p<0.001). The decreases in PTPase activities from these groups of rats were further supported by chromatography on a Mono Q column. These data support the view that inhibition of PTPases plays a role in the insulin-mimetic action of vanadate.     

A polyphenol rescues lipid induced insulin resistance in skeletal muscle cells and adipocytes

Skeletal muscle and adipose tissues are known to be two important insulin target sites. Therefore, lipid induced insulin resistance in these tissues greatly contributes in the development of type 2 diabetes (T2D). Ferulic acid (FRL) purified from the leaves of Hibiscus mutabilis, showed impressive effects in preventing saturated fatty acid (SFA) induced defects in skeletal muscle cells. Impairment of insulin signaling molecules by SFA was significantly waived by FRL. SFA markedly reduced insulin receptor β (IRβ) in skeletal muscle cells, this was affected due to the defects in high mobility group A1 (HMGA1) protein obtruded by phospho-PKCε and that adversely affects IRβ mRNA expression. FRL blocked PKCε activation and thereby permitted HMGA1 to activate IRβ promoter which improved IR expression deficiency. In high fat diet (HFD) fed diabetic rats, FRL reduced blood glucose level and enhanced lipid uptake activity of adipocytes isolated from adipose tissue. Importantly, FRL suppressed fetuin-A (FetA) gene expression, that reduced circulatory FetA level and since FetA is involved in adipose tissue inflammation, a significant attenuation of proinflammatory cytokines occurred. Collectively, FRL exhibited certain unique features for preventing lipid induced insulin resistance and therefore promises a better therapeutic choice for T2D.     

Pyruvate dehydrogenase activity in several tissues of genetically obese hyperglycemic mice

The active form (PDHa) and total activity of pyruvate dehydrogenase (PDH) were measured in homogenates from heart muscle, epididymal fat pads and liver of genetically obese hyperglycemic mice and compared with similar data derived from lean controls or Swiss albino mice. Both PDHa and total PDH activities were similar in heart muscle from all mice with a precipitous decrease in the PDHa upon fasting. Adipose tissue and liver of obese mice had a PDHa level that was almost two-fold higher than either lean control or Swiss albino mice. Fasting for 24 hours decreased the elevated activity of PDHa in adipose tissue and liver in obese mice to a value that was comparable to lean control or Swiss albino mice, fasted similarly. The elevation in both the active form and total activity of pyruvate dehydrogenase in livers from obese mice could explain the increased provision of acetyl-CoA units necessary for the accelerated hepatic lipogenesis observed with this mouse, a model for human obesity and insulin resistance.     

Lipogenesis in rat tissues following carbohydrate refeeding: Spleen lipogenesis is modulated by insulin

Intraperitoneal administration of [1,2-¹⁴C]-acetate to Wistar rats was used to assess tissue lipogenic rates after estimating the incorporation of the label into the tissular lipid fractions. Refeeding the animals with glucose (after an overnight fast) induced an increase in white adipose tissue (4.5 fold), liver (4.1 fold), small intestine (1.9 fold), carcass (2.9 fold) and spleen (3.7 fold) lipogenesis (expressed as the radioactivity present in the lipid fraction corrected by the plasma circulating radioactivity). No changes were found following refeeding in either brain or brown adipose tissue. Administration of mannoheptulose (an inhibitor of insulin secretion) to refed rats completely abolished the increased lipogenesis in white adipose tissue, liver, carcass, spleen and small intestine, thus suggesting that insulin secretion is involved in this phenomenon. This is the first report showing that spleen lipogenesis may be modulated by refeeding via insulin secretion and suggests an important role of this organ on the in vivo lipogenic response of the organism after carbohydrate refeeding. (Mol Cell Biochem 175: 149–152, 1997)     

PPARγ2 Pro12Ala polymorphism is associated with improved lipoprotein lipase functioning in adipose tissue of insulin resistant obese women

Lipoprotein lipase (LPL) plays a pivotal role in lipid metabolism, contributes to metabolic disorders related to insulin action and body weight regulation, and is influenced by inflammation. The Pro12Ala polymorphism of the peroxisome proliferator-activated receptor (PPAR)γ2 gene seems to influence LPL functioning, but its role in obesity and insulin resistance status, which usually coexist in the clinical setting, has not been explored. Our aim was to analyze the association of obesity and insulin resistance with adipose LPL activity and expression, and the influence of the PPARγ2 Pro12Ala polymorphism. A cross-sectional study was conducted in 58 reproductive-age women who underwent elective abdominal surgery. Free-fatty acids, glucose, insulin, and selected adipokines were measured in fasting blood samples. DNA was isolated and the polymorphism genotyped. Biopsies of abdominal subcutaneous adipose tissue obtained during surgery were used to determine enzymatic LPL activity and expression; and expression of selected cytokines. Overweight/obese women presented lower LPL activity (P = 0.022) and higher circulating TNF-α (P = 0.020) than controls. Insulin resistant women also showed borderline lower LPL activity than non-resistant (P = 0.052), but adiposity and inflammatory molecules were comparable. Nevertheless, LPL activity was higher in Pro12Ala carriers than in non-carriers after adjusting for obesity, insulin resistance and inflammation. Likewise, adipose LPL expression was increased in carriers while expression of cytokines was decreased. Our data suggest that insulin resistance is associated with low adipose LPL activity independently of obesity, but the PPARγ2 Pro12Ala polymorphism seems to protect the LPL functioning of obese insulin resistant women, likely through regulating inflammation in adipose tissue.     

Dynamics of protein-tyrosine phosphatases in rat adipocytes

Protein-tyrosine phosphatases (PTPases) play a key role in maintaining the steady-state tyrosine phosphorylation of the insulin receptor (IR) and its substrate proteins such as insulin receptor substrate 1 (IRS-1). However, the PTPase(s) that inactivate IR and IRS-1 under physiological conditions remain unidentified. Here, we analyze the subcellular distribution in rat adipocytes of several PTPases thought to be involved in the counterregulation of insulin signaling. We found that the transmembrane enzymes, protein-tyrosine phosphatase (PTP)-alpha and leukocyte common antigen-related (LAR), were detected predominantly in the plasma membrane and to a lesser extent in the heavy microsomes, a distribution similar to that of insulin receptor. PTP-1B and IRS-1 were present in light microsomes and cytosol, whereas SHPTP2/Syp was exclusively cytosolic. Insulin induced a redistribution of PTP-alpha from the plasma membrane to the heavy microsomes in a parallel fashion with the receptor. The distribution of PTP-1B in the light microsomes from resting adipocytes was similar to that of IRS-1 as determined by sucrose velocity gradient fractionation. Analysis of the catalytic activity of partially purified rat adipocyte PTP-alpha and LAR and recombinant PTP-1B showed that all three PTPases dephosphorylate IR. When a mix of IR/IRS-1 was used as a substrate, PTP-1B was particularly effective in dephosphorylating IRS-1. Considering that IR and IRS-1 can be dephosphorylated in internal membrane compartments from rat adipocytes (Kublaoui, B., Lee, J., and Pilch, P.F. (1995) J. Biol. Chem. 270, 59-65) and that PTP-alpha and PTP-1B are the respective PTPases in these fractions, we conclude that these PTPases are responsible for the counterregulation of insulin signaling there, whereas both LAR and PTP-alpha may act upon cell surface insulin receptors.     

Lipid-Overloaded Enlarged Adipocytes Provoke Insulin Resistance Independent of Inflammation

In obesity, adipocyte hypertrophy and proinflammatory responses are closely associated with the development of insulin resistance in adipose tissue. However, it is largely unknown whether adipocyte hypertrophy per se might be sufficient to provoke insulin resistance in obese adipose tissue. Here, we demonstrate that lipid-overloaded hypertrophic adipocytes are insulin resistant independent of adipocyte inflammation. Treatment with saturated or monounsaturated fatty acids resulted in adipocyte hypertrophy, but proinflammatory responses were observed only in adipocytes treated with saturated fatty acids. Regardless of adipocyte inflammation, hypertrophic adipocytes with large and unilocular lipid droplets exhibited impaired insulin-dependent glucose uptake, associated with defects in GLUT4 trafficking to the plasma membrane. Moreover, Toll-like receptor 4 mutant mice (C3H/HeJ) with high-fat-diet-induced obesity were not protected against insulin resistance, although they were resistant to adipose tissue inflammation. Together, our in vitro and in vivo data suggest that adipocyte hypertrophy alone may be crucial in causing insulin resistance in obesity.     

Changes of insulin binding in rat tissues after exposure to stress.

The effects of various stressors on insulin receptors in adipose, liver and skeletal muscle tissues were studied in rats exposed to acute or repeated stress. Adult male rats were exposed to immobilization (IMO) for 2.5 hours daily for 1, 7 and 42 days, or to hypokinesia (HK) for 1, 7 and 21 days. We determined the values of specific insulin binding (SIB) and insulin receptor binding capacity (IR) of plasma cell membranes from adipose, liver and muscle tissue (IMO groups), or insulin binding to isolated adipocytes and hepatocytes (HK groups). A significant decrease of SIB and IR was observed in rats exposed to acute stress (1x IMO) in muscle, adipose and liver tissues. However, in animals exposed to repeated stress (7x and 42x IMO), SIB and IR were diminished in the muscle tissue, whereas no significant changes were noted in the liver and adipose tissue. When tissue samples were collected 3-24 hours after exposure to IMO stress, no changes of SIB and IR were found in liver and adipose tissue, but insulin binding was lowered in skeletal muscles. In animals exposed to HK for one day, a decrease of SIB and IR was found in isolated adipocytes, but no changes in insulin binding were noted in the liver tissue. In rats exposed to HK for 7 and 21 days, values of IR were similar as in control group. Our results indicate a) the different changes of IR in the liver, fat and muscle tissues after exposure to stress situations, b) a long-term decrease of insulin binding in muscles of rats exposed to repeated IMO stress, and c) the return of reduced SIB and IR (induced by acute stress) to control values in the liver and adipose tissue after a short recovery period.     

Genetic dissection of retinoid esterification and accumulation in the liver and adipose tissue

Approximately 80–90% of all retinoids in the body are stored as retinyl esters (REs) in the liver. Adipose tissue also contributes significantly to RE storage. The present studies, employing genetic and nutritional interventions, explored factors that are responsible for regulating RE accumulation in the liver and adipose tissue and how these influence levels of retinoic acid (RA) and RA-responsive gene expression. Our data establish that acyl-CoA:retinol acyltransferase (ARAT) activity is not involved in RE synthesis in the liver, even when mice are nutritionally stressed by feeding a 25-fold excess retinol diet or upon ablation of cellular retinol-binding protein type I (CRBPI), which is proposed to limit retinol availability to ARATs. Unlike the liver, where lecithin:retinol acyltransferase (LRAT) is responsible for all RE synthesis, this is not true for adipose tissue where Lrat-deficient mice display significantly elevated RE concentrations. However, when CrbpI is also absent, RE levels resemble wild-type levels, suggesting a role for CrbpI in RE accumulation in adipose tissue. Although expression of several RA-responsive genes is elevated in Lrat-deficient liver, employing a sensitive liquid chromatography tandem mass spectrometry protocol and contrary to what has been assumed for many years, we did not detect elevated concentrations of all-trans-RA. The elevated RA-responsive gene expression was associated with elevated hepatic triglyceride levels and decreased expression of Pparδ and its downstream Pdk4 target, suggesting a role for RA in these processes in vivo.