The mitochondrial pyruvate dehydrogenase complex (PDC) is inactivated in many tissues during starvation and diabetes. We investigated carbohydrate oxidation (CHO) and the regulation of the PDC in lean and obese Zucker diabetic fatty (ZDF) rats during fed and starved conditions as well as during an oral glucose load without and with pharmacologically reduced levels of free fatty acids (FFA) to estimate the relative contribution of FFA on glucose tolerance, CHO, and PDC activity. The increase in total PDC activity (20-45%) was paralleled by increased protein levels ( approximately 2-fold) of PDC subunits in liver and muscle of obese ZDF rats. Pyruvate dehydrogenase kinase-4 (PDK4) protein levels were higher in obese rats, and consequently PDC activity was reduced. Although PDK4 protein levels were rapidly downregulated (57-62%) in both lean and obese animals within 2 h after glucose challenge, CHO over 3 h as well as the peak of PDC activity (1 h after glucose load) in liver and muscle were significantly lower in obese rats compared with lean rats. Similar differences were obtained with pharmacologically suppressed FFA by nicotinic acid, but with significantly improved glucose tolerance in obese rats, as well as increased CHO and delta increases in PDC activity (0-60 min) both in muscle and liver. These results demonstrated the suppressive role of FFA acids on the measured parameters. Furthermore, the results clearly demonstrate a rapid reactivation of PDC in liver and muscle of lean and obese rats after a glucose load and show that PDC activity is significantly lower in obese ZDF rats.

Objective: The severity of obesity is often more determined by the distribution of fat depots rather than by body weight itself. Therefore, the effect of rimonabant on fat distribution pattern was investigated in female candy-fed Wistar rats. Design: Female Wistar rats were fed a high fat, high carbohydrate (candy-) diet for 12 weeks. During the last 6 weeks rats were treated with rimonabant. Food intake and body weight development were investigated, as well as effects on total body fat, especially visceral fat and ectopic lipid accumulation in skeletal muscle and liver, determined by in vivo magnetic resonance imaging/magnetic resonance spectroscopy. Results: Candy-diet increased body weight, which was predominantly due to the increased total fat mass with predominance of visceral fat accumulation. Treatment with rimonabant fully reversed the weight gain and fat deposition in the visceral cavity and skeletal muscle, in contrast to pair feeding. In spite of an only transient reduction of food intake, body weight reduction, as well as normalized body fat, reduced visceral fat and intramyocellular lipids were maintained over the treatment period. Conclusions: We conclude that additional factors other than reduced caloric intake must be responsible for the improvements in these lipid parameters. The complete cluster of results is consistent with increased lipid oxidation caused by rimonabant.

Intramyocellular lipid content (IMCL) serves as a good biomarker of skeletal muscle insulin resistance (IR). However, intracellular fatty acid metabolites [malonyl-CoA, long-chain acyl-CoA (LCACoA)] rather than IMCL are considered to be responsible for IR. This study aimed to investigate dynamics of IMCL and fatty acid metabolites during fed-to-starved-to-refed transition in lean and obese (IR) Zucker diabetic fatty rats in the following different muscle types: soleus (oxidative), extensor digitorum longus (EDL, intermediary), and white tibialis anterior (wTA, glycolytic). In the fed state, IMCL was significantly elevated in obese compared with lean rats in all three muscle types (soleus: 304%, EDL: 333%, wTA: 394%) in the presence of elevated serum triglycerides but similar levels of free fatty acids (FFA), malonyl-CoA, and total LCACoAs. During starvation, IMCL in soleus remained relatively constant, whereas in both rat groups IMCL increased significantly in wTA and EDL after comparable dynamics of starvation-induced FFA availability. The decreases of malonyl-CoA in wTA and EDL during starvation were more pronounced in lean than in obese rats, although there were no changes in soleus muscles for both groups. The concomitant increase in IMCL with the fall of malonyl-CoA support the concept that, as a reaction to starvation-induced FFA availability, muscle will activate lipid oxidation more the lower its oxidative capacity and then store the rest as IMCL.

AIMS/HYPOTHESIS:Insulin resistance in skeletal muscle is a hallmark of type 2 diabetes. Therefore, we sought to identify and validate genes involved in the development of insulin resistance in skeletal muscle.MATERIALS:Differentially regulated genes in skeletal muscle of male obese insulin-resistant, and lean insulin-sensitive Zucker diabetic fatty (ZDF) rats were determined using Affymetrix microarrays. Based on these data, various aspects of glucose disposal, insulin signalling and fatty acid composition were analysed in a muscle cell line overexpressing stearoyl-CoA desaturase 1 (SCD1).RESULTS:Gene expression profiling in insulin-resistant skeletal muscle revealed the most pronounced changes in gene expression for genes involved in lipid metabolism. Among these, Scd1 showed increased expression in insulin-resistant animals, correlating with increased amounts of palmitoleoyl-CoA. This was further investigated in a muscle cell line that overexpressed SCD1 and accumulated lipids, revealing impairments of glucose uptake and of different steps of the insulin signalling cascade. We also observed differential effects of high-glucose and fatty acid treatment on glucose uptake and long-chain fatty acyl-CoA profiles, and in particular an accumulation of palmitoleoyl-CoA in cells overexpressing SCD1.CONCLUSIONS/INTERPRETATION:Insulin-resistant skeletal muscle of ZDF rats is characterised by a specific gene expression profile with increased levels of Scd1. An insulin-resistant phenotype similar to that obtained by treatment with palmitate and high glucose can be induced in vitro by overexpression of SCD1 in muscle cells. This supports the hypothesis that elevated SCD1 expression is a possible cause of insulin resistance and type 2 diabetes.

Basement membrane (BM) formation was functionally dissected in 3d-cocultures of human keratinocytes (HK) and fibroblasts (human/mouse, HF/MFf) by either blocking interactions or implementing molecular deficiencies. This was supposed to complement knockout mouse studies, where loss or functional defects of collagen-IV, laminins, nidogen, or perlecan are causing embryonic or neonatal death. HK or HaCaT cells were grown on collagen gels harboring hf or mf from normal or ko-mice. To block nidogen-binding to laminin-10 the corresponding laminin-fragment (gamma1-iii3-5, L-gamma-f) was applied. BM-formation was surveyed by immunofluorescence (IF), regular (EM) and immuno-electron microscopy (IEM). In 3d-cocultures of HK and HF L-gamma-f blocked deposition of nidogen, laminin-10, and perlecan, while collagen-IV appeared normal. Although the hemidesmosome components laminin-5, BP180, and integrin alpha6beta4 were only mildly affected, EM and IEM revealed complete absence of BM, hemidesmosomes, and basal insertion of keratin filaments. To eliminate nidogen, made by fibroblasts, MF from nidogen1/nidogen2 ko-mice or crossbreds were employed. In 3d-cocultures with HaCaT cells nidogen1/2 (??/++)-MF abolished nidogen1-staining, but (??/+?)-mf reduced also largely nidogen2, collagen-IV, and drastically laminin-10. Total absence of nidogen (??/??) also deleted collagen-IV & laminin-5, integrins e.g. alpha6beta4 appearing still normal (IF). BM-formation could be entirely rescued by applying recombinant nidogens. In skin, perlecan can be apparently synthesized by both keratinocytes & fibroblasts. Accordingly, deficiency in either cell type did not affect BM-formation, demonstrated by combining either perlecan (?/?)-mf or HaCaT anti-sense-perlecan cells with respective normal partner cells. Thus, in this skin model BM-components are efficiently transported to their actual assembly site.

The physiological dynamics of intramyocellular lipids (IMCLs) in different muscle types and of hepatocellular lipids (HepCLs) are still uncertain. The dynamics of IMCLs in the soleus, tibialis anterior, and extensor digitorum longus (EDL) muscles and HepCL during fed, 12- to 72-h starved, and refed conditions were measured in vivo by (1)H-magnetic resonance spectroscopy (MRS) in Wistar rats. Despite significant elevations of free fatty acids (FFAs) during starvation, HepCLs and IMCLs in soleus remained constant. In tibialis anterior and EDL, however, IMCLs increased significantly by 170 and 450% after 72 h of starvation, respectively. After refeeding, elevated IMCLs dropped immediately in both muscles. Total muscle long-chain acyl-CoAs (LCACoAs) remained constant during the study period. Hepatic palmitoleoyl-CoA (C16:1) decreased significantly during starvation while total hepatic LCACoAs increased significantly. Consistent with constant values for FFAs, HepCLs, IMCLs, and muscle LCACoAs from 12-72 h of starvation, insulin sensitivity did not change. We conclude that during starvation-induced adipocytic lipolysis, oxidative muscles dispose elevated FFAs by oxidation, while nonoxidative ones neutralize FFAs by reesterification. Both mechanisms might prevent impairment of insulin signaling by maintaining low levels of LCACoAs. Hepatic palmitoleoyl-CoA might have a special role in lipid metabolism due to its unique dynamic profile during starvation.

The investigation of intramyocellular lipids (IMCLs) with proton MR spectroscopy ((1)H-MRS) in humans has recently received increasing attention. IMCL levels correlate with insulin resistance and are affected by diet and exercise, making IMCL an interesting marker for metabolic investigations. In the present in vivo study, the feasibility of using (1)H MRS for the detection of IMCL in rats is demonstrated, and the influence of various factors, such as age, gender, muscle type, and rat strain, on IMCL levels is systematically analyzed. In healthy Wistar and Sprague Dawley (SD) rats, the highest ratios of IMCL/tCr were found in young rats, and IMCL/tCr decreased with increasing age. In addition, IMCL concentration was clearly influenced by gender and muscle type. Insulin-resistant, male, obese, Zucker diabetic fatty (ZDF) rats showed significantly higher IMCL levels than Wistar or SD rats. In conclusion, although IMCL levels are clearly influenced by insulin resistance, several other factors influence IMCL levels, such as age, gender, muscle type, and rat strain. Therefore, when using IMCL as a surrogate marker for insulin resistance, it is necessary to carefully compare results with age- and gender-matched controls, and to use identical conditions.

1. Steroids are known to act as permissive factors in hepatocytes. This study shows that dexamethasone (DEX) is a permissive factor for induction of CYP2B1/2, CYP3A1, CYP2A1 and probably also CYP2C11 in cultures with primary rat hepatocytes. 2. The induction factor of phenobarbital (PB)-induced formation of 16 β -hydroxytestosterone (OHT), a testosterone biotransformation product predominantly formed by CYP2B1, is increased 18-fold by the addition of 32 nM DEX to the culture medium. Interestingly, higher concentrations of DEX up to 1000 nM led to a concentration-dependent maximally 5-fold decrease (p = 0.002) of phenobarbital-induced 16 β -OHT formation compared with the effect observed with 32 nM DEX. Thus, DEX shows permissive and suppressive effects on enzyme induction depending on the concentration of the glucocorticoid. 3. Qualitatively similar but smaller permissive and suppressive effects of DEX were observed for PB-induced CYP3A1 activity as evidenced by formation of 2 β -, 6 β - and 15 β -OHT. 4. DEX is a permissive factor for induction of CYP2A1 activity by 3-methylcholanthrene (3MC), as evidenced by the formation of 7 α -OHT. Without addition of DEX, 3MC did not induce formation of 7 α -OHT, whereas an almost 3-fold induction occurred in the presence of DEX. In contrast to CYP2B and CYP3A, concentrations up to 1000 nM DEX were not suppressive for the induction of CYP2A1. 5. We described recently a technique that allows preparation of cultures from cryopreserved hepatocytes. An almost identical influence of dexamethasone on enzyme induction was observed here in cultures from cryopreserved compared with freshly isolated hepatocytes. 6. Cultures with primary hepatocyte cultures represent a well-established technique for the study of drug-drug interactions. However, a large interlaboratory variation is known. Our study provides evidence that differences in glucocorticoid concentration in the culture medium contribute to this variation.

The use of primary hepatocytes is now well established for both studies of drug metabolism and enzyme induction. Cryopreservation of primary hepatocytes decreases the need for fresh liver tissue. This is especially important for research with human hepatocytes because availability of human liver tissue is limited. In this review, we summarize our research on optimization and validation of cryopreservation techniques.The critical elements for successful cryopreservation of hepatocytes are (1) the freezing protocol, (2) the concentration of the cryoprotectant [10% dimethylsulfoxide (DMSO)], (3) slow addition and removal of DMSO, (4) carbogen equilibration during isolation of hepatocytes and before cryopreservation, and (5) removal of unvital hepatocytes by Percoll centrifugation after thawing. Hepatocytes of human, monkey, dog, rat, and mouse isolated and cryopreserved by our standard procedure have a viability greater than or equal to 80%. Metabolic capacity of cryopreserved hepatocytes determined by testosterone hydroxylation, 7-ethoxyresorufin-O-deethylase (EROD), 7-ethoxycoumarin-O-deethylase (ECOD), glutathione S-transferase, UDP-glucuronosyl transferase, sulfotransferase, and epoxide hydrolase activities is greater than or equal to 60% of freshly isolated cells. Cryopreserved hepatocytes in suspension were successfully applied in short-term metabolism studies and as a metabolizing system in mutagenicity investigations. For instance, the complex pattern of benzo[a]pyrene metabolites including phase II metabolites formed by freshly isolated and cryopreserved hepatocytes was almost identical. For the study of enzyme induction, a longer time period and therefore cryopreserved hepatocyte cultures are required. We present a technique with cryopreserved hepatocytes that allows the induction of testosterone metabolism with similar induction factors as for fresh cultures. However, enzyme activities of induced hepatocytes and solvent controls were smaller in the cryopreserved cells.In conclusion, cryopreserved hepatocytes held in suspension can be recommended for short-term metabolism or toxicity studies. Systems with cryopreserved hepatocyte cultures that could be applied for studies of enzyme induction are already in a state allowing practical application, but may be further optimized.

The use of hepatocyte cultures is well established for the study of drug-drug interactions. However, the major hindrance for the use of human hepatocyte cultures is that human hepatocytes are only occasionally available. This problem could be overcome by cryopreservation. Although cryopreserved hepatocytes have been recommended for short term applications in suspension, studies on induction of enzyme activity, requiring a more prolonged maintenance of cryopreserved hepatocytes in culture, represent a new field of research. In the present study, we established a technique that allows preparation of rat hepatocyte co-cultures, using cryopreserved hepatocytes. After incubation with phenobarbital (0.75 mM; 72 h) induction factors for the isoenzyme-dependent regio and stereoselective testosterone hydroxylations were 1.6, 2.2, 1.0, 2.1, 5.6, 2.4, 3.6, 4.5 and 0.9 for 2alpha-, 2beta-, 6alpha-, 6beta-, 7alpha-, 15beta-, 16alpha- and 16beta-hydroxytestosterone and 4-androsten-3,17 dione. Regarding induction factors of less than 2-fold, as questionable these induction factors were similar to those of cultures with freshly isolated hepatocytes and the induction pattern of the individual hydroxylation products was similar to the in vivo situation. In addition 3-methylcholanthrene (5 microM; 72 h) induced exclusively the formation of 7alpha-hydroxytestosterone (6.6-fold) in cultures with cryopreserved hepatocytes. This specificity also correlates to that obtained in rats. Although these induction factors were clearly satisfactory in cryopreserved cultures, the absolute activities of the main testosterone hydroxylation products were reduced when compared to fresh cultures. For instance, 6beta-hydroxytestosterone, the main metabolite in solvent controls was reduced to 79%, 7alpha-hydroxytestosterone, the main metabolite after induction with 3-MC, was reduced to 66% and 16beta-hydroxytestosterone, the main metabolite after induction with PB, was reduced to 52%. Similarly, EROD activity after induction with 3-methylcholanthrene in cryopreserved cultures was reduced to 62%, compared with that in fresh cultures. Although further optimization and validation is required, the data show that cytochrome P450 activities can clearly be induced in co-cultures of cryopreserved hepatocytes, in a fashion which for the investigated inducers, is similar to that in cultures from freshly isolated hepatocytes and similar to the in vivo situation.

Fibrosis and cirrhosis of the liver are often the result of chronic liver damage by a variety of different agents. Pathological accumulation of collagen, disruption of the lobular structure, and impaired hepatocellular function frequently lead to systemic involvement and fatal complications. Drugs inhibiting collagen hydroxylation and accumulation are expected to improve this situation, making prolyl 4-hydroxylase (P4H), the key enzyme of intracellular collagen processing, a rational target for pharmacological intervention. S 4682, a novel inhibitor of purified P4H (Ki = 155 nmol/L), reduced hydroxyproline (Hyp) synthesis in chicken embryo calvaria (IC50 = 8.2 micromol/L) and in cultured hepatic stellate cells (HSC) (IC50 = 39 micromol/L). S 4682 inhibited hepatic collagen hydroxylation in vivo after metabolic labeling with [14C]proline. In the CCl4 model of chronic hepatic injury, characterized by histologically and biochemically evident fibrosis and highly elevated levels of serum procollagen type III N-peptide, S 4682 reduced hepatic collagen accumulation, decreased prevalence of ascites, and lowered serum procollagen type III N-peptide (PIIINP) levels. The hepatic Hyp content of drug-treated animals was closely correlated with serum levels of PIIINP S 4682 had no influence on Hyp content of heart, lung, and kidney.

A major nidogen binding site of mouse laminin was previously localized to about three EGF‐like repeats (Nos 3–5) of its B2 chain domain III [M. Gerl et al. (1991) Eur. J. Biochem., 202, 167]. The corresponding cDNA was amplified by polymerase chain reaction and inserted into a eukaryotic expression vector tagged with a signal peptide. Stably transfected human kidney cell clones were shown to process and secrete the resulting fragment B2III3‐5 in substantial quantities. It possessed high binding activity for recombinant nidogen in ligand assays, with an affinity comparable with that of authentic laminin fragments. In addition, complexes of B2III3‐5 and nidogen could be efficiently converted into a covalent complex by cross‐linking reagents. Proteolytic degradation of the covalent complex demonstrated the association of B2III3‐5 with a approximately 80 residue segment of nidogen domain G3 to which laminin binding has previously been attributed. The correct formation of most of the 12 disulfide bridges in B2III3‐5 was indicated from its protease resistance and the complete loss of cross‐reacting epitopes as well as of nidogen‐binding activity after reduction and alkylation. Smaller fragments were prepared by the same recombinant procedure and showed that combinations of EGF‐like repeats 3–4 and 4–5 and the single repeat 4 but not repeats 3 or 5 possess full nidogen‐binding activity. This identifies repeat 4 as the only binding structure. The sequence of repeat 4 is well conserved in the human and in part in the Drosophila laminin B2 chain.(ABSTRACT TRUNCATED AT 250 WORDS)