Adipose tissue and serum CCDC80 in obesity and its association with related metabolic disease.

Coiled-coil domain-containing 80 (CCDC80) is an adipocyte-secreted protein that modulates glucose homeostasis in response to diet-induced obesity in mice. The objective of this study is to analyze the link between human CCDC80 and obesity. CCDC80 protein expression was assessed in paired visceral (VAT) and subcutaneous (SAT) adipose tissue from 10 subjects (BMI range 22.4-38.8 kg/m2). Circulating CCDC80 levels were quantified in serum samples from two independent cross-sectional cohorts comprising 33 lean and 15 obese (cohort 1) and 32 morbid obese (cohort 2) male subjects. Insulin sensitivity, insulin secretion and blood neutrophil count were quantified in serum samples from both cohorts. Additionally, circulating free IGF-1 levels and oral glucose tolerance tests (OGTT) were assessed in cohort 1 whereas C-reactive protein levels and degree of atherosclerosis and hepatic steatosis were studied in cohort 2. In lean subjects, total CCDC80 protein content assessed by immunoblotting was lower in VAT than in SAT. In obese patients, CCDC80 was increased in VAT (P<0.05), but equivalent in SAT compared with lean counterparts. In cohort 1, serum CCDC80 correlated negatively with the acute insulin response to glucose and IGF1 levels, and positively with blood neutrophil count, independently of BMI, but not with insulin sensitivity. In cohort 2, serum CCDC80 was positively linked to the inflammatory biomarker C-reactive protein (r=0.46; P=0.009), atherosclerosis (carotid intima-media thickness, r=0.62; P<0.001) and hepatic steatosis (ANOVA P=0.025). Overall, these results suggest for the first time that CCDC80 may be a component of the obesity-altered secretome in VAT and could act as an adipokine whose circulant levels are linked to glucose tolerance derangements and related to inflammation-associated chronic complications.

Oleate prevents saturated-fatty-acid-induced ER stress, inflammation and insulin resistance in skeletal muscle cells through an AMPK-dependent mechanism.

AIMS/HYPOTHESIS: Although the substitution of saturated fatty acids with oleate has been recommended in the management of type 2 diabetes mellitus, the mechanisms by which oleate improves insulin resistance in skeletal muscle cells are not completely known. Here, we examined whether oleate, through activation of AMP-activated protein kinase (AMPK), prevented palmitate-induced endoplasmic reticulum (ER) stress, which is involved in the link between lipid-induced inflammation and insulin resistance. METHODS: Studies were conducted in mouse C2C12 myotubes and in the human myogenic cell line LHCN-M2. To analyse the involvement of AMPK, activators and inhibitors of this kinase and overexpression of a dominant negative AMPK construct (K45R) were used. RESULTS: Palmitate increased the levels of ER stress markers, whereas oleate did not. In palmitate-exposed cells incubated with a lower concentration of oleate, the effects of palmitate were prevented. The induction of ER stress markers by palmitate was prevented by the presence of the AMPK activators AICAR and A-769662. Moreover, the ability of oleate to prevent palmitate-induced ER stress and inflammation (nuclear factor-kappa B [NF-κB] DNA-binding activity and expression and secretion of IL6) as well as insulin-stimulated Akt phosphorylation and 2-deoxyglucose uptake was reversed in the presence of the AMPK inhibitor compound C or by overexpression of a dominant negative AMPK construct. Finally, palmitate reduced phospho-AMPK levels, whereas this was not observed in oleate-exposed cells or in palmitate-exposed cells supplemented with oleate. CONCLUSIONS/INTERPRETATION: Overall, these findings indicate that oleate prevents ER stress, inflammation and insulin resistance in palmitate-exposed skeletal muscle cells by activating AMPK.

Plasma PTX3 protein levels inversely correlate with insulin secretion and obesity, whereas visceral adipose tissue PTX3 gene expression is increased in obesity.

Plasma acutephase protein pentraxin 3 (PTX3) concentration is dysregulated in human obesity and metabolic syndrome. Here, we explore its relationship with insulin secretion and sensitivity, obesity markers, and adipose tissue PTX3 gene expression. Plasma PTX3 protein levels were analyzed in a cohort composed of 27 lean [body mass index (BMI) ≤ 25 kg/m(2)] and 48 overweight (BMI 25-30 kg/m(2)) men (cohort 1). In this cohort, plasma PTX3 was negatively correlated with fasting triglyceride levels and insulin secretion after intravenous and oral glucose administration. Plasma PTX3 protein and PTX3 gene expression in visceral (VAT) and subcutaneous (SAT) whole adipose tissue and adipocyte and stromovascular fractions were analyzed in cohort 2, which was composed of 19 lean, 28 overweight, and 15 obese subjects (BMI >30 kg/m(2)). An inverse association with body weight and waist/hip ratio was observed in cohort 2. In VAT depots, PTX3 mRNA levels were higher in subjects with BMI >25 kg/m(2) than in lean subjects, positively correlated with IL-1ß mRNA levels, and higher in the adipocyte than stromovascular fraction. Human preadipocyte SGBS cell line was used to study PTX3 production in response to factors that obesity entails. In SGBS adipocytes, PTX3 gene expression was enhanced by IL-1ß and TNFα but not IL-6 or insulin. In conclusion, the negative correlation between PTX3 and glucose-stimulated insulin secretion suggests a role for PTX3 in metabolic control. PTX3 gene expression is upregulated in VAT depots in obesity, despite lower plasma PTX3 protein, and by some proinflammatory cytokines in cultured adipocytes.

New emerging role of protein-tyrosine phosphatase 1B in the regulation of glycogen metabolism in basal and TNF-α-induced insulin-resistant conditions in an immortalised muscle cell line isolated from mice.

AIMS/HYPOTHESIS: Protein-tyrosine phosphatase 1B (PTP1B) negatively regulates insulin action, promoting attenuation of the insulin signalling pathway. The production of this phosphatase is enhanced in insulin-resistant states, such as obesity and type 2 diabetes, where high levels of proinflammatory cytokines (TNF-α, IL-6) are found. In these metabolic conditions, insulin action on glycogen metabolism in skeletal muscle is greatly impaired. We addressed the role of PTP1B on glycogen metabolism in basal and insulin-resistant conditions promoted by TNF-α. METHODS: We studied the effect of TNF-α in the presence and absence of insulin on glycogen content and synthesis, glycogen synthase (GS) and glycogen phosphorylase (GP) activities and on glycogen synthesis and degradation signalling pathways. For this purpose we used immortalised cell lines isolated from skeletal muscle from mice lacking PTP1B. RESULTS: Absence of PTP1B caused activation of GS and GP with a net glycogenolytic effect, reflected in lower amounts of glycogen and activation of the glycogenolytic signalling pathway, with higher rates of phosphorylation of cyclic adenosine monophosphate-dependent kinase (PKA), phosphorylase kinase (PhK) and GP phosphorylation. Nevertheless, insulin action was strongly enhanced in Ptp1b (also known as Ptpn1)(-/-) cells in terms of glycogen content, synthesis, GS activation rates and GS Ser641 dephosphorylation. Treatment with TNF-α augmented the activity ratios of both GS and GP, and impaired insulin stimulation of glycogen synthesis in wild-type myocytes, whereas Ptp1b (-/-) myocytes restored this inhibitory effect. We report a glycogenolytic effect of TNF-α, as demonstrated by greater activation of the degradation signalling cascade PKA/PhK/GP. In our model, this effect is mediated by the activation of PKA. CONCLUSIONS/INTERPRETATION: We provide new data about the role of PTP1B in glycogen metabolism and confirm the beneficial effect that absence of the phosphatase confers against an insulin-resistant condition.

Overexpression of UCP3 in cultured human muscle lowers mitochondrial membrane potential, raises ATP/ADP ratio, and favors fatty acid vs. glucose oxidation.

The skeletal muscle mitochondrial uncoupling protein-3 (UCP3) promotes substrate oxidation, but direct evidence for its metabolic role is lacking. Here, we show that UCP3 overexpression in cultured human muscle cells decreased mitochondrial membrane potential (DYm). Despite this, the ATP content was not significantly decreased compared with control cells, whereas ADP content was reduced and thus the ATP/ADP ratio raised. This finding was contrasts with the effect caused by the chemical protonophoric uncoupler, CCCP, which lowered DYm, ATP, and the ATP/ADP ratio. UCP3-overexpression enhanced oxidation of oleate, regardless of the presence of glucose, whereas etomoxir, which blocks fatty acid entry to mitochondria, suppressed the UCP3 effect. Glucose oxidation was stimulated in UCP3-overexpressing cells, but this effect was inhibited by oleate. UCP3 caused weak increase of both 2-Deoxyglucose uptake and glycolytic rate, which differed from the marked stimulation by CCCP. We concluded that UCP3 promoted nutrient oxidation by lowering DYm and enhanced fatty acid-dependent inhibition of glucose oxidation. Unlike the uncoupler CCCP, however, UCP3 raised the ATP/ADP ratio and modestly increased glucose uptake and glycolysis. We propose that this differential effect provides a biological significance to UCP3, which is up-regulated in metabolic stress situations where it could be involved in nutrient partitioning.

Treatment based on a combination of the CYP2B1/cyclophosphamide system and p53 delivery enhances tumour regression in human pancreatic cancer.

BACKGROUND: Strategies based on the introduction of pro-drug activating enzymes or the restoration of tumour suppressor genes have been proposed as encouraging methods to improve the efficiency of treatments in pancreatic cancer. The in situ bioactivation of cyclophosphamide by cytochrome p450-2B1 and subsequent p53 delivery were examined. MATERIALS AND METHODS: NP-18 cell line derived from a human pancreatic adenocarcinoma was treated in vitro with a combination of the Adenovirus-CYP2B1/cyclophosphamide and adenoviral-mediated wt-p53 reintroduction. Cell viability and cytometric cell cycle profiles were analyzed to evaluate the sensitivity of NP-18 cells to this treatment. The efficiency of this combination was assessed in an in vivo model consisting of xenografts into the subcutaneous tissue of Balb/c mice by tumour growth, histological analysis and cell cycle determinations. RESULTS: Ad-CYP2B1/cyclophosphamide or Ad-p53 treatments led to a marked decrease in cell viability of NP-18 cells. Combination of both treatments elicited a higher loss of cell viability and marked increases in sub-G1 population in cell cycle profiles. Animals treated with the combination strategy showed a quick reduction of tumour volumes due to the bioactivation of cyclophosphamide by CYP2B1 and sustained growth inhibition throughout the period evaluated after p53 delivery. Only this group of animals presented statistically significant differences with respect to control and cyclophosphamide-treated groups (P < 0.05). CONCLUSIONS: These results indicate that in situ bioactivation of cyclophosphamide by CYP2B1 and the recognition of the damaged DNA by p53 increase tumour regressions and may be a promising therapy for solid tumour therapy in man.

Hepatic glycogen synthesis is highly sensitive to phosphorylase activity: evidence from metabolic control analysis.

We used metabolic control analysis to determine the flux control coefficient of phosphorylase on glycogen synthesis in hepatocytes by titration with a specific phosphorylase inhibitor (CP-91149) or by expression of muscle phosphorylase using recombinant adenovirus. The muscle isoform was used because it is catalytically active in the b-state. CP-91149 inactivated phosphorylase with sequential activation of glycogen synthase. It increased glycogen synthesis by 7-fold at 5 mm glucose and by 2-fold at 20 mm glucose with a decrease in the concentration of glucose causing half-maximal rate (S(0.5)) from 26 to 19 mm. Muscle phosphorylase was expressed in hepatocytes mainly in the b-state. Low levels of phosphorylase expression inhibited glycogen synthesis by 50%, with little further inhibition at higher enzyme expression, and caused inactivation of glycogen synthase that was reversed by CP-91149. At endogenous activity, phosphorylase has a very high (greater than unity) negative control coefficient on glycogen synthesis, regardless of whether it is determined by enzyme inactivation or overexpression. This high control is attenuated by glucokinase overexpression, indicating dependence on other enzymes with high control. The high control coefficient of phosphorylase on glycogen synthesis affirms that phosphorylase is a strong candidate target for controlling hyperglycemia in type 2 diabetes in both the absorptive and postabsorptive states.

DAG accumulation from saturated fatty acids desensitizes insulin stimulation of glucose uptake in muscle cells.

The increased availability of saturated lipids has been correlated with development of insulin resistance, although the basis for this impairment is not defined. This work examined the interaction of saturated and unsaturated fatty acids (FA) with insulin stimulation of glucose uptake and its relation to the FA incorporation into different lipid pools in cultured human muscle. It is shown that basal or insulin-stimulated 2-deoxyglucose uptake was unaltered in cells preincubated with oleate, whereas basal glucose uptake was increased and insulin response was impaired in palmitate- and stearate-loaded cells. Analysis of the incorporation of FA into different lipid pools showed that palmitate, stearate, and oleate were similarly incorporated into phospholipids (PL) and did not modify the FA profile. In contrast, differences were observed in the total incorporation of FA into triacylglycerides (TAG): unsaturated FA were readily diverted toward TAG, whereas saturated FA could accumulate as diacylglycerol (DAG). Treatment with palmitate increased the activity of membrane-associated protein kinase C, whereas oleate had no effect. Mixture of palmitate with oleate diverted the saturated FA toward TAG and abolished its effect on glucose uptake. In conclusion, our data indicate that saturated FA-promoted changes in basal glucose uptake and insulin response were not correlated to a modification of the FA profile in PL or TAG accumulation. In contrast, these changes were related to saturated FA being accumulated as DAG and activating protein kinase C. Therefore, our results suggest that accumulation of DAG may be a molecular link between an increased availability of saturated FA and the induction of insulin resistance.

Overexpression of protein targeting to glycogen in cultured human muscle cells stimulates glycogen synthesis independent of glycogen and glucose 6-phosphate levels.

There is growing evidence that glycogen targeting subunits of protein phosphatase-1 play a critical role in regulation of glycogen metabolism. In the current study, we have investigated the effects of adenovirus-mediated overexpression of a specific glycogen targeting subunit known as protein targeting to glycogen (PTG) in cultured human muscle cells. PTG was overexpressed both in muscle cells cultured at high glucose (glycogen replete) or in cells incubated for 18 h in the absence of glucose and then incubated in high glucose (glycogen re-synthesizing). In both glycogen replete and glycogen resynthesizing cells, PTG overexpression caused glycogen to be synthesized at a linear rate 1-5 days after viral treatment, while in cells treated with a virus lacking a cDNA insert (control virus), glycogen content reached a plateau at day 1 with no further increase. In the glycogen replete PTG overexpressing cells, glycogen content was 20 times that in controls at day 5. Furthermore, in cells undergoing glycogen resynthesis, PTG overexpression caused a doubling of the initial rate of glycogen synthesis over the first 24 h relative to cells treated with control virus. In both sets of experiments, the effects of PTG on glycogen synthesis were correlated with a 2-3-fold increase in glycogen synthase activity state, with no changes in glycogen phosphorylase activity. The alterations in glycogen synthase activity were not accompanied by changes in the intracellular concentration of glucose 6-phosphate. We conclude that PTG overexpression activates glycogen synthesis in a glucose 6-phosphate-independent manner in human muscle cells while overriding glycogen-mediated inhibition. Our findings suggest that modulation of PTG expression in muscle may be a mechanism for enhancing muscle glucose disposal and improving glucose tolerance in diabetes.

Increased glucose disposal induced by adenovirus-mediated transfer of glucokinase to skeletal muscle in vivo.

In non-insulin-dependent diabetes mellitus, insulin-stimulated glucose uptake is impaired in muscle, contributing in a major way to development of hyperglycemia. We previously showed that expression of the glucose phosphorylating enzyme glucokinase (GK) in cultured human myocytes improved glucose storage and disposal, suggesting that GK delivery to muscle in situ could potentially enhance glucose clearance. Here we have tested this idea directly by intramuscular delivery of an adenovirus containing the liver GK cDNA (AdCMV-GKL) into one hind limb. We injected an adenovirus containing the beta-galactosidase gene (AdCMV-lacZ) into the hind limb of newborn rats. beta-Galactosidase activity was localized in muscle for as long as 1 month after delivery, with a large percentage of fibers staining positive in the gastrocnemius. Using the same approach with AdCMV-GKL, GK protein content was increased from zero to 50-400% of the GK in normal liver sample, and total glucose phosphorylating activity was increased in GK-expressing muscles relative to the counterpart uninfected muscle. Expression of GK in muscle improved glucose tolerance rather than changing basal glycemic control. Glucose levels were reduced by approximately 35% 10 min after administration of a glucose bolus to fed animals treated with AdCMV-GKL relative to AdCMV-lacZ-treated controls. The enhanced rate of glucose clearance was reflected in increases in muscle 2-deoxy glucose uptake and blood lactate levels. We conclude that restricted expression of GK in muscle leads to an enhanced capacity for muscle glucose disposal and whole body glucose tolerance under conditions of maximal glucose-insulin stimulation, suggesting that under these conditions glucose phosphorylation becomes rate-limiting. Our findings also show that gene delivery to a fraction of the whole body is sufficient to improve glucose disposal, providing a rationale for the development of new therapeutic strategies for treatment of diabetes.-Jiménez-Chillarón, J. C., Newgard, C. B., Gómez-Foix, A. M. Increased glucose disposal induced by adenovirus-mediated transfer of glucokinase to skeletal muscle in vivo.

Genetic background determines the response to adenovirus-mediated wild-type p53 expression in pancreatic tumor cells.

The development of new therapies is particularly urgent with regard to pancreatic tumors. Gene therapy approaches involving p53 replacement are promising due to the central role of p53 in the cellular response to DNA damage and the high incidence of p53 mutations in pancreatic tumors. Adenoviruses containing wild-type (wt) p53 cDNA (Ad5CMV-p53) were introduced into four human pancreatic cell lines to examine the impact caused by exogenous wt p53 on these cells. Introduction of wt p53 in mutant p53 cells (NP-9, NP-18, and NP-31) caused marked falls in cell proliferation and rises in the level of apoptosis. In contrast, overexpression of p53 did not induce apoptosis in NP-29 (wt p53). The presence of p16 contributes to the induction of apoptosis, as demonstrated by introduction of the wt p16 gene (Ad5RSV-p16). Analysis of cell cycle and apoptosis in etoposide-treated cells corroborated the inability of NP-29 to die by apoptosis, suggesting that this wt p53 cell line lacks p53 downstream functions in the apoptosis pathway. Taken together, our results indicate that the effects elicited by exogenous p53 protein depend upon the molecular alterations related to p53 actions on cell cycle and apoptosis. Therefore, knowledge of the genetic background of tumor cells is crucial to the development of efficient therapies based on the introduction of tumor suppressor genes.

Enhanced pancreatic tumor regression by a combination of adenovirus and retrovirus-mediated delivery of the herpes simplex virus thymidine kinase gene.

We have evaluated the effectiveness of combining the different characteristics of retrovirus and adenovirus to apply the herpes simplex virus thymidine kinase gene (HSVtk) and ganciclovir (GCV) treatment for gene therapy of pancreatic cancer. Transduction of NP-18 human pancreatic cells in culture by either the adenoviral vector (ADV/tk) or the retroviral vector (Rv/tk) followed by GCV treatment resulted in a GCV dose-dependent cytotoxic effect. A bystander effect was determined, both in NP-18 cultures and in xenogeneic cell mixtures of NP-18 and PA317 cells. Studies in vivo indicated that the effectiveness of tumor regression after HSVtk gene transfer and GCV treatment was dependent first on the tumor size at the time of viral injection and secondly, in large tumors, on the type of virus administered. The administration of the viral combination (ADV/tk + vector producer cells VPC-Rv/tk) was the best approach tested and resulted in a dramatic reduction in tumor mass after 4 days of GCV treatment which was maintained for the treatment period. Remarkably, two animals presented a complete eradication of the tumor. Thus, the HSVtk/GCV system when administered using a viral combination (ADV/tk + VPC-Rv/tk), may be a promising suicide gene therapy for pancreatic carcinomas.

Adenovirus-mediated introduction of DNA into pig sperm and offspring.

The ability of adenoviral vectors to transfer DNA into boar spermatozoa and to offspring was tested. Exposure of spermatozoa to adenovirus bearing the E. coli lacZ gene resulted in the transfer of the gene to the head of the spermatozoa. Treatment did not affect either viability or acrosomal integrity of boar sperm. Of the 2-to 8-cell embryos obtained after in vitro fertilization with adenovirus-exposed sperm, 21.7% expressed the LacZ product. Four out of 56 piglets (about 7%) obtained after artificial insemination with adenovirus-exposed spermatozoa were positive in PCR analyses, even though none of the piglets showed the LacZ gene after southern blot analysis. RT-PCR analysis performed in tissues from two positive stillborn piglets showed the presence of the LacZ mRNA in all of the tissues tested. The offspring obtained after mating two positive animals did not show LacZ gene presence. Our results indicate that adenovirus could be a feasible mechanism for the delivery of DNA into spermatozoa, even though the transfer of the transgene may be limited to the first generation.

Glycogen depletion rather than glucose 6-P increments controls early glycogen recovery in human cultured muscle.

In glycogen-containing muscle, glycogenesis appears to be controlled by glucose 6-phosphate (6-P) provision, but after glycogen depletion, an autoinhibitory control of glycogen could be a determinant. We analyzed in cultured human muscle the contribution of glycogen depletion versus glucose 6-P in the control of glycogen recovery. Acute deglycogenation was achieved by engineering cells to overexpress glycogen phosphorylase (GP). Cells treated with AdCMV-MGP adenovirus to express 10 times higher active GP showed unaltered glycogen relative to controls at 25 mM glucose, but responded to 6-h glucose deprivation with more extensive glycogen depletion. Glycogen synthase (GS) activity ratio was double in glucose-deprived AdCMV-MGP cells compared with controls, despite identical glucose 6-P. The GS activation peak (30 min) induced by glucose reincubation dose dependently correlated with glucose 6-P concentration, which reached similar steady-state levels in both cell types. GS activation was significantly blunted in AdCMV-MGP cells, whereas it strongly correlated, with an inverse relationship, with glycogen content. An initial (0-1 h) rapid insulin-independent glycogen resynthesis was observed only in AdCMV-MGP cells, which progressed up to glycogen levels approximately 150 micrograms glucose/mg protein; control cells, which did not deplete glycogen below this concentration, showed a 1-h lag time for recovery. In summary, acute deglycogenation, as achieved by GP overexpression, caused the activation of GS, which inversely correlated with glycogen replenishment independent of glucose 6-P. During glycogen recovery, the activation promoted by acute deglycogenation rendered GS effective for controlling glycogenesis, whereas the transient activation of GS induced by the glucose 6-P rise had no impact on the resynthesis rate. We conclude that the early insulin-independent glycogen resynthesis is dependent on the activation of GS due to GP-mediated exhaustion of glycogen rather than glucose 6-P provision.

Expression of glucokinase in cultured human muscle cells confers insulin-independent and glucose concentration-dependent increases in glucose disposal and storage.

Insulin resistance, as is found in skeletal muscle of individuals with obesity and NIDDM, appears to involve a reduced capacity of the hormone to stimulate glucose uptake and/or phosphorylation. The glucose phosphorylation step, as catalyzed by hexokinase II, has been described as rate limiting for glucose disposal in muscle, but overexpression of this enzyme under control of a muscle-specific promoter in transgenic mice has had limited metabolic impact. In the current study, we investigated in a cultured muscle model whether expression of glucokinase, which in contrast to hexokinase II is not inhibited by glucose-6-phosphate (G-6-P), would have a pronounced metabolic impact. We used a recombinant adenovirus containing the cDNA-encoding rat liver glucokinase (AdCMV-GKL) to increase the glucose phosphorylating activity in cultured human muscle cells by fourfold. G-6-P levels increased in AdCMV-GKL-treated cells in a glucose concentration-dependent manner over the range of 1-30 mmol/l, whereas the much smaller increases in G-6-P in control cells were maximal at glucose concentrations <5 mmol/l. Further, cells expressing glucokinase accumulated 17 times more 2-deoxyglucose-6-phosphate than control cells. In AdCMV-GKL-treated cells, the time-dependent rise in G-6-P correlated with an increase in the activity ratio of glycogen synthase. AdCMV-GKL-treated cells also exhibited a 2.5- to 3-fold increase in glycogen content and a four- to fivefold increase in glycolytic flux, proportional to the increase in glucose phosphorylating capacity. All of these observations were made in the absence of insulin. Thus we concluded that expression of glucokinase in cultured human muscle cells results in proportional increases in insulin-independent glucose disposal, and that muscle glucose storage and utilization becomes controlled in a glucose concentration-dependent manner in AdCMV-GKL-treated cells. These results encourage testing whether delivery of glucokinase to muscle in vivo has an impact on glycemic control, which could be a method for circumventing the failure of insulin to stimulate glucose uptake and/or phosphorylation in muscle normally in insulin-resistant subjects.

Reduction of islet amylin expression and basal secretion by adenovirus-mediated delivery of amylin antisense cDNA.

Reduction of amylin content and secretion in rat islets was attempted by transduction with an adenovirus bearing a 0.2-kb fragment of rat amylin cDNA inserted in the antisense orientation (AdCMV-alpha amylin). Exposure of islets to AdCMV-alpha amylin at a multiplicity of infection (moi) of 200 (1.2 x 10(7) pfu/ml) reduced amylin mRNA levels by 37 +/- 5% (p < 0.005), whereas infection with an adenovirus expressing the reporter gene of beta-galactosidase (AdCMV-lacz) did not modify amylin expression. Transduction with the antisense construct was specifically associated with the decrease (30 +/- 6%; p < 0.001) in the amylin content. Insulin content was unaltered in AdCMV-alpha amylin islets compared to AdCMV-lacz-transduced or untransduced cells. Basal amylin secretion (2.8 mM glucose) was 36 +/- 3% (p < 0.005) lower in AdCMV-alpha amylin islets than in untransduced or AdCMV-lacz-transduced islets. In contrast, no difference in amylin secretion in response to high glucose concentrations (16.7 mM) was detected in AdCMV-alpha amylin-transduced islets. Thus, a reduction of amylin content and basal secretion in islet cells can be achieved by the adenovirus-mediated expression of antisense RNA.

Overexpression of glycogen phosphorylase increases GLUT4 expression and glucose transport in cultured skeletal human muscle.

Skeletal muscle glucose utilization, a major factor in the control of whole-body glucose tolerance, is modulated in accordance with the muscle metabolic demand. For instance, it is increased in chronic contraction or exercise training in association with elevated expression of GLUT4 and hexokinase II (HK-II). In this work, the contribution of increased metabolic flux to the regulation of the glucose transport capacity was analyzed in cultured human skeletal muscle engineered to overexpress glycogen phosphorylase (GP). Myocytes treated with an adenovirus-bearing muscle GP cDNA (AdCMV-MGP) expressed 10 times higher GP activity and exhibited a twofold increase in the Vmax for 2-deoxy-D-[3H]glucose (2-DG) uptake, with no effect on the apparent Km. The stimulatory effect of insulin on 2-DG uptake was also markedly enhanced in AdCMV-MGP-treated cells, which showed maximal insulin stimulation 2.8 times higher than control cells. No changes in HKII total activity or the intracellular compartmentalization were found. GLUT4, protein, and mRNA were raised in AdCMV-MGP-treated cells, suggesting pretranslational activation. GLUT4 was immunodetected intracellularly with a perinuclear predominance. Culture in glucose-free or high-glucose medium did not alter GLUT4 protein content in either control cells or AdCMV-MGP-treated cells. Control and GP-overexpressing cells showed similar autoinhibition of glucose transport, although they appeared to differ in the mechanism(s) involved in this effect. Whereas GLUT1 protein increased in control cells when they were switched from a high-glucose to a glucose-free medium, GLUT1 remained unaltered in GP-expressing cells upon glucose deprivation. Therefore, the increased intracellular metabolic (glycogenolytic-glycolytic) flux that occurs in muscle cells overexpressing GP causes an increase in GLUT4 expression and enhances basal and insulin-stimulated glucose transport, without significant changes in the autoinhibition of glucose transport. This mechanism of regulation may be operative in the postexercise situation in which GLUT4 expression is upregulated in coordination with increased glycolytic flux and energy demand.

Metabolic impact of adenovirus-mediated overexpression of the glucose-6-phosphatase catalytic subunit in hepatocytes.

Glucose-6-phosphatase (G6Pase) catalyzes the hydrolysis of glucose 6-phosphate (Glu-6-P) to free glucose and, as the last step in gluconeogenesis and glycogenolysis in liver, is thought to play an important role in glucose homeostasis. G6Pase activity appears to be conferred by a set of proteins localized to the endoplasmic reticulum, including a glucose-6-phosphate translocase, a G6Pase phosphohydrolase or catalytic subunit, and glucose and inorganic phosphate transporters in the endoplasmic reticulum membrane. In the current study, we used a recombinant adenovirus containing the cDNA encoding the G6Pase catalytic subunit (AdCMV-G6Pase) to evaluate the metabolic impact of overexpression of the enzyme in primary hepatocytes. We found that AdCMV-G6Pase-treated liver cells contain significantly less glycogen and Glu-6-P, but unchanged UDP-glucose levels, relative to control cells. Further, the glycogen synthase activity state was closely correlated with Glu-6-P levels over a wide range of glucose concentrations in both G6Pase-overexpressing and control cells. The reduction in glycogen synthesis in AdCMV-G6Pase-treated hepatocytes is therefore not a function of decreased substrate availability but rather occurs because of the regulatory effects of Glu-6-P on glycogen synthase activity. We also found that AdCMV-G6Pase-treated-cells had significantly lower rates of lactate production and [3-3H]glucose usage, coupled with enhanced rates of gluconeogenesis and Glu-6-P hydrolysis. We conclude that overexpression of the G6Pase catalytic subunit alone is sufficient to activate flux through the G6Pase system in liver cells. Further, hepatocytes treated with AdCMV-G6Pase exhibit a metabolic profile resembling that of liver cells from patients or animals with non-insulin-dependent diabetes mellitus, suggesting that dysregulation of the catalytic subunit of G6Pase could contribute to the etiology of the disease.

Adenovirus-mediated gene transfer into human hepatocytes: analysis of the biochemical functionality of transduced cells.

The use of replication-defective adenoviruses to deliver transgenes into hepatocytes seems to be a promising approach to human liver gene therapy. However, the effects that the adenovirus-mediated expression of a foreign gene could have on the expression of other hepatic characteristic genes have not yet been properly examined. We have investigated this problem by using human hepatocytes infected with a recombinant E-1 defective adenovirus that carried a modified lacZ gene. The analysis of the biochemical functionality of transduced cells showed that the use of adenovirus: (1) was a very efficient way to introduce a foreign gene into human hepatocytes (80% transduced cells after 1 h contact, at an MOI of 15; approximately 100% transduced cells at an MOI of 20); (2) allowed the expression of the transgene to levels that enabled cells effectively to use lactose as an energy source; (3) does not affect urea synthesis, plasma protein synthesis and xenobiotic biotransformation activities (1A2, 2A1, 2B6, 3A3/5). Glycolysis was moderately increased (approximately 20%), while gluconeogenesis decreased (approximately 20%) in transduced hepatocyte; moreover, (4) the expression of inducible genes (acute-phase plasma proteins, CYPs) was not impaired in transduced human hepatocytes upon stimulation with IL-6 or methylcholantrene. The results of this research support the idea that efficient expression of transgenes can be achieved in human hepatocytes by means of adenoviral transduction, without altering these characteristic hepatic biochemical functions.