Liver glycogen reduces food intake and attenuates obesity in a high-fat diet-fed mouse model.

We generated mice that overexpress protein targeting to glycogen (PTG) in the liver (PTG(OE)), which results in an increase in liver glycogen. When fed a high-fat diet (HFD), these animals reduced their food intake. The resulting effect was a lower body weight, decreased fat mass, and reduced leptin levels. Furthermore, PTG overexpression reversed the glucose intolerance and hyperinsulinemia caused by the HFD and protected against HFD-induced hepatic steatosis. Of note, when fed an HFD, PTG(OE) mice did not show the decrease in hepatic ATP content observed in control animals and had lower expression of neuropeptide Y and higher expression of proopiomelanocortin in the hypothalamus. Additionally, after an overnight fast, PTG(OE) animals presented high liver glycogen content, lower liver triacylglycerol content, and lower serum concentrations of fatty acids and ß-hydroxybutyrate than control mice, regardless of whether they were fed an HFD or a standard diet. In conclusion, liver glycogen accumulation caused a reduced food intake, protected against the deleterious effects of an HFD, and diminished the metabolic impact of fasting. Therefore, we propose that hepatic glycogen content be considered a potential target for the pharmacological manipulation of diabetes and obesity.

Neuronal glycogen synthesis contributes to physiological aging.

Glycogen is a branched polymer of glucose and the carbohydrate energy store for animal cells. In the brain, it is essentially found in glial cells, although it is also present in minute amounts in neurons. In humans, loss-of-function mutations in laforin and malin, proteins involved in suppressing glycogen synthesis, induce the presence of high numbers of insoluble polyglucosan bodies in neuronal cells. Known as Lafora bodies (LBs), these deposits result in the aggressive neurodegeneration seen in Lafora's disease. Polysaccharide-based aggregates, called corpora amylacea (CA), are also present in the neurons of aged human brains. Despite the similarity of CA to LBs, the mechanisms and functional consequences of CA formation are yet unknown. Here, we show that wild-type laboratory mice also accumulate glycogen-based aggregates in the brain as they age. These structures are immunopositive for an array of metabolic and stress-response proteins, some of which were previously shown to aggregate in correlation with age in the human brain and are also present in LBs. Remarkably, these structures and their associated protein aggregates are not present in the aged mouse brain upon genetic ablation of glycogen synthase. Similar genetic intervention in Drosophila prevents the accumulation of glycogen clusters in the neuronal processes of aged flies. Most interestingly, targeted reduction of Drosophila glycogen synthase in neurons improves neurological function with age and extends lifespan. These results demonstrate that neuronal glycogen accumulation contributes to physiological aging and may therefore constitute a key factor regulating age-related neurological decline in humans.

Glucose-6-phosphate-mediated activation of liver glycogen synthase plays a key role in hepatic glycogen synthesis.

The liver responds to an increase in blood glucose levels in the postprandial state by uptake of glucose and conversion to glycogen. Liver glycogen synthase (GYS2), a key enzyme in glycogen synthesis, is controlled by a complex interplay between the allosteric activator glucose-6-phosphate (G6P) and reversible phosphorylation through glycogen synthase kinase-3 and the glycogen-associated form of protein phosphatase 1. Here, we initially performed mutagenesis analysis and identified a key residue (Arg(582)) required for activation of GYS2 by G6P. We then used GYS2 Arg(582)Ala knockin (+/R582A) mice in which G6P-mediated GYS2 activation had been profoundly impaired (60-70%), while sparing regulation through reversible phosphorylation. R582A mutant-expressing hepatocytes showed significantly reduced glycogen synthesis with glucose and insulin or glucokinase activator, which resulted in channeling glucose/G6P toward glycolysis and lipid synthesis. GYS2(+/R582A) mice were modestly glucose intolerant and displayed significantly reduced glycogen accumulation with feeding or glucose load in vivo. These data show that G6P-mediated activation of GYS2 plays a key role in controlling glycogen synthesis and hepatic glucose-G6P flux control and thus whole-body glucose homeostasis.

Tungstate reduces the expression of gluconeogenic enzymes in STZ rats.

AIMS: Oral administration of sodium tungstate has shown hyperglycemia-reducing activity in several animal models of diabetes. We present new insights into the mechanism of action of tungstate. METHODS: We studied protein expression and phosphorylation in the liver of STZ rats, a type I diabetes model, treated with sodium tungstate in the drinking water (2 mg/ml) and in primary cultured-hepatocytes, through Western blot and Real Time PCR analysis. RESULTS: Tungstate treatment reduces the expression of gluconeogenic enzymes (PEPCK, G6Pase, and FBPase) and also regulates transcription factors accountable for the control of hepatic metabolism (c-jun, c-fos and PGC1α). Moreover, ERK, p90rsk and GSK3, upstream kinases regulating the expression of c-jun and c-fos, are phosphorylated in response to tungstate. Interestingly, PKB/Akt phosphorylation is not altered by the treatment. Several of these observations were reproduced in isolated rat hepatocytes cultured in the absence of insulin, thereby indicating that those effects of tungstate are insulin-independent. CONCLUSIONS: Here we show that treatment with tungstate restores the phosphorylation state of various signaling proteins and changes the expression pattern of metabolic enzymes.

Deleterious effects of neuronal accumulation of glycogen in flies and mice.

Under physiological conditions, most neurons keep glycogen synthase (GS) in an inactive form and do not show detectable levels of glycogen. Nevertheless, aberrant glycogen accumulation in neurons is a hallmark of patients suffering from Lafora disease or other polyglucosan disorders. Although these diseases are associated with mutations in genes involved in glycogen metabolism, the role of glycogen accumulation remains elusive. Here, we generated mouse and fly models expressing an active form of GS to force neuronal accumulation of glycogen. We present evidence that the progressive accumulation of glycogen in mouse and Drosophila neurons leads to neuronal loss, locomotion defects and reduced lifespan. Our results highlight glycogen accumulation in neurons as a direct cause of neurodegeneration.

A functional role for tumor cell heterogeneity in a mouse model of small cell lung cancer.

Small cell lung cancer (SCLC) is the lung neoplasia with the poorest prognosis, due to its high metastatic potential and chemoresistance upon relapse. Using the previously described mouse model for SCLC, we found that the tumors are often composed of phenotypically different cells with either a neuroendocrine or a mesenchymal marker profile. These cells had a common origin because they shared specific genomic aberrations. The transition from neuroendocrine to mesenchymal phenotype could be achieved by the ectopic expression of oncogenic Ras(V12). Crosstalk between mesenchymal and neuroendocrine cells strongly influenced their behavior. When engrafted as a mixed population, the mesenchymal cells endowed the neuroendocrine cells with metastatic capacity, illustrating the potential relevance of tumor cell heterogeneity in dictating tumor properties.

Hepatic overexpression of a constitutively active form of liver glycogen synthase improves glucose homeostasis.

In this study, we tested the efficacy of increasing liver glycogen synthase to improve blood glucose homeostasis. The overexpression of wild-type liver glycogen synthase in rats had no effect on blood glucose homeostasis in either the fed or the fasted state. In contrast, the expression of a constitutively active mutant form of the enzyme caused a significant lowering of blood glucose in the former but not the latter state. Moreover, it markedly enhanced the clearance of blood glucose when fasted rats were challenged with a glucose load. Hepatic glycogen stores in rats overexpressing the activated mutant form of liver glycogen synthase were enhanced in the fed state and in response to an oral glucose load but showed a net decline during fasting. In order to test whether these effects were maintained during long term activation of liver glycogen synthase, we generated liver-specific transgenic mice expressing the constitutively active LGS form. These mice also showed an enhanced capacity to store glycogen in the fed state and an improved glucose tolerance when challenged with a glucose load. Thus, we conclude that the activation of liver glycogen synthase improves glucose tolerance in the fed state without compromising glycogenolysis in the postabsorptive state. On the basis of these findings, we propose that the activation of liver glycogen synthase may provide a potential strategy for improvement of glucose tolerance in the postprandial state.

Immune response in lung cancer mouse model mimics human anti-Hu reactivity.

Most patients with paraneoplastic encephalomyelitis/sensory neuronopathy PEM/SN have small-cell lung cancer (SCLC) and develop antibodies against neuronal-specific Hu proteins, which are abnormally expressed in the tumor. Anti-Hu reactivity is present in ~16% of SCLC patients without PEM/SN. Here we test the hypothesis that engineered SCLC-prone mice may exhibit anti-Hu reactivity. We show that tumors from SCLC-prone mice misexpress Hu proteins, and 14% of mice harbor anti-Hu antibodies. Mice appear to show reactivity prior to clinical diagnosis of SCLC. This mouse model system will be useful to study SCLC-associated autoimmunity, its diagnostic value, and the potential protective role of oncoantigen-directed autoantibodies.

Enhancement of gemcitabine-induced apoptosis by restoration of p53 function in human pancreatic tumors.

Human pancreatic adenocarcinomas are highly resistant to conventional treatment modalities, specially to chemotherapy. Among the genes that modulate apoptosis in response to cytotoxic drugs, the role of p53 has been demonstrated to be of paramount importance. Moreover, p53 is mutated in close to 50% of pancreatic cancer, which renders attractive the reintroduction of this gene as a way to enhance the action of chemotherapeutics. In this paper, gemcitabine, the most effective drug for the treatment of pancreatic tumors, has been selected to develop a new combination approach in vivo based on an administration schedule previously optimized in vitro. In a human xenograft model, the sequential administration of gemcitabine and p53 resulted in potent tumor growth inhibition. Statistical differences were observed with respect to the growth of tumors receiving only gemcitabine or p53. Moreover, the chemosensitization observed in tumors treated with the combination gemcitabine-p53 correlated with differential histological features such as important increases in intratumoral fibrosis and apoptotic levels, when compared with unimodal treatments. Taken together, our data indicate that reintroduction of p53 function in human pancreatic tumors in vivo allows to restore molecular pathways improving the response to gemcitabine. It may constitute a useful step towards a better clinical treatment of patients harboring pancreatic cancer.

Adenovirus-mediated retinoblastoma 94 gene transfer induces human pancreatic tumor regression in a mouse xenograft model.

PURPOSE: Gene transfer of a truncated variant of the retinoblastoma (RB) gene encoding a M(r) 94000 protein that lacks the NH(2)-terminal 112 amino acid residues, termed RB94, has been shown to inhibit proliferation of several human tumor cell types. We have assessed its therapeutic effectiveness on pancreatic cancer, one of the most aggressive and therapy-resistant types of cancer. For this purpose, preclinical studies aimed to evaluate the therapeutic potential of RB94 gene transfer in pancreatic cancer were carried out. EXPERIMENTAL DESIGN: We have compared the antiproliferative effects of adenovirus-mediated gene transfer of RBwt and RB94 at the in vitro and in vivo levels in three RB-positive human pancreatic tumor cell lines: (a). NP-9; (b). NP-18; and (c). NP-31. We have also examined their effects on cell cycle and their capacity to induce apoptosis. RESULTS: In vitro results indicate that RB94 gene transfer has stronger antiproliferative effects compared with RBwt. RB94 transduction correlated with accumulation at the S-G(2) phase of the cell cycle in the three cell lines tested and induction of apoptosis in two of them. In vivo studies show significant decreases in the growth rate of tumors treated with Ad-RB94 when compared with those treated with Ad-RBwt. Moreover, terminal deoxynucleotidyl transferase-mediated nick end labeling analyses of Ad-RB94-treated tumor sections revealed that only RB94 is able to significantly induce apoptosis. CONCLUSIONS: RB94 gene expression has antiproliferative effects also in human pancreatic tumor cells, being more effective than wild-type RB in preventing tumor growth.

CDK9 is constitutively expressed throughout the cell cycle, and its steady-state expression is independent of SKP2.

CDK9 is a CDC2-related kinase and the catalytic subunit of the positive-transcription elongation factor b and the Tat-activating kinase. It has recently been reported that CDK9 is a short-lived protein whose levels are regulated during the cell cycle by the SCF(SKP2) ubiquitin ligase complex (R. E. Kiernan et al., Mol. Cell. Biol. 21:7956-7970, 2001). The results presented here are in contrast to those observations. CDK9 protein levels remained unchanged in human cells entering and progressing through the cell cycle from G(0), despite dramatic changes in SKP2 expression. CDK9 levels also remained unchanged in cells exiting from mitosis and progressing through the next cell cycle. Similarly, the levels of CDK9 protein did not change as cells exited the cell cycle and differentiated along various lineages. In keeping with these observations, the kinase activity associated with CDK9 was found to not be regulated during the cell cycle. We have also found that endogenous CDK9 is a very stable protein with a half-life (t(1/2)) of 4 to 7 h, depending on the cell type. In contrast, when CDK9 is overexpressed, it is not stabilized and is rapidly degraded, with a t(1/2) of less than 1 h, depending on the level of expression. Treatment of cells with proteasome inhibitors blocked the degradation of short-lived proteins, such as p27, but did not affect the expression of endogenous CDK9. Ectopic overexpression of SKP2 led to reduction of p27 protein levels but had no effect on the expression of endogenous CDK9. Finally, downregulation of endogenous SKP2 gene expression by interfering RNA had no effect on CDK9 protein levels, whereas p27 protein levels increased dramatically. Therefore, the SCF(SKP2) ubiquitin ligase does not regulate CDK9 expression in a cell cycle-dependent manner.

SKP2 associates with p130 and accelerates p130 ubiquitylation and degradation in human cells.

p130 is a member of the retinoblastoma family of pocket proteins, which includes pRB and p107. Unlike pRB and p107, p130 protein levels decrease dramatically following its hyperphosphorylation starting in the mid-G1 phase of the cell cycle. However, the mechanism leading to p130 downregulation is unknown. We have found that the proteasome inhibitor, lactacystin, inhibited p130 downregulation in T98G cells progressing through the G1/S transition and S phase and that p130 is multiubiquitylated in 293 cells. We have previously shown that ectopic expression of both cyclin D and E induces phosphorylation and downregulation of p130. Since the SKP1/Cul1/SKP2 E3 ubiquitin ligase complex mediates ubiquitylation of substrates previously phosphorylated by cyclin-dependent kinases, we investigated the potential role of this ubiquitin ligase in mediating p130 downregulation. We found that p130 interacts with SKP1, Cul-1 and SKP2 in human 293 cells. We also found that ectopic coexpression of SKP2 and p130 leads to dose-dependent downregulation of p130, reduces p130 protein half-life and induces p130 ubiquitylation in these cells. Moreover, adenoviral-mediated expression of SKP2 accelerates downregulation of endogenous hyperphosphorylated p130 in mitogen-stimulated T98G cells and primary WI38 fibroblasts. We conclude that p130 is a substrate of the SCF(SKP2) ubiquitin ligase and this E3 ligase regulates p130 abundance during the cell cycle.

G1 cyclin/cyclin-dependent kinase-coordinated phosphorylation of endogenous pocket proteins differentially regulates their interactions with E2F4 and E2F1 and gene expression.

Mitogenic stimulation leads to activation of G(1) cyclin-dependent kinases (CDKs), which phosphorylate pocket proteins and trigger progression through the G(0)/G(1) and G(1)/S transitions of the cell cycle. However, the individual role of G(1) cyclin-CDK complexes in the coordinated regulation of pocket proteins and their interaction with E2F family members is not fully understood. Here we report that individually or in concert cyclin D1-CDK and cyclin E-CDK complexes induce distinct and coordinated phosphorylation of endogenous pocket proteins, which also has distinct consequences in the regulation of pocket protein interactions with E2F4 and the expression of p107 and E2F1, both E2F-regulated genes. The up-regulation of these two proteins and the release of p130 and pRB from E2F4 complexes allows formation of E2F1 complexes not only with pRB but also with p130 and p107 as well as the formation of p107-E2F4 complexes. The formation of these complexes occurs in the presence of active cyclin D1-CDK and cyclin E-CDK complexes, indicating that whereas phosphorylation plays a role in the abrogation of certain pocket protein/E2F interactions, these same activities induce the formation of other complexes in the context of a cell expressing endogenous levels of pocket and E2F proteins. Of note, phosphorylated p130 "form 3," which does not interact with E2F4, readily interacts with E2F1. Our data also demonstrate that ectopic overexpression of either cyclin is sufficient to induce mitogen-independent growth in human T98G and Rat-1 cells, although the effects of cyclin D1 require downstream activation of cyclin E-CDK2 activity. Interestingly, in T98G cells, cyclin D1 induces cell cycle progression more potently than cyclin E. This suggests that cyclin D1 activates pathways independently of cyclin E that ensure timely progression through the cell cycle.