The Adaptor Protein NumbL Is Involved in the Control of Glucolipotoxicity-Induced Pancreatic Beta Cell Apoptosis.

Avoiding the loss of functional beta cell mass is critical for preventing or treating diabetes. Currently, the molecular mechanisms underlying beta cell death are partially understood, and there is a need to identify new targets for developing novel therapeutics to treat diabetes. Previously, our group established that Mig6, an inhibitor of EGF signaling, mediates beta cell death under diabetogenic conditions. The objective here was to clarify the mechanisms linking diabetogenic stimuli to beta cell death by investigating Mig6-interacting proteins. Using co-immunoprecipitation and mass spectrometry, we evaluated the binding partners of Mig6 under both normal glucose (NG) and glucolipotoxic (GLT) conditions in beta cells. We identified that Mig6 interacted dynamically with NumbL, whereas Mig6 associated with NumbL under NG, and this interaction was disrupted under GLT conditions. Further, we demonstrated that the siRNA-mediated suppression of NumbL expression in beta cells prevented apoptosis under GLT conditions by blocking the activation of NF-κB signaling. Using co-immunoprecipitation experiments, we observed that NumbL's interactions with TRAF6, a key component of NFκB signaling, were increased under GLT conditions. The interactions among Mig6, NumbL, and TRAF6 were dynamic and context-dependent. We proposed a model wherein these interactions activated pro-apoptotic NF-κB signaling while blocking pro-survival EGF signaling under diabetogenic conditions, leading to beta cell apoptosis. These findings indicated that NumbL should be further investigated as a candidate anti-diabetic therapeutic target.

Lack of liver glycogen causes hepatic insulin resistance and steatosis in mice.

Disruption of the Gys2 gene encoding the liver isoform of glycogen synthase generates a mouse strain (LGSKO) that almost completely lacks hepatic glycogen, has impaired glucose disposal, and is pre-disposed to entering the fasted state. This study investigated how the lack of liver glycogen increases fat accumulation and the development of liver insulin resistance. Insulin signaling in LGSKO mice was reduced in liver, but not muscle, suggesting an organ-specific defect. Phosphorylation of components of the hepatic insulin-signaling pathway, namely IRS1, Akt, and GSK3, was decreased in LGSKO mice. Moreover, insulin stimulation of their phosphorylation was significantly suppressed, both temporally and in an insulin dose response. Phosphorylation of the insulin-regulated transcription factor FoxO1 was somewhat reduced and insulin treatment did not elicit normal translocation of FoxO1 out of the nucleus. Fat overaccumulated in LGSKO livers, showing an aberrant distribution in the acinus, an increase not explained by a reduction in hepatic triglyceride export. Rather, when administered orally to fasted mice, glucose was directed toward hepatic lipogenesis as judged by the activity, protein levels, and expression of several fatty acid synthesis genes, namely, acetyl-CoA carboxylase, fatty acid synthase, SREBP1c, chREBP, glucokinase, and pyruvate kinase. Furthermore, using cultured primary hepatocytes, we found that lipogenesis was increased by 40% in LGSKO cells compared with controls. Of note, the hepatic insulin resistance was not associated with increased levels of pro-inflammatory markers. Our results suggest that loss of liver glycogen synthesis diverts glucose toward fat synthesis, correlating with impaired hepatic insulin signaling and glucose disposal.

Muscle glycogen remodeling and glycogen phosphate metabolism following exhaustive exercise of wild type and laforin knockout mice.

Glycogen, the repository of glucose in many cell types, contains small amounts of covalent phosphate, of uncertain function and poorly understood metabolism. Loss-of-function mutations in the laforin gene cause the fatal neurodegenerative disorder, Lafora disease, characterized by increased glycogen phosphorylation and the formation of abnormal deposits of glycogen-like material called Lafora bodies. It is generally accepted that the phosphate is removed by the laforin phosphatase. To study the dynamics of skeletal muscle glycogen phosphorylation in vivo under physiological conditions, mice were subjected to glycogen-depleting exercise and then monitored while they resynthesized glycogen. Depletion of glycogen by exercise was associated with a substantial reduction in total glycogen phosphate and the newly resynthesized glycogen was less branched and less phosphorylated. Branching returned to normal on a time frame of days, whereas phosphorylation remained suppressed over a longer period of time. We observed no change in markers of autophagy. Exercise of 3-month-old laforin knock-out mice caused a similar depletion of glycogen but no loss of glycogen phosphate. Furthermore, remodeling of glycogen to restore the basal branching pattern was delayed in the knock-out animals. From these results, we infer that 1) laforin is responsible for glycogen dephosphorylation during exercise and acts during the cytosolic degradation of glycogen, 2) excess glycogen phosphorylation in the absence of laforin delays the normal remodeling of the branching structure, and 3) the accumulation of glycogen phosphate is a relatively slow process involving multiple cycles of glycogen synthesis-degradation, consistent with the slow onset of the symptoms of Lafora disease.

Hepatic glucose uptake and disposition during short-term high-fat vs. high-fructose feeding.

In dogs consuming a high-fat and -fructose diet (52 and 17% of total energy, respectively) for 4 wk, hepatic glucose uptake (HGU) in response to hyperinsulinemia, hyperglycemia, and portal glucose delivery is markedly blunted with reduction in glucokinase (GK) protein and glycogen synthase (GS) activity. The present study compared the impact of selective increases in dietary fat and fructose on liver glucose metabolism. Dogs consumed weight-maintaining chow (CTR) or hypercaloric high-fat (HFA) or high-fructose (HFR) diets diet for 4 wk before undergoing clamp studies with infusion of somatostatin and intraportal insulin (3-4 times basal) and glucagon (basal). The hepatic glucose load (HGL) was doubled during the clamp using peripheral vein (Pe) glucose infusion in the first 90 min (P1) and portal vein (4 mg·kg(-1)·min(-1)) plus Pe glucose infusion during the final 90 min (P2). During P2, HGU was 2.8 ± 0.2, 1.0 ± 0.2, and 0.8 ± 0.2 mg·kg(-1)·min(-1) in CTR, HFA, and HFR, respectively (P < 0.05 for HFA and HFR vs. CTR). Compared with CTR, hepatic GK protein and catalytic activity were reduced (P < 0.05) 35 and 56%, respectively, in HFA, and 53 and 74%, respectively, in HFR. Liver glycogen concentrations were 20 and 38% lower in HFA and HFR than CTR (P < 0.05). Hepatic Akt phosphorylation was decreased (P < 0.05) in HFA (21%) but not HFR. Thus, HFR impaired hepatic GK and glycogen more than HFA, whereas HFA reduced insulin signaling more than HFR. HFA and HFR effects were not additive, suggesting that they act via the same mechanism or their effects converge at a saturable step.

Flywheel resistance exercise to maintain muscle oxidative potential during unloading.

BACKGROUND: As spaceflight compromises skeletal muscle oxidative and aerobic work capacity, this study assessed the efficacy of resistance exercise (RE) to counteract muscle metabolic perturbations induced by 5 wk unilateral lower limb unloading (UL). METHODS: There were 21 men and women (30-56 yr) who were randomly assigned to either UL with (Group, Grp; UL+RE; N = 10) or without (Grp UL; N = 11) concurrent RE. Iso-inertial RE comprised four sets of seven maximal coupled concentric-eccentric knee extensions executed 2-3 times per week. Percutaneous biopsies were obtained from m. vastus lateralis before and after either intervention. Levels of mRNA expression of factors regulating skeletal muscle oxidative capacity i.e., peroxisome proliferator-activated receptor gamma coactivator-1 (PGC-1alpha) and vascular endothelial growth factor (VEGF), and glycolytic capacity, i.e., phosphofructokinase (PFK), glycogen phosphorylase and synthase, hexokinase, and phosphorylase kinase alpha1, were subsequently analyzed. RESULTS: Grp UL showed decreased (36%) PGC-1alpha expression, increased (1.5-fold) PFK expression, and a trend toward decreased VEGF post-intervention. Grp UL+RE showed no changes. DISCUSSION: These results suggest that 5 wk unloading reduces skeletal muscle oxidative capacity and increases glycolytic enzyme activity. More importantly, only 12 bouts of high-force, low-volume resistance exercise attenuated these responses. Thus, the current resistance exercise paradigm emphasizing eccentric overload effectively counteracts unwarranted metabolic alterations induced by 5 wk unloading and may, therefore, aid in maintaining skeletal muscle integrity and endurance, and hence astronaut health and fitness during spaceflight.

Trefoil Factor 2 Expressed by the Murine Pancreatic Acinar Cells Is Required for the Development of Islets and for ß-Cell Function During Aging.

Exocrine-to-endocrine cross talk in the pancreas is crucial to maintain ß-cell function. However, the molecular mechanisms underlying this cross talk are largely undefined. Trefoil factor 2 (Tff2) is a secreted factor known to promote the proliferation of ß-cells in vitro, but its physiological role in vivo in the pancreas is unknown. Also, it remains unclear which pancreatic cell type expresses Tff2 protein. We therefore created a mouse model with a conditional knockout of Tff2 in the murine pancreas. We find that the Tff2 protein is preferentially expressed in acinar but not ductal or endocrine cells. Tff2 deficiency in the pancreas reduces ß-cell mass on embryonic day 16.5. However, homozygous mutant mice are born without a reduction of ß-cells and with acinar Tff3 compensation by day 7. When mice are aged to 1 year, both male and female homozygous and male heterozygous mutants develop impaired glucose tolerance without affected insulin sensitivity. Perifusion analysis reveals that the second phase of glucose-stimulated insulin secretion from islets is reduced in aged homozygous mutant compared with controls. Collectively, these results demonstrate a previously unknown role of Tff2 as an exocrine acinar cell-derived protein required for maintaining functional endocrine ß-cells in mice.

Dose-dependent progression of multiple low dose streptozotocin-induced diabetes in mice.

This study investigated the effects of different multiple low doses of streptozotocin (STZ), namely 35 and 55 mg/kg, on the onset and progression of diabetes in mice. Both doses are commonly used in research, and while both induced a loss of beta cell mass, they had distinct effects on whole glucose tolerance, beta cell function and gene transcription. Mice treated with 55 mg/kg became rapidly glucose intolerant, whereas those treated with 35 mg/kg had a slower onset and remained glucose tolerant for up to a week before becoming equally glucose intolerant as the 55 mg/kg group. Beta cell mass loss was similar between the two groups, but the 35 mg/kg-treated mice had improved glucose-stimulated insulin secretion in gold-standard hyperglycemic clamp studies. Transcriptomic analysis revealed that the 55 mg/kg dose caused disruptions in nearly five times as many genes as the 35 mg/kg dose in isolated pancreatic islets. Pathways that were downregulated in both doses were more downregulated in the 55 mg/kg-treated mice, while pathways that were upregulated in both doses were more upregulated in the 35 mg/kg treated mice. Moreover, we observed a differential downregulation in the 55 mg/kg-treated islets of beta cell characteristic pathways, such as exocytosis or hormone secretion. On the other hand, apoptosis was differentially upregulated in 35 mg/kg-treated islets, suggesting different transcriptional mechanisms in the onset of STZ-induced damage in the islets. This study demonstrates that the two STZ doses induce distinctly mechanistic progressions for the loss of functional beta cell mass.

Pancreatic loss of Mig6 alters murine endocrine cell fate and protects functional beta cell mass in an STZ-induced model of diabetes.

Background: Maintaining functional beta cell mass (BCM) to meet glycemic demands is essential to preventing or reversing the progression of diabetes. Yet the mechanisms that establish and regulate endocrine cell fate are incompletely understood. We sought to determine the impact of deletion of mitogen-inducible gene 6 (Mig6), a negative feedback inhibitor of epidermal growth factor receptor (EGFR) signaling, on mouse endocrine cell fate. The extent to which loss of Mig6 might protect against loss of functional BCM in a multiple very low dose (MVLD) STZ-induced model of diabetes was also determined. Methods: Ten-week-old male mice with whole pancreas (Pdx1:Cre, PKO) and beta cell-specific (Ins1:Cre, BKO) knockout of Mig6 were used alongside control (CON) littermates. Mice were given MVLD STZ (35 mg/kg for five days) to damage beta cells and induce hyperglycemia. In vivo fasting blood glucose and glucose tolerance were used to assess beta cell function. Histological analyses of isolated pancreata were utilized to assess islet morphology and beta cell mass. We also identified histological markers of beta cell replication, dedifferentiation, and death. Isolated islets were used to reveal mRNA and protein markers of beta cell fate and function. Results: PKO mice had significantly increased alpha cell mass with no detectable changes to beta or delta cells. The increase in alpha cells alone did not impact glucose tolerance, BCM, or beta cell function. Following STZ treatment, PKO mice had 18±8% higher BCM than CON littermates and improved glucose tolerance. Interestingly, beta cell-specific loss of Mig6 was insufficient for protection, and BKO mice had no discernable differences compared to CON mice. The increase in BCM in PKO mice was the result of decreased beta cell loss and increased beta cell replication. Finally, STZ-treated PKO mice had more Ins+/Gcg+ bi-hormonal cells compared to controls suggesting alpha to beta cell transdifferentiation. Conclusions: Mig6 exerted differential effects on alpha and beta cell fate. Pancreatic loss of Mig6 reduced beta cell loss and promoted beta cell growth following STZ. Thus, suppression of Mig6 may provide relief of diabetes.

Longitudinal analysis of a dietary mouse model of non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH).

Non-alcoholic fatty liver disease (NAFLD), and resultant non-alcoholic steatohepatitis (NASH), incidence and prevalence are rising globally due to increasing rates of obesity and diabetes. Currently, there are no approved pharmacological treatments for NAFLD, highlighting a need for additional mechanistic studies to develop prevention and/or therapeutic strategies. Diet-induced preclinical models of NAFLD can be used to examine the dynamic changes that occur during NAFLD development and progression throughout the lifespan. To date, most studies utilizing such models have focused exclusively on terminal time points and have likely missed critical early and late changes that are important for NAFLD progression (i.e, worsening). We performed a longitudinal analysis of histopathological, biochemical, transcriptomic, and microbiome changes that occurred in adult male mice fed either a control diet or a NASH-promoting diet (high in fat, fructose, and cholesterol) for up to 30 weeks. We observed progressive development of NAFLD in mice fed the NASH diet compared to the control diet. Differential expression of immune-related genes was observed at an early stage of diet-induced NAFLD development (10 weeks) and persisted into the later stages of the disease (20 and 30 weeks). Differential expression of xenobiotic metabolism related genes was observed at the late stage of diet-induced NAFLD development (30 weeks). Microbiome analysis revealed an increased abundance of Bacteroides at an early stage (10 weeks) that persisted into the later stages of the disease (20 and 30 weeks). These data provide insight into the progressive changes that occur during NAFLD/NASH development and progression in the context of a typical Western diet. Furthermore, these data are consistent with what has been reported in patients with NAFLD/NASH, supporting the preclinical use of this diet-induced model for development of strategies to prevent or treat the disease.

Integration of metabolic flux with hepatic glucagon signaling and gene expression profiles in the conscious dog.

Glucagon rapidly and profoundly simulates hepatic glucose production (HGP), but for reasons which are unclear, this effect normally wanes after a few hours, despite sustained plasma glucagon levels. This study characterized the time course and relevance (to metabolic flux) of glucagon mediated molecular events in the livers of conscious dogs. Glucagon was either infused into the hepato-portal vein at a 6-fold basal rate in the presence of somatostatin and basal insulin, or it was maintained at a basal level in control studies. In one control group glucose remained at basal while in the other glucose was infused to match the hyperglycemia that occurred in the hyperglucagonemic group. Elevated glucagon caused a rapid (30 min) but only partially sustained increase in hepatic cAMP over 4h, a continued elevation in G6P, and activation and deactivation of glycogen phosphorylase and synthase activities, respectively. Net hepatic glycogenolysis and HGP increased rapidly, peaking at 30 min, then returned to baseline over the next 3h (although glucagons stimulatory effect on HGP was sustained relative to the hyperglycemic control group). Hepatic gluconeogenic flux did not increase due to lack of glucagon effect on substrate supply to the liver. Global gene expression profiling highlighted glucagon-regulated activation of genes involved in cellular respiration, metabolic processes, and signaling, and downregulation of genes involved in extracellular matrix assembly and development.

Genetic depletion of the malin E3 ubiquitin ligase in mice leads to lafora bodies and the accumulation of insoluble laforin.

Approximately 90% of cases of Lafora disease, a fatal teenage-onset progressive myoclonus epilepsy, are caused by mutations in either the EPM2A or the EPM2B genes that encode, respectively, a glycogen phosphatase called laforin and an E3 ubiquitin ligase called malin. Lafora disease is characterized by the formation of Lafora bodies, insoluble deposits containing poorly branched glycogen or polyglucosan, in many tissues including skeletal muscle, liver, and brain. Disruption of the Epm2b gene in mice resulted in viable animals that, by 3 months of age, accumulated Lafora bodies in the brain and to a lesser extent in heart and skeletal muscle. Analysis of muscle and brain of the Epm2b(-/-) mice by Western blotting indicated no effect on the levels of glycogen synthase, PTG (type 1 phosphatase-targeting subunit), or debranching enzyme, making it unlikely that these proteins are targeted for destruction by malin, as has been proposed. Total laforin protein was increased in the brain of Epm2b(-/-) mice and, most notably, was redistributed from the soluble, low speed supernatant to the insoluble low speed pellet, which now contained 90% of the total laforin. This result correlated with elevated insolubility of glycogen and glycogen synthase. Because up-regulation of laforin cannot explain Lafora body formation, we conclude that malin functions to maintain laforin associated with soluble glycogen and that its absence causes sequestration of laforin to an insoluble polysaccharide fraction where it is functionally inert.

Impaired glucose tolerance and predisposition to the fasted state in liver glycogen synthase knock-out mice.

Conversion to glycogen is a major fate of ingested glucose in the body. A rate-limiting enzyme in the synthesis of glycogen is glycogen synthase encoded by two genes, GYS1, expressed in muscle and other tissues, and GYS2, primarily expressed in liver (liver glycogen synthase). Defects in GYS2 cause the inherited monogenic disease glycogen storage disease 0. We have generated mice with a liver-specific disruption of the Gys2 gene (liver glycogen synthase knock-out (LGSKO) mice), using Lox-P/Cre technology. Conditional mice carrying floxed Gys2 were crossed with mice expressing Cre recombinase under the albumin promoter. The resulting LGSKO mice are viable, develop liver glycogen synthase deficiency, and have a 95% reduction in fed liver glycogen content. They have mild hypoglycemia but dispose glucose less well in a glucose tolerance test. Fed, LGSKO mice also have a reduced capacity for exhaustive exercise compared with mice carrying floxed alleles, but the difference disappears after an overnight fast. Upon fasting, LGSKO mice reach within 4 h decreased blood glucose levels attained by control floxed mice only after 24 h of food deprivation. The LGSKO mice maintain this low blood glucose for at least 24 h. Basal gluconeogenesis is increased in LGSKO mice, and insulin suppression of endogenous glucose production is impaired as assessed by euglycemic-hyperinsulinemic clamp. This observation correlates with an increase in the liver gluconeogenic enzyme phosphoenolpyruvate carboxykinase expression and activity. This mouse model mimics the pathophysiology of glycogen storage disease 0 patients and highlights the importance of liver glycogen stores in whole body glucose homeostasis.

Starch binding domain-containing protein 1/genethonin 1 is a novel participant in glycogen metabolism.

Stbd1 is a protein of previously unknown function that is most prevalent in liver and muscle, the major sites for storage of the energy reserve glycogen. The protein is predicted to contain a hydrophobic N terminus and a C-terminal CBM20 glycan binding domain. Here, we show that Stbd1 binds to glycogen in vitro and that endogenous Stbd1 locates to perinuclear compartments in cultured mouse FL83B or Rat1 cells. When overexpressed in COSM9 cells, Stbd1 concentrated at enlarged perinuclear structures, co-localized with glycogen, the late endosomal/lysosomal marker LAMP1 and the autophagy protein GABARAPL1. Mutant Stbd1 lacking the N-terminal hydrophobic segment had a diffuse distribution throughout the cell. Point mutations in the CBM20 domain did not change the perinuclear localization of Stbd1, but glycogen was no longer concentrated in this compartment. Stable overexpression of glycogen synthase in Rat1WT4 cells resulted in accumulation of glycogen as massive perinuclear deposits, where a large fraction of the detectable Stbd1 co-localized. Starvation of Rat1WT4 cells for glucose resulted in dissipation of the massive glycogen stores into numerous and much smaller glycogen deposits that retained Stbd1. In vitro, in cells, and in animal models, Stbd1 consistently tracked with glycogen. We conclude that Stbd1 is involved in glycogen metabolism by binding to glycogen and anchoring it to membranes, thereby affecting its cellular localization and its intracellular trafficking to lysosomes.

Metabolic adaptations in skeletal muscle after 84 days of bed rest with and without concurrent flywheel resistance exercise.

As metabolic changes in human skeletal muscle after long-term (simulated) spaceflight are not well understood, this study examined the effects of long-term microgravity, with and without concurrent resistance exercise, on skeletal muscle oxidative and glycolytic capacity. Twenty-one men were subjected to 84 days head-down tilt bed rest with (BRE; n = 9) or without (BR; n = 12) concurrent flywheel resistance exercise. Activity and gene expression of glycogen synthase, glycogen phosphorylase (GPh), hexokinase, phosphofructokinase-1 (PFK-1), and citrate synthase (CS), as well as gene expression of succinate dehydrogenase (SDH), vascular endothelial growth factor (VEFG), peroxisome proliferator-activated receptor gamma coactivator-1 (PGC-1α), and myostatin, were analyzed in samples from m. vastus lateralis collected before and after bed rest. Activity and gene expression of enzymes controlling oxidative metabolism (CS, SDH) decreased in BR but were partially maintained in BRE. Activity of enzymes regulating anaerobic glycolysis (GPh, PFK-1) was unchanged in BR. Resistance exercise increased the activity of GPh. PGC-1α and VEGF expression decreased in both BR and BRE. Myostatin increased in BR but decreased in BRE after bed rest. The analyses of these unique samples indicate that long-term microgravity induces marked alterations in the oxidative, but not the glycolytic, energy system. The proposed flywheel resistance exercise was effective in counteracting some of the metabolic alterations triggered by 84-day bed rest. Given the disparity between gene expression vs. enzyme activity in several key metabolic markers, posttranscriptional mechanisms should be explored to fully evaluate metabolic adaptations to long-term microgravity with/without exercise countermeasures in human skeletal muscle.

Effect of Systemic Iron Overload and a Chelation Therapy in a Mouse Model of the Neurodegenerative Disease Hereditary Ferritinopathy.

Mutations in the ferritin light chain (FTL) gene cause the neurodegenerative disease neuroferritinopathy or hereditary ferritinopathy (HF). HF is characterized by a severe movement disorder and by the presence of nuclear and cytoplasmic iron-containing ferritin inclusion bodies (IBs) in glia and neurons throughout the central nervous system (CNS) and in tissues of multiple organ systems. Herein, using primary mouse embryonic fibroblasts from a mouse model of HF, we show significant intracellular accumulation of ferritin and an increase in susceptibility to oxidative damage when cells are exposed to iron. Treatment of the cells with the iron chelator deferiprone (DFP) led to a significant improvement in cell viability and a decrease in iron content. In vivo, iron overload and DFP treatment of the mouse model had remarkable effects on systemic iron homeostasis and ferritin deposition, without significantly affecting CNS pathology. Our study highlights the role of iron in modulating ferritin aggregation in vivo in the disease HF. It also puts emphasis on the potential usefulness of a therapy based on chelators that can target the CNS to remove and redistribute iron and to resolubilize or prevent ferritin aggregation while maintaining normal systemic iron stores.

Hepatic glucose metabolism in late pregnancy: normal versus high-fat and -fructose diet.

Net hepatic glucose uptake (NHGU) is an important contributor to postprandial glycemic control. We hypothesized that NHGU is reduced during normal pregnancy and in a pregnant diet-induced model of impaired glucose intolerance/gestational diabetes mellitus (IGT/GDM). Dogs (n = 7 per group) that were nonpregnant (N), normal pregnant (P), or pregnant with IGT/GDM (pregnant dogs fed a high-fat and -fructose diet [P-HFF]) underwent a hyperinsulinemic-hyperglycemic clamp with intraportal glucose infusion. Clamp period insulin, glucagon, and glucose concentrations and hepatic glucose loads did not differ among groups. The N dogs reached near-maximal NHGU rates within 30 min; mean ± SEM NHGU was 105 ± 9 µmol·100 g liver⁻¹·min⁻¹. The P and P-HFF dogs reached maximal NHGU in 90-120 min; their NHGU was blunted (68 ± 9 and 16 ± 17 µmol·100 g liver⁻¹·min⁻¹, respectively). Hepatic glycogen synthesis was reduced 20% in P versus N and 40% in P-HFF versus P dogs. This was associated with a reduction (>70%) in glycogen synthase activity in P-HFF versus P and increased glycogen phosphorylase (GP) activity in both P (1.7-fold greater than N) and P-HFF (1.8-fold greater than P) dogs. Thus, NHGU under conditions mimicking the postprandial state is delayed and suppressed in normal pregnancy, with concomitant reduction in glycogen storage. NHGU is further blunted in IGT/GDM. This likely contributes to postprandial hyperglycemia during pregnancy, with potential adverse outcomes for the fetus and mother.

Liver glycogen loading dampens glycogen synthesis seen in response to either hyperinsulinemia or intraportal glucose infusion.

The purpose of this study was to determine the effect of liver glycogen loading on net hepatic glycogen synthesis during hyperinsulinemia or hepatic portal vein glucose infusion in vivo. Liver glycogen levels were supercompensated (SCGly) in two groups (using intraportal fructose infusion) but not in two others (Gly) during hyperglycemic-normoinsulinemia. Following a 2-h control period during which fructose infusion was stopped, there was a 2-h experimental period in which the response to hyperglycemia plus either 4× basal insulin (INS) or portal vein glucose infusion (PoG) was measured. Increased hepatic glycogen reduced the percent of glucose taken up by the liver that was deposited in glycogen (74 ± 3 vs. 53 ± 5% in Gly+INS and SCGly+INS, respectively, and 72 ± 3 vs. 50 ± 6% in Gly+PoG and SCGly+PoG, respectively). The reduction in liver glycogen synthesis in SCGly+INS was accompanied by a decrease in both insulin signaling and an increase in AMPK activation, whereas only the latter was observed in SCGly+PoG. These data indicate that liver glycogen loading impairs glycogen synthesis regardless of the signal used to stimulate it.

Portal vein glucose entry triggers a coordinated cellular response that potentiates hepatic glucose uptake and storage in normal but not high-fat/high-fructose-fed dogs.

The cellular events mediating the pleiotropic actions of portal vein glucose (PoG) delivery on hepatic glucose disposition have not been clearly defined. Likewise, the molecular defects associated with postprandial hyperglycemia and impaired hepatic glucose uptake (HGU) following consumption of a high-fat, high-fructose diet (HFFD) are unknown. Our goal was to identify hepatocellular changes elicited by hyperinsulinemia, hyperglycemia, and PoG signaling in normal chow-fed (CTR) and HFFD-fed dogs. In CTR dogs, we demonstrated that PoG infusion in the presence of hyperinsulinemia and hyperglycemia triggered an increase in the activity of hepatic glucokinase (GK) and glycogen synthase (GS), which occurred in association with further augmentation in HGU and glycogen synthesis (GSYN) in vivo. In contrast, 4 weeks of HFFD feeding markedly reduced GK protein content and impaired the activation of GS in association with diminished HGU and GSYN in vivo. Furthermore, the enzymatic changes associated with PoG sensing in chow-fed animals were abolished in HFFD-fed animals, consistent with loss of the stimulatory effects of PoG delivery. These data reveal new insight into the molecular physiology of the portal glucose signaling mechanism under normal conditions and to the pathophysiology of aberrant postprandial hepatic glucose disposition evident under a diet-induced glucose-intolerant condition.

A cyclic guanosine monophosphate-dependent pathway can regulate net hepatic glucose uptake in vivo.

We previously showed that hepatic nitric oxide regulates net hepatic glucose uptake (NHGU), an effect that can be eliminated by inhibiting hepatic soluble guanylate cyclase (sGC), suggesting that the sGC pathway is involved in the regulation of NHGU. The aim of the current study was to determine whether hepatic cyclic guanosine monophosphate (cGMP) reduces NHGU. Studies were performed on conscious dogs with transhepatic catheters. A hyperglycemic-hyperinsulinemic clamp was established in the presence of portal vein glucose infusion. 8-Br-cGMP (50 µg/kg/min) was delivered intraportally, and either the glucose load to the liver (CGMP/GLC; n = 5) or the glucose concentration entering the liver (CGMP/GCC; n = 5) was clamped at 2× basal. In the control group, saline was given intraportally (SAL; n = 10), and the hepatic glucose concentration and load were doubled. 8-Br-cGMP increased portal blood flow, necessitating the two approaches to glucose clamping in the cGMP groups. NHGU (mg/kg/min) was 5.8 ± 0.5, 2.7 ± 0.5, and 4.8 ± 0.3, whereas the fractional extraction of glucose was 11.0 ± 1, 5.5 ± 1, and 8.5 ± 1% during the last hour of the study in SAL, CGMP/GLC, and CGMP/GCC, respectively. The reduction of NHGU in response to 8-Br-cGMP was associated with increased AMP-activated protein kinase phosphorylation. These data indicate that changes in liver cGMP can regulate NHGU under postprandial conditions.

Hexokinase 2, glycogen synthase and phosphorylase play a key role in muscle glycogen supercompensation.

BACKGROUND: Glycogen-depleting exercise can lead to supercompensation of muscle glycogen stores, but the biochemical mechanisms of this phenomenon are still not completely understood. METHODS: Using chronic low-frequency stimulation (CLFS) as an exercise model, the tibialis anterior muscle of rabbits was stimulated for either 1 or 24 hours, inducing a reduction in glycogen of 90% and 50% respectively. Glycogen recovery was subsequently monitored during 24 hours of rest. RESULTS: In muscles stimulated for 1 hour, glycogen recovered basal levels during the rest period. However, in those stimulated for 24 hours, glycogen was supercompensated and its levels remained 50% higher than basal levels after 6 hours of rest, although the newly synthesized glycogen had fewer branches. This increase in glycogen correlated with an increase in hexokinase-2 expression and activity, a reduction in the glycogen phosphorylase activity ratio and an increase in the glycogen synthase activity ratio, due to dephosphorylation of site 3a, even in the presence of elevated glycogen stores. During supercompensation there was also an increase in 5'-AMP-activated protein kinase phosphorylation, correlating with a stable reduction in ATP and total purine nucleotide levels. CONCLUSIONS: Glycogen supercompensation requires a coordinated chain of events at two levels in the context of decreased cell energy balance: First, an increase in the glucose phosphorylation capacity of the muscle and secondly, control of the enzymes directly involved in the synthesis and degradation of the glycogen molecule. However, supercompensated glycogen has fewer branches.