Composition and energy harvesting capacity of the gut microbiota: relationship to diet, obesity and time in mouse models.

BACKGROUND AND AIMS: Increased efficiency of energy harvest, due to alterations in the gut microbiota (increased Firmicutes and decreased Bacteroidetes), has been implicated in obesity in mice and humans. However, a causal relationship is unproven and contributory variables include diet, genetics and age. Therefore, we explored the effect of a high-fat (HF) diet and genetically determined obesity (ob/ob) for changes in microbiota and energy harvesting capacity over time. METHODS: Seven-week-old male ob/ob mice were fed a low-fat diet and wild-type mice were fed either a low-fat diet or a HF-diet for 8 weeks (n=8/group). They were assessed at 7, 11 and 15 weeks of age for: fat and lean body mass (by NMR); faecal and caecal short-chain fatty acids (SCFA, by gas chromatography); faecal energy content (by bomb calorimetry) and microbial composition (by metagenomic pyrosequencing). RESULTS: A progressive increase in Firmicutes was confirmed in both HF-fed and ob/ob mice reaching statistical significance in the former, but this phylum was unchanged over time in the lean controls. Reductions in Bacteroidetes were also found in ob/ob mice. However, changes in the microbiota were dissociated from markers of energy harvest. Thus, although the faecal energy in the ob/ob mice was significantly decreased at 7 weeks, and caecal SCFA increased, these did not persist and faecal acetate diminished over time in both ob/ob and HF-fed mice, but not in lean controls. Furthermore, the proportion of the major phyla did not correlate with energy harvest markers. CONCLUSION: The relationship between the microbial composition and energy harvesting capacity is more complex than previously considered. While compositional changes in the faecal microbiota were confirmed, this was primarily a feature of high-fat feeding rather than genetically induced obesity. In addition, changes in the proportions of the major phyla were unrelated to markers of energy harvest which changed over time. The possibility of microbial adaptation to diet and time should be considered in future studies.

Inhibition or deletion of the lipopolysaccharide receptor Toll-like receptor-4 confers partial protection against lipid-induced insulin resistance in rodent skeletal muscle.

AIMS/HYPOTHESIS: A role for increased activity of the innate immune system in the pathogenesis of insulin resistance is supported by a number of studies. The current study assessed the potential role of the lipopolysaccharide receptor known as Toll-like receptor-4 (TLR-4), a component of the innate immune system, in mediating lipid-induced insulin resistance in skeletal muscle. METHODS: The effects of TLR-4 inhibition/deletion on lipid-induced insulin resistance was determined in skeletal muscle of TLR-4 null mice in vivo and in rat L6 myotubes in vitro. RESULTS: In mice, acute hyperlipidaemia induced skeletal muscle insulin resistance, but a deletion of TLR-4 conferred significant protection against these effects. In L6 myotubes, inhibition of TLR-4 activity substantially reduced the capacity of the saturated fatty acid palmitate to induce insulin resistance. Importantly, palmitate activated the nuclear factor kappaB (NFkappaB) pathway in L6 myotubes and mouse skeletal muscle, and these effects were blocked by inhibition of TLR-4 in L6 myotubes and absence of TLR-4 in skeletal muscle. Furthermore, inhibition of the NFkappaB pathway downstream of TLR-4 in L6 myotubes also protected against the induction of insulin resistance by palmitate. CONCLUSIONS/INTERPRETATION: Inhibition or absence of TLR-4 confers protection against the detrimental effects of lipids on skeletal muscle insulin action, and these effects are associated with a prevention of the activation of the NFkappaB pathway by lipids. Importantly, inhibition of the NFkappaB pathway in myotubes downstream of TLR-4 also protects against lipid-induced insulin resistance, suggesting a mechanism by which reduced TLR-4 activity confers beneficial effects on insulin action.

Blunted fasting-induced decreases in plasma and CSF leptin concentrations in obese rats: the role of increased leptin secretion.

Fasting substantially decreases plasma leptin concentration. However, in obesity, the fasting-induced decrease in leptin concentration is blunted or absent. The objective of the current study was to determine in diet-induced obese (DIO) rats the contribution of altered leptin secretion and leptin expression to the blunted nutritional regulation of leptin concentration. Fasting-induced decreases in plasma leptin concentration were greater in lean (standard chow (SC)) rats compared to DIO (approximately 79 vs approximately 48%, P<0.05 for DIO vs SC). CSF leptin was significantly reduced by fasting in SC (approximately 46%) but not in DIO. Fasting decreased adipose tissue leptin secretion in both SC and DIO, but the decrease was greater in SC (average approximately 74%) compared to DIO (average approximately 44%, P<0.05, DIO vs SC). Additionally, leptin secretion in fasted DIO was substantially greater (average approximately 175%) than in fasted SC (P<0.05). Adipose tissue leptin protein content was increased by approximately 80% in DIO compared to SC (P<0.05). We conclude that a probable cause of blunted fasting-induced decreases in leptin concentration in obesity is elevated leptin secretion, which possibly results from increased leptin gene expression.

A modest glucokinase overexpression in the liver promotes fed expression levels of glycolytic and lipogenic enzyme genes in the fasted state without altering SREBP-1c expression.

Hepatic genes crucial for carbohydrate and lipid homeostasis are regulated by insulin and glucose metabolism. However, the relative contributions of insulin and glucose to the regulation of metabolic gene expression are poorly defined in vivo. To address this issue, adenovirus-mediated hepatic overexpression of glucokinase was used to determine the effects of increased hepatic glucose metabolism on gene expression in fasted or ad libitum fed rats. In the fasted state, a 3 fold glucokinase overexpression was sufficient to mimic feeding-induced increases in pyruvate kinase and acetyl CoA carboxylase mRNA levels, demonstrating a primary role for glucose metabolism in the regulation of these genes in vivo. Conversely, glucokinase overexpression was unable to mimic feeding-induced alterations of fatty acid synthase, glucose-6-phosphate dehydrogenase, carnitine palmitoyl transferase I or PEPCK mRNAs, indicating insulin as the primary regulator of these genes. Interestingly, glucose-6-phosphatase mRNA was increased by glucokinase overexpression in both the fasted and fed states, providing evidence, under these conditions, for the dominance of glucose over insulin signaling for this gene in vivo. Importantly, glucokinase overexpression did not alter sterol regulatory element binding protein 1-c mRNA levels in vivo and glucose signaling did not alter the expression of this gene in primary hepatocytes. We conclude that a modest hepatic overexpression of glucokinase is sufficient to alter expression of metabolic genes without changing the expression of SREBP-1c.

Nutritional regulation of hypothalamic leptin receptor gene expression is defective in diet-induced obesity.

Leptin action in the hypothalamus plays a critical role in maintaining normal food intake and body weight. Hyperleptinaemia is associated with obesity in humans and animal models, suggesting a state of leptin resistance. Although the mechanism of leptin resistance is not clearly understood, alterations in leptin receptor (Ob-R) gene expression have been proposed as a potential mechanism mediating modifications in leptin action in obesity and during changes in nutritional status (fed/fasted). The current study examined the effects of diet-induced obesity (DIO) made by feeding rats a high fat diet for 9 weeks, and nutritional status on levels of long form (Ob-Rb) and total (Ob-Rtot) Ob-R mRNA expression in the hypothalamus. In the fed state, hypothalamic Ob-Rb mRNA and Ob-Rtot mRNA levels were similar in DIO and control standard chow fed rats (SC) despite hyperleptinaemia in DIO rats. However, although an overnight fast moderately increased hypothalamic Ob-Rb mRNA levels in SC rats, fasting did not increase Ob-Rb mRNA levels in DIO rats. To address the possibility that elevated leptin concentration in DIO rats may mediate an alteration in OB-R mRNA levels, we examined the effects of adenovirus-mediated hyperleptinaemia on Ob-R gene expression in SC rats. Despite substantially elevated plasma and cerebrospinal fluid concentrations of leptin, hypothalamic Ob-R mRNA levels were similar in both groups. In conclusion, the current study demonstrates that DIO is associated with a loss of nutritional regulation of hypothalamic Ob-R mRNA levels, and that hyperleptinaemia is not sufficient to alter Ob-R mRNA expression.

Organizing glucose disposal: emerging roles of the glycogen targeting subunits of protein phosphatase-1.

Glucose is stored in mammalian tissues in the form of glycogen. Glycogen levels are markedly reduced in liver or muscle cells of patients with insulin-resistant or insulin-deficient forms of diabetes, suggesting that impaired glycogen synthesis may contribute to development of hyperglycemia. Recently, interest in this area has been further stimulated by new insights into the spatial organization of metabolic enzymes within cells and the importance of such organization in regulation of glycogen metabolism. It is now clear that a four-member family of glycogen targeting subunits of protein phosphatase-1 (PP1) plays a major role in coordinating these events. These proteins target PP1 to the glycogen particle and also bind differentially to glycogen synthase, glycogen phosphorylase, and phosphorylase kinase, thereby serving as molecular scaffolds. Moreover, the various glycogen-targeting subunits have distinct tissue expression patterns and can influence regulation of glycogen metabolism in response to glycogenic and glycogenolytic signals. The purpose of this article is to summarize new insights into the structure, function, regulation, and metabolic effects of the glycogen-targeting subunits of PP1 and to evaluate the possibility that these molecules could serve as therapeutic targets for lowering of blood glucose in diabetes.

Correction of diet-induced hyperglycemia, hyperinsulinemia, and skeletal muscle insulin resistance by moderate hyperleptinemia.

Human obesity and high fat feeding in rats are associated with the development of insulin resistance and perturbed carbohydrate and lipid metabolism. It has been proposed that these metabolic abnormalities may be reversible by interventions that increase plasma leptin. Up to now, studies in nongenetic animal models of obesity and in human obesity have concentrated on multiple injection therapy with mixed results. Our study sought to determine whether a sustained, moderate increase in plasma leptin, achieved by administration of a recombinant adenovirus containing the leptin cDNA (AdCMV-leptin) would be effective in reversing the metabolic abnormalities of the obese phenotype. Wistar rats fed a high-fat diet (HF) were heavier (P < 0.05), had increased fat mass and intramuscular triglycerides (mTG), and had elevated plasma glucose, insulin, triglyceride, and free fatty acids compared with standard chow-fed (SC) control animals (all P < 0.01). HF rats also had impaired glucose tolerance and were markedly insulin resistant, as demonstrated by a 40% reduction in insulin-stimulated muscle glucose uptake (P < 0.001). Increasing plasma leptin levels to 29.0 +/- 1.5 ng/ml (from 7.0 +/- 1.4 ng/ml, P < 0.001) for a period of 6 days decreased adipose mass by 40% and normalized plasma glucose and insulin levels. In addition, insulin-stimulated skeletal muscle glucose uptake was normalized in hyperleptinemic rats, an effect that correlated closely with a 60% (P < 0.001) decrease in mTG. Importantly, HF rats that received a control adenovirus containing the beta-galactosidase cDNA and were calorically matched to AdCMV-leptin-treated animals remained hyperglycemic, hyperinsulinemic, insulin resistant, and maintained elevated mTG. We conclude that a gene-therapeutic intervention that elevates plasma leptin moderately for a sustained period reverses diet-induced hyperglycemia, hyperinsulinemia, and skeletal muscle insulin resistance, and that these improvements are tightly linked to leptin-induced reductions in mTG.

Activation of direct and indirect pathways of glycogen synthesis by hepatic overexpression of protein targeting to glycogen.

Glycogen-targeting subunits of protein phosphatase-1, such as protein targeting to glycogen (PTG), direct the phosphatase to the glycogen particle, where it stimulates glycogenesis. We have investigated the metabolic impact of overexpressing PTG in liver of normal rats. After administration of PTG cDNA in a recombinant adenovirus, animals were fasted or allowed to continue feeding for 24 hours. Liver glycogen was nearly completely depleted in fasted control animals, whereas glycogen levels in fasted or fed PTG-overexpressing animals were 70% higher than in fed controls. Nevertheless, transgenic animals regulated plasma glucose, triglycerides, FFAs, ketones, and insulin normally in the fasted and fed states. Fasted PTG-overexpressing animals receiving an oral bolus of [U-(13)C]glucose exhibited a large increase in hepatic glycogen content and a 70% increase in incorporation of [(13)C]glucose into glycogen. However, incorporation of labeled glucose accounted for only a small portion of the glycogen synthesized in PTG-overexpressing animals, consistent with our earlier finding that PTG promotes glycogen synthesis from gluconeogenic precursors. We conclude that hepatic PTG overexpression activates both direct and indirect pathways of glycogen synthesis. Because of its ability to enhance glucose storage without affecting other metabolic indicators, the glycogen-targeting subunit may prove valuable in controlling blood glucose levels in diabetes.

Role of Ca(2+) fluctuations in L6 myotubes in the regulation of the hexokinase II gene.

Expression of the hexokinase (HK) II gene in skeletal muscle is upregulated by electrically stimulated muscle contraction and moderate-intensity exercise. However, the molecular mechanism by which this occurs is unknown. Alterations in intracellular Ca(2+) homeostasis accompany contraction and regulate gene expression in contracting skeletal muscle. Therefore, as a first step in understanding the exercise-induced increase in HK II, the ability of Ca(2+) to increase HK II mRNA was investigated in cultured skeletal muscle cells, namely L6 myotubes. Exposure of cells to the ionophore A-23187 resulted in an approximately threefold increase in HK II mRNA. Treatment of cells with the extracellular Ca(2+) chelator EGTA did not alter HK II mRNA, nor was it able to prevent the A-23187-induced increase. Treatment of cells with the intracellular Ca(2+) chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM) also resulted in an approximately threefold increase in HK II mRNA in the absence of ionophore, which was similar to the increase in HK II mRNA induced by the combination of BAPTA-AM and A-23187. In summary, a rise in intracellular Ca(2+) is not necessary for the A-23187-induced increase in HK II mRNA, and increases in HK II mRNA occur in response to treatments that decrease intracellular Ca(2+) stores. Depletion of intracellular Ca(2+) stores may be one mechanism by which muscle contraction increases HK II mRNA.

Metabolic impact of glucokinase overexpression in liver: lowering of blood glucose in fed rats is accompanied by hyperlipidemia.

The balance between hepatic glucose uptake and production is perturbed in both major forms of diabetes. It has been suggested that pharmacologic or genetic methods for enhancing glucokinase (GK) enzymatic activity in liver might be a means of increasing glucose disposal and lowering blood glucose in diabetic patients. To better evaluate this possibility, we used a recombinant adenovirus containing the cDNA encoding GK (AdCMV-GKL) to achieve overexpression of the enzyme at different levels in liver of normal rats. In a first set of experiments, in rats fasted for 18 h, AdCMV-GKL infusion caused a 211% increase in hepatic GK activity relative to animals infused with a control virus (AdCMV-betaGAL). AdCMV-GKL-treated fasted rats exhibited no significant changes in circulating glucose, free fatty acids (FFAs), lactate, beta-hydroxybutyrate, or insulin levels relative to controls, whereas triglyceride (TG) levels were slightly increased (53%). In a second set of studies, in rats fed ad libitum, GK was overexpressed in liver by 3- and 6.4-fold. Animals with the lower degree of GK overexpression exhibited no significant changes in circulating glucose, FFAs, insulin, TG, or lactate levels relative to controls that received a virus encoding a catalytically inactive mutant GK (AdCMV-GK203), but did show a modest increase in lactate (58%) relative to AdCMV-betaGAL-infused controls. In contrast, the higher level of GK overexpression caused a 38% decrease in blood glucose levels and a 67% decrease in circulating insulin levels relative to AdCMV-GK203-infused animals. The decline in glucose levels was accompanied by a 190% increase in circulating TG and a 310% increase in circulating FFAs; total plasma cholesterol was unaffected. Finally, fasted animals treated with AdCMV-GKL had 5.4 times as much liver glycogen as AdCMV-betaGAL-treated controls; no significant increases in liver glycogen were observed at either level of GK overexpression in ad libitum-fed rats relative to fed controls. In sum, levels of hepatic GK overexpression associated with a decline in blood glucose are accompanied by equally dramatic increases in FFAs and TG, raising concerns about manipulation of liver GK activity as a viable strategy for treatment of diabetes.

Sparing effect of leptin on liver glycogen stores in rats during the fed-to-fasted transition.

The effect of moderate hyperleptinemia ( approximately 20 ng/ml) on liver and skeletal muscle glycogen metabolism was examined in Wistar rats. Animals were studied approximately 90 h after receiving recombinant adenoviruses encoding rat leptin (AdCMV-leptin) or beta-galactosidase (AdCMV-betaGal). Liver and skeletal muscle glycogen levels in the fed and fasted (18 h) states were similar in AdCMV-leptin- and AdCMV-betaGal-treated rats. However, after delivery of a glucose bolus, liver glycogen levels were significantly greater in AdCMV-leptin compared with AdCMV-betaGal rats (P < 0.05). To investigate the mechanism(s) of these differences, glycogen levels were measured immediately after the cessation of a 3- or 6-h glucose infusion or 3, 6, and 9 h after the cessation of a 6-h glucose infusion. Similar increases in liver and skeletal muscle glycogen occurred in hyperleptinemic and control rats in response to glucose infusions. However, 3 and 6 h after the cessation of a glucose infusion, liver glycogen levels were approximately twofold greater (P < 0.05) in AdCMV-leptin-treated compared with AdCMV-betaGal-treated animals. Skeletal muscle glycogen levels were similar in AdCMV-leptin-treated and AdCMV-betaGal-treated animals at the same time points. Glycogen phosphorylase, phosphodiesterase 3B, and glycogen synthase activities were unaltered by hyperleptinemia. We conclude that moderate increases in plasma leptin levels decrease liver glycogen degradation during the fed-to-fasted transition.

Metabolic engineering with recombinant adenoviruses.

Fuel homeostasis in mammals is accomplished by the interplay between tissues and organs with distinct metabolic roles. These regulatory mechanisms are disrupted in obesity and diabetes, leading to a renewed emphasis on discovery of molecular and pharmacologic methods for reversing metabolic disorders. In this chapter, we review the use of recombinant adenoviral vectors as tools for delivering metabolic regulatory genes to cells in culture and to tissues of intact animals. Included are studies on the use of these vectors for gaining insights into the biochemical mechanisms that regulate glucose-stimulated insulin secretion from pancreatic islet beta-cells. We also highlight their use for understanding the function of newly discovered genes that regulate glycogen metabolism in liver and other tissues, and for evaluating "candidate" genes such as glucose-6-phosphatase, which may contribute to development of metabolic dysfunction in pancreatic islets and liver. Finally, we discuss the use of adenoviral and related vectors for causing chronic increases in the levels of circulating hormones. These examples serve to highlight the power of viral gene transfer vectors as tools for understanding metabolic regulatory mechanisms.

Perturbation of fuel homeostasis caused by overexpression of the glucose-6-phosphatase catalytic subunit in liver of normal rats.

The terminal step in hepatic gluconeogenesis is catalyzed by glucose-6-phosphatase, an enzyme activity residing in the endoplasmic reticulum and consisting of a catalytic subunit (glucose-6-phosphatase (G6Pase)) and putative accessory transport proteins. We show that Zucker diabetic fatty rats (fa/fa), which are known to exhibit impaired suppression of hepatic glucose output, have 2.4-fold more glucose-6-phosphatase activity in liver than lean controls. To define the potential contribution of increased hepatic G6Pase to development of diabetes, we infused recombinant adenoviruses containing the G6Pase cDNA (AdCMV-G6Pase) or the beta-galactosidase gene into normal rats. Animals were studied by one of three protocols as follows: protocol 1, fed ad libitum for 7 days; protocol 2, fed ad libitum for 5 days, fasted overnight, and subjected to an oral glucose tolerance test; protocol 3, fed ad libitum for 4 days, fasted for 48 h, subjected to oral glucose tolerance test, and then allowed to refeed overnight. Hepatic glucose-6-phosphatase enzymatic activity was increased by 1.6-3-fold in microsomes isolated from AdCMV-G6Pase-treated animals in all three protocols, and the resultant metabolic profile was similar in each case. AdCMV-G6Pase-treated animals exhibited several of the abnormalities associated with early stage non-insulin-dependent diabetes mellitus, including glucose intolerance, hyperinsulinemia, decreased hepatic glycogen content, and increased peripheral (muscle) triglyceride stores. These animals also exhibited significant decreases in circulating free fatty acids and triglycerides, changes not normally associated with the disease. Our studies show that overexpression of G6Pase in liver is sufficient to perturb whole animal glucose and lipid homeostasis, possibly contributing to the development of metabolic abnormalities associated with diabetes.

Overexpression of protein targeting to glycogen (PTG) in rat hepatocytes causes profound activation of glycogen synthesis independent of normal hormone- and substrate-mediated regulatory mechanisms.

Protein targeting to glycogen (PTG), also known as PPP1R5, is a widely expressed member of a growing family of proteins that target protein phosphatase-1 (PP-1) to glycogen particles. Because PTG also binds to glycogen synthase and phosphorylase kinase, it has been suggested that it serves as a "scaffold" for efficient activation of glycogen synthesis. However, very little is known about the metabolic effects of PTG. In this study, we have used recombinant adenovirus to overexpress PTG in primary rat hepatocytes, a cell type with high glycogenic capacity. We find that overexpression of PTG potently activates glycogen synthesis in cultured hepatocytes. Surprisingly, the glycogenic effect of PTG is observed even in the complete absence of carbohydrates or insulin in the culture medium. Furthermore, glycogenolytic agents such as forskolin or glucagon are largely ineffective at activating glycogen degradation in PTG overexpressing hepatocytes, even though large increases in cAMP levels are demonstrated. These metabolic effects of PTG overexpression are accompanied by a 3.6-fold increase in glycogen synthase activation state and a 40% decrease in glycogen phosphorylase activity. Our results are consistent with a model in which PTG overexpression "locks" the hepatocyte in a glycogenic mode, presumably via its ability to promote interaction of enzymes of glycogen metabolism with PP-1.

The repression of hormone-activated PEPCK gene expression by glucose is insulin-independent but requires glucose metabolism.

Phosphoenolpyruvate carboxykinase (PEPCK) is a rate-controlling enzyme in hepatic gluconeogenesis, and it therefore plays a central role in glucose homeostasis. The rate of transcription of the PEPCK gene is increased by glucagon (via cAMP) and glucocorticoids and is inhibited by insulin. Under certain circumstances glucose also decreases PEPCK gene expression, but the mechanism of this effect is poorly understood. The glucose-mediated stimulation of a number of glycolytic and lipogenic genes requires the expression of glucokinase (GK) and increased glucose metabolism. HL1C rat hepatoma cells are a stably transfected line of H4IIE rat hepatoma cells that express a PEPCK promoter-chloramphenicol acetyltransferase fusion gene that is regulated in the same manner as the endogenous PEPCK gene. These cells do not express GK and do not normally exhibit a response of either the endogenous PEPCK gene, or of the trans-gene, to glucose. A recombinant adenovirus that directs the expression of glucokinase (AdCMV-GK) was used to increase glucose metabolism in HL1C cells to test whether increased glucose flux is also required for the repression of PEPCK gene expression. In AdCMV-GK-treated cells glucose strongly inhibits hormone-activated transcription of the endogenous PEPCK gene and of the expressed fusion gene. The glucose effect on PEPCK gene promoter activity is blocked by 5 mM mannoheptulose, a specific inhibitor of GK activity. The glucose analog, 2-deoxyglucose mimics the glucose response, but this effect does not require GK expression. 3-O-methylglucose is ineffective. Glucose exerts its effect on the PEPCK gene within 4 h, at physiologic concentrations, and with an EC50 of 6.5 mM, which approximates the Km of glucokinase. The effects of glucose and insulin on PEPCK gene expression are additive, but only at suboptimal concentrations of both agents. The results of these studies demonstrate that, by inhibiting PEPCK gene transcription, glucose participates in a feedback control loop that governs its production from gluconeogenesis.

Analysis of insulin-stimulated skeletal muscle glucose uptake in conscious rat using isotopic glucose analogs.

An isotopic method was used in conscious rats to determine the roles of glucose transport and the transsarcolemmal glucose gradient (TSGG) in control of basal and insulin-stimulated muscle glucose uptake. Rats received an intravenous 3-O-[3H]methylglucose (3-O-[3H]MG) infusion from -100 to 40 min and a 2-deoxy-[3H]glucose infusion from 0 to 40 min to calculate a glucose metabolic index (Rg). Insulin was infused from -100 to 40 min at rates of 0.0, 0.6, 1.0, and 4.0 mU.kg-1.min-1, and glucose was clamped at basal concentrations. The ratios of soleus intracellular to extracellular 3-O-[3H]MG concentration and soleus glucose concentrations were used to estimate the TSGG using principles of glucose counter-transport. Tissue glucose concentrations were compared in well-perfused, slow-twitch muscle (soleus) and poorly perfused, fast-twitch muscle (vastus lateralis, gastrocnemius). Data show that 1) small increases in insulin increase soleus Rg without decreasing TSGG, suggesting that muscle glucose delivery and phosphorylation can accommodate the increased flux; 2) due to a limitation in soleus glucose phosphorylation and possibly delivery, insulin at high physiological levels decreases TSGG, and at supraphysiological insulin levels the TSGG is not significantly different from 0; 3) maximum Rg is maintained even though TSGG decreases with increasing insulin levels, indicating that glucose transport continues to increase and is not rate limiting for maximal insulin-stimulated glucose uptake; and 4) muscle consisting of fast-twitch fibers that are poorly perfused exhibits a 35-45% fall in tissue glucose with insulin, suggesting that glucose delivery is a major limitation in sustaining the TSGG. In conclusion, control of glucose uptake is distributed between glucose transport and factors that determine the TSGG. Insulin stimulation of glucose transport increases the demands on the factors that maintain glucose delivery to the muscle membrane and glucose phosphorylation inside the muscle.

Regulation of hexokinase II activity and expression in human muscle by moderate exercise.

A single bout of exercise increases the rate of insulin-stimulated glucose uptake and metabolism in skeletal muscle. Exercise also increases insulin-stimulated glucose 6-phosphate in skeletal muscle, suggesting that exercise increases hexokinase activity. Within 3 h, exercise increases hexokinase II (HK II) mRNA and activity in skeletal muscle from rats. It is not known, however, if a single bout of moderate-intensity exercise increases HK II expression in humans. The present study was undertaken to answer this question. Six subjects had percutaneous biopsies of the vastus lateralis muscle before and 3 h after a single 3-h session of moderate-intensity aerobic (60% of maximal oxygen consumption) exercise. Glycogen synthase, HK I, and HK II activities as well as HK I and HK II mRNA content were determined from the muscle biopsy specimens. The fractional velocity of glycogen synthase was increased by 446 +/- 84% after exercise (P < 0.005). Hexokinase II activity in the soluble fraction of the homogenates increased from 1.2 +/- 0.4 to 4.5 +/- 1.6 pmol.min-1.microgram-1 (P < 0.05) but was unchanged in the particulate fraction (4.3 +/- 1.3 vs. 5.3 +/- 1.5). HK I activity in neither the soluble nor particulate fraction changed after exercise. Relative to a 28S rRNA control signal, HK II mRNA increased from 0.091 +/- 0.02 to 0.195 +/- 0.037 (P < 0.05), whereas HK I mRNA was unchanged (0.414 +/- 0.061 vs. 0.498 +/- 0.134, P < 0.20). The increase in HK II activity after moderate exercise in healthy subjects could be one factor responsible for the enhanced rate of insulin-stimulated glucose uptake seen after exercise.

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 expression of the catalytic subunit of glucose-6-phosphatase in INS-1 cells. Effects on glucose cycling, glucose usage, and insulin secretion.

Glucose-6-phosphatase (Glu-6-Pase) catalyzes the terminal step of gluconeogenesis, the conversion of glucose 6-phosphate (Glu-6-P) to free glucose. This enzyme activity is thought to be conferred by a complex of proteins residing in the endoplasmic reticulum (ER), including a Glu-6-P translocase that transports Glu-6-P into the lumen of the ER, a phosphohydrolase catalytic subunit residing in the lumen, and putative glucose and inorganic phosphate transporters that allow exit of the products of the reaction. In this study, we have investigated the effect of adenovirus-mediated overexpression of the Glu-6-Pase catalytic subunit on glucose metabolism and insulin secretion, using a well differentiated insulinoma cell line, INS-1. We found that the overexpressed Glu-6-Pase catalytic subunit was normally glycosylated, correctly sorted to the ER, and caused a 10-fold increase in Glu-6-Pase enzymatic activity in in vitro assays. Consistent with these findings, a 4.2-fold increase in 3H2O incorporation into glucose was observed in INS-1 cells treated with the recombinant adenovirus containing the Glu-6-Pase catalytic subunit cDNA (AdCMV-Glu-6-Pase). 3-[3H]Glucose usage was decreased by 32% in AdCMV-Glu-6-Pase-treated cells relative to controls, resulting in a proportional 30% decrease in glucose-stimulated insulin secretion. Our findings indicate that overexpression of the Glu-6-Pase catalytic subunit significantly impacts glucose metabolism and insulin secretion in islet beta-cells. However, INS-1 cells treated with AdCMV-Glu-6-Pase do not exhibit the severe alterations of beta-cell function and metabolism associated with islets from rodent models of obesity and non-insulin-dependent diabetes mellitus, suggesting the involvement of genes in addition to the catalytic subunit of Glu-6-Pase in the etiology of such beta-cell dysfunction.