GCN2 is required to increase fibroblast growth factor 21 and maintain hepatic triglyceride homeostasis during asparaginase treatment.

The antileukemic agent asparaginase triggers the amino acid response (AAR) in the liver by activating the eukaryotic initiation factor 2 (eIF2) kinase general control nonderepressible 2 (GCN2). To explore the mechanism by which AAR induction is necessary to mitigate hepatic lipid accumulation and prevent liver dysfunction during continued asparaginase treatment, wild-type and Gcn2 null mice were injected once daily with asparaginase or phosphate buffered saline for up to 14 days. Asparaginase induced mRNA expression of multiple AAR genes and greatly increased circulating concentrations of the metabolic hormone fibroblast growth factor 21 (FGF21) independent of food intake. Loss of Gcn2 precluded mRNA expression and circulating levels of FGF21 and blocked mRNA expression of multiple genes regulating lipid synthesis and metabolism including Fas, Ppara, Pparg, Acadm, and Scd1 in both liver and white adipose tissue. Furthermore, rates of triglyceride export and protein expression of apolipoproteinB-100 were significantly reduced in the livers of Gcn2 null mice treated with asparaginase, providing a mechanistic basis for the increase in hepatic lipid content. Loss of AAR-regulated antioxidant defenses in Gcn2 null livers was signified by reduced Gpx1 gene expression alongside increased lipid peroxidation. Substantial reductions in antithrombin III hepatic expression and activity in the blood of asparaginase-treated Gcn2 null mice indicated liver dysfunction. These results suggest that the ability of the liver to adapt to prolonged asparaginase treatment is influenced by GCN2-directed regulation of FGF21 and oxidative defenses, which, when lost, corresponds with maladaptive effects on lipid metabolism and hemostasis.

General control nonderepressible 2 (GCN2) kinase protects oligodendrocytes and white matter during branched-chain amino acid deficiency in mice.

Branched-chain amino acid (BCAA) catabolism is regulated by branched-chain α-keto acid dehydrogenase, an enzyme complex that is inhibited when phosphorylated by its kinase (BDK). Loss of BDK function in mice and humans causes BCAA deficiency and epilepsy with autistic features. In response to amino acid deficiency, phosphorylation of eukaryotic initiation factor 2α (eIF2∼P) by general control nonderepressible 2 (GCN2) activates the amino acid stress response. We hypothesized that GCN2 functions to protect the brain during chronic BCAA deficiency. To test this idea, we generated mice lacking both Gcn2 and Bdk (GBDK) and examined the development of progeny. GBDK mice appeared normal at birth, but they soon stopped growing, developed severe ataxia, tremor, and anorexia, and died by postnatal day 15. BCAA levels in brain were diminished in both Bdk(-/-) and GBDK pups. Brains from Bdk(-/-) pups exhibited robust eIF2∼P and amino acid stress response induction, whereas these responses were absent in GBDK mouse brains. Instead, myelin deficiency and diminished expression of myelin basic protein were noted in GBDK brains. Genetic markers of oligodendrocytes and astrocytes were also reduced in GBDK brains in association with apoptotic cell death in white matter regions of the brain. GBDK brains further demonstrated reduced Sod2 and Cat mRNA and increased Tnfα mRNA expression. The data are consistent with the idea that loss of GCN2 during BCAA deficiency compromises glial cell defenses to oxidative and inflammatory stress. We conclude that GCN2 protects the brain from developing a lethal leukodystrophy in response to amino acid deficiencies.

Leucine and protein metabolism in obese Zucker rats.

Branched-chain amino acids (BCAAs) are circulating nutrient signals for protein accretion, however, they increase in obesity and elevations appear to be prognostic of diabetes. To understand the mechanisms whereby obesity affects BCAAs and protein metabolism, we employed metabolomics and measured rates of [1-(14)C]-leucine metabolism, tissue-specific protein synthesis and branched-chain keto-acid (BCKA) dehydrogenase complex (BCKDC) activities. Male obese Zucker rats (11-weeks old) had increased body weight (BW, 53%), liver (107%) and fat (∼300%), but lower plantaris and gastrocnemius masses (-21-24%). Plasma BCAAs and BCKAs were elevated 45-69% and ∼100%, respectively, in obese rats. Processes facilitating these rises appeared to include increased dietary intake (23%), leucine (Leu) turnover and proteolysis [35% per g fat free mass (FFM), urinary markers of proteolysis: 3-methylhistidine (183%) and 4-hydroxyproline (766%)] and decreased BCKDC per g kidney, heart, gastrocnemius and liver (-47-66%). A process disposing of circulating BCAAs, protein synthesis, was increased 23-29% by obesity in whole-body (FFM corrected), gastrocnemius and liver. Despite the observed decreases in BCKDC activities per gm tissue, rates of whole-body Leu oxidation in obese rats were 22% and 59% higher normalized to BW and FFM, respectively. Consistently, urinary concentrations of eight BCAA catabolism-derived acylcarnitines were also elevated. The unexpected increase in BCAA oxidation may be due to a substrate effect in liver. Supporting this idea, BCKAs were elevated more in liver (193-418%) than plasma or muscle, and per g losses of hepatic BCKDC activities were completely offset by increased liver mass, in contrast to other tissues. In summary, our results indicate that plasma BCKAs may represent a more sensitive metabolic signature for obesity than BCAAs. Processes supporting elevated BCAA]BCKAs in the obese Zucker rat include increased dietary intake, Leu and protein turnover along with impaired BCKDC activity. Elevated BCAAs/BCKAs may contribute to observed elevations in protein synthesis and BCAA oxidation.

Molecular characterization of skeletal muscle atrophy in the R6/2 mouse model of Huntington's disease.

Huntington's disease (HD), a neurodegenerative disorder caused by mutant huntingtin, is characterized by a catabolic phenotype. To determine the mechanisms underlying muscle wasting, we examined key signal transduction pathways governing muscle protein metabolism, apoptosis, and autophagy in R6/2 mice, a well-characterized transgenic model of HD. R6/2 mice exhibited increased adiposity, elevated energy expenditure, and decreased body weight and lean mass without altered food intake. Severe skeletal muscle wasting accounted for a majority of the weight loss. Protein synthesis was unexpectedly increased 19% in gastrocnemius muscle, which was associated with overactivation of basal and refeeding-stimulated mammalian target of rapamycin (mTOR) signaling, elevated Akt expression and Ser(473) phosphorylation, and decreased AMPK Thr(172) phosphorylation. Moreover, mRNA abundance of atrogenes muscle ring finger-1 and atrophy F-box, was markedly attenuated during fasting and refeeding, and the urinary excretion of 3-methylhistidine was decreased, arguing against a role for the ubiquitin proteasome-mediated proteolysis in the atrophy. In contrast, mRNA expression of several caspase genes and genes involved in the extrinsic or intrinsic apoptotic pathway, caspase-3/7, -8, and -9 activity, protein abundance of caspase-3 and -9, Fas, and Fadd, and cytochrome c release were elevated. Protein expressions of LC3B-I and -II, beclin-I, and atg5 and -7 in muscle were upregulated. Thus, mutant huntingtin in skeletal muscle results in increased protein synthesis and mTOR signaling, which is countered by activation of the apoptotic and autophagic pathways, contributing to an overall catabolic phenotype and the severe muscle wasting.

Transamination is required for {alpha}-ketoisocaproate but not leucine to stimulate insulin secretion.

It remains unclear how α-ketoisocaproate (KIC) and leucine are metabolized to stimulate insulin secretion. Mitochondrial BCATm (branched-chain aminotransferase) catalyzes reversible transamination of leucine and α-ketoglutarate to KIC and glutamate, the first step of leucine catabolism. We investigated the biochemical mechanisms of KIC and leucine-stimulated insulin secretion (KICSIS and LSIS, respectively) using BCATm(-/-) mice. In static incubation, BCATm disruption abolished insulin secretion by KIC, D,L-α-keto-ß-methylvalerate, and α-ketocaproate without altering stimulation by glucose, leucine, or α-ketoglutarate. Similarly, during pancreas perfusions in BCATm(-/-) mice, glucose and arginine stimulated insulin release, whereas KICSIS was largely abolished. During islet perifusions, KIC and 2 mM glutamine caused robust dose-dependent insulin secretion in BCATm(+/+) not BCATm(-/-) islets, whereas LSIS was unaffected. Consistently, in contrast to BCATm(+/+) islets, the increases of the ATP concentration and NADPH/NADP(+) ratio in response to KIC were largely blunted in BCATm(-/-) islets. Compared with nontreated islets, the combination of KIC/glutamine (10/2 mM) did not influence α-ketoglutarate concentrations but caused 120 and 33% increases in malate in BCATm(+/+) and BCATm(-/-) islets, respectively. Although leucine oxidation and KIC transamination were blocked in BCATm(-/-) islets, KIC oxidation was unaltered. These data indicate that KICSIS requires transamination of KIC and glutamate to leucine and α-ketoglutarate, respectively. LSIS does not require leucine catabolism and may be through leucine activation of glutamate dehydrogenase. Thus, KICSIS and LSIS occur by enhancing the metabolism of glutamine/glutamate to α-ketoglutarate, which, in turn, is metabolized to produce the intracellular signals such as ATP and NADPH for insulin secretion.

Disruption of BCAA metabolism in mice impairs exercise metabolism and endurance.

Exercise enhances branched-chain amino acid (BCAA) catabolism, and BCAA supplementation influences exercise metabolism. However, it remains controversial whether BCAA supplementation improves exercise endurance, and unknown whether the exercise endurance effect of BCAA supplementation requires catabolism of these amino acids. Therefore, we examined exercise capacity and intermediary metabolism in skeletal muscle of knockout (KO) mice of mitochondrial branched-chain aminotransferase (BCATm), which catalyzes the first step of BCAA catabolism. We found that BCATm KO mice were exercise intolerant with markedly decreased endurance to exhaustion. Their plasma lactate and lactate-to-pyruvate ratio in skeletal muscle during exercise and lactate release from hindlimb perfused with high concentrations of insulin and glucose were significantly higher in KO than wild-type (WT) mice. Plasma and muscle ammonia concentrations were also markedly higher in KO than WT mice during a brief bout of exercise. BCATm KO mice exhibited 43-79% declines in the muscle concentration of alanine, glutamine, aspartate, and glutamate at rest and during exercise. In response to exercise, the increments in muscle malate and alpha-ketoglutarate were greater in KO than WT mice. While muscle ATP concentration tended to be lower, muscle IMP concentration was sevenfold higher in KO compared with WT mice after a brief bout of exercise, suggesting elevated ammonia in KO is derived from the purine nucleotide cycle. These data suggest that disruption of BCAA transamination causes impaired malate/aspartate shuttle, thereby resulting in decreased alanine and glutamine formation, as well as increases in lactate-to-pyruvate ratio and ammonia in skeletal muscle. Thus BCAA metabolism may regulate exercise capacity in mice.

Adipose tissue branched chain amino acid (BCAA) metabolism modulates circulating BCAA levels.

Whereas the role of adipose tissue in glucose and lipid homeostasis is widely recognized, its role in systemic protein and amino acid metabolism is less well-appreciated. In vitro and ex vivo experiments suggest that adipose tissue can metabolize substantial amounts of branched chain amino acids (BCAAs). However, the role of adipose tissue in regulating BCAA metabolism in vivo is controversial. Interest in the contribution of adipose tissue to BCAA metabolism has been renewed with recent observations demonstrating down-regulation of BCAA oxidation enzymes in adipose tissue in obese and insulin-resistant humans. Using gene set enrichment analysis, we observe alterations in adipose-tissue BCAA enzyme expression caused by adipose-selective genetic alterations in the GLUT4 glucose-transporter expression. We show that the rate of adipose tissue BCAA oxidation per mg of tissue from normal mice is higher than in skeletal muscle. In mice overexpressing GLUT4 specifically in adipose tissue, we observe coordinate down-regulation of BCAA metabolizing enzymes selectively in adipose tissue. This decreases BCAA oxidation rates in adipose tissue, but not in muscle, in association with increased circulating BCAA levels. To confirm the capacity of adipose tissue to modulate circulating BCAA levels in vivo, we demonstrate that transplantation of normal adipose tissue into mice that are globally defective in peripheral BCAA metabolism reduces circulating BCAA levels by 30% (fasting)-50% (fed state). These results demonstrate for the first time the capacity of adipose tissue to catabolize circulating BCAAs in vivo and that coordinate regulation of adipose-tissue BCAA enzymes may modulate circulating BCAA levels.

Protein phosphatase 2Cm is a critical regulator of branched-chain amino acid catabolism in mice and cultured cells.

The branched-chain amino acids (BCAA) are essential amino acids required for protein homeostasis, energy balance, and nutrient signaling. In individuals with deficiencies in BCAA, these amino acids can be preserved through inhibition of the branched-chain-alpha-ketoacid dehydrogenase (BCKD) complex, the rate-limiting step in their metabolism. BCKD is inhibited by phosphorylation of its E1alpha subunit at Ser293, which is catalyzed by BCKD kinase. During BCAA excess, phosphorylated Ser293 (pSer293) becomes dephosphorylated through the concerted inhibition of BCKD kinase and the activity of an unknown intramitochondrial phosphatase. Using unbiased, proteomic approaches, we have found that a mitochondrial-targeted phosphatase, PP2Cm, specifically binds the BCKD complex and induces dephosphorylation of Ser293 in the presence of BCKD substrates. Loss of PP2Cm completely abolished substrate-induced E1alpha dephosphorylation both in vitro and in vivo. PP2Cm-deficient mice exhibited BCAA catabolic defects and a metabolic phenotype similar to the intermittent or intermediate types of human maple syrup urine disease (MSUD), a hereditary disorder caused by defects in BCKD activity. These results indicate that PP2Cm is the endogenous BCKD phosphatase required for nutrient-mediated regulation of BCKD activity and suggest that defects in PP2Cm may be responsible for a subset of human MSUD.

Leucine supplementation of drinking water does not alter susceptibility to diet-induced obesity in mice.

Branched-chain amino acids (BCAA), Leu, and the signaling pathways they regulate have been reported to either improve or worsen adiposity and insulin sensitivity. Therefore, it is unclear whether dietary supplementation of Leu would be beneficial. To help address this question, we examined the effect of adding Leu (150 mmol/L; Expt. 1 and Expt. 2) or BCAA (109 mmol/L of each; Expt. 3) to the drinking water on diet-induced obesity (induced with a 60-kJ% fat diet) in singly housed C57BL6/J male mice for at least 14 wk. Liquid and solid food intakes were evaluated weekly along with body weight. During the last few weeks, several blood samples were taken at different times for plasma glucose, total cholesterol, or Leu measurements. Metabolic rate by indirect calorimetry, locomotor activity by light beam breaking, body composition by H1-NMR, and insulin tolerance were also determined. Compared with control, supplementation did not affect body weight, food intake, oxygen consumption, locomotor activity, body composition, insulin tolerance, or total cholesterol. In fed mice, this method of Leu supplementation only increased plasma Leu by 76% when the supplemented group was compared with control. On the other hand, after overnight food deprivation, the plasma Leu did not differ between these 2 groups, even though the mice in the supplemented group had continuous access to Leu-containing water during the solid food deprivation. Taken together, the results do not provide evidence that either Leu or BCAA supplementation of drinking water ameliorates diet-induced obesity in mice, although it may improve glycemia.

Skeletal and cardiac myopathy in HIV-1 transgenic rats.

The mechanism by which human immunodeficiency virus (HIV)-1 infection in humans leads to the erosion of lean body mass is poorly defined. Therefore, the purpose of the present study was to determine whether transgenic (Tg) rats that constitutively overexpress HIV-1 viral proteins exhibit muscle wasting and to elucidate putative mechanisms. Over 7 mo, Tg rats gained less body weight than pair-fed controls exclusively as a result of a proportional reduction in lean, not fat, mass. Fast- and slow-twitch muscle atrophy in Tg rats did not result from a reduction in the in vivo-determined rate of protein synthesis. In contrast, urinary excretion of 3-methylhistidine, as well as the content of atrogin-1 and the 14-kDa actin fragment, was elevated in gastrocnemius of Tg rats, suggesting increased muscle proteolysis. Similarly, Tg rats had reduced cardiac mass, which was independent of a change in protein synthesis. This decreased cardiac mass was associated with a reduction in stroke volume, but cardiac output was maintained by a compensatory increase in heart rate. The HIV-induced muscle atrophy was associated with increased whole body energy expenditure, which was not due to an elevated body temperature or secondary bacterial infection. Furthermore, the atrophic response could not be attributed to the development of insulin resistance, decreased levels of circulating amino acids, or increased tissue cytokines. However, skeletal muscle and, to a lesser extent, circulating insulin-like growth factor I was reduced in Tg rats. Although hepatic injury was implicated by increased plasma levels of aspartate and alanine aminotransferases, hepatic protein synthesis was not different between control and Tg rats. Hence, HIV-1 Tg rats develop atrophy of cardiac and skeletal muscle, the latter of which results primarily from an increased protein degradation and may be related to the marked reduction in muscle insulin-like growth factor I.

Non-invasive detection of fatty liver in dairy cows by digital analyses of hepatic ultrasonograms.

During early lactation, many dairy cows develop fatty liver, which is associated with decreased health and reproductive performance. Currently, fatty liver can be detected reliably only by using liver biopsy followed by chemical or histological analysis, which is not practical in most on-farm situations. We tested whether digital analyses of hepatic ultrasonograms can be used to detect non-invasively fatty liver and estimate liver triacylglycerol content. A total of 49 liver biopsies and ultrasonograms were taken from 29 dairy cows within 2 weeks postpartum. The usefulness of 17 first- or second-order parameters from digital analysis of B-mode ultrasonograms were evaluated by discriminant, correlation, and regression analyses. A group of linear combinations of the 17 parameters correctly classified 40 of 49 samples into normal liver as well as mild, moderate and severe fatty liver when cut-off values were 1%, 5% and 10% and correctly classified 45 of 49 samples when cut-off values were 5% and 10% triacylglycerol of wet weight. A linear combination of 16 image parameters estimated triacylglycerol concentrations of 38 of the 39 liver samples below the cut-off value of 10% within 2.5% of liver wet weight, and a linear combination of 3 parameters estimated triacylglycerol concentrations of the 10 liver samples above the cut-off value of 10% within 2% of liver wet weight. Therefore, ultrasound imaging followed by digital analysis of sonograms has potential to non-invasively detect fatty liver and estimate liver triacylglycerol content.

Obesity-related elevations in plasma leucine are associated with alterations in enzymes involved in branched-chain amino acid metabolism.

Elevations in branched-chain amino acids (BCAAs) in human obesity were first reported in the 1960s. Such reports are of interest because of the emerging role of BCAAs as potential regulators of satiety, leptin, glucose, cell signaling, adiposity, and body weight (mTOR and PKC). To explore loss of catabolic capacity as a potential contributor to the obesity-related rises in BCAAs, we assessed the first two enzymatic steps, catalyzed by mitochondrial branched chain amino acid aminotransferase (BCATm) or the branched chain alpha-keto acid dehydrogenase (BCKD E1alpha subunit) complex, in two rodent models of obesity (ob/ob mice and Zucker rats) and after surgical weight loss intervention in humans. Obese rodents exhibited hyperaminoacidemia including BCAAs. Whereas no obesity-related changes were observed in rodent skeletal muscle BCATm, pS293, or total BCKD E1alpha or BCKD kinase, in liver BCKD E1alpha was either unaltered or diminished by obesity, and pS293 (associated with the inactive state of BCKD) increased, along with BCKD kinase. In epididymal fat, obesity-related declines were observed in BCATm and BCKD E1alpha. Plasma BCAAs were diminished by an overnight fast coinciding with dissipation of the changes in adipose tissue but not in liver. BCAAs also were reduced by surgical weight loss intervention (Roux-en-Y gastric bypass) in human subjects studied longitudinally. These changes coincided with increased BCATm and BCKD E1alpha in omental and subcutaneous fat. Our results are consistent with the idea that tissue-specific alterations in BCAA metabolism, in liver and adipose tissue but not in muscle, may contribute to the rise in plasma BCAAs in obesity.

Disruption of BCATm in mice leads to increased energy expenditure associated with the activation of a futile protein turnover cycle.

Leucine is recognized as a nutrient signal; however, the long-term in vivo consequences of leucine signaling and the role of branched-chain amino acid (BCAA) metabolism in this signaling remain unclear. To investigate these questions, we disrupted the BCATm gene, which encodes the enzyme catalyzing the first step in peripheral BCAA metabolism. BCATm(-/-) mice exhibited elevated plasma BCAAs and decreased adiposity and body weight, despite eating more food, along with increased energy expenditure, remarkable improvements in glucose and insulin tolerance, and protection from diet-induced obesity. The increased energy expenditure did not seem to be due to altered locomotor activity, uncoupling proteins, sympathetic activity, or thyroid hormones but was strongly associated with food consumption and an active futile cycle of increased protein degradation and synthesis. These observations suggest that elevated BCAAs and/or loss of BCAA catabolism in peripheral tissues play an important role in regulating insulin sensitivity and energy expenditure.

The messenger RNA profiles in liver, hypothalamus, white adipose tissue, and skeletal muscle of female Zucker diabetic fatty rats after topiramate treatment.

Topiramate (TPM) is a novel neurotherapeutic agent approved for the treatment of epilepsy and for migraine prophylaxis. It has been observed that in obese-associated, type 2 diabetic rodent models, TPM treatment reduced the body weight gain, improved insulin sensitivity, and enhanced glucose-regulated insulin release. A long-term treatment with TPM thus ameliorated obesity and diabetic syndromes in female Zucker diabetic fatty rats and db/db mice. The molecular mechanisms of TPM antiobesity and antidiabetic effects remain unknown. We have applied DNA microarray technology to explore genes that might be involved in the mechanisms by which TPM improves insulin sensitivity and blood glucose handling, as well as body weight control. In female Zucker diabetic fatty rats, 7-day TPM treatment significantly reduced the plasma levels of glucose and triglyceride in a dose-dependent manner. The DNA microarray data revealed that TPM treatment altered messenger RNA profiles in liver, hypothalamus, white adipose tissue, and skeletal muscle. The most marked effect of TPM on gene expression occurred in liver with those genes related with metabolic enzymes and signaling regulatory proteins involved in energy metabolism. TPM treatment decreased messenger RNA amounts for sterol regulatory element binding protein-1c, stearoyl-coenzyme A (CoA) desaturase-1, choline kinase, and fatty acid CoA ligase, long chain 4. TPM also up-regulated 3 cholesterol synthesis genes. In addition, the short-term effect of TPM on gene expression was examined at 16 hours after a single administration. TPM markedly reduced hepatic expression of genes related with fatty acid synthesis, eg, stearoyl-CoA desaturase and acetyl-CoA carboxylase. TPM also changed genes related with fatty acid beta-oxidation, increased 3-2-trans-enoyl-CoA isomerase and mitochondrial acyl-CoA thioesterase, and decreased fatty acid CoA ligase (long chain 2 and long chain 5). These gene expression changes were independent of food intake as shown by pair feeding. Our results suggest that TPM regulates hepatic expression of genes involved in lipid metabolism, which could be part of the mechanisms by which TPM reduces plasma triglyceride levels in obese diabetic rodents.

Free fatty acids produce insulin resistance and activate the proinflammatory nuclear factor-kappaB pathway in rat liver.

To study mechanisms by which free fatty acids (FFAs) cause hepatic insulin resistance, we have used euglycemic-hyperinsulinemic clamping with and without infusion of lipid/heparin (to raise or to lower plasma FFAs) in alert male rats. FFA-induced hepatic insulin resistance was associated with increased hepatic diacylglycerol content (+210%), increased activities of two serine/threonine kinases (protein kinase C-delta and inhibitor of kappaB [IkappaB] kinase-beta), increased activation of the proinflammatory nuclear factor-kappaB (NF-kappaB) pathway (IkappaB kinase-beta, +640%; IkappaB-alpha, -54%; and NF-kappaB, +73%), and increased expression of inflammatory cytokines (tumor necrosis factor-alpha, +1,700% and interleukin-1beta, +440%) and plasma levels of monocyte chemoattractant protein-1 (+220%). We conclude that FFAs caused hepatic insulin resistance, which can produce overproduction of glucose and hyperglycemia, and initiated inflammatory processes in the liver that could potentially result in the development of steatohepatitis.

Antisense oligonucleotides targeted against glucocorticoid receptor reduce hepatic glucose production and ameliorate hyperglycemia in diabetic mice.

Abstract Specific blockade of glucocorticoid receptor (GCCR) action in the liver without affecting the hypothalamus-pituitary-adrenal axis could be a novel pharmaceutical approach to treat type 2 diabetes. In the present study, we applied an antisense oligonucleotide (ASO) against GCCR (ASO-GCCR) to reduce the expression of liver GCCR and examined its impact on the diabetic syndrome in ob / ob and db / db mice. A 3-week treatment regimen of ASO-GCCR (25 mg/kg IP, twice per week) markedly reduced liver GCCR messenger RNA and protein expression with no alteration of GCCR messenger RNA expression in the hypothalamus, pituitary, or adrenal gland. The ASO-GCCR treatment lowered blood glucose levels by 45% and 23% in ob / ob and db / db mice, respectively, compared with those observed in the control group. The ASO-GCCR-treated mice also showed significant enhancement of insulin-mediated inhibition of hepatic glucose production during a euglycemic-hyperinsulinemic clamp as well as marked reduction of phosphoenolpyruvate carboxykinase and glucose 6-phosphatase activity compared with control mice. The ASO-GCCR treatment did not change peripheral insulin sensitivity during the clamp. The ob / ob mice treated with ASO-GCCR had no significant difference in the plasma corticosterone and corticotropin levels compared with control mice. Lean mice receiving a similar treatment regimen of ASO-GCCR exhibited no change in blood glucose levels, oral glucose tolerance tests, or insulin tolerance tests. Our results demonstrate that selective inhibition of GCCR expression in the liver by the ASO-GCCR treatment reduced hepatic glucose production and improved blood glucose control under diabetic conditions.

Reduction in glucagon receptor expression by an antisense oligonucleotide ameliorates diabetic syndrome in db/db mice.

Excess glucagon levels contribute to the hyperglycemia associated with type 2 diabetes. Reducing glucagon receptor expression may thus ameliorate the consequences of hyperglucagonemia and improve blood glucose control in diabetic patients. This study describes the antidiabetic effects of a specific glucagon receptor antisense oligonucleotide (GR-ASO) in db/db mice. The ability of GR-ASOs to inhibit glucagon receptor mRNA expression was demonstrated in primary mouse hepatocytes by quantitative real-time RT-PCR. Intraperitoneal administration of GR-ASO at a dosage of 25 mg/kg twice a week in db/db mice for 3 weeks resulted in 1) decreased glucagon receptor mRNA expression in liver; 2) decreased glucagon-stimulated cAMP production in hepatocytes isolated from GR-ASO-treated db/db mice; 3) significantly reduced blood levels of glucose, triglyceride, and free fatty acids; 4) improved glucose tolerance; and 5) a diminished hyperglycemic response to glucagon challenge. Neither lean nor db/db mice treated with GR-ASO exhibited hypoglycemia. Suppression of GR expression was also associated with increased ( approximately 10-fold) levels of plasma glucagon. No changes were observed in pancreatic islet cytoarchitecture, islet size, or alpha-cell number. However, alpha-cell glucagon levels were increased significantly. Our studies support the concept that antagonism of glucagon receptors could be an effective approach for controlling blood glucose in diabetes.

Mechanisms by which liver-specific PEPCK knockout mice preserve euglycemia during starvation.

Liver-specific PEPCK knockout mice, which are viable despite markedly abnormal lipid metabolism, exhibit mild hyperglycemia in response to fasting. We used isotopic tracer methods, biochemical measurements, and nuclear magnetic resonance spectroscopy to show that in mice lacking hepatic PEPCK, 1) whole-body glucose turnover is only slightly decreased; 2) whole-body gluconeogenesis from phosphoenolpyruvate, but not from glycerol, is moderately decreased; 3) tricarboxylic acid cycle activity is globally increased, even though pyruvate cycling and anaplerosis are decreased; 4) the liver is unable to synthesize glucose from lactate/pyruvate and produces only a minimal amount of glucose; and 5) glycogen synthesis in both the liver and muscle is impaired. Thus, although mice without hepatic PEPCK have markedly impaired hepatic gluconeogenesis, they are able to maintain a near-normal blood glucose concentration while fasting by increasing extrahepatic gluconeogenesis coupled with diminishing whole-body glucose utilization.