Hepatic Glucose Overproduction Research Articles

Sirt1 Mediates Vitamin D Deficiency-Driven Gluconeogenesis in the Liver via mTorc2/Akt Signaling.

As an active form of vitamin D (VD), 1,25-dihydroxyvitamin D (1,25(OH)2D3) is involved in the development of many metabolic diseases, such as diabetes, autoimmune diseases, and tumours. While prospective epidemiological studies have consistently implicated VD deficiency in the regulation of glucose metabolism and insulin sensitivity, the specific mechanism remains unclear. Here, we generated 1α(OH)ase-null mice (targeted ablation of the 25-hydroxyvitamin D 1α hydroxylase enzyme) and found that these mice developed hepatic glucose overproduction, glucose intolerance, and hepatic insulin resistance accompanied by reduced Sirtuin 1 (Sirt1) expression. The chromatin immunoprecipitation (ChIP) and a luciferase reporter assay revealed that 1,25(OH)2D3-activated VD receptor (VDR) directly interacted with one VD response element (VDRE) in the Sirt1 promoter to upregulate Sirt1 transcription, triggering a cascade of serine/threonine kinase (AKT) phosphorylation at S473 and FOXO1 phosphorylation at S256. This phosphorylation cascade reduced the expression of gluconeogenic genes, eventually attenuating glucose overproduction in the liver. In addition, a signaling pathway was found to modulate gluconeogenesis involving VDR, Sirt1, Rictor (a component of mTOR complex 2 [mTorc2]), AKT, and FOXO1, and Sirt1 and FOXO1 were identified as key modulators of dysregulated gluconeogenesis due to VD deficiency.

Leptin resistance before and after obesity: evidence that tissue glucose uptake underlies adipocyte enlargement and liver steatosis/steatohepatitis in Zucker rats from early-life stages

Leptin resistance occurs in obese patients, but its independent contribution to adiposity and the accompanying metabolic diseases, i.e., diabetes, liver steatosis, and steatohepatitis, remains to be established. This study was conducted in an extreme model of leptin resistance to investigate mechanisms initiating diabetes, fat expansion, liver steatosis, and inflammatory disease, focusing on the involvement of glucose intolerance and organ-specific glucose uptake in brown and subcutaneous adipose tissues (BAT, SAT) and in the liver. We studied preobese and adult Zucker rats (fa/fa, fa/+ ) during fasting or glucose loading to assess glucose tolerance. Relevant pancreatic and intestinal hormonal levels were measured by Milliplex. Imaging of 18F-fluorodeoxyglucose by positron emission tomography was used to quantify glucose uptake in SAT, BAT, and liver, and evaluate its relationship with adipocyte size and biopsy-proven nonalcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH). Preobese fa/fa pups showed impaired glucose tolerance, adipocyte enlargement, hepatic microsteatosis, and lobular inflammation, with elevated hepatic post-glucose load glucose uptake and production. Adult fa/fa rats had more severe glucose intolerance, fasting hyperglycemia, hormonal abnormalities, elevated glucose uptake in SAT and BAT, and more markedly in the liver, together with macrosteatosis, and highly prevalent hepatic inflammation. Organ glucose uptake was proportional to the degree of fat accumulation and tissue inflammation and was able to dissect healthy from NAFLD and NAFLD/NASH livers. Most severe NASH livers showed a decline in glucose uptake and liver enzymes. In fa/fa Zucker rats, leptin resistance leads to glucose intolerance, mainly due to hepatic glucose overproduction, preceding obesity, and explaining pancreatic and intestinal hormonal changes and fat accumulation in adipocytes and hepatocytes. Our data support the involvement of liver glucose uptake in the pathogenesis of liver inflammatory disease. Its potential as more generalized biomarker or diagnostic approach remains to be established outside of our leptin-receptor-deficient rat model.

MicroRNA-185-5p inhibits hepatic gluconeogenesis and reduces fasting blood glucose levels by suppressing G6Pase.

Aims/hypothesis: MicroRNAs (miRNAs) are known to contribute to many metabolic diseases, including type 2 diabetes. This study aimed to investigate the roles and molecular mechanisms of miR-185-5p in the regulation of hepatic gluconeogenesis.Methods: MicroRNA high-throughput sequencing was performed to identify differentially expressed miRNAs. High-fat diet-induced obese C57BL/6 mice and db/db mice, a genetic mouse model for diabetes, were used for examining the regulation of hepatic gluconeogenesis. Quantitative reverse transcriptase PCR and Western blotting were performed to measure the expression levels of various genes and proteins. Luciferase reporter assays were used to determine the regulatory roles of miR-185-5p on G6Pase expression.Results: Hepatic miR-185-5p expression was significantly decreased during fasting or insulin resistance. Locked nucleic acid (LNA)-mediated suppression of miR-185-5p increased blood glucose and hepatic gluconeogenesis in healthy mice. In contrast, overexpression of miR-185-5p in db/db mice alleviated blood hyperglycemia and decreased gluconeogenesis. At the molecular level, miR-185-5p directly inhibited G6Pase expression by targeting its 3'-untranslated regions. Furthermore, metformin, an anti-diabetic drug, could upregulate miR-185-5p expression to suppress G6Pase, leading to hepatic gluconeogenesis inhibition.Conclusions/interpretation: Our findings provided a novel insight into the role of miR-185-5p that suppressed hepatic gluconeogenesis and alleviated hyperglycemia by targeting G6Pase. We further identified that the /G6Pase axis mediated the inhibitory effect of metformin on hepatic gluconeogenesis. Thus, miR-185-5p might be a therapeutic target for hepatic glucose overproduction and fasting hyperglycemia.

1806-P: Vitamin D Deficiency Impairs mTORC2/Akt Signaling through Downregulating SIRT1 and Results in Increased Hepatic Gluconeogenesis

1,25(OH)2D3 as an active form of vitamin D is involved in the development of many metabolic-related diseases including diabetes. While prospective epidemiological studies have shown that vitamin D deficiency is implicated in the regulation of glucose metabolism, the specific mechanism still remains unclear. Here, we generated 1α(OH)ase-null mice and discovered that these mice developed hepatic glucose overproduction and hepatic insulin resistance accompanied by decreased expression of Sirt1. ChIP and Luciferase assay confirmed that 1,25(OH)2D3 activating vitamin D receptor (VDR) directly interacts with one vitamin D response element located in Sirt1 promoter to up-regulate its transcription, triggering a cascade of phosphorylation of Akt at S473 and FOXO1 at S256 and resulting in decreased transcription of the gluconeogenic genes glucose-6-phosphatase (G6pase) and phosphoenolpyruvate carboxykinase (PCK1), eventually hepatic glucose overproduction. We have identified a signaling pathway involving VDR, Sirt1, Rictor (a component of mTOR complex 2 [mTorc2]), Akt, and FoxO1 that regulates gluconeogenesis, and identified Sirt1 and FoxO1 as key modulators of increased gluconeogenesis induced by vitamin D deficiency. Our work demonstrates a novel mechanism of 1,25(OH)2D3 deficiency-induced hyperglycemia mediated through Sirt1 downregulation. Disclosure Q. Yuan: None. J. Yang: None. M. Sun: None. S. Tang: None. M. Dong: None. L. Mao: None. Funding National Natural Science Foundation of China (81400789)

Carvedilol improves glucose tolerance and insulin sensitivity in treatment of adrenergic overdrive in high fat diet-induced obesity in mice.

Catecholamine excess reflecting an adrenergic overdrive of the sympathetic nervous system (SNS) has been proposed to link to hyperleptinemia in obesity and may contribute to the development of metabolic disorders. However, relationship between the catecholamine level and plasma leptin in obesity has not yet been investigated. Moreover, whether pharmacological blockade of the adrenergic overdrive in obesity by the third-generation beta-blocker agents such as carvedilol could help to prevent metabolic disorders is controversial and remains to be determined. Using the high fat diet (HFD)-induced obese mouse model, we found that basal plasma norepinephrine, the principal catecholamine as an index of SNS activity, was persistently elevated and highly correlated with plasma leptin concentration during obesity development. Targeting the adrenergic overdrive from this chronic norepinephrine excess in HFD-induced obesity with carvedilol, a third-generation beta-blocker with vasodilating action, blunted the HFD-induced hepatic glucose over-production by suppressing the induction of gluconeogenic enzymes, and enhanced the muscular insulin signaling pathway. Furthermore, carvedilol treatment in HFD-induced obese mice decreased the enlargement of white adipose tissue and improved the glucose tolerance and insulin sensitivity without affecting body weight and blood glucose levels. Our results suggested that catecholamine excess in obesity might directly link to the hyperleptinemic condition and the therapeutic targeting of chronic adrenergic overdrive in obesity with carvedilol might be helpful to attenuate obesity-related metabolic disorders.

Sodium–glucose co‐transporter 2 inhibitors: a review of their use in older people with type 2 diabetes mellitus

AbstractTraditional treatment options for type 2 diabetes mellitus (T2DM) target the primary pathophysiological defects of increased insulin resistance, hepatic glucose overproduction and progressive pancreatic beta‐cell dysfunction. Sodium–glucose co‐transporter 2 (SGLT2) inhibitors are the newest class of medication for T2DM and provide an alternative, insulin‐independent, pathway for addressing hyperglycaemia, by reducing renal glucose reabsorption. SGLT2 inhibitors are associated with a low risk of hypoglycaemia and recent trials have indicated, for empagliflozin at least, reductions in cardiovascular morbidity and mortality and a slower progression of kidney disease. These characteristics make SGLT2 inhibitors attractive second‐line agents. Adverse effects include an increased risk of genitourinary tract infections, hypotension, fractures and acute renal impairment. Common events in hospitalised older patients, such as volume depletion, indwelling catheter use, potential renal insults (e.g. aminoglycosides or intravenous contrast administration) and unpredictable haemodynamics, may exacerbate these adverse effects, suggesting SGLT2 inhibitors may be best avoided in this setting. Clinical heterogeneity of the older patient cohort calls for individualised management. As there is a lack of evidence to guide diabetes management in older people, this review aims to summarise the available evidence from clinical trials and meta‐analyses and provide a practical perspective on how best to use SGLT2 inhibitors in older patients.

MicroRNA-451 Negatively Regulates Hepatic Glucose Production and Glucose Homeostasis by Targeting Glycerol Kinase–Mediated Gluconeogenesis

MicroRNAs (miRNAs) are a new class of regulatory molecules implicated in type 2 diabetes, which is characterized by insulin resistance and hepatic glucose overproduction. We show that miRNA-451 (miR-451) is elevated in the liver tissues of dietary and genetic mouse models of diabetes. Through an adenovirus-mediated gain- and loss-of-function study, we found that miR-451 negatively regulates hepatic gluconeogenesis and blood glucose levels in normal mice and identified glycerol kinase (Gyk) as a direct target of miR-451. We demonstrate that miR-451 and Gyk regulate hepatic glucose production, the glycerol gluconeogenesis axis, and the AKT-FOXO1-PEPCK/G6Pase pathway in an opposite manner; Gyk could reverse the effect of miR-451 on hepatic gluconeogenesis and AKT-FOXO1-PEPCK/G6Pase pathway. Moreover, overexpression of miR-451 or knockdown of Gyk in diabetic mice significantly inhibited hepatic gluconeogenesis, alleviated hyperglycemia, and improved glucose tolerance. Further studies showed that miR-451 is upregulated by glucose and insulin in hepatocytes; the elevation of hepatic miR-451 in diabetic mice may contribute to inhibiting Gyk expression. This study provides the first evidence that miR-451 and Gyk regulate the AKT-FOXO1-PEPCK/G6Pase pathway and play critical roles in hepatic gluconeogenesis and glucose homeostasis and identifies miR-451 and Gyk as potential therapeutic targets against hyperglycemia in diabetes.

Prediabetes as a toxic environment for the initiation of microvascular and macrovascular complications.

Prediabetes is a state characterized by impaired fasting glucose or impaired glucose tolerance. Evidence is increasingly demonstrating that prediabetes is a toxic state, in addition to being a harbinger of future development of diabetes mellitus. This minireview discusses the pathophysiology and clinical significance of prediabetes, and approach to its management, in the context of the worldwide diabetes epidemic. The pathophysiologic defects underlying prediabetes include insulin resistance, β cell dysfunction, increased lipolysis, inflammation, suboptimal incretin effect, and possibly hepatic glucose overproduction. Recent studies have revealed that the long-term complications of diabetes may manifest in some people with prediabetes; these complications include classical microvascular and macrovascular disorders, and our discussion explores the role of glycemia in their development. Finally, landmark intervention studies in prediabetes, including lifestyle modification and pharmacologic treatment, are reviewed.

A Single Amino Acid Mutation (R104P) in the E/DRY Motif of GPR40 Impairs Receptor Function.

Type 2 Diabetes Mellitus with insulin resistance, pancreatic β cell dysfunction, and hepatic glucose overproduction is increasing in epidemic proportions worldwide. G protein-coupled receptor 40 (GPR40), a clinically proven anti-diabetic drug target, is mainly expressed in pancreatic β cells and insulin-secreting cell lines. Long chain fatty acids (LCFA) increase intracellular calcium concentration and amplify glucose-stimulated insulin secretion by activating GPR40. Here we report that the arginine 104 (R104) is critical for the normal function of GPR40. Mutation of R104 to Proline (R104P) results in complete loss of the receptor function. Linoleic acid, ligand of GPR40, could not elicit calcium increase and ERK phosphorylation in cells expressing this mutant receptor. Further study indicated the R104P mutation reduces cell surface localization of GPR40 without affecting the expression of the protein. The small portion of GPR40 R104P mutant that is still located on the membrane has no physiological function, and does not internalize in response to linoleic acid stimulation. These data demonstrate that R104 in GPR40 is critically involved in the normal receptor functions. Interestingly, R104P is a registered single-nucleotide polymorphism of GPR40. The relationship of this GPR40 variant and type 2 diabetes warrants further investigation.

Computational identification of novel natural inhibitors of glucagon receptor for checking type II diabetes mellitus.

BackgroundInteraction of the small peptide hormone glucagon with glucagon receptor (GCGR) stimulates the release of glucose from the hepatic cells during fasting; hence GCGR performs a significant function in glucose homeostasis. Inhibiting the interaction between glucagon and its receptor has been reported to control hepatic glucose overproduction and thus GCGR has evolved as an attractive therapeutic target for the treatment of type II diabetes mellitus.ResultsIn the present study, a large library of natural compounds was screened against 7 transmembrane domain of GCGR to identify novel therapeutic molecules that can inhibit the binding of glucagon with GCGR. Molecular dynamics simulations were performed to study the dynamic behaviour of the docked complexes and the molecular interactions between the screened compounds and the ligand binding residues of GCGR were analysed in detail. The top scoring compounds were also compared with already documented GCGR inhibitors- MK-0893 and LY2409021 for their binding affinity and other ADME properties. Finally, we have reported two natural drug like compounds PIB and CAA which showed good binding affinity for GCGR and are potent inhibitor of its functional activity.ConclusionThis study contributes evidence for application of these compounds as prospective small ligand molecules against type II diabetes. Novel natural drug like inhibitors against the 7 transmembrane domain of GCGR have been identified which showed high binding affinity and potent inhibition of GCGR

The proliferative gene cyclin D1 and gluconeogenesis--could suppressing glucose production also promote cancer?

Hepatic insulin resistance manifested as impaired suppression of glucose production is a key metabolic derangement that contributes to fasting hyperglycemia and increased HbA1c levels in type 2 diabetes (1). Genetic manipulation to overexpress the key enzymes in gluconeogenesis (particularly PEPCK) in preclinical models has shown that a primary defect in this system can result in increased hepatic glucose production (2,3) but, importantly, can lead to defects in insulin resistance in other tissues (muscle/fat) and impairments in insulin secretion (4,5). Despite this, medications that specifically target hepatic glucose overproduction in diabetes are lacking. Currently, the only medication (other than insulin) that targets the liver is the biguanide metformin, and despite being used for more than half a century, the mechanism by which it does this is still not completely understood (6–9). Thus, understanding the mechanism causing hepatic insulin resistance will lead to more effective and targeted therapeutic options. Cyclin D1 is involved in cell cycle regulation and DNA synthesis and has been shown to be upregulated in a variety of tumors (including breast cancer). In addition, a number of articles has shown that cyclin D1 can affect metabolic processes, particularly glycolysis, lipogenesis, and mitochondrial function (10–12). Specifically, the loss of cyclin D1 caused an upregulation of hexokinase II, pyruvate kinase, fatty acid synthase, and acetyl-CoA carboxylase, leading to increased lipogenesis. …

Effects of small interfering RNA-mediated hepatic glucagon receptor inhibition on lipid metabolism in db/db mice

Hepatic glucose overproduction is a major characteristic of type 2 diabetes. Because glucagon is a key regulator for glucose homeostasis, antagonizing the glucagon receptor (GCGR) is a possible therapeutic strategy for the treatment of diabetes mellitus. To study the effect of hepatic GCGR inhibition on the regulation of lipid metabolism, we generated siRNA-mediated GCGR knockdown (si-GCGR) in the db/db mouse. The hepatic knockdown of GCGR markedly reduced plasma glucose levels; however, total plasma cholesterol was increased. The detailed lipid analysis showed an increase in the LDL fraction, and no change in VLDL HDL fractions. Further studies showed that the increase in LDL was the result of over-expression of hepatic lipogenic genes and elevated de novo lipid synthesis. Inhibition of hepatic glucagon signaling via siRNA-mediated GCGR knockdown had an effect on both glucose and lipid metabolism in db/db mice.

Induction of Cytosolic Phospholipase A2α Is Required for Adipose Neutrophil Infiltration and Hepatic Insulin Resistance Early in the Course of High-Fat Feeding

In established obesity, inflammation and macrophage recruitment likely contribute to the development of insulin resistance. In the current study, we set out to explore whether adipose tissue infiltration by neutrophils that occurs early (3 days) after initiating a high-fat diet (HFD) could contribute to the early occurrence of hepatic insulin resistance and to determine the role of cytosolic phospholipase A2α (cPLA2α) in this process. The 3-day HFD caused a significant upregulation of cPLA2α in periepididymal fat and in the liver. A specific antisense oligonucleotide (AS) effectively prevented cPLA2α induction, neutrophil infiltration into adipose tissue (likely involving MIP-2), and protected against 3-day HFD–induced impairment in hepatic insulin signaling and glucose over-production from pyruvate. To sort out the role of adipose neutrophil infiltration independent of cPLA2α induction in the liver, mice were injected intraperitoneally with anti–intracellular adhesion molecule-1 (ICAM-1) antibodies. This effectively prevented neutrophil infiltration without affecting cPLA2α or MIP-2, but like AS, prevented impairment in hepatic insulin signaling, the enhanced pyruvate-to-glucose flux, and the impaired insulin-mediated suppression of hepatic glucose production (assessed by clamp), which were induced by the 3-day HFD. Adipose tissue secretion of tumor necrosis factor-α (TNF-α) was increased by the 3-day HFD, but not if mice were treated with AS or ICAM-1 antibodies. Moreover, systemic TNF-α neutralization prevented 3-day HFD–induced hepatic insulin resistance, suggesting its mediatory role. We propose that an acute, cPLA2α-dependent, neutrophil-dominated inflammatory response of adipose tissue contributes to hepatic insulin resistance and glucose overproduction in the early adaptation to high-fat feeding.

Hepatic Sirt1 deficiency in mice impairs mTorc2/Akt signaling and results in hyperglycemia, oxidative damage, and insulin resistance

Insulin resistance is a major risk factor for type 2 diabetes mellitus. The protein encoded by the sirtuin 1 (Sirt1) gene, which is a mouse homolog of yeast Sir2, is implicated in the regulation of glucose metabolism and insulin sensitivity; however, the underlying mechanism remains elusive. Here, using mice with a liver-specific null mutation of Sirt1, we have identified a signaling pathway involving Sirt1, Rictor (a component of mTOR complex 2 [mTorc2]), Akt, and Foxo1 that regulates gluconeogenesis. We found that Sirt1 positively regulates transcription of the gene encoding Rictor, triggering a cascade of phosphorylation of Akt at S473 and Foxo1 at S253 and resulting in decreased transcription of the gluconeogenic genes glucose-6-phosphatase (G6pase) and phosphoenolpyruvate carboxykinase (Pepck). Liver-specific Sirt1 deficiency caused hepatic glucose overproduction, chronic hyperglycemia, and increased ROS production. This oxidative stress disrupted mTorc2 and impaired mTorc2/Akt signaling in other insulin-sensitive organs, leading to insulin resistance that could be largely reversed with antioxidant treatment. These data delineate a pathway through which Sirt1 maintains insulin sensitivity and suggest that treatment with antioxidants might provide protection against progressive insulin resistance in older human populations.

Plant derived therapeutics for the treatment of Metabolic Syndrome

Type 2 diabetes is a progressive disorder whose pathophysiology consists of pancreatic dysfunction, hepatic glucose overproduction and insulin resistance in peripheral tissues such as adipose tissue and muscle. Currently, clinical strategies to achieve improved metabolic control endorse the use of a combination of agents from multiple pharmacologic classes and which target specific pathophysiologic defects. Given the role of insulin resistance in the pathophysiology, the improvement of insulin sensitivity for treatment of the metabolic syndrome or type 2 diabetes remains as a primary clinical strategy. Recent data, however, have questioned the safety of the current pharmacologic agents used to enhance insulin sensitivity. As such, alternative strategies, e.g., nutritional supplementation with over-the-counter agents, are extensively practiced by a large number of patients and are frequently done so without the knowledge of the provider. Based on historical human use, there has been great interest in plant extracts (botanicals) as a source for nutritional supplements intended as adjunctive therapies for human diseases. Specifically, isolated compounds identified from plant sources, i.e. phytochemicals or bioactives, have served as a source for therapeutic agents for many diseases, including malignancy, infectious diseases and diabetes. Interestingly, the development of one of the most commonly used anti-hyperglycemic agents in the world today, i.e. metformin, can be traced back to a botanical source. However, the concern with most nutritional supplements, including those considered „natural“ (e.g. botanicals) by the consumer, is the paucity of data in humans in regard to efficacy to improve metabolic abnormalities. Thus, there remains considerable controversy regarding the use of botanical supplements for human health. Despite the stated concerns for botanical supplements, there are a number of botanicals that have shown considerable promise for human use but they have to be carefully validated in controlled clinical trials.

Proteome, Phosphoproteome, and Hydroxyproteome of Liver Mitochondria in Diabetic Rats at Early Pathogenic Stages

It has been proposed that mitochondrial dysfunction is involved in the pathogenesis of type 2 diabetes (T2D). To dissect the underlying mechanisms, we performed a multiplexed proteomics study on liver mitochondria isolated from a spontaneous diabetic rat model before/after they were rendered diabetic. Altogether, we identified 1091 mitochondrial proteins, 228 phosphoproteins, and 355 hydroxyproteins. Mitochondrial proteins were found to undergo expression changes in a highly correlated fashion during T2D development. For example, proteins involved in beta-oxidation, the tricarboxylic acid cycle, oxidative phosphorylation, and other bioenergetic processes were coordinately up-regulated, indicating that liver cells confronted T2D by increasing energy expenditure and activating pathways that rid themselves of the constitutively increased flux of glucose and lipid. Notably, activation of oxidative phosphorylation was immediately related to the overproduction of reactive oxygen species, which caused oxidative stress within the cells. Increased oxidative stress was also evidenced by our post-translational modification profiles such that mitochondrial proteins were more heavily hydroxylated during T2D development. Moreover, we observed a distinct depression of antiapoptosis and antioxidative stress proteins that might reflect a higher apoptotic index under the diabetic stage. We suggest that such changes in systematic metabolism were causally linked to the development of T2D. Comparing proteomics data against microarray data, we demonstrated that many T2D-related alterations were unidentifiable by either proteomics or genomics approaches alone, underscoring the importance of integrating different approaches. Our compendium could help to unveil pathogenic events in mitochondria leading to T2D and be useful for the discovery of diagnosis biomarker and therapeutic targets of T2D.

SAR Study of β-Aminoacyl-Containing Cyclic Hydrazide Derivatives as DPP-IV Inhibitors

Department of Chemistry, Sogang University, Seoul 121-742, KoreaReceived August 3, 2008In continuation of our efforts to further derivatize dipeptidyl peptidase IV (DPP-IV) inhibitors, a series of β-aminoacyl-containing 5-, 6- and 7- membered cyclic hydrazide derivatives was synthesized. All thecompounds were evaluated for their ability to inhibit DPP-IV, and an optimum structural unit on basic skeletonis identified to show good in vitro activity.Key Words : Dipeptidyl peptidase IV, Diabetes, Cyclic hydrazideIntroductionA non-insulin dependent diabetes mellitus (NIDDM) ischaracterized by chronic hyperglycemia, and belongs to agroup of metabolic disorders with multiple etiologies. It isvery common and may result from insulin resistance, inade-quate secretion of insulin, hepatic glucose overproduction,or glucose intolerance.

Differing mechanisms of hepatic glucose overproduction in triiodothyronine-treated rats vs. Zucker diabetic fatty rats by NMR analysis of plasma glucose

The metabolic mechanism of hepatic glucose overproduction was investigated in 3,3'-5-triiodo-l-thyronine (T3)-treated rats and Zucker diabetic fatty (ZDF) rats (fa/fa) after a 24-h fast. 2H2O and [U-13C3]propionate were administered intraperitoneally, and [3,4-13C2]glucose was administered as a primed infusion for 90 min under ketamine-xylazine anesthesia. 13C NMR analysis of monoacetone glucose derived from plasma glucose indicated that hepatic glucose production was twofold higher in both T3-treated rats and ZDF rats compared with controls, yet the sources of glucose overproduction differed significantly in the two models by 2H NMR analysis. In T3-treated rats, the hepatic glycogen content and hence the contribution of glycogenolysis to glucose production was essentially zero; in this case, excess glucose production was due to a dramatic increase in gluconeogenesis from TCA cycle intermediates. 13C NMR analysis also revealed increased phosphoenolpyruvate carboxykinase flux (4x), increased pyruvate cycling flux (4x), and increased TCA flux (5x) in T3-treated animals. ZDF rats had substantial glycogen stores after a 24-h fast, and consequently nearly 50% of plasma glucose originated from glycogenolysis; other fluxes related to the TCA cycle were not different from controls. The differing mechanisms of excess glucose production in these models were easily distinguished by integrated 2H and 13C NMR analysis of plasma glucose.

A Novel Glucagon Receptor Antagonist Inhibits Glucagon-Mediated Biological Effects

Glucagon maintains glucose homeostasis during the fasting state by promoting hepatic gluconeogenesis and glycogenolysis. Hyperglucagonemia and/or an elevated glucagon-to-insulin ratio have been reported in diabetic patients and animals. Antagonizing the glucagon receptor is expected to result in reduced hepatic glucose overproduction, leading to overall glycemic control. Here we report the discovery and characterization of compound 1 (Cpd 1), a compound that inhibits binding of 125I-labeled glucagon to the human glucagon receptor with a half-maximal inhibitory concentration value of 181 +/- 10 nmol/l. In CHO cells overexpressing the human glucagon receptor, Cpd 1 increased the half-maximal effect for glucagon stimulation of adenylyl cyclase with a KDB of 81 +/- 11 nmol/l. In addition, Cpd 1 blocked glucagon-mediated glycogenolysis in primary human hepatocytes. In contrast, a structurally related analog (Cpd 2) was not effective in blocking glucagon-mediated biological effects. Real-time measurement of glycogen synthesis and breakdown in perfused mouse liver showed that Cpd 1 is capable of blocking glucagon-induced glycogenolysis in a dosage-dependent manner. Finally, when dosed in humanized mice, Cpd 1 blocked the rise of glucose levels observed after intraperitoneal administration of exogenous glucagon. Taken together, these data suggest that Cpd 1 is a potent glucagon receptor antagonist that has the capability to block the effects of glucagon in vivo.

Mutation/polymorphism scanning of glucose-6-phosphatase gene promoter in noninsulin-dependent diabetes mellitus patients.

Glucose-6-phosphatase (G6Pase) catalyzes the rate-limiting step of gluconeogenesis, and hepatic G6Pase activity is increased in diabetes. We have cloned and analyzed the human G6Pase gene promoter region and identified putative regulatory sequences for insulin, cAMP, glucocorticoid, and hepatocyte nuclear factors. The promoter region of the G6Pase gene was analyzed in 154 noninsulin-dependent diabetes mellitus patients and 90 control subjects by PCR-single strand conformation polymorphism and direct sequencing methods. Polymorphisms were not found in any subjects. The results suggested that in noninsulin-dependent diabetic patients, the major cause of the hepatic glucose overproduction was not attributed to dysregulation of the G6Pase gene due to mutation/polymorphism of its promoter region.