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Aim: To develop an obese, insulin‐resistant cynomolgus monkey model of non‐alcoholic steatohepatitis (NASH) with fibrosis with a high fat/high cholesterol (HFHC) diet (with or without high fructose) and test its responsiveness to caloric restriction or pioglitazone. Methods: First, two groups of monkeys (n = 24/group) with histologically proven NASH and fibrosis were fed the HFHC diet for 17 weeks. The treatment group was subjected to a 40% caloric restriction (CR) and had their diet switched from the HFHC diet to a chow diet (DSCR). Paired liver biopsies were taken before and 17 weeks after DSCR. Subsets of monkeys (nine/group) had whole liver fat content assessed by MRI. Next, two groups of monkeys with histologically proven NASH and fibrosis were treated with vehicle (n = 9) or pioglitazone (n = 20) over 24 weeks. Results: The HFHC and DSCR groups lost 0.9% and 11.4% of body weight, respectively. After 17 weeks, non‐alcoholic fatty liver disease activity score (NAS) improvement was observed in 66.7% of the DSCR group versus 12.5% of the HFHC group (P <.001). Hepatic fat was reduced to 5.2% in the DSCR group versus 23.0% in the HFHC group (P =.0001). After 24 weeks, NAS improvement was seen in 30% of the pioglitazone group versus 0% of the vehicle group (P =.08). Conclusions: Both weight loss induced by DSCR and treatment with pioglitazone improve the histological features of NASH in a diet‐induced cynomolgus monkey model. This model provides a translational preclinical model for testing novel NASH therapies. [ABSTRACT FROM AUTHOR]

The hepatic energy state, defined by adenine nucleotide levels, couples metabolic pathways with energy requirements. This coupling is fundamental in the adaptive response to many conditions and is impaired in metabolic disease. We have found that the hepatic energy state is substantially reduced following exercise, fasting, and exposure to other metabolic stressors in C57BL/6 mice. Glucagon receptor signaling was hypothesized to mediate this reduction because increased plasma levels of glucagon are characteristic of metabolic stress and because this hormone stimulates energy consumption linked to increased gluconeogenic flux through cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) and associated pathways. We developed what we believe to be a novel hyperglucagonemic-euglycemic clamp to isolate an increment in glucagon levels while maintaining fasting glucose and insulin. Metabolic stress and a physiological rise in glucagon lowered the hepatic energy state and amplified AMP-activated protein kinase signaling in control mice, but these changes were abolished in glucagon receptor--null mice and mice with liver-specific PEPCK-C deletion. 129X1/Sv mice, which do not mount a glucagon response to hypoglycemia, displayed an increased hepatic energy state compared with C57BL/6 mice in which glucagon was elevated. Taken together, these data demonstrate in vivo that the hepatic energy state is sensitive to glucagon receptor activation and requires PEPCK-C, thus providing new insights into liver metabolism., Introduction The energy state in the cell is defined by adenine nucleotide levels and is critically coupled to nearly all metabolic processes (1). In the cell, the adenine nucleotides ATP, [...]

A portal venous 5-aminoimidazole-4-carboxamide-1-[beta]-D-fibofuranoside infusion that results in hepatic 5-aminoimidazole-4-carboxamide-1-[beta]-D-ribosyl-5-monophosphate (ZMP) concentrations of ~4 [micro]mol/g liver increases hepatic glycogenolysis and glucose output. ZMP is an AMP analog that mimics the regulatory actions of this nucleotide. The aim of this study was to measure hepatic AMP concentrations in response to increasing energy requirements to test the hypothesis that AMP achieves concentrations during exercise, consistent with a role in stimulation of hepatic glucose metabolism. Male C57BL/6J mice (27.4 [+ or -] 0.4 g) were subjected to 35 min of rest [sedentary (SED), n = 8], underwent short-term (ST, 35 min) moderate (20 m/min, 5% grade) exercise (n = 8), or underwent treadmill exercise under similar conditions but until exhaustion (EXH, n = 8). Hepatic AMP concentrations were 0.82 [+ or -] 0.05, 1.17 [+ or -] 0.11, and 2.52 [+ or -] 0.16 [micro]mol/g liver in SED, ST, and EXH mice, respectively (P < 0.05). Hepatic energy charge was 0.66 [+ or -] 0.01, 0.58 [+ or -] 0.02, and 0.33 [+ or -] 0.22 in SED, ST, and EXH mice, respectively (P < 0.05). Hepatic glycogen was 11.6 [+ or -] 1.0, 8.8 [+ or -] 2.2, and 0.0 [+ or -] 0.1 mg/g liver in SED, ST, and EXH mice, respectively (P < 0.05). Hepatic AMPK ([Thr.sup.172]) phosphorylation was 1.00 [+ or -] 0.14, 1.96 [+ or -] 0.16, and 7.44 [+ or -] 0.63 arbitrary units in SED, ST, and EXH mice, respectively (P < 0.05). Thus exercise increases hepatic AMP concentrations. These data suggest that the liver is highly sensitive to metabolic demands, as evidenced by dramatic changes in cellular energy indicators (AMP) and sensors thereof (AMP-activated protein kinase). In conclusion, AMP is sensitively regulated, consistent with it having an important role in hepatic metabolism. adenosine 5'-monophosphate; adenosine 5'-monophosphate-activated protein kinase; glycogen

This study aimed to test whether stimulation of net hepatic glucose output (NHGO) by increased concentrations of the AMP analog, 5-aminoimidazole-4-carboxamide-l-[beta]-D-ribosyl-5-monophosphate, can be suppressed by pharmacological insulin levels. Dogs had sampling (artery, portal vein, hepatic vein) and infusion (vena cava, portal vein) catheters and flow probes (hepatic artery, portal vein) implanted > 16 days before study. Protocols consisted of equilibration (- 130 to -30 min), basal (-30 to 0 min), and hyperinsulinemic-euglycemic (0-150 min) periods. At time (t) = 0 min, somatostatin was infused, and basal glucagon was replaced via the portal vein. Insulin was infused in the portal vein at either 2 (INS2) or 5 (INS5) mU*[kg.sup.-1]*[min.sup.-1]. At t = 60 min, 1 mg*[kg.sup.-1]*[min.sup.-1] portal venous 5-aminoimidazole-4-carbox-amide-l-[beta]-D-ribofuranoside (AICAR) infusion was initiated. Arterial insulin rose ~9- and ~27-fold in INS2 and INS5, respectively. Glucagon, catecholamines, and cortisol did not change throughout the study. NHGO was completely suppressed before t = 60 min. Intraportal AICAR stimulated NHGO by 1.9 [+ or -] 0.5 and 2.0 [+ or -] 0.5 mg*[kg.sup.-1]*[min.sup.-1] in INS2 and INS5, respectively. AICAR stimulated tracer-determined endogenous glucose production similarly in both groups. Intraportal AICAR infusion significantly increased hepatic acetyl-CoA carboxylase (ACC, [Ser.sup.79]) phosphorylation in INS2. Hepatic ACC ([Ser.sup.79]) phosphorylation, however, was not increased in INS5. Thus intraportal AICAR infusion renders hepatic glucose output insensitive to pharmacological insulin. The effectiveness of AICAR in countering the suppressive effect of pharmacological insulin on NHGO occurs even though AICAR-stimulated ACC phosphorylation is completely blocked. hepatic glucose production; glycogenolysis; AMP-activated protein kinase; adenosine 5'-monophosphate; 5-aminoimidazole-4-carboxamide-1-[beta]-D-ribosyl-5-monophosphate

The purpose of this study was to determine whether the sedentary dog is able to autoregulate glucose production ([R.sub.a]) in response to non-insulin-induced changes (16 days before study. Protocols consisted of basal (-30 to 0 min) and bilateral renal arterial phloridzin infusion (0-180 min) periods. Somatostatin was infused, and glucagon and insulin were replaced to basal levels. In one protocol (Phl [+ or -] Glc), glucose was allowed to fall from t = 0-90 min. This was followed by a period when glucose was infused to restore euglycemia (90-150 min) and a period when glucose was allowed to fall again (150-180 min). In a second protocol (EC), glucose was infused to compensate for the renal glucose loss due to phloridzin and maintain euglycemia from t = 0-180 min. Arterial insulin, glucagon, cortisol, and catecholamines remained at basal in both protocols. In Phl [+ or -] Glc, glucose fell by ~20 mg/dl by t = 90 min with phloridzin infusion. [R.sub.a] did not change from basal in Phl [+ or -] Glc despite the fall in glucose for the first 90 min. [R.sub.a] was significantly suppressed with restoration of euglycemia from t = 90-150 min (P < 0.05) and returned to basal when glucose was allowed to fall from t = 150-180 min. [R.sub.a] did not change from basal in EC. In conclusion, the liver autoregulates [R.sub.a] in response to small changes in glucose independently of changes in pancreatic hormones at rest. However, the liver of the resting dog is more sensitive to a small increment, rather than decrement, in arterial glucose. glucose turnover; phloridzin

Aim: The aim of this study was to evaluate amino acids as glucagon receptor (GCGR)‐specific biomarkers in rodents and cynomolgus monkeys in the presence of agonism of both glucagon‐like peptide‐1 receptor (GLP1R) and GCGR with a variety of dual agonist compounds. Materials and methods: Primary hepatocytes, rodents (normal, diet‐induced obese and GLP1R knockout) and cynomolgus monkeys were treated with insulin (hepatocytes only), glucagon (hepatocytes and cynomolgus monkeys), the GLP1R agonist, dulaglutide, or a variety of dual agonists with varying GCGR potencies. Results: A long‐acting dual agonist, Compound 2, significantly decreased amino acids in both wild‐type and GLP1R knockout mice in the absence of changes in food intake, body weight, glucose or insulin, and increased expression of hepatic amino acid transporters. Dulaglutide, or a variant of Compound 2 lacking GCGR agonism, had no effect on amino acids. A third variant with ~31‐fold less GCGR potency than Compound 2 significantly decreased amino acids, albeit to a significantly lesser extent than Compound 2. Dulaglutide (with saline infusion) had no effect on amino acids, but an infusion of glucagon dose‐dependently decreased amino acids on the background of GLP1R engagement (dulaglutide) in cynomolgus monkeys, as did Compound 2. Conclusions: These results show that amino acids are sensitive and translatable GCGR‐specific biomarkers. [ABSTRACT FROM AUTHOR]

We evaluated the hepatic and nonhepatic responses to glucose-responsive insulin (GRI). Eight dogs received GRI or regular human insulin (HI) in random order. A primed, continuous intravenous infusion of [3-3H]glucose began at -120 min. Basal sampling (-30 to 0 min) was followed by two study periods (150 min each), clamp period 1 (P1) and clamp period 2 (P2). At 0 min, somatostatin and GRI (36 ± 3 pmol/kg/min) or HI (1.8 pmol/kg/min) were infused intravenously; basal glucagon was replaced intraportally. Glucose was infused intravenously to clamp plasma glucose at 80 mg/dL (P1) and 240 mg/dL (P2). Whole-body insulin clearance and insulin concentrations were not different in P1 versus P2 with HI, but whole-body insulin clearance was 23% higher and arterial insulin 16% lower in P1 versus P2 with GRI. Net hepatic glucose output was similar between treatments in P1. In P2, both treatments induced net hepatic glucose uptake (HGU) (HI mean ± SEM 2.1 ± 0.5 vs. 3.3 ± 0.4 GRI mg/kg/min). Nonhepatic glucose uptake in P1 and P2, respectively, differed between treatments (2.6 ± 0.3 and 7.4 ± 0.6 mg/kg/min with HI vs. 2.0 ± 0.2 and 8.1 ± 0.8 mg/kg/min with GRI). Thus, glycemia affected GRI but not HI clearance, with resultant differential effects on HGU and nonHGU. GRI holds promise for decreasing hypoglycemia risk while enhancing glucose uptake under hyperglycemic conditions. [ABSTRACT FROM AUTHOR]

As therapeutic recombinant fusion proteins become more widely applicable for the treatment of various types of diseases, there is an increased demand for universal methods such as liquid chromatography (LC)--mass spectrometry (MS) for the determination of their pharmacokinetic properties, particularly their catabolism. The most common approach of analyzing proteins by LC--MS is to digest them into peptides, which can serve as surrogates of the protein. Alternatively, we have developed a novel high-resolution mass spectrometry (HRMS) based approach for analyzing large-molecule proteins at the intact level in biological samples without digestion. We established an immunoaffinity capture LC--HRMS method to quantify the intact parent molecule while simultaneously identifying catabolites for recombinant fusion proteins. We describe this method using dulaglutide, a glucagon-like peptide 1 (GLP1)--Fc fusion protein. Two proteolytic sites within the GLP1 peptide sequence of dulaglutide were identified using this novel LC--HRMS analysis in vivo in mice. These proteolytic sites were identified with the intact molecule being quantified simultaneously. Together with the trypsin digestion based LC--MS/MS analysis using surrogate peptides from different domains of the analyte, an insightful understanding of the pharmacokinetics and in vivo biotransformation of dulaglutide was obtained. Thus, this method enables simultaneous acquisition of both intact drug concentration and important catabolite information for this recombinant fusion protein, providing valuable insight into the integrity of the molecule and its catabolism in vivo. This is critical for designing and screening novel protein therapeutics and for understanding their pharmacokinetics and pharmacodynamics. With continuing advancement of LC--HRMS and software, this method can be very beneficial in drug discovery and development. [ABSTRACT FROM AUTHOR]

Hyperinsulinemic-euglycemic clamps are considered the "gold standard" for assessing whole body insulin sensitivity. When used in combination with tracer dilution techniques and physiological insulin concentrations, insulin sensitization can be dissected and attributed to hepatic and peripheral (primarily muscle) effects. Non-human primates (NHPs), such as rhesus monkeys, are the closest pre-clinical species to humans, and thus serve as an ideal model for testing of compound efficacy to support translation to human efficacy. We determined insulin infusion rates that resulted in high physiological insulin concentrations that elicited maximal pharmacodynamic responses during hyperinsulinemic-euglycemic clamps. These rates were then used with [U-13C]-D-glucose, to assess and document the degrees of hepatic and peripheral insulin resistance between healthy and insulin-resistant, dysmetabolic NHPs. Next, dysmetabolic NHPs were treated for 28 days with pioglitazone (3 mg/kg) and again had their insulin sensitivity assessed, illustrating a significant improvement in hepatic and peripheral insulin sensitivity. This coincided with a significant increase in insulin clearance, and normalization of circulating adiponectin. In conclusion, we have determined a physiological clamp paradigm (similar to humans) for assessing glucose turnover in NHPs. We have also demonstrated that insulin-resistant, dysmetabolic NHPs respond to the established insulin sensitizer, pioglitazone, thus confirming their use as an ideal pre-clinical translational model to assess insulin sensitizing compounds. [ABSTRACT FROM AUTHOR]

This study aimed to test whether stimulation of net hepatic glucose output (NHGO) by increased concentrations of the AMP analog, 5-amino-imidazole-4-carboxamide-1-β-D-ribosyl-5-monophosphate, can be suppressed by pharmacological insulin levels. Dogs had sampling (artery, portal vein, hepatic vein) and infusion (vena cava, portal vein) catheters and flow probes (hepatic artery, portal vein) implanted » 16 days betbre study. Protocols consisted of equilibration (-130 to -30 min), basal (-30 to 0 min), and hyperinsulinemic-euglycemic (0–150 min) periods. At time (t) = 0 min, somatostatin was infused, and basal glucagon was replaced via the portal vein. Insulin was infused in the portal vein at either 2 (INS2) or 5 (INS5) mU·kg-1·min-1. At t = 60 min, 1 mg·kg-1·min-1 portal venous 5-aminoimidazole-4-carboxamide-l-β-D-ribofuranoside (AICAR) infusion was initiated. Arterial insulin rose ∼9- and ∼27-fold in INS2 and INS5, respectively. Glucagon, catecholamines, and cortisol did not change throughout the study. NHGO was completely suppressed before t = 60 min. Intraportal AICAR stimulated NHGO by 1.9 ± 0.5 and 2.0 ± 0.5 mg·kg-1·min-1 in INS2 and INS5, respectively. AICAR stimulated tracer-determined endogenous glucose production similarly in both groups. Intraportal AICAR infusion significantly increased hepatic acetyl-CoA carboxylase (ACC, Ser79) phosphorylation in INS2. Hepatic ACC (Ser79) phosphorylation, however, was not increased in INS5. Thus intraportal AICAR infusion renders hepatic glucose output insensitive to pharmacological insulin. The effectiveness of AICAR in countering the suppressive effect of pharmacological insulin on NHGO occurs even though AICAR-stimulated ACC phosphorylation is completely blocked. [ABSTRACT FROM AUTHOR]

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AMP-activated protein kinase (AMPK) plays a key role in regulating metabolism, serving as a metabolic master switch. The aim of this study was to assess whether increased concentrations of the AMP analog, 5-aminoimidazole-4-carboxamide-1-β-D-ribosyl-5-monophosphate, in the liver would create a metabolic response consistent with an increase in whole-body metabolic need. Dogs had sampling (artery, portal vein, hepatic vein) and infusion (vena cava, portal vein) catheters and flow probes (hepatic artery, portal vein) implanted >16 days before a study. Protocols consisted of equilibration (-130 to -30 min), basal (-30 to 0 min), and hyperinsulinemic-euglycemic or -hypoglycemic clamp periods (0-150 min). At t = 0 min, somatostatin was infused and glucagon was replaced in the portal vein at basal rates. An intraportal hyperinsulinemic (2 mU ⋅ kg-1⋅ min-1) infusion was also initiated at this time. Glucose was clamped at hypoglycemic or euglycemic levels in the presence (H-AIC, n = 6; E-AIC, n = 6) or absence (H-SAL, n = 6; E-SAL, n = 6) of a portal venous 5-aminoimidazole-4-carboxamide-ribofuranoside (AICAR) infusion (1 mg ⋅ kg-1 ⋅ min-1) initiated at t = 60 min. In the presence of intraportal saline, glucose was infused into the vena cava to match glucose levels seen with intraportal AICAR. Glucagon remained fixed at basal levels, whereas insulin rose similarly in all groups. Glucose fell to 50 ± 2 mg/dl by t = 60 min in hypoglycemic groups and remained at 105 ± 3 mg/dl in euglycemic groups. Endogenous glucose production (Ra) was similarly suppressed among groups in the presence of euglycemia or hypoglycemia before t = 60 min and remained suppressed in the H-SAL and E-SAL groups. However, intraportal AICAR infusion stimulated Ra to increase by 2.5 ± 1.0 and 3.4 ± 0.4 mg ⋅ kg1 ⋅ min-1 in the E-AIC and H-AIC groups, respectively. Arteriovenous measurement of net hepatic glucose output showed similar results. AICAR stimulated hepatic glycogen to decrease by 5 ± 3 and 19 ± 5 mg/g tissue (P < 0.05) in the presence of euglycemia and hypoglycemia, respectively. AICAR significantly increased net hepatic lactate output in the presence of hypoglycemia. Thus, intraportal AICAR infusion caused marked stimulation of both hepatic glucose output and net hepatic glycogenolysis, even in the presence of high levels of physiological insulin. This stimulation of glucose output by AICAR was equally marked in the presence of both euglycemia and hypoglycemia. However, hypoglycemia amplified the net hepatic glycogenolytic response to AICAR by approximately fourfold. Diabetes 54:373-382, 2005 [ABSTRACT FROM AUTHOR]

Hyperinsulinemia during exercise in people with diabetes requiring exogenous insulin is a major clinical problem. The aim of this study was to assess the significance of portal vein versus arterial insulin to hepatic effects of hyperinsulinemia during exercise. Dogs had sampling (artery, portal vein, and hepatic vein) and infusion (vena cava and portal vein) catheters and flow probes (hepatic artery and portal vein) implanted >16 days before a study. Protocols consisted of equilibration (-130 to -30 min), basal (-30 to 0 min), and treadmill exercise (0-150 min) periods. Somatostatin was infused and glucagon and insulin were replaced in the portal vein to achieve basal arterial and portal vein levels at rest and simulated levels during the first 60 min of exercise. From 60 to 150 min of exercise, the simulated insulin infusion was sustained (C; n = 7), modified to selectively create a physiologic increment in arterial insulin (Pe; n = 7), or altered to increase arterial insulin as in Pe but with a concomitant increase in portal insulin (PePo; n = 7). Euglycemic clamps were performed in all studies. Portal and arterial insulin were 15 ± 2 and 4 ± 1 µU/ml (mean ± SE of all groups), respectively, at t = 60 min in all groups. Insulin levels were unchanged for the remainder of the exercise period in C. Arterial insulin was increased from 3 ± 1 to 14 ± 2 µU/ml, whereas portal insulin did not change in Pe after t = 60 min. Arterial insulin was increased from 3 ± 1 to 15 ± 2 µU/ml, and portal insulin was increased from 16 ± 3 to 33 ± 3 µU/ml in PePo after t = 60 min. Endogenous glucose production (Ra) rose similarly from basal during the first 60 min of exercise in all groups (mean ± SE of all groups was from 2.2 ± 0.1 to 6.8 ± 0.5 mg · kg-1 · min-1. The increase in Ra was sustained for the remainder of the exercise period in C. Ra was suppressed by ∼40%, but only after 60 min of hyperinsulinemia, and by ∼20% after 90 min of hyperinsulinemia in Pe. In contrast, the addition of portal venous hyperinsulinemia caused ∼90% suppression of Ra within 20 min and for the remainder of the experiment in PePo. Measurements of net hepatic glucose output were similar to Ra responses in all groups. Arterial free fatty acids (FFAs), a stimulus of Ra, were increased to 1,255 ± 258 µmol/l in C but were only 459 ± 67 and 312 ± 42 µmol/l in Pe and PePo, respectively, by 150 min of exercise. Thus, during exercise, the exquisite sensitivity of Ra to hyperinsulinemia is due entirely to portal venous hyperinsulinemia during the first 60 min, after which peripheral hyperinsulinemia may control ∼20-40%, possibly as a result of inhibition of the exercise-induced increase in FFA. [ABSTRACT FROM AUTHOR]

Adenine nucleotides define the cell energy status and sensitively regulate signaling pathways and enzymes that control nutrient flux. The liver plays a key role in energy metabolism as it is the site of energy producing and consuming pathways involved in not only cell, but whole-body metabolism. The aim was to assess in C57BL/6J mice (a) the role of adenine nucleotides during nutrient stress and diabetes, (b) whether adenine nucleotides potentially play a role in coordinating glucagun (GGN) action, and (c) whether changes in adenine nucleotides are consistent with a role in AMPK signaling. Hepatic energy charge (HEC) was calculated from adenine nucleotides [(ATP+0.5ADP)/ (ATP+ADP+AMP)] measured by HPLC. HEC decreased markedly from 0.72±0.02 in 5h fasted control mice to 0.22±0.01, 0.43±0.05, 0.20±0.02, and 0.33±0.22 in 5h fasted high-fat fed, 16h fasted, exercised, and streptozotocin-induced diabetic mice while the skeletal muscle energy charge (SEC) did not differ from the value in 5h fasted control mice (0.94±0.02). Elevated GGN is common to each of these conditions, thus we tested the role of this hormone using a novel phloridzin-glucose clamp technique (phlor-clamp). Wild type (WT) and GGN receptor knockout (KO) littermate mice were catheterized and infused with 10 ng/kg/min GGN (GGN) or saline (SAL) for 2h to in the conscious state while maintaining plasma glucose at basal (∼150 mg/dL). During the phlor-clamp, WT-GGN mice insulin and plasma glucose were unchanged despite increased GGN (51±8 to >1000 pg/mL) and HEC fell to 0.37±0.07. In WT-SAL, KO-SAL, and KO-GGN there were no changes in plasma glucose and insulin during the phlor-clamp and HEC was 0.66±0.02, 0.72±0.01, and 0.68±0.02. SEC was unaffected by GGN or genotype. AMPK activity increased 59% in WT-GGN compared to WT-SAL mice based on P-AMPK[Thr172]. In summary, (a) in contrast to muscle, liver experiences profound changes in energy status in response to nutrient stress and diabetes; (b) GGN receptor mediated processes play a role in regulation of adenine nucleotide metabolism; and (c) GGN potently activates AMPK in vivo and this may be secondary to changes in adenine nucleotides. The marked changes in energy status of the liver create a key role for adenine nucleotides in nutrient flux and should be considered in interpreting experiments designed to study liver. [ABSTRACT FROM AUTHOR]