Peripherally delivered hepatopreferential insulin analog insulin-406 mimics the hypoglycaemia-sparing effect of portal vein human insulin infusion in dogs.

AIMS: We previously quantified the hypoglycaemia-sparing effect of portal vs peripheral human insulin delivery. The current investigation aimed to determine whether a bioequivalent peripheral vein infusion of a hepatopreferential insulin analog, insulin-406, could similarly protect against hypoglycaemia. MATERIALS AND METHODS: Dogs received human insulin infusions into either the hepatic portal vein (PoHI, n = 7) or a peripheral vein (PeHI, n = 7) for 180 minutes at four-fold the basal secretion rate (6.6 pmol/kg/min) in a previous study. Insulin-406 (Pe406, n = 7) was peripherally infused at 6.0 pmol/kg/min, a rate determined to decrease plasma glucose by the same amount as with PoHI infusion during the first 60 minutes. Glucagon was fixed at basal concentrations, mimicking the diminished α-cell response seen in type 1 diabetes. RESULTS: Glucose dropped quickly with PeHI infusion, reaching 41 ± 3 mg/dL at 60 minutes, but more slowly with PoHI and Pe406 infusion (67 ± 2 and 72 ± 4 mg/dL, respectively; P < 0.01 vs PeHI for both). The hypoglycaemic nadir (c. 40 mg/dL) occurred at 60 minutes with PeHI infusion vs 120 minutes with PoHI and Pe406 infusion. ΔAUCepinephrine during the 180-minute insulin infusion period was two-fold higher with PeHI infusion compared with PoHI and Pe406 infusion. Glucose production (mg/kg/min) was least suppressed with PeHI infusion (Δ = 0.79 ± 0.33) and equally suppressed with PoHI and Pe406 infusion (Δ = 1.16 ± 0.21 and 1.18 ± 0.17, respectively; P = NS). Peak glucose utilization (mg/kg/min) was highest with PeHI infusion (4.94 ± 0.17) and less with PoHI and Pe406 infusion (3.58 ± 0.58 and 3.26 ± 0.08, respectively; P < 0.05 vs Pe for both). CONCLUSIONS: Peripheral infusion of hepatopreferential insulin can achieve a metabolic profile that closely mimics portal insulin delivery, which reduces the risk of hypoglycaemia compared with peripheral insulin infusion.

Chronic overeating impairs hepatic glucose uptake and disposition.

Dogs consuming a hypercaloric high-fat and -fructose diet (52 and 17% of total energy, respectively) or a diet high in either fructose or fat for 4 wk exhibited blunted net hepatic glucose uptake (NHGU) and glycogen deposition in response to hyperinsulinemia, hyperglycemia, and portal glucose delivery. The effect of a hypercaloric diet containing neither fructose nor excessive fat has not been examined. Dogs with an initial weight of ≈25 kg consumed a chow and meat diet (31% protein, 44% carbohydrate, and 26% fat) in weight-maintaining (CTR; n = 6) or excessive (Hkcal; n = 7) amounts for 4 wk (cumulative weight gain 0.0 ± 0.3 and 1.5 ± 0.5 kg, respectively, P < 0.05). They then underwent clamp studies with infusions of somatostatin and intraportal insulin (4× basal) and glucagon (basal). The hepatic glucose load was doubled with peripheral (Pe) glucose infusion for 90 min (P1) and intraportal glucose at 4 mg·kg(-1)·min(-1) plus Pe glucose for the final 90 min (P2). NHGU was blunted (P < 0.05) in Hkcal during both periods (mg·kg(-1)·min(-1); P1: 1.7 ± 0.2 vs. 0.3 ± 0.4; P2: 3.6 ± 0.3 vs. 2.3 ± 0.4, CTR vs. Hkcal, respectively). Terminal hepatic glucokinase catalytic activity was reduced nearly 50% in Hkcal vs. CTR (P < 0.05), although glucokinase protein did not differ between groups. In Hkcal vs. CTR, liver glycogen was reduced 27% (P < 0.05), with a 91% increase in glycogen phosphorylase activity (P < 0.05) but no significant difference in glycogen synthase activity. Thus, Hkcal impaired NHGU and glycogen synthesis compared with CTR, indicating that excessive energy intake, even if the diet is balanced and nutritious, negatively impacts hepatic glucose metabolism.

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

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

Biologic comparison of inhaled insulin formulations: Exubera™ and novel spray-dried engineered particles of dextran-10.

Inhaled peptides and proteins have promise for respiratory and systemic disease treatment. Engineered spray-dried powder formulations have been shown to stabilize peptides and proteins and optimize aerosol properties for pulmonary delivery. The current study was undertaken to investigate the in vitro and in vivo inhalation performance of a model spray-dried powder of insulin and dextran 10 in comparison to Exubera™. Dextrans are a class of glucans that are generally recognized as safe with optimum glass transition temperatures well suited for spray drying. A 70% insulin particle loading was prepared by formulating with 30% (w/v) dextran 10. Physical characterization revealed a "raisin like" particle. Both formulations were generated to produce a similar bimodal particle size distribution of less than 3.5 µm MMAD. Four female Beagle dogs were exposed to each powder in a crossover design. Similar presented and inhaled doses were achieved with each powder. Euglycemia was achieved in each dog prior and subsequent to dosing and blood samples were drawn out to 245 min post-exposure. Pharmacokinetic analyses of post-dose insulin levels were similar for both powders. Respective dextran 10-insulin and Exubera exposures were similar producing near identical area under the curve (AUC), 7,728 ± 1,516 and 6,237 ± 2,621; concentration maximums (C max), 126 and 121 (µU/mL), and concentration-time maximums, 20 and 14 min, respectively. These results suggest that dextran-10 and other dextrans may provide a novel path for formulating peptides and proteins for pulmonary delivery.

Effects of intraportal exenatide on hepatic glucose metabolism in the conscious dog.

Incretins improve glucose metabolism through multiple mechanisms. It remains unclear whether direct hepatic effects are an important part of exenatide's (Ex-4) acute action. Therefore, the objective of this study was to determine the effect of intraportal delivery of Ex-4 on hepatic glucose production and uptake. Fasted conscious dogs were studied during a hyperglycemic clamp in which glucose was infused into the hepatic portal vein. At the same time, portal saline (control; n = 8) or exenatide was infused at low (0.3 pmol·kg⁻¹·min⁻¹, Ex-4-low; n = 5) or high (0.9 pmol·kg⁻¹·min⁻¹, Ex-4-high; n = 8) rates. Arterial plasma glucose levels were maintained at 160 mg/dl during the experimental period. This required a greater rate of glucose infusion in the Ex-4-high group (1.5 ± 0.4, 2.0 ± 0.7, and 3.7 ± 0.7 mg·kg⁻¹·min⁻¹ between 30 and 240 min in the control, Ex-4-low, and Ex-4-high groups, respectively). Plasma insulin levels were elevated by Ex-4 (arterial: 4,745 ± 428, 5,710 ± 355, and 7,262 ± 1,053 µU/ml; hepatic sinusoidal: 14,679 ± 1,700, 15,341 ± 2,208, and 20,445 ± 4,020 µU/ml, 240 min, area under the curve), whereas the suppression of glucagon was nearly maximal in all groups. Although glucose utilization was greater during Ex-4 infusion (5.92 ± 0.53, 6.41 ± 0.57, and 8.12 ± 0.54 mg·kg⁻¹·min⁻¹), when indices of hepatic, muscle, and whole body glucose uptake were expressed relative to circulating insulin concentrations, there was no indication of insulin-independent effects of Ex-4. Thus, this study does not support the notion that Ex-4 generates acute changes in hepatic glucose metabolism through direct effects on the liver.

A high-fat, high-fructose diet accelerates nutrient absorption and impairs net hepatic glucose uptake in response to a mixed meal in partially pancreatectomized dogs.

The aim of this study was to elucidate the impact of a high-fat, high-fructose diet (HFFD; fat, 52%; fructose, 17%), in the presence of a partial (~65%) pancreatectomy (PPx), on the response of the liver and extrahepatic tissues to an orally administered, liquid mixed meal. Adult male dogs were fed either a nonpurified, canine control diet (CTR; fat, 26%; no fructose; n = 5) or a HFFD (n = 5) for 8 wk. Diets were provided in a quantity to maintain neutral or positive energy balance in CTR or HFFD, respectively. Dogs underwent a sham operation or PPx at wk 0, portal and hepatic vein catheterization at wk 6, and a mixed meal test at wk 8. Postprandial glucose concentrations were significantly greater in the HFFD group (14.5 ± 2.0 mmol/L) than in the CTR group (9.2 ± 0.5 mmol/L). Impaired glucose tolerance in HFFD was due in part to accelerated gastric emptying and glucose absorption, as indicated by a more rapid rise in arterial plasma acetaminophen and the rate of glucose output by the gut, respectively, in HFFD than in CTR. It was also attributable to lower net hepatic glucose uptake (NHGU) in the HFFD group (5.5 ± 3.9 µmol · kg(-1) · min(-1)) compared to the CTR group (26.6 ± 7.0 µmol · kg(-1) · min(-1)), resulting in lower hepatic glycogen synthesis (GSYN) in the HFFD group (10.8 ± 5.4 µmol · kg(-1) · min(-1)) than in the CTR group (30.4 ± 7.0 µmol · kg(-1) · min(-1)). HFFD also displayed aberrant suppression of lipolysis by insulin. In conclusion, HFFD feeding accelerates gastric emptying and diminishes NHGU and GSYN, thereby impairing glucose tolerance following a mixed meal challenge. These data reveal a constellation of deleterious metabolic consequences associated with consumption of a HFFD for 8 wk.

Chronic consumption of a high-fat/high-fructose diet renders the liver incapable of net hepatic glucose uptake.

The objective of this study was to assess the response of a large animal model to high dietary fat and fructose (HFFD). Three different metabolic assessments were performed during 13 wk of feeding an HFFD (n = 10) or chow control (CTR, n = 4) diet: oral glucose tolerance tests (OGTTs; baseline, 4 and 8 wk), hyperinsulinemic-euglycemic clamps (HIEGs; baseline and 10 wk) and hyperinsulinemic-hyperglycemic clamps (HIHGs, 13 wk). The ΔAUC for glucose during the OGTTs more than doubled after 4 and 8 wk of HFFD feeding, and the average glucose infusion rate required to maintain euglycemia during the HIEG clamps decreased by ≈30% after 10 wk of HFFD feeding. These changes did not occur in the CTR group. The HIHG clamps included experimental periods 1 (P1, 0-90 min) and 2 (P2, 90-180 min). During P1, somatostatin, basal intraportal glucagon, 4 × basal intraportal insulin, and peripheral glucose (to double the hepatic glucose load) were infused; during P2, glucose was also infused intraportally (4.0 mg·kg(-1)·min(-1)). Net hepatic glucose uptake during P1 and P2 was -0.4 ± 0.1 [output] and 0.2 ± 0.8 mg·kg(-1)·min(-1) in the HFFD group, respectively, and 1.8 ± 0.8 and 3.5 ± 1.0 mg·kg(-1)·min(-1) in the CTR group, respectively (P < 0.05 vs. HFFD during P1 and P2). Glycogen synthesis through the direct pathway was 0.5 ± 0.2 and 1.5 ± 0.4 mg·kg(-1)·min(-1) in the HFFD and CTR groups, respectively (P < 0.05 vs. HFFD). In conclusion, chronic consumption of an HFFD diminished the sensitivity of the liver to hormonal and glycemic cues and resulted in a marked impairment in NHGU and glycogen synthesis.

Effect of 11 beta-hydroxysteroid dehydrogenase-1 inhibition on hepatic glucose metabolism in the conscious dog.

Inactive cortisone is converted to active cortisol within the liver by 11 beta-hydroxysteroid dehydrogenase-1 (11 beta-HSD1), and impaired regulation of this process may be related to increased hepatic glucose production (HGP) in individuals with type 2 diabetes. The primary aim of this study was to investigate the effect of acute 11 beta-HSD1 inhibition on HGP and fat metabolism during insulin deficiency. Sixteen conscious, 42-h-fasted, lean, healthy dogs were studied. Somatostatin was infused to create insulin deficiency, and the animals were treated with a specific 11 beta-HSD1 inhibitor (compound 531) or placebo for 5 h. 11 beta-HSD1 inhibition completely suppressed hepatic cortisol production, and this attenuated the increase in HGP that occurred during insulin deficiency. PEPCK and glucose-6-phosphatase expression were decreased when 11 beta-HSD1 was inhibited, but gluconeogenic flux was unchanged, implying an effect on glycogenolysis. Since inhibition of hepatic cortisol production reduces HGP during insulin deficiency, 11 beta-HSD1 is a potential therapeutic target for the treatment of excess glucose production that occurs in diabetes.

Insulin's direct effects on the liver dominate the control of hepatic glucose production.

Insulin inhibits glucose production through both direct and indirect effects on the liver; however, considerable controversy exists regarding the relative importance of these effects. The first aim of this study was to determine which of these processes dominates the acute control of hepatic glucose production (HGP). Somatostatin and portal vein infusions of insulin and glucagon were used to clamp the pancreatic hormones at basal levels in the nondiabetic dog. After a basal sampling period, insulin infusion was switched from the portal vein to a peripheral vein. As a result, the arterial insulin level doubled and the hepatic sinusoidal insulin level was reduced by half. While the arterial plasma FFA level and net hepatic FFA uptake fell by 40-50%, net hepatic glucose output increased more than 2-fold and remained elevated compared with that in the control group. The second aim of this study was to determine the effect of a 4-fold rise in head insulin on HGP during peripheral hyperinsulinemia and hepatic insulin deficiency. Sensitivity of the liver was not enhanced by increased insulin delivery to the head. Thus, this study demonstrates that the direct effects of insulin dominate the acute regulation of HGP in the normal dog.

Inhaled insulin is associated with prolonged enhancement of glucose disposal in muscle and liver in the canine.

Diabetic patients treated with inhaled insulin exhibit reduced fasting plasma glucose levels. In dogs, insulin action in muscle is enhanced for as long as 3 h after insulin inhalation. This study was designed to determine whether this effect lasts for a prolonged duration such that it could explain the effect observed in diabetic patients. Human insulin was administered via inhalation (Exubera; n = 9) or infusion (Humulin R; n = 9) in dogs using an infusion algorithm that yielded matched plasma insulin kinetics between the two groups. Somatostatin was infused to prevent insulin secretion, and glucagon was infused to replace basal plasma levels of the hormone. Glucose was infused into the portal vein at 4 mg/kg/min and into a peripheral vein to maintain the arterial plasma glucose level at 160 mg/dl. Arterial and hepatic sinusoidal insulin and glucose levels were virtually identical in the two groups. Notwithstanding, glucose utilization was greater when insulin was administered by inhalation. At its peak, the peripheral glucose infusion rate was 4 mg/kg/min greater in the inhalation group, and a 50% difference between groups persisted over 8 h. Inhalation of insulin caused a greater increase in nonhepatic glucose uptake in the first 3 h after inhalation; thereafter, net hepatic glucose uptake was greater. Inhalation of insulin was associated with greater than expected (based on insulin levels) glucose disposal. This may explain the reduced fasting glucose concentrations observed in humans after administration of certain inhaled insulin formulations compared with subcutaneous insulin.

Inhalation of human insulin (exubera) augments the efficiency of muscle glucose uptake in vivo.

This study assessed the site of increased glucose uptake resulting from insulin inhalation, quantified its effect under steady-state glucose concentrations, and identified the time to onset of effect. Human insulin was administered to 13 beagles via inhalation (Exubera [insulin human (rDNA origin)] Inhalation Powder; n = 7) or infusion into the inferior vena cava (Humulin R; n = 6) using an algorithm to match plasma insulin levels and kinetics for both groups. Somatostatin and glucagon were infused. Glucose was delivered into the portal vein (4 mg x kg(-1) x min(-1)) and a peripheral vein, as needed, to maintain arterial plasma glucose levels at 180 mg/dl. Hepatic exposure to insulin and glucose and liver glucose uptake were similar in both groups. Despite comparable arterial insulin and glucose levels, hind-limb glucose uptake increased 2.4-fold after inhalation compared with infusion due to increased muscle glucose uptake. Glucose infusion rate, nonhepatic glucose uptake, and tracer-determined glucose disposal were about twice as great compared with intravenous insulin. The effect appeared after 1 h, persisting at least as long as arterial insulin levels remained above basal. Pulmonary administration of insulin increases nonhepatic glucose uptake compared with infusion, and skeletal muscle is the likely site of that effect.

The effect of vagal cooling on canine hepatic glucose metabolism in the presence of hyperglycemia of peripheral origin.

We examined the role of vagus nerves in the transmission of the portal glucose signal in conscious dogs. At time 0, somatostatin infusion was started along with intraportal insulin and glucagon at 4-fold basal and basal rates, respectively. Glucose was infused via a peripheral vein to create hyperglycemia ( approximately 2 fold basal). At t = 90, hollow coils around the vagus nerves were perfused with -10 degrees C or 37 degrees C solution in the vagally cooled (COOL) and sham-cooled (SHAM) groups, respectively (n = 6 per group). Effectiveness of vagal blockade was demonstrated by increase in heart rate during perfusion in the COOL vs SHAM groups (183 +/- 3 vs 102 +/- 5 beats per minute, respectively) and by prolapse of the third eyelid in the COOL group. Arterial plasma insulin (22 +/- 2 and 24 +/- 3 micro U/mL) and glucagon (37 +/- 5 and 40 +/- 4 pg/mL) concentrations did not change significantly between the first experimental period and the coil perfusion period in either the SHAM or COOL group, respectively. The hepatic glucose load throughout the entire experiment was 46 +/- 1 and 50 +/- 2 mg . kg(-1) . min(-1) in the SHAM and COOL groups, respectively. Net hepatic glucose uptake (NHGU) did not differ in the SHAM and COOL groups before (2.2 +/- 0.5 and 2.9 +/- 0.8 mg . kg(-1) . min(-1), respectively) or during the cooling period (3.0 +/- 0.5 and 3.4 +/- 0.6 mg . kg(-1) . min(-1), respectively). Likewise, net hepatic glucose fractional extraction and nonhepatic glucose uptake and clearance were not different between groups during coil perfusion. Interruption of vagal signaling in the presence of hyperinsulinemia and hyperglycemia resulting from peripheral glucose infusion did not affect NHGU, further supporting our previous suggestion that vagal input to the liver is not a primary determinant of NHGU.

Inhalation of insulin (Exubera) is associated with augmented disposal of portally infused glucose in dogs.

The results of the present study, using the conscious beagle dog, demonstrate that inhaled insulin (INH; Exubera) provides better glycemic control during an intraportal glucose load than identical insulin levels induced by insulin (Humulin) infusion into the inferior vena cava (IVC). In the INH group (n = 13), portal glucose infusion caused arterial plasma glucose to rise transiently (152 +/- 9 mg/dl), before it returned to baseline (65 min) for the next 2 h. Net hepatic glucose uptake was minimal, whereas nonhepatic uptake rose to 12.5 +/- 0.5 mg x kg(-1) x min(-1) (65 min). In the IVC group (n = 9), arterial glucose rose rapidly (172 +/- 6 mg/dl) and transiently fell to 135 +/- 13 mg/dl (65 min) before returning to 165 +/- 15 mg/dl (125 min). Plasma glucose excursions and hepatic glucose uptake were much greater in the IVC group, whereas nonhepatic uptake was markedly less (8.6 +/- 0.9 mg x kg(-1) x min(-1); 65 min). Insulin kinetics and areas under the curve were identical in both groups. These data suggest that inhalation of Exubera results in a unique action on nonhepatic glucose clearance.

Selective antagonism of the hepatic glucocorticoid receptor reduces hepatic glucose production.

A liver-selective glucocorticoid (GC) receptor antagonist (A-348441) was used to determine the effect of reduced hepatic GC signaling on hepatic glucose production. Fasted conscious dogs were studied in the presence (GRA, n = 6) or absence (CON, n = 6) of the intraduodenally administered GC receptor antagonist (100 mg/kg). All dogs were maintained on a pancreatic clamp and in a euglycemic state for 7 hours to ensure that any changes in glucose metabolism were the direct result of the effects of A-348441, which was given at the start of a 5-hour experimental period. In the GRA group, the arterial plasma insulin level was 4.6 +/- 0.7 and 4.8 +/- 0.6 microU/mL during the basal and the last 30 minutes of the experimental periods, respectively. In the CON group, it was 4.0 +/- 0.3 and 4.5 +/- 0.5 microU/mL in the 2 periods, respectively. The arterial plasma glucagon level was 49 +/- 4 and 46 +/- 3 pg/mL in the 2 periods in the GRA group, and 45 +/- 3 and 42 +/- 3 pg/mL in the CON group. Net hepatic glucose balance progressively decreased in the GRA group from 1.31 +/- 0.18 to 0.49 +/- 0.30 mg/kg per minute, whereas in the CON group, net hepatic glucose balance was 1.17 +/- 0.09 and 1.43 +/- 0.18 mg/kg per minute during the basal and last 30 minutes of the experimental periods, respectively. No significant change in net renal or gut glucose balance or nonhepatic glucose uptake was observed in either group. This study demonstrates that the GC receptor plays an important role in the regulation of basal hepatic glucose production and represents a significant potential therapeutic target.

Hepatic glycogen can regulate hypoglycemic counterregulation via a liver-brain axis.

Liver glycogen is important for the counterregulation of hypoglycemia and is reduced in individuals with type 1 diabetes (T1D). Here, we examined the effect of varying hepatic glycogen content on the counterregulatory response to low blood sugar in dogs. During the first 4 hours of each study, hepatic glycogen was increased by augmenting hepatic glucose uptake using hyperglycemia and a low-dose intraportal fructose infusion. After hepatic glycogen levels were increased, animals underwent a 2-hour control period with no fructose infusion followed by a 2-hour hyperinsulinemic/hypoglycemic clamp. Compared with control treatment, fructose infusion caused a large increase in liver glycogen that markedly elevated the response of epinephrine and glucagon to a given hypoglycemia and increased net hepatic glucose output (NHGO). Moreover, prior denervation of the liver abolished the improved counterregulatory responses that resulted from increased liver glycogen content. When hepatic glycogen content was lowered, glucagon and NHGO responses to insulin-induced hypoglycemia were reduced. We conclude that there is a liver-brain counterregulatory axis that is responsive to liver glycogen content. It remains to be determined whether the risk of iatrogenic hypoglycemia in T1D humans could be lessened by targeting metabolic pathway(s) associated with hepatic glycogen repletion.

Insulin sensitively controls the glucagon response to mild hypoglycemia in the dog.

In the present study, we examined how the arterial insulin level alters the alpha-cell response to a fall in plasma glucose in the conscious overnight fasted dog. Each study consisted of an equilibration (-140 to -40 min), a control (-40 to 0 min), and a test period (0 to 180 min), during which BAY R 3401 (10 mg/kg), a glycogen phosphorylase inhibitor, was administered orally to decrease glucose output in each of four groups (n = 5). In group 1, saline was infused. In group 2, insulin was infused peripherally (3.6 pmol. kg(- 1). min(-1)), and the arterial plasma glucose level was clamped to the level seen in group 1. In group 3, saline was infused, and euglycemia was maintained. In group 4, insulin (3.6 pmol. kg(-1). min(-1)) was given, and euglycemia was maintained by glucose infusion. In group 1, drug administration decreased the arterial plasma glucose level (mmol/l) from 5.8 +/- 0.2 (basal) to 5.2 +/- 0.3 and 4.4 +/- 0.3 by 30 and 90 min, respectively (P < 0.01). Arterial plasma insulin levels (pmol/l) and the hepatic portal-arterial difference in plasma insulin (pmol/l) decreased (P < 0.01) from 78 +/- 18 and 90 +/- 24 to 24 +/- 6 and 12 +/- 6 over the first 30 min of the test period. The arterial glucagon levels (ng/l) and the hepatic portal-arterial difference in plasma glucagon (ng/l) rose from 43 +/- 5 and 5 +/- 2 to 51 +/- 5 and 10 +/- 5 by 30 min (P < 0.05) and to 79 +/- 16 and 31 +/- 15 (P < 0.05) by 90 min, respectively. In group 2, in response to insulin infusion, arterial insulin (pmol/l) was elevated from 48 +/- 6 to 132 +/- 6 to an average of 156 +/- 6. The hepatic portal-arterial difference in plasma insulin was eliminated, indicating a complete inhibition of endogenous insulin release. The arterial glucagon level (ng/l) and the hepatic portal-arterial difference in plasma glucagon (ng/l) did not rise significantly (40 +/- 5 and 7 +/- 4 at basal, 44 +/- 4 and 9 +/- 4 at 90 min, and 44 +/- 8 and 15 +/- 7 at 180 min). In group 3, when euglycemia was maintained, the insulin and glucagon levels and the hepatic portal-arterial difference remained constant. In group 4, the arterial plasma glucose level remained basal (5.9 +/- 1.1 mmol/l) throughout, whereas insulin infusion increased the arterial insulin level to an average of 138 +/- 6 pmol/l. The hepatic portal-arterial difference in plasma insulin was again eliminated. Arterial glucagon level (ng/l) and the hepatic portal-arterial difference in plasma glucagon (ng/l) did not change significantly (43 +/- 2 and 9 +/- 2 at basal, 39 +/- 3 and 9 +/- 2 at 90 min, and 37 +/- 3 and 7 +/- 2 at 180 min). Thus, a difference of approximately 120 pmol/l in arterial insulin completely abolished the response of the alpha-cell to mild hypoglycemia.

Direct and indirect effects of insulin on glucose uptake and storage by the liver.

Studies were conducted in conscious 42-h-fasted dogs to determine how much of insulin's effect on hepatic glucose uptake arises from its direct hepatic action versus its indirect (extrahepatic) action. Each experiment consisted of equilibration, basal, and experimental periods. During the latter, somatostatin, basal intraportal glucagon, portal glucose (21.3 micromol x kg(-1) x min(-1)), and peripheral glucose (to double the hepatic glucose load) were infused. During the experimental period, insulin was infused intraportally at a basal rate (BI, n = 6), at a fourfold basal rate (PoI, n = 6), or via a peripheral vein to create a selective increase in the arterial insulin level similar to that in PoI (PeI, n = 6). Arterial and hepatic sinusoidal insulin levels (in picomoles per liter) during the experimental period were 31 +/- 5 and 113 +/- 15 in BI, 97 +/- 11 and 394 +/- 66 in PoI, and 111 +/- 13 and 96 +/- 9 in PeI, respectively. Net hepatic glucose uptake (NHGU) averaged 7.0 +/- 1.1 micromol x kg(-1) x min(-1), 15.7 +/- 2.7 micromol x kg(-1) x min(-1) (P < 0.05 vs. BI), and 12.0 +/- 2.4 micromol x kg(-1) x min(-1) (P < 0.05 vs. BI) in BI, PoI, and PeI, respectively. Net hepatic carbon retention was 4.4 +/- 1.2 micromol glucose equivalents. kg(-1) x min(-1), 12.3 +/- 2.5 micromol glucose equivalents x kg(-1) x min(-1) (P < 0.05 vs. BI, P < 0.05 vs. PeI), and 7.1 +/- 1.0 micromol glucose equivalents x kg(-1) x min(-1) (P < 0.05 vs. BI) in BI, PoI, and PeI, respectively. Both direct and indirect insulin actions increase NHGU, but the rise in hepatic sinusoidal insulin appears critical for efficient storage of glucose as hepatic glycogen.

Portal serotonin infusion and glucose disposal in conscious dogs.

Whether serotonin (5-hydroxytryptamine [5-HT]) enhances net hepatic glucose uptake (NHGU) during glucose infusion was examined in conscious 42-h-fasted dogs, using arteriovenous difference and tracer ([3-(3)H]glucose) techniques. Experiments consisted of equilibration (-120 to -30 min), basal (-30 to 0 min), and experimental (0-390 min) periods. During the experimental period, somatostatin, fourfold basal intraportal insulin, basal intraportal glucagon, and peripheral glucose (to double the hepatic glucose load) were infused. In one group of dogs (SER; n = 8), saline was infused intraportally from 0 to 90 min (P1), and 5-HT was infused intraportally at 10, 20, and 40 microg.kg(-1).min(-1) from 90 to 150 (P2), 150 to 210 (P3), and 210 to 270 (P4) min, respectively. In the other group (SAL; n = 8), saline was infused intraportally from 0 to 270 min. NHGU in SAL was 12.4 +/- 2.3, 14.9 +/- 2.7, 13.4 +/- 2.1, and 15.1 +/- 1.8 micromol.kg(-1).min(-1) in P1 to P4, respectively, whereas NHGU in SER averaged 13.2 +/- 3.0, 16.4 +/- 2.4, 19.0 +/- 2.4 (P < 0.05 vs. SAL), and 22.0 +/- 2.9 micromol.kg(-1).min(-1) (P < 0.05 vs. SAL). Nonhepatic glucose uptake ( micromol.kg(-1).min(-1)) in SAL was 31.7 +/- 4.9, 43.9 +/- 5.1, 55.1 +/- 5.6, and 66.2 +/- 8.6 during P1 to P4, respectively, whereas in SER, the corresponding values were 26.1 +/- 5.7, 31.6 +/- 9.4, 35.1 +/- 7.6 (P < 0.05 vs. SAL), and 34.7 +/- 7.7 (P < 0.05 vs. SAL). Intraportal 5-HT enhances NHGU but blunts nonhepatic glucose uptake, raising the possibility that hepatic-targeted 5-HT or 5-HT receptor agonists might reduce postprandial hyperglycemia.

Effects of insulin deficiency or excess on hepatic gluconeogenic flux during glycogenolytic inhibition in the conscious dog.

The direct acute effects of insulin on the regulation of hepatic gluconeogenic flux to glucose-6-phosphate (G6P) in vivo may be masked by the hormone's effects on net hepatic glycogenolytic flux and the resulting changes in glycolysis. To investigate this possibility, we used a glycogen phosphorylase inhibitor (BAY R3401) to inhibit glycogen breakdown in the overnight-fasted dog, and the effects of complete insulin deficiency or a fourfold rise in the plasma insulin level were assessed during a 5-h experimental period. Hormone levels were controlled using somatostatin with portal insulin and glucagon infusion. After the control period, plasma insulin infusion 1) was discontinued, creating insulin deficiency; 2) increased fourfold; or 3) was continued at the basal rate. During insulin deficiency, glucose production and the plasma level and net hepatic uptake of nonesterified free fatty acids increased, whereas during hyperinsulinemia they decreased. Net hepatic lactate uptake increased sixfold during insulin deficiency and 2.5-fold during hyperinsulinemia. Net hepatic gluconeogenic flux increased more than fourfold during insulin deficiency but was not reduced by hyperinsulinemia. We conclude that in the absence of appreciable glycogen breakdown, an acute gluconeogenic effect of hypoinsulinemia becomes manifest, whereas inhibition of the process by a physiologic rise in insulin was not evident.

Inhalation of insulin in dogs: assessment of insulin levels and comparison to subcutaneous injection.

Pulmonary insulin delivery is being developed as a more acceptable alternative to conventional subcutaneous administration. In 15 healthy Beagle dogs (average weight 9.3 kg), we compared insulin distribution in arterial, deep venous, and hepatic portal circulation. Dogs received 0.36 units/kg s.c. regular human insulin (n = 6) or 1 mg (2.8 units/kg) or 2 mg (5.6 units/kg) dry-powder human inhaled insulin (n = 3 and 6, respectively). Postinhalation of inhaled insulin (1 or 2 mg), arterial insulin levels quickly rose to a maximum of 55 +/- 6 or 92 +/- 9 microU/ml, respectively, declining to typical fasting levels by 3 h. Portal levels were lower than arterial levels at both doses, while deep venous levels were intermediate to arterial and portal levels. In contrast, subcutaneous insulin was associated with a delayed and lower peak arterial concentration (55 +/- 8 microU/ml at 64 min), requiring 6 h to return to baseline. Peak portal levels for subcutaneous insulin were comparable to those for 1 mg and significantly less than those for 2 mg inhaled insulin, although portal area under the curve (AUC) was comparable for the subcutaneous and 2-mg groups. The highest insulin levels with subcutaneous administration were seen in the deep venous circulation. Interestingly, the amount of glucose required for maintaining euglycemia was highest with 2 mg inhaled insulin. We conclude that plasma insulin AUC for the arterial insulin level (muscle) and hepatic sinusoidal insulin level (liver) is comparable for 2 mg inhaled insulin and 0.36 units/kg subcutaneous insulin. In addition, arterial peak concentration following insulin inhalation is two times greater than subcutaneous injection; however, the insulin is present in the circulation for half the time.