Free fatty acids impair hepatic insulin extraction in vivo.

Hyperinsulinemia is a common finding in obesity and results from insulin hypersecretion and impaired hepatic insulin extraction. In vitro studies have shown that free fatty acids (FFAs), which are often elevated in obesity, can impair insulin binding and degradation in isolated rat hepatocytes. To investigate whether FFAs impair hepatic insulin extraction (E(H)) in vivo, either saline (SAL) or 10% Intralipid (0.03 ml x kg(-1) x min(-1)) plus heparin (0.44 U x kg(-1) x min(-1)) (IH) was infused into normal dogs to elevate FFA levels. Insulin was infused intraportally at 18 pmol x kg(-1) x min(-1) for 150 min (period A, high insulin dose), and then at 2.4 pmol x kg(-1) x min(-1) for another 150 min (period B, low insulin dose). After the low portal insulin dose, additional insulin was infused peripherally at 8.4 pmol x kg(-1) x min(-1) for 120 min (period C) to assess the clearance of insulin from the peripheral plasma. In 16 paired experiments, FFA levels were 1,085 +/- 167, 1,491 +/- 240, 1,159 +/- 221 micromol/l (IH) and 221 +/- 44, 329 +/- 72, 176 +/- 44 micromol/l (SAL) in periods A, B, and C, respectively. Peripheral insulin levels were greater with IH (P < 0.001) than with SAL in all periods (1,620 +/- 114, 126 +/- 12, 1,050 +/- 72 pmol/l for IH vs. 1,344 +/- 168, 96 +/- 4.2, 882 +/- 60 pmol/l for SAL). Glucose clearance was impaired by IH in all periods (P < 0.05), whereas glucose production was slightly increased by IH during period B. Peripheral insulin clearance (Cl) and E(H) were calculated from the insulin infusion rate and insulin concentration data in each period by taking into account the nonlinearity of insulin kinetics. Cl was lower (P < 0.01) with IH (9.6 +/- 0.6, 12.0 +/- 0.9, 10.2 +/- 0.6 ml x kg(-1) x min(-1)) than with SAL (11.2 +/- 1, 13.6 +/- 0.7, 11.9 +/- 0.9 ml x kg(-1) x min(-1)) in periods A, B, and C. E(H) was also lower (P < 0.05) with IH (25 +/- 4, 40 +/- 5, 32 +/- 5%) than with SAL (30 +/- 2.8, 47 +/- 3, 38 +/- 3%). We conclude that FFAs can impair hepatic insulin extraction in vivo at high and low insulin levels, an effect that may contribute to the peripheral hyperinsulinemia of obesity.

Glucagon-like peptide 1 increases insulin sensitivity in depancreatized dogs.

To determine whether glucagon-like peptide (GLP)-1 increases insulin sensitivity in addition to stimulating insulin secretion, we studied totally depancreatized dogs to eliminate GLP-1's incretin effect. Somatostatin was infused (0.8 microg x kg(-1) x min(-1)) to inhibit extrapancreatic glucagon in dogs, and basal glucagon was restored by intraportal infusion (0.65 ng x kg(-1) x min(-1)). To simulate the residual intraportal insulin secretion in type 2 diabetes, basal intraportal insulin infusion was given to obtain plasma glucose concentrations of approximately 10 mmol/l. Glucose was clamped at this level for the remainder of the experiment, which included peripheral insulin infusion (high dose, 5.4 pmol x kg(-1) x min(-1), or low dose, 0.75 pmol x kg(-1) x min(-1)) with or without GLP-1(7-36) amide (1.5 pmol x kg(-1) x min(-1)). Glucose production and utilization were measured with 3-[3H]glucose, using radiolabeled glucose infusates. In 12 paired experiments with six dogs at the high insulin dose, GLP-1 infusion resulted in higher glucose requirements than saline (60.9+/-11.0 vs. 43.6+/-8.3 micromol x kg(-1) x min(-1), P< 0.001), because of greater glucose utilization (72.6+/-11.0 vs. 56.8+/-9.7 micromol x kg(-1) x min(-1), P<0.001), whereas the suppression of glucose production was not affected by GLP-1. Free fatty acids (FFAs) were significantly lower with GLP-1 than saline (375.3+/-103.0 vs. 524.4+/-101.1 micromol/l, P<0.01), as was glycerol (77.9+/-17.5 vs. 125.6+/-51.8 micromol/l, P<0.05). GLP-1 receptor gene expression was found using reverse transcriptase-polymerase chain reaction of poly(A)-selected RNA in muscle and adipose tissue, but not in liver. Low levels of GLP-1 receptor gene expression were also found in adipose tissue using Northern blotting. In 10 paired experiments with five dogs at the low insulin dose, GLP-1 infusion did not affect glucose utilization or FFA and glycerol suppression when compared with saline, suggesting that GLP-1's effect on insulin action was dependent on the insulin dose. In conclusion, in depancreatized dogs, GLP-1 potentiates insulin-stimulated glucose utilization, an effect that might be contributed in part by GLP-1 potentiation of insulin's antilipolytic action.

Direct and indirect effects of insulin in suppressing glucose production in depancreatized dogs: role of glucagon.

We have previously shown that during glucose clamps in moderately hyperglycemic depancreatized dogs: 1) peripheral insulin infusion, resulting in greater systemic insulinemia and greater suppression of glucagon than equidose portal infusion, inhibited glucose production (GP) to a greater extent; and 2) portal and half-dose peripheral infusions, resulting in matched peripheral insulinemia and similar suppression of glucagon, inhibited GP equally. These findings are consistent with an indirect effect of insulin in suppressing GP in diabetic dogs, which might be partly mediated by the differential suppression of glucagon. To address this question, we performed the experimental protocols of the previous study under conditions of constant glucagon levels (approximately 550 ng/liter), achieved by a high rate portal glucagon infusion (5 ng/kg.min). As in the previous study (basal glucagon levels, approximately 170 ng/liter), we used depancreatized dogs and assessed GP with HPLC-purified [6(-3)H]glucose. After obtaining constant basal hyperglycemia (approximately 10 mM) with portal infusions of insulin (4.8 +/- 0.5 pmol/kg.min) and glucagon, an additional infusion of insulin was administered for 180 min, either portally (portal; n = 7) or peripherally (peripheral; n = 8) at the same rate (5.4 pmol/kg.min) or at half that rate peripherally (1/2 periph; n = 5). Plasma glucose and glucose specific activities were clamped at basal levels. Systemic insulin levels increased by 215 +/- 16,310 +/- 26, and 184 +/- 15 pM, and estimated hepatic insulin levels increased by 398 +/- 20, 310 +/- 26, and 184 +/- 15 pM with portal, peripheral, and 1/2 periph, respectively. GP was suppressed to the same extent with portal and peripheral (53 +/- 6% and 50 +/- 6%), but less with 1/2 periph (35 +/- 5%). FFA levels were suppressed to a greater extent with peripheral than portal or 1/2 periph, whereas the responses of lactate alanine and glycerol to insulin infusion were similar in the three groups. Thus, in the present report, unlike in our previous study, 1) suppression of GP was proportional to the hepatic insulin levels; and 2) systemic insulin levels did not dominate suppression of GP. We, therefore, conclude that in hyperglycemic depancreatized dogs 1) glucagon, at concentrations seen in poorly controlled diabetes, can unmask a direct effect of hepatic insulin levels on GP; and 2) the suppression of glucagon may play a role in the peripheral effect of exogenously delivered insulin on GP. This is the first in vivo study to show that the main direct effect of insulin on the liver is to counteract the effect of glucagon.

Determinants of glucose turnover in the pathophysiology of diabetes: an in vivo analysis in diabetic dogs.

Hyperglycaemia in diabetes results from a combination of increased hepatic glucose production and decreased metabolic clearance of glucose. Our report summarizes recent work conducted in our laboratory to investigate the regulatory factors involved in the control of glucose turnover in diabetes. The action of insulin, both directly and indirectly, in regulating glucose turnover in diabetic dogs is considered. 1) In the depancreatized diabetic dog, peripheral rather than portal insulin levels determine the suppression of hepatic glucose production via indirect mechanisms such as limiting, precursors for gluconeogenesis and/or inhibiting glucagon secretion. 2) The differential effects of insulin and insulin-like growth factor I on glucose turnover may be dependent on a decline in glycaemia since previously observed differential effects on glucose turnover were masked under conditions of clamped hyperglycaemia in the depancreatized dog. 3) In a paradoxical dichotomous fashion, hyperglycaemia both contributes to, and compensates for, defective glucose clearance in diabetes. Acute restoration of euglycaemia significantly improves glucose clearance at rest and normalizes the exercise-induced increment in clearance in alloxan-diabetic dogs. 4) Our model of centrally-induced stress also shows that an increase in glucose utilization and clearance is largely independent of changes in insulin and that the combined effects of catecholamines and glucagon are responsible for increasing glucose production.

Insulin acutely suppresses glucose production by both peripheral and hepatic effects in normal dogs.

To determine whether the predominant effect of insulin in suppressing tracer-determined glucose production (Ra) is hepatic or peripheral, we infused insulin peripherally (PER) and portally (POR) at both low (0.75 pmol.kg-1.min-1) and high physiological rates (2.7 pmol.kg-1.min-1) during euglycemic clamps in normal dogs. We also infused insulin peripherally at one-half these rates (1/2 PER) to match the peripheral insulin levels in POR and thus obtain a selective POR vs. 1/2 PER difference in hepatic insulin levels. At the high-rate insulin infusion, peripheral insulin levels were greatest with PER (PER = 212 +/- 10 pM, n = 5; POR = 119 +/- 5 pM, n = 6; 1/2 PER = 122 +/- 5 pM, n = 6). Calculated hepatic insulin levels were greatest with POR (POR = 227 +/- 13 pM, PER = 206 +/- 19 pM, 1/2 PER = 123 +/- 8 pM). High-dose PER yielded a greater suppression of Ra than POR (79 +/- 18 vs. 56 +/- 6%, P < .001). Ra was only suppressed by 45 +/- 6% with 1/2 PER (P < 0.01 vs. POR on 6 paired experiments). Free fatty acid (FFA) was suppressed by 57 +/- 8% with PER and only by 33 +/- 5 and 37 +/- 2% with POR and 1/2 PER, respectively. The low-dose PER and POR yielded an equal Ra suppression (PER = 46 +/- 9%, POR = 43 +/- 4%). Only 1/2 PER was associated with a lower suppression of Ra (36 +/- 8, P < 0.05 vs. POR). FFA showed similar suppression in all three groups (approximately 25%). Using both insulin infusion rates, the percent Ra suppression per unit difference in peripheral insulin was approximately twofold greater than that per unit difference in hepatic insulin. These results suggest that, during euglycemic clamps without somatostatin in normal dogs, Ra suppression is mediated by both peripheral and hepatic effects of insulin and that peripheral insulin, at least at high physiological infusion rates, is more potent than hepatic insulin in suppressing Ra.