Elevated plasma non-esterified fatty acid levels and insulin secretion in non-diabetic relatives of type 2 diabetic patients

OBJECTIVE: High non-esterified fatty acid (NEFA) levels impair glucose-stimulated insulin secretion from islets derived from non-diabetic Zucker rats that are genetically predisposed to diabetes. We therefore examined the effect of elevated plasma NEFA levels on insulin secretion in non-diabetic first-degree relatives of type 2 diabetic patients who are at increased risk of developing diabetes. SUBJECTS AND STUDY DESIGN: Normal glucose tolerant relatives (n = 9) and control subjects with no family history of diabetes were pair-matched for age, sex, body mass index (BMI), insulin sensitivity and early insulin response during an oral glucose tolerance test (OGTT). Plasma NEFA levels were raised from 0 to 340 minutes by the infusion of 20% Intralipid and heparin. From 180 minutes, insulin secretion rates (IRSs) were assessed by stepped low-dose glucose infusion. RESULTS: The mean (geometric mean +/- 95% CI) NEFA levels were comparable between relatives and control subjects (2.7 [2.1-3.6] and 2.1 [1.7-2.7] mmol/l, paired analysis, NS). Similarly, plasma glucose levels achieved at each glucose infusion step were comparable between the groups. However, there were no significant differences between the groups for ISR throughout the study. CONCLUSIONS: Sustained elevation of plasma non-esterified fatty acid levels does not decrease insulin secretion in non-diabetic relatives of type 2 diabetic patients, and is therefore unlikely to be important in the development of the impaired pancreatic beta-cell function in type 2 diabetes mellitus.     

Effect of increased plasma non-esterified fatty acids (NEFAs) on arginine-stimulated insulin secretion in obese humans

Aims/hypothesis: We have shown previously that the increase of plasma non-esterified fatty acids for 48 h results in decreased glucose-stimulated insulin secretion in lean and non-diabetic obese subjects. It is currently not known if a prolonged increase in non-esterified fatty acids also impairs the insulin secretory response to non-glucose secretagogues. Methods: Heparin and intralipid (to increase plasma non-esterified fatty acid concentrations by about two- to fourfold) or normal saline was infused intravenously for 48 h in 14 non-diabetic obese subjects. On the third day in both studies, insulin, C-peptide, proinsulin, and insulin secretion rate were assessed in response to an intravenous arginine infusion at fasting glucose concentration and a second arginine infusion after a 60-min 11 mmol/l hyperglycaemic clamp. Results: There were no significant differences detected in acute (5 min) or total (90 min) arginine-stimulated C-peptide or insulin secretion response in the heparin-intralipid study compared with the control group at fasting glucose or during hyperglycaemia. Conclusion/interpretation: We have shown that a prolonged increase in plasma NEFA does not blunt arginine-stimulated insulin secretion or plasma insulin concentrations in non-diabetic obese subjects. These findings suggest that the previously demonstrated NEFA-induced impairment in insulin secretory response to glucose cannot be generalized for non-glucose secretagogues. [Diabetologia (2001) 44: 1989–1997] Received: 27 November 2001 and in revised form: 2 August 2001     

The impact of acute elevation of non-esterified fatty acids on insulin sensitivity and secretion in women with former gestational diabetes

OBJECTIVES: Elevations in non-esterified fatty acids (NEFA) have been shown to decrease insulin action and secretion, and are a risk factor for the development of Type 2 diabetes. As women who have had gestational diabetes (GDM) are at increased risk of diabetes, we examined the effect of an acute elevation of NEFA on insulin secretion and action in these women. PATIENTS AND DESIGN: Nineteen women with recent former GDM and 19 age- and BMI-matched postpartum healthy control subjects underwent a 40-min intravenous glucose tolerance test, with and without a preceding 2-h infusion of 20% Intralipid. Insulin action was assessed by glucose disappearance (Kg) and insulin sensitivity (SI); insulin secretion by first phase insulin release (FPIR) and disposition index (DI). RESULTS: NEFA levels were similarly elevated in both groups by the Intralipid infusion (up to 1.140 +/- 0.03 mm). As expected, the lipid infusion significantly reduced Kg (2.15 +/- 0.13 vs. 1.69 +/- 0.09/min, P < 0.001) and SI (3.14 +/- 0.28 vs. 2.13 +/- 0.17/min/mUl/min, P < 0.001) in all subjects, and these were significant within the GDM and control subgroups. FPIR was elevated in the Intralipid study in the total group of women (4.50 +/- 0.50 vs. 5.02 +/- 0.53, P = 0.02), but DI was significantly reduced (12.13 +/- 1.1 vs. 8.83 +/- 0.7, P < 0.001). There was no significant difference, however, in the absolute or percentage change in Kg, SI or FPIR with lipid infusion between the GDM and control groups. GDM status was not a predictor of the response of Kg, SI or FPIR to lipid infusion, rather, adiposity (% fat), average fasting NEFA levels and basal disposition index were associated. CONCLUSION: These data suggest that women with former gestational diabetes, in contrast to other prediabetic states, are not more susceptible to the deleterious effects of an acute elevation in nonesterified fatty acids than matched control subjects.     

The effects of a low-dose intravenous insulin infusion upon plasma glucose and non-esterified fatty acid levels in very obese and non-obese human subjects

Summary After an overnight fast, the effects of a 30-min low-dose intravenous insulin infusion (2.6 units/h) upon plasma glucose and non-esterified fatty acids were compared in 29 very obese patients and 17 nonobese controls. The dose of insulin was chosen so as to have its sole or predominant hypoglycaemic effect upon hepatic glucose release. The proportional fall from basal values at 30 min of both plasma glucose and non-esterified fatty acids was significantly greater in the controls and there was no difference between males and females. In the controls the fall in plasma glucose and non-esterified fatty acids was significantly and inversely correlated with the basal plasma insulin level. Neither index of insulin sensitivity was significantly related with the basal plasma insulin in the obese subjects. Weight loss in the obese subjects led to increased insulin sensitivity; in particular, the degree of change in insulin-induced nonesterified fatty acids was significantly related to the percentage change in weight. Despite their extreme degree of obesity, the distributions of basal plasma insulin levels and the indices of insulin sensitivity in the obese subjects overlapped with those of the nonobese controls.     

Effect of normal pregnancy on glucose assimilation,insulin and non-esterified fatty acids levels

Summary Insulin and non-esterified fatty acid (NEFA) levels were determined following a rapid intravenous injection of glucose in a control group as well as in early and late normal pregnancy. The peak level of plasma insulin occuring within the first six minutes of the test was higher in early pregnancy than in the control group. This difference thereafter was blunted. — The highest insulinic response was obtained in late pregnancy and was maintained throughout the whole test. — There were no differences in NEFA concentrations in the control and the early pregnant group. — NEFA levels were the highest in late pregnancy. — The hypothesis we have considered to explain these variations is a double one. It brings into play an enzymatic destruction of insulin by the placenta early in pregnancy. At this time the CGP-HPL levels are not yet sufficient to exhibit an antagonistic action on the maternal glucose tolerance. This would induce a pancreatic hyperplasia very early during pregnancy. This hyperplasia helps to explain why, after the same glucose stimulus, the insulin response is transiently higher during early pregnancy. The increase in circulating insulin would explain the improvement of the glucose tolerance noted at this time. — On the contrary, in late pregnancy, the high levels of CGP-HPL increase the NEFA levels to a maximum, and consequently lower the tissue utilisation of glucose. The insulin produced in greater quantity is deviated from its usual utilization and remains at a higher level and for a longer period in the circulation.

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Auswirkungen der normalen Schwangerschaft auf die Glucoseaufnahme und die Konzentrationen des Insulins und der freien Fettsäuren im Blut

Zusammenfassung Bestimmungen des Insulins und der nicht veresterten Fettsäuren wurden bei einer Kontrollgruppe sowie im Beginn und am Ende normaler Schwangerschaften nach vorheriger intravenöser Glucosebelastung vorgenommen. — Am Anfang der Schwangerschaft wurde ein stärkerer Anstieg des Insulingehaltes als bei Kontrollpersonen beobachtet. Nach der 6. Minute glich er sich aus. — Am Ende der Schwangerschaft war der Anstieg des Insulinspiegels am ausgeprägtesten. Er blieb während des ganzen Testes erhöht. — Im Beginn der Schwangerschaft entsprachen die Werte der nichtveresterten Fettsäuren denen der Vergleichsgruppe, hingegen waren sie am Ende der Schwangerschaft erhöht. — Die Hypothese, mit der wir diese Beobachtungen zu erklären versuchen, betrifft zwei verschiedene Mechanismen. Einerscits nehmen wir an, daß der durch die Placenta bedingte enzymatische Insulinabbau schon früh während der Schwangerschaft wirksam wird. Zu diesem Zeitpunkt sind die CGP-HPL-Spiegel noch zu niedrig, um eine antagonistische Wirkung auf die mütterliche Glucosetoleranz auszuüben. Dieser enzymatische Insulinabbau würde eine Hyperplasie und Hyperaktivität der Langerhans'schen Inseln bedingen. Hierdurch wäre die Tatsache, daß die gleiche Glucosebelastung im Anfang der Schwangerschaft vorübergehend eine stärkere Insulinsekretion verursacht, verständIich. Der höhere Insulinspiegel im Kreislauf würde die Verbesserung der Glucosetoleranz zu diesem Zeitpunkt erklären. — Andererseits verursachen am Ende der Schwangerschaft die hohen CGP-HPL-Werte einen maximalen Wert der nichtveresterten Fettsäuren. Dieser verursacht seinerseits wieder eine Verminderung der Glucoseutilisation in den Geweben. Das in größeren Mengen sezernierte und in seiner natürlichen Stoffwechselwirkung behinderte Insulin verbleibt somit länger und in höherer Konzentration im Kreislauf.

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Effet de la grossesse normale sur l'assimilation du glucose, sur les taux d'insuline et d'acides gras non estérifiés

Résumé Des dosages d'insuline et d'acides gras non estérifiés (NEFA) ont été pratiqués dans un groupe témoin non gravide, en début et en fin de grossesse normale après injection de glucose par voie intraveineuse. — En début de grossesse, il existe déjà une réponse insulinique supérieure à celle du groupe témoin. Cette différence ne persiste pas au-delà de la 6e minute de l'épreuve. — En fin de grossesse, la riposte insulinique est la plus élevée et reste élevée pendant toute l'épreuve. — Les NEFA sont augmentés en fin de grossesse et ne diffèrent pas du groupe témoin en début de grossesse. — L'hypothèse envisagée pour expliquer ces variations est double. Elle fait intervenir précocement au cours de la grossesse une destruction enzymatique de l'insuline par le placenta. Ceci à un moment où les taux de CGP-HPL ne sont pas encore suffisamment élevés pour exercer une action antagoniste sur la tolérance glucosée maternelle. Cette destruction plus importante provoquerait une hyperplasie et une hyperactivité des îlots de Langerhans précocement au cours de la grossesse. Ceci permettrait de comprendre pourquoi, après un même stimulus glucose, la réponse insulinique est, en début de grossesse, transitoirement plus élevée. L'augmentation de l'insuline circulante expliquant, à son tour, l'amélioration de la tolérance glucidique enregistrée à ce moment. En fin de grossesse par contre, les taux élevés de CGP-HPL augmentant au maximum le taux des NEFA, diminuent l'utilisation tissulaire du glucose. L'insuline élaborée en plus grande quan tité, déviée de son utilisation métabolique habituelle, persistera à des doses plus élevées pendant un temps plus long dans la circulation.

     

Maternal plasma polyunsaturated fatty acid levels during pregnancy and childhood lipid and insulin levels

Background and aims

Maternal polyunsaturated fatty acid (PUFA) levels are associated with cord blood lipid and insulin levels. Not much is known about the influence of maternal PUFAs during pregnancy on long-term offspring lipid and insulin metabolism. We examined the associations of maternal plasma n-3 and n-6 PUFA levels during pregnancy with childhood lipid and insulin levels.

Acute elevation of free fatty acid levels leads to hepatic insulin resistance in obese subjects

Raised levels of free fatty acids (FFA) compete with glucose for utilization by insulin-sensitive tissues, and, therefore, they may induce insulin resistance in the normal subject. The influence of experimental elevations in FFA levels on glucose metabolism in native insulin-resistant states is not known. We studied seven women with moderate obesity (63% above their ideal body weight) but normal glucose tolerance with the use of the insulin clamp technique with or without an infusion of Intralipid + heparin. Upon raising plasma insulin levels to approximately 60 microU/mL while maintaining euglycemia, whole body glucose utilization (3H-3-glucose) rose similarly without (from 66 +/- 7 to 113 +/- 11 mg/min m2, P less than .02) or with (from 70 +/- 7 to 137 +/- 19 mg/min m2, P less than .02) concomitant lipid infusion. In contrast, endogenous glucose production was considerably (73%) suppressed (from 66 +/- 7 to 15 +/- 8 mg/min m2, P less than .001) during the clamp without lipid, but declined only marginally (from 70 +/- 7 to 48 +/- 7 mg/min m2, NS) with lipid administration. The difference between the control and the lipid study was highly significant (P less than .02), and amounted to an average of 3.8 g of relative glucose overproduction during the second hour of the clamp. Blood levels of lactate rose by 34 +/- 15% (.1 greater than P greater than .05) in the control study but only by 17 +/- 10% (NS) during lipid infusion. Blood pyruvate concentrations fell in both sets of experiments (by approximately 45% at the end of the study) with similar time courses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hypertriglyceridaemia in subjects with normal and abnormal glucose tolerance: relative contributions of insulin secretion,insulin resistance and suppression of plasma non-esterified fatty acids

Summary Although plasma insulin and triglyceride concentrations are positively correlated in many studies, the relationships between insulin resistance, insulin secretion and hypertriglyceridaemia remain unclear. To study these associations, subjects between the ages of 40 and 64 were randomly selected from a general practice register and invited to attend for a standard oral glucose tolerance test for measurement of insulin, triglyceride and non-esterified fatty acid concentrations. The study comprised 1122 subjects who were not previously known to have diabetes and who completed the test. Using the World Health Organisation criteria, 51 subjects were classified to have non-insulin-dependent diabetes mellitus, 188 had impaired glucose tolerance and 883 subjects had normal glucose tolerance. Triglyceride concentrations in subjects with glucose intolerance were elevated compared to those in control subjects, even after adjustment for age, obesity and gender (p<0.001 for subjects with diabetes and p<0.01 for those with impaired glucose tolerance compared to normal subjects). In separate multiple regression analyses for males and females, the most important determinants of the plasma triglyceride concentration were the area under the non-esterified fatty acid suppression curve (p<0.001 in both genders) and the waist-hip ratio (p<0.001 for men and <0.01 for women). The fasting insulin concentration was independently associated with triglyceride concentration in women only (p<0.01). The most important determinant of the area under the non-esterified fatty acid suppression curve in men was the 30-min insulin increment, a measure of insulin secretion, (p<0.001) whereas for women age (p<0.001) and the body mass index (p<0.01) were the most important.Abbreviations NIDDM non-insulin-dependent diabetes mellitus - IGT impaired glucose tolerance - NEFA non-esterified fatty acid     

妊娠糖尿病患者血游离脂肪酸与β细胞功能及胰岛素抵抗的关系

检测36例孕24～36周妊娠糖尿病患者和同期30例健康孕妇凌晨3:00、5:00及75 g葡萄糖负荷后血游离脂肪酸和胰岛素水平,比较两组胰岛素敏感性和胰岛素分泌功能.结果提示凌晨及糖负荷后高游离脂肪酸均与妊娠糖尿病患者胰岛素抵抗相关[胰岛素:(7.18±3.19)对(5.05±1.80)mIU/L(3:00)、(8.19±4.42)对(5.31±1.82)mIU/L(5:00)、(59.18±30.85)对(40.52±15.07)mIU/L(糖负荷后);游离脂肪酸:(0.39±0.20)对(0.23±0.11)mmol/L(3:00)、(0.46±0.17)对(0.29±0.12)mmol/L(5:00)、(0.19±0.13)对(0.09±0.06)mmol/L(糖负荷后);均P<0.01],糖负荷后30 min(早期相)的高水平的游离脂肪酸可能与妊娠糖尿病患者早期胰岛素分泌功能缺陷有关.     

Stimulatory effect of increased non-esterified fatty acid concentrations on proinsulin processing in healthy humans

Aims/hypothesis. To assess the effect of increased concentrations of non-esterified fatty acids (NEFA) on proinsulin processing in healthy humans.¶Methods. We did a hyperglycaemic clamp (130 min duration, 8 mmol/l glucose, with a 5-g arginine bolus at min 120) before and after a 5-h infusion of Intralipid/heparin in 14 healthy subjects. Of the subjects eight underwent a saline control experiment. The proinsulin : insulin (PI:I) ratio immediately after the arginine bolus (122.5 to 125 min) was considered to provide an estimate for the conversion of proinsulin to insulin in the beta cell.¶Results. Concentrations of NEFA were 757 ± 86 μmol/l and 1669 ± 134 μmol/l (p < 0.001) after the 5-h infusion of saline or Intralipid, respectively. Insulin secretion rates were no different between the Intralipid and saline infusions (p = 0.73). There was no statistically significant difference for either the proinsulin concentration or the PI:I ratio during glucose stimulation alone (0 to 120 min). In response to arginine, in contrast, proinsulin remained unchanged during the saline infusion (from 31 ± 6 to 29 ± 7 pmol/l, p = 0.50) but decreased during 5 h of lipid infusion from (21 ± 3 to 15 ± 2 pmol/l, p = 0.02). The PI:I ratio in response to the arginine bolus was higher during the saline infusion (2.0 ± 0.2 % vs 1.7 ± 0.2 %, p = 0.04) but decreased during the Intralipid infusion (from 1.6 ± 0.2 % to 1.2 ± 0.1 %, p = 0.04).¶Conclusion/interpretation. The statistically significantly lower PI:I ratio in response to arginine during experimentally increased concentrations of NEFA suggests that NEFA increase the conversion of proinsulin to insulin in humans in vivo. [Diabetologia (2000) 43: 1368–1373]     

Lack of effect of calcium infusion on blood glucose and plasma insulin levels in patients with insulinoma

In vitro calcium plays a fundamental role in regulating insulin secretion. On the other hand, the influence of calcium excess on insulin release in vivo is not clearly defined. Recently, calcium infusion has been proposed as a provocative test for the diagnosis of insulin-secreting tumors. A 2-h infusion of calcium gluconate was performed (4 mg/kg . h) in six patients with islet cell adenoma. As a result, mean calcium plasma levels increased from 9.6 +/- 0.4 to 11.6 +/- 0.8 mg/100 ml. During calcium infusion, blood glucose and plasma insulin concentrations remained unchanged. These observations suggest that calcium fails to stimulate basal insulin secretion even in cases of organic hyperinsulinism. They show that calcium infusion is not helpful as a provocative test in the diagnosis of insulinoma.     

Impaired non-esterified fatty acid suppression to intravenous glucose during late pregnancy persists postpartum in gestational diabetes: a dominant role for decreased insulin secretion rather than insulin resistance

Aims/hypothesis Non-esterified fatty acids are implicated in the pathogenesis of gestational (GDM) and type 2 diabetes. We examined the relationship between NEFA dynamics, insulin resistance and beta cell dysfunction in women with GDM in late pregnancy and postpartum.Methods A total of 19 Caucasian women with GDM and 19 healthy pregnant women matched for BMI and age underwent an IVGTT in the third trimester and 4 months postpartum, deriving values for insulin sensitivity (SI), insulin secretion (AIRg) and disposition index (DI). NEFA levels were measured serially.Results In pregnancy, the GDM women had similar SI but reduced AIRg and DI compared with control subjects. The GDM group demonstrated significantly slower NEFA suppression, which was attributable to the GDM women who required insulin during pregnancy (n=7) and who had markedly reduced AIRg and K_NEFA (NEFA disappearance constant) compared with their matched controls. In contrast, GDM subjects not requiring insulin (n=12) had similar NEFA suppression curves and AIRg to control subjects. Postpartum, GDM subjects demonstrated reduced SI and DI. The impaired suppression of NEFA persisted postpartum, but again only in the subgroup of GDM subjects who had required insulin during pregnancy. Furthermore, K_NEFA correlated with AIRg and DI in both states, but not with SI.Conclusions/interpretation Impaired NEFA suppression occurs in GDM subjects both in late pregnancy and postpartum in response to IVGTT-induced endogenous insulin secretion. The impaired NEFA suppression is present in GDM women with the most severe beta cell dysfunction (who had required insulin during pregnancy) and is related to their insulin secretory dysfunction rather than their reduced SI.     

Human triglyceride-rich lipoproteins impair glucose metabolism and insulin signalling in L6 skeletal muscle cells independently of non-esterified fatty acid levels

AIMS/HYPOTHESIS: Elevated fasting and postprandial plasma levels of triglyceride-rich lipoproteins (TGRLs), i.e. VLDL/remnants and chylomicrons/remnants, are a characteristic feature of insulin resistance and are considered a consequence of this state. The aim of this study was to investigate whether intact TGRL particles are capable of inducing insulin resistance. METHODS: We studied the effect of highly purified TGRLs on glycogen synthesis, glycogen synthase activity, glucose uptake, insulin signalling and intramyocellular lipid (IMCL) content using fully differentiated L6 skeletal muscle cells. RESULTS: Incubation with TGRLs diminished insulin-stimulated glycogen synthesis, glycogen synthase activity, glucose uptake and insulin-stimulated phosphorylation of Akt and glycogen synthase kinase 3. Insulin-stimulated tyrosine phosphorylation of IRS-1, and IRS-1- and IRS-2-associated phosphatidylinositol 3-kinase (PI3K) activity were not impaired by TGRLs, suggesting that these steps were not involved in the lipoprotein-induced effects on glucose metabolism. The overall observed effects were time- and dose-dependent and paralleled IMCL accumulation. NEFA concentration in the incubation media did not increase in the presence of TGRLs indicating that the effects observed were solely due to intact lipoprotein particles. Moreover, co-incubation of TGRLs with orlistat, a potent active-site inhibitor of various lipases, did not alter TGRL-induced effects, whereas co-incubation with receptor-associated protein (RAP), which inhibits interaction of TGRL particles with members of the LDL receptor family, reversed the TGRL-induced effects on glycogen synthesis and insulin signalling. CONCLUSIONS/INTERPRETATION: Our data suggest that the accumulation of TGRLs in the blood stream of insulin-resistant patients may not only be a consequence of insulin resistance but could also be a cause for it.     

Involvement of non-esterified fatty acid oxidation in glucocorticoid-induced peripheral insulin resistance in vivo in rats

Summary The mechanism by which glucocorticoids induce insulin resistance was studied in normal rats administered for 2 days with corticosterone then tested by euglycaemic hyperinsulinaemic clamps. Corticosterone administration induced a slight hyperglycaemia, hyperinsulinaemia and increased non-esterified fatty acid levels. It impaired insulin-stimulated total glucose utilization (corticosterone 15.7±0.7; controls 24.6±0.8 mg·kg⁻¹·min⁻¹), as well as residual hepatic glucose production (corticosterone 4.9±1.0; controls 2.0±0.7 mg·kg⁻¹·min⁻¹). During the clamps, insulin did not decrease the elevated non-esterified fatty acid levels in corticosterone-administered rats (corticosterone 1.38±0.15, controls 0.22±0.04 mmol/l). Corticosterone administration decreased the in vivo insulin-stimulated glucose utilization index by individual muscles by 62±6%, and the de novo glycogen synthesis by 78±2% (n=8–9 muscles). GLUT4 protein and mRNA levels were either unchanged or slightly increased by corticosterone administration. Inhibition of lipid oxidation by etomoxir prevented corticosterone-induced muscle but not hepatic insulin resistance. In conclusion, glucocorticoid-induced muscle insulin resistance is due to excessive nonesterified fatty acid oxidation, possibly via increased glucose fatty-acid cycle ultimately inhibiting glucose transport, or via decreased glycogen synthesis, or by a direct effect on glucose transporter translocation or activity or both.     

Diurnal variation in the effects of insulin on blood glucose,plasma non-esterified fatty acids and growth hormone

Insulin 0.05 mu/kg body weight was injected intravenously into 14 subjects both at 8 a.m. and 5 p.m. in random order 12 hrs after a 50 g glucose meal. Fasting glucose levels were similar in both cases but the 48percent plus or minus 10 percent fall in blood glucose in the morning was significantly greater (p smaller than 0.001) than that of 34 percent plus or minus 7 percent in the afternoon. Fasting plasma NEFA, however, varied markedly between 477 plus or minus 150 muEqlL in tth morning and 725 plus or minus 195 muEqlL in the afternoon (p smaller than 0.001) and the fall after insulin injection (64 percent plus or minus 14 percent) was greater in the afternoon than in the morning (47 percent plus or minus 15 percent) (p smaller than 0.001). There was an inverse relationship between proportional glucose disappearance and proportional NEFA disappearance (p smaller than 0.001). The calculated caloric change in plasma, the sum of the falls in glucose and NEFA, were very similar in both morning (2.2 plus or minus 0.5 Cals/1) and afternoon (2.3 plus or minus 0.5 Cals/1), i.e., in spite of the variations of glucose and NEFA metabolism produced by insulin at different times, the nett effect, in terms of energy, was the same. Plasma growth hormone response in the afternoon was found to be enhanced compared with the morning values, although the degree of hypoglycaemia was greater in the morning.