Metabolic Effects of Suppression of Nonesterified Fatty Acid Levels With Acipimox in Obese NIDDM Subjects G.R. FULCHER, M. WALKER, C. CATALANO, L AGIUS, AND K.G.M.M. ALBERTI

NEFAs characteristically are elevated in obese NIDDM patients in both the basal state and after insulin. This elevation might aggravate glycemic control both by decreasing peripheral glucose disposal (glucose-fatty acid cycle), and by increasing HGO. Thus, lowering plasma NEFA levels might improve carbohydrate metabolism. We therefore measured HGO and fuel use (by indirect calorimetry) both in the basal state and during the last 30 min of a hyperinsulinemic clamp (0.025U kg~ 1 h"1) in 8 obese NIDDM patients (BMI 34.8 ± 1 . 0 kg/m2) after complete overnight suppression of plasma NEFA levels with acipimox, a new nicotinic acid analogue. After acipimox, mean basal plasma NEFA and glycerol levels were lower than control values (0.11 ± 0.02 vs. 0.65 ± 0.04 mM, P < 0.001; and 16 ± 3 vs. 68 ± 7 |xM, P = 0.004, respectively) and were accompanied by a fall in lipid oxidation (acipimox vs. placebo: 16.1 ± 1 . 2 vs. 38.8 ± 2.4 mg • m~2 • min~ 1 ; P < 0.001) and a rise in glucose oxidation (91.1 ± 6.2 vs. 54.1 ± 9.0 mg m" 2 min~ 1 ; P= 0.002). Basal HGO and fasting plasma glucose levels were lower (94.1 ± 9.2 vs. 118.5 ± 9.5 mg m" 2 min" 1 , P= 0.01; and 8.3 ± 1.2 vs. 9.8 ± 1.2 mM; P < 0.001), respectively. Serum insulin levels were similar during the clamps (44.2 ± 4.9 vs. 48.2 ± 5.7 JLU/L; NS), but

despite this, HGO was suppressed more after acipimox (18.2 ± 7.6 vs. 49.7 ± 12.9 mg m" 2 min" 1 ; P < 0.01), and the metabolic clearance rate for glucose was higher (101.98 ± 19.34 vs. 75.43 ± 9.94 ml m~ 2 • min~ 1 ; P < 0.05). In conclusion, prolonged overnight suppression of lipolysis and lipid oxidation in obese NIDDM lowers fasting blood glucose and

From the Department of Medicine, The Medical School, Framlington Place, University of Newcastle Upon Tyne, Newcastle-Upon-Tyne, United Kingdom. Address correspondence and reprint requests to Dr. G. R. Fulcher, Department of Endocrinology, The Royal North Shore Hospital, Pacific Highway, St Leonards, 2065, Sydney, Australia. Received for publication 5 September 1991 and accepted in revised form 3 March 1992. NEFA, nonesterified fatty acids; HGO, hepatic glucose output; NIDDM, non-insulin-dependent diabetes; NS, not significant; BMI, body mass index; fla, rate of appearance; Rd, rate of disappearance; MCR, metabolic clearance rate; SSPG, final mean concentrations of plasma glucose.

1400

HGO and increases peripheral and hepatic sensitivity to insulin in obese NIDDM patients. Diabetes 41:1400-08, 1992

N

IDDM is characterized by decreased insulin sensitivity of liver (1,2) and muscle (3-6). This decrease is most marked in patients who are also obese (7). Although plasma insulin levels are frequently normal or increased in absolute terms, the regulation of both carbohydrate and fat metabolism is clearly abnormal (8-10). Hyperglycemia is accompanied by raised plasma levels of NEFA (9,11), and the regulation of NEFA turnover, oxidation, and nonoxidative disposal (reesterification) is impaired (10). Fatty acid oxidation rates also are increased (9). A strong interrelationship exists between the changes in lipid metabolism on the one hand and those of carbohydrate metabolism on the other (10). This is found in both peripheral tissues (skeletal muscle) and the liver. In muscle, a glucose-fatty acid cycle (12) has been known for many years. The oxidation of fatty acids decreases glucose uptake and utilization through substrate competition, and apparent peripheral insulin action is diminished. In vivo studies are consistent with this hypothesis (13). At the same time, the rate of fatty acid oxidation determines endogenous glucose production. Thus, good correlations are obtained in vivo between raised fasting plasma NEFA levels, lipid oxidation rates, and basal HGO (3,10,13). Because an increased rate of gluconeogenesis is responsible for fasting hyperglycemia in NIDDM patients (14), and because fatty acids stimulate gluconeogenesis in vitro (15), it is reasonable to hypothesize a causal association between the increased rate of fatty acid oxidation on the one hand and fasting hyperglycemia on the other. These data raise the possibility that NEFA-lowering drugs might have hypoglycemic effects. Theoretically, increased peripheral glucose utilization and decreased

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G.R. FULCHER AND ASSOCIATES

endogenous glucose production could result from a decreased flux of plasma NEFA to both muscle and liver, and blood glucose levels therefore could be lowered. Although nicotinic acid has been used for many years as a lipid-lowering agent, its effects on carbohydrate metabolism remain unclear. In long-term studies of diabetic patients taking nicotinic acid, glycemic control is frequently worse (16), and consequently, its use in this population is limited (17). In contrast, good experimental evidence in rodents indicates that acutely lowering plasma NEFA levels can affect carbohydrate metabolism profoundly; a fall in HGO and fasting blood glucose levels and an increase in peripheral glucose disposal has been demonstrated (18,19). Disappointingly, these changes do not occur acutely in humans. Although an increase in peripheral glucose uptake may occur, this is matched by an increase in hepatic glucose production. Fasting blood glucose levels do not change (20). Thus, nicotinic acid has been reported to increase (16), decrease (18,19), or have no effect (20) on fasting glycemia. An explanation for this apparent discrepancy is that both the duration of inhibition of lipolysis and the availability of hepatic glycogen may be key to the changes in carbohydrate metabolism (21,22). Thus, in NIDDM patients, an evening dose of insulin lowers both fasting plasma glucose levels and HGO, changes strongly correlated with the overnight decrease in plasma levels of NEFA (23). Because insulin inhibits glycogenolysis directly and gluconeogenesis indirectly (through the supply of fatty acids to the liver for oxidation), such changes might be anticipated. In contrast, lowering fatty acid oxidation rates with nicotinic acid is likely to increase glycogen breakdown (24), and an immediate fall in blood glucose levels therefore would be unlikely. It is possible, however, that a prolonged inhibition of lipid oxidation might accelerate glycogen depletion. Under such conditions, a further decrease in the rate of gluconeogenesis might be expected to lower HGO and fasting glucose levels. We therefore used the antilipolytic drug acipimox, a long-acting nicotinic acid analogue (25), to determine the changes, if any, in fasting plasma glucose levels, HGO, and fuel use that occur after complete overnight suppression of lipolysis in obese NIDDM patients. RESEARCH DESIGN AND METHODS Informed written consent was obtained from 8 obese NIDDM patients (6 women, 2 men) who were recruited from the diabetic outpatient clinic of the Royal Victoria Infirmary, Newcastle-Upon-Tyne. The age of the patients was 57 ± 4 yr (mean ± SE), and the BMI was 34.8 ± 1.0 kg/m2. All patients were treated with a standard diet, and four patients were treated additionally with diet and oral hypoglycemic agents. Of these, the two patients taking metformin ceased this medication 2 wk before the studies, and the two patients taking sulphonylureas ceased all medication 48 h before each study. All patients were well, had no major complications of NIDDM, and were not taking other drugs likely to interfere with lipid metabolism.

DIABETES, VOL. 41, NOVEMBER 1992

Studv protocol.

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Acipimox / Placebo Hepatic glucose output Isoglycaemic clamp. Indirect calorimetry. FIG. 1. Outline of study protocol.

The study was approved by the Ethical Committee of the University of Newcastle Upon Tyne. Study protocol. An outline of the study protocol is shown in Fig. 1. For the 72 h before each study, patients were placed on a weight-maintaining diet providing at least 200 g of carbohydrate/day. Fifty percent of calories were provided as carbohydrate, 30% as fat, and 20% as protein. The final meal was served in the Clinical Investigation Unit at 2000 on the evening before the study, and patients then fasted until 1200 the following day. Acipimox (250 mg) or placebo was administered in doubleblind random fashion at 1900, 0100, and 0600. A venous blood sample was drawn at 1900 (before the first dose) to determine comparability at presentation between studies and again at 0600 (before the last dose), at the start of the basal period. At 0700, an i.v. cannula was inserted retrogradely into a dorsal hand vein that was heated in a thermoregulated plexiglas box set at a temperature of 50°C for arterialized blood sampling. A second cannula was inserted in the contralateral antecubital vein for infusion. A primed (32 |xCi) continuous (0.32 |xCi/min) infusion of tritiated glucose then was commenced, and endogenous glucose production was measured throughout the study. After a 2-h basal period for the equilibration of isotope, a hyperinsulinemic (0.025 U • kg" 1 • h~1) isoglycemic clamp was started and continued for 180 min. Plasma glucose was clamped - 1 0 % below basal levels to decrease endogenous insulin secretion. The insulin infusion rate was chosen to ensure that lipolysis and HGO would not be suppressed completely during the clamp. In this way, relative changes between the studies caused by treatment with acipimox could be assessed. Finally, indirect calorimetry was performed in the last 30 min of the basal period (0830-0900), and in the last 30 min of the clamp (1130-1200) with a Deltatrac indirect calorimeter (Datex, Helsinki, Finland). Urinewas collected throughout the study period for the measurement of urinary nitrogen and glucose. One patient could not complete the second clamp study for technical reasons. For statistical comparisons, her basal data were included for analysis, but comparisons between clamps, and between the basal and clamp periods, were based on data from the remaining seven subjects only.

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OVERNIGHT FATTY ACID SUPPRESSION IN OBESE NIDDM

TABLE 1 Mean ± SE blood glucose, serum insulin, cholesterol, and triglyceride concentrations at presentation (1900) Acipimox Blood glucose (mM) Serum insulin (|xU/L) Serum cholesterol (mM) Serum triglycerides (mM)

7.4 19.7 6.8 2.36

±1.1 ±2.6 ± 0.6 ± 0.43

Placebo 7.4 19.9 6.5 2.35

± ± ± ±

0.9 2.9 0.5 0.47

There were no statistical differences between presenting values on each study day.

200

Hepatic glucose output.

180 160 140 120

Placebo

100 Acipimox

80 60 40

Analytical methods. Plasma and blood glucose were measured with a glucose analyzer (Yellow Springs Instruments, Yellow Springs, OH). Serum insulin (26) and plasma glucagon (27) were measured by radioimmunoassay. Plasma NEFA (28) and blood metabolites (29) were measured enzymatically using a Cobas Bio (Roche, Welwyn Garden City, UK) centrifugal analyzer, and serum triglycerides by the glycerol kinase method, with kit no. 0714445 supplied by Roche. Glucose specific activity was determined by standard techniques. Briefly, after deproteinization according to Somogyi (30), plasma was freeze-dried to remove tritiated water and then resuspended in distilled water. Radioactivity was measured in a liquid scintillation spectrometer after the addition of 5 ml of scintillation liquid (Optiphase HiSafe II, Pharmacia). Urinary nitrogen was measured by the Kjeldahl method (31). Calculations. The total Ra and Rd of glucose were calculated with the equations of Steele in their derivative form (32). Rd values presented in the text are corrected for urinary glucose loss. Values obtained during the last 30 min of the basal period and the last 30 min of the clamp were used for statistical analysis, and non-steady state was assumed for these periods. The glucose space was assumed to be 200 ml/kg, and the glucose pool fraction, 0.65 (33). Endogenous glucose production was obtained by subtracting the amount of glucose infused from the isotopically determined total Ra of glucose. Substrate oxidation was calculated with the equations of Frayn (34). Because the study design did not allow blood glucose to be clamped at the same concentrations, glucose clearance was calculated by dividing glucose uptake (after correction for urinary glucose loss) by the plasma glucose concentration (35-37). Statistical analyses. Values are means ± SE. Comparisons between studies were made by Student's paired t tests, and 3-hydroxybutyrate values were log-transformed before analysis. RESULTS

The initial blood glucose, serum insulin, cholesterol, and triglyceride values were closely similar on the two study nights (Table 1). Patients were in reasonable long-term glycemic control (HbA1 7.8 ± 0.4%; reference range 5.0-7.5%) and were mildly hyperlipidemic. Hormone concentrations. Serum insulin levels were lower after the overnight fast with acipimox (7.5 ± 1.2 vs.

1402

20 0 Basal

Clamp

FIG. 2. Change in HGO after acipimox and placebo before basal (Basal) and in the final 30 min of clamp (Clamp). * P= 0.01, acipimox vs. placebo.

12.1 ± 1.4 |JLU/L; P = 0.002), but steady-state levels during the hyperinsulinemic clamps were similar (acipimox vs. placebo 44.2 ± 4.9 vs. 48.2 ± 5.7 |JLU/L; NS). Plasma glucagon concentrations were similar both at the start and in the final 30 min of the basal period (0700 39 ± 4 vs. 37 ± 9 ng/L, NS; 0830 to 0900 33 ± 3 vs. 28 ± 6 ng/L, NS). Carbohydrate metabolism. Plasma concentration. Blood glucose levels were similar at the start of the study (Table 1). After the overnight fast, plasma glucose levels were significantly lower with acipimox treatment (8.3 ±1.2 vs. 9.8 ±1.2 mM; P < 0.001). Because plasma glucose was clamped at -0.7 mM below basal values during both studies, the SSPG was lower after acipimox than placebo (7.6 ± 1.4 vs. 9.1 ± 1.4 mM, P < 0.001). HGO. Basal HGO (Fig. 2) was significantly lower after acipimox (94.1 ± 9.2 vs. 118.5 ± 9.5 mg • m~2 • min" 1 ; P = 0.01). During the hyperinsulinemic clamps, despite similar insulin levels, HGO fell more after acipimox (83.1 ± 6.1 vs. 60.7 ± 6.6%; P < 0.01), and final values (150 to 180 min) were lower (18.2 ± 7.6 vs. 49.7 ± 12.9 mg • rrT2 • min~1; P = 0.01). Total body glucose uptake. During the basal period, the MCRs for glucose were similar during both studies (68 ± 8 vs. 70 ± 5 ml • m" 2 • min" 1 ; NS). During the clamp with placebo, the rate of infused glucose matched the suppression of hepatic glucose output so that Rd did not change. During the clamp with acipimox, however, despite a greater overall suppression of HGO, there was an insignificant increase in total glucose disposal (Table 2). The MCR of glucose, however, was significantly higher (acipimox vs. placebo 102 ± 19 vs. 75 ± 10 ml • rrT2 • min" 1 ; P < 0.05), as was the rate of exogenous glucose infusion during the final 30 min of the clamp (106.7 ±13.4 vs. 64.3 ± 6.2 mg • m~2 • min~1; P = 0.01). Glucose disposal. Glucose oxidation was higher in the basal period after acipimox (91.1 ±6.2 vs. 54.1 ± 9 mg • m" 2 • min" 1 ; P = 0.002) and increased in both stud-

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TABLE 2 Mean glucose disposal (Rd) and carbohydrate oxidation rates (Glu ox) during basal period and clamp with acipimox and placebo Acipimox

Placebo

TABLE 3 Mean ± SE fasting (0600) venous plasma NEFA, blood glycerol, and 3-hydroxybutyrate levels after acipimox and placebo in obese NIDDM patients

P

Basal period 99.6 ±11.0 117.3 ±9.1 NS Rd (mg • rrr -mm" 2 54.1 ± 9.0 0.002 Glu ox (mg • m 2 • min 1) 91.1 ±6.2

Plasma NEFA (mM) Blood glycerol (u,M) Blood 3-hydroxybutyrate (|xM)

Clamp Rd (mg • m"^ • min"1) 124.0 ±18.0 117.0 ±16.0 NS Glu ox (mg • rrr 2 • min~1) 111.0 ± 9.7 76.2 ± 7.4 0.01

* Acipimox vs. placebo.

ies in response to insulin (Table 2). The rate of glucose oxidation during the acipimox clamp remained significantly higher than during the control study (111.0 ± 9.7 vs. 76.2 ± 7.4 mg • m~2 • min" 1 ; P = 0.01). The amount of glucose metabolized by nonoxidative pathways (the difference between glucose disposal and glucose oxidation rates) was less after acipimox in both the basal period (8.6 ± 12.1 vs. 63.1 ± 14.2mg • m" 2 • min" 1 ; P = 0.01) and during the clamp (150 to 180 min 12.7 ± 21.5 vs. 40.5 ± 20.0 mg • m~2 • min" 1 ; NS), although the difference between clamp studies was not statistically significant. Lipid metabolism Plasma concentrations. Although concentrations of triglycerides were lower in 7 of the 8 subjects after acipimox (Fig. 3), overall, the mean values were not significantly different (acipimox vs. placebo 1.83 ± 0.28 vs. 2.18 ± 0.4 mM, P = 0.1). Serum cholesterol values were unchanged (acipimox vs. placebo 6.58 ± 0.46 vs. 6.44 ± 0.47 mM; NS). Acipimox caused marked overnight suppression of plasma NEFA, blood glycerol, and 3-hydroxybutyrate levels compared with placebo (Table 3). The changes in plasma NEFA and blood glycerol and 3-hydroxybutyrate

5 50% fall in the basal rate of lipid oxidation as the flux of NEFA to liver and muscle was diminished. Because the tissue uptake of NEFA depends largely on plasma concentration, an acute inhibition of lipolysis would be sufficient to explain these changes. An alternative possibility is that acipimox also might directly inhibit the pathways of fatty acid oxidation. In fact, in vivo rat studies with both acipimox and the antilipolytic agent 3,5-dimethyl-pyrazole have shown that both these drugs produce a specific decrease in the hepatic peroxisomal oxidation of fatty acids, a significant depletion of liver glycogen, and a fall in plasma glucose (41). The relevance of such changes to man is uncertain, but it is possible that the significant decrease in lipid oxidation rates reflects both a sustained (14-h) decrease in substrate flux to the liver and a direct inhibition of the oxidative pathways of fat metabolism. The marked fall in lipid oxidation rates was accompanied by a 68% rise in the basal rate of glucose oxidation. This is entirely consistent with the known close interrelationship between the metabolism of carbohydrate and fat (3,10,13,39,42-45). Thus, when the rate of fatty acid oxidation is increased, the oxidation of pyruvate is inhibited, and consequently, the rate of glycolysis is slowed. This glucose-fatty acid cycle operates in both muscle and liver (12,40). Conversely, an increase in the rates of glycolysis and glucose oxidation should occur when fatty acid oxidation is suppressed after acipimox. Once again, we emphasize that a detailed understanding of the mechanisms of action of acipimox is unavailable, and in particular, it is not clear whether these findings specifically reflect metabolic changes in muscle or in liver. In fact, recent data show that despite a significant increase in carbohydrate oxidation after acipimox, the activities of muscle pyruvate dehydrogenase and phosphofructokinase do not change (46). It is therefore entirely possible that at fasting insulin concentrations, the major metabolic effects of acipimox on carbohydrate metabolism are centered in liver and that the effects in muscle, if any, are relatively minor. Specifically, direct or indirect effects of acipimox on the glycolytic pathway in muscle appear unlikely under basal conditions. In our study, the fall in the rate of basal lipid oxidation was not associated with an increase in glucose clearance. This suggests that at fasting insulin levels in obese NIDDM patients, glucose disposal is not influenced directly by changes in lipid metabolism. Although this is contrary to the findings of Balasse and Neef (20) and Paul et al. (47), all of whom were able to demonstrate increased glucose oxidation and clearance acutely after nicotinic acid, other published data would appear to

DIABETES, VOL. 41, NOVEMBER 1992

support our findings. Thus, Vaag et al. found that glucose disposal did not increase after acipimox at basal insulin concentrations despite a significant decrease in fatty acid oxidation and an increase in carbohydrate oxidation (46). Furthermore, when normal, nondiabetic subjects are studied under hyperglycemic, insulinopenic conditions (simulating the diabetic state), basal peripheral glucose uptake is not inhibited by an acute increase in plasma levels of NEFA (13). In fact, such changes can only be produced in the presence of hyperinsulinemia (13). Similarly, despite an increase in NEFA levels as NIDDM becomes more severe (48,49), the basal MCR of glucose remains constant over a wide range of fasting plasma glucose levels (8). Taken together, it is therefore highly unlikely that changes in fat metabolism significantly alter peripheral glucose disposal in the basal state in diabetic man. The discrepancy between these findings and those of Balasse and Neef (20) and Paul et al. (47) is not explained, but differences in the study population, duration of fatty acid suppression, and pharmacological differences between preparations are possible explanations. Two important aspects of HGO and its regulation are relevant to our findings. Firstly, it is an increase in gluconeogenesis rather than glycogenolysis that determines the rate of fasting HGO and therefore plasma glucose levels in NIDDM patients (14). Secondly, basal rates of fatty acid oxidation correlate significantly with basal HGO (3,10,13,42,44,50). This result was also found in our study (r- 0.52; P< 0.05). One explanation that links these two findings has been provided by in vitro studies that demonstrate that the oxidation of fat has a strong stimulatory effect on gluconeogenesis both by providing a continued source of ATP and by activating key gluconeogenic enzymes (15,51). Under these circumstances, it is perhaps not surprising that HGO was significantly lower after acipimox, and that fasting plasma glucose values also were decreased significantly. At the same time, we (unpublished observations) and others (20,47) have not previously found an acute change in basal plasma glucose levels after either acipimox or nicotinic acid, respectively, when lipolysis was inhibited for a shorter period of time (