Insulin fails to alter plasma LCFA metabolism in muscle perfused at similar glucose uptake

Alice J. Yee and Lorraine P. Turcotte

Department of Kinesiology, University of Southern California, Los Angeles, California 90089


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Insulin has been shown to alter long-chain fatty acid (LCFA) metabolism and malonyl-CoA production in muscle. However, these alterations may have been induced, in part, by the accompanying insulin-induced changes in glucose uptake. Thus, to determine the effects of insulin on LCFA metabolism independently of changes in glucose uptake, rat hindquarters were perfused with 600 µM palmitate and [1-14C]palmitate and with either 20 mM glucose and no insulin (G) or 6 mM glucose and 250 µU/ml of insulin (I). As dictated by our protocol, glucose uptake was not significantly different between the G and I groups (10.3 ± 0.6 vs. 11.0 ± 0.5 µmol · g-1 · h-1; P > 0.05). Total palmitate uptake and oxidation were not significantly different (P > 0.05) between the G (10.1 ± 1.0 and 0.8 ± 0.1 nmol · min-1 · g-1) and I (10.2 ± 0.6 and 1.1 ± 0.2 nmol · min-1 · g-1) groups. Preperfusion muscle triglyceride and malonyl-CoA levels were not significantly different between the G and I groups and did not change significantly during the perfusion (P > 0.05). Similarly, muscle triglyceride synthesis was not significantly different between groups (P > 0.05). These results demonstrate that the presence of insulin under conditions of similar glucose uptake does not alter LCFA metabolism and suggest that cellular mechanisms induced by carbohydrate availability, but independent of insulin, may be important in the regulation of muscle LCFA metabolism.

malonyl-coenzyme A; fatty acid oxidation; fatty acid uptake; triglyceride synthesis; intramuscular triglycerides; long-chain fatty acids


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IT IS WELL ESTABLISHED that insulin has potent lipogenic effects in adipose tissue (2, 12). Although the effects of insulin on lipid metabolism in muscle have not been studied extensively, it is generally accepted that insulin has similar antioxidative lipogenic effects in muscle (8, 13). However, in those studies, supraphysiological levels of insulin were used, and this may have been associated with undocumented increases in glucose uptake. Thus the effects of insulin on long-chain fatty acid (LCFA) metabolism documented in these studies may have been induced, in part, by the significant increases in glucose flux rather than by insulin alone. Therefore, there is a need to determine the effect of insulin on LCFA metabolism independent of changes in glucose uptake.

Malonyl-CoA has been proposed by some, but not all, to be a regulator of LCFA oxidation in muscle (4, 5, 16, 17, 22, 26). In both perfused and incubated rat muscle and in humans, it has been shown by some that malonyl-CoA levels increase under conditions of hyperglycemia, hyperinsulinemia, or both (1, 4, 7, 19, 27). High glucose levels have been shown to be associated with an increase in malonyl-CoA levels, possibly via an increase in citrate levels (18, 20). Alternatively, it has been suggested that insulin may directly impact on malonyl-CoA production possibly via inhibition of AMP-activated protein kinase activity (19, 27). However, because of the concomitant increase in glucose uptake expected with the use of supraphysiological levels of insulin, it is not clear whether the increase in insulin concentration or the increase in glucose uptake was the main factor regulating the observed changes in malonyl-CoA in those studies. Consequently, there is a need to determine the effect of insulin on malonyl-CoA production independently of changes in glucose uptake.

Thus the purpose of this study was to determine the independent effects of insulin on LCFA metabolism and malonyl-CoA levels under conditions of similar glucose uptake and palmitate delivery. Muscle LCFA metabolism was assessed by measuring uptake, oxidation, and triglyceride synthesis by use of the perfused rat hindquarter preparation. In addition, malonyl-CoA concentrations were assessed to determine whether changes in LCFA oxidation occurred via alterations in malonyl-CoA levels.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animal preparation. Male Wistar rats were housed in pairs and maintained on a 12:12-h light-dark cycle. They received regular rat chow and water ad libitum. The rats were randomly assigned to the glucose (G: n = 10) or insulin (I: n = 10) group. Body weights were not significantly different between groups (305.6 ± 10.6 and 295.1 ± 10.2 g for the G and I groups, respectively; P > 0.05).

Hindquarter perfusion. Rats were anesthetized intraperitoneally with ketamine-xylazine (80 and 12 mg/kg body wt, respectively) and prepared surgically for hindquarter perfusion as previously described (24). Before the perfusion catheters were inserted, heparinized saline (150 IU) was administered into the inferior vena cava. The rats were killed with an intracardial injection of pentobarbital sodium (83 mg/kg body wt) immediately before the catheters were inserted, and the preparation was placed in a perfusion apparatus as previously described (24).

The initial perfusate (200 ml) consisted of Krebs-Henseleit solution, 1-day-old washed bovine erythrocytes (hematocrit, 30%), 5% bovine serum albumin (Cohn fraction V; Sigma, St. Louis, MO), 600 µM albumin-bound palmitate, 4 µCi of albumin-bound [1-14C]palmitate, and either 20 mM glucose and no insulin in the G group or 6 mM glucose and 250 µU/ml insulin in the I group. The perfusate (37°C) was continuously gassed with a mixture of 95% O2-5% CO2, which yielded arterial pH values of 7.2-7.3 and arterial PO2 and PCO2 values that were typically 102-140 and 36-43 Torr, in both the G and I groups, respectively.

The first 25 ml of perfusate that passed through the hindquarter were discarded. The left gastrocnemius-plantaris-soleus muscle group was clamped in situ with aluminum clamps precooled in liquid N2. The left iliac vessels were then tied off, and a clamp was fixed tightly around the proximal part of the leg to prevent bleeding. After an equilibration period of 20 min, the right leg was perfused at rest for 40 min at a perfusate flow of 5 ml/min (0.30 and 0.29 ml · min-1 · g-1 perfused muscle in the G and I groups, respectively; P > 0.05). After equilibration, arterial and venous perfusate samples were taken at 10, 20, 30, and 40 min for analysis of [14C]LCFA and 14CO2 radioactivities, as well as LCFA, glucose, and lactate concentrations. Arterial and venous perfusate samples for determinations of PCO2, PO2, and pH were taken at 10 and 40 min. Arterial perfusate samples were taken for determination of hematocrit and hemoglobin before equilibration. Mean perfusion pressures were 62.1 ± 9.2 and 73.2 ± 11.1 mmHg in the G and I groups, respectively (P > 0.05). At the end of the perfusion, the muscle group of the right leg was taken out and treated as described above.

Blood sample analyses. Arterial and venous perfusate samples were analyzed for glucose, lactate, and LCFA concentrations as well as for [14C]LCFA and 14CO2 radioactivities. Blood samples for glucose and lactate were put into 200 µM ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA, pH 7) and immediately analyzed using the YSI 1500 glucose and lactate analyzers (Yellow Springs Instrument, Yellow Springs, OH), respectively. Samples for LCFA were put in 200 µM EGTA (pH 7) and centrifuged, and the supernatant was kept frozen until analyzed. LCFA concentrations were determined spectrophotometrically using the WAKO NEFA-C test (Biochemical Diagnostics, Edgewood, NY). Plasma [14C]LCFA and 14CO2 radioactivities were determined as previously described in detail (24). Perfusate samples for the determination of PCO2, PO2, pH, and hemoglobin were collected anaerobically, placed on ice, and measured within 5 min of collection with an ABL-5 acid-base laboratory (Radiometer America, Westlake, OH) and spectrophotometrically (Sigma).

Muscle sample analyses. Muscle triglyceride concentration was determined as glycerol residues after extraction and separation of the muscle samples, as previously described (23). Malonyl-CoA levels were determined using neutralized perchloric acid extracts prepared from freeze-clamped muscle samples and analyzed as previously described (25).

Calculations and statistics. Palmitate delivery, fractional and total palmitate uptake, and percent and total palmitate oxidation were calculated as previously described (24, 25). Both percent and total palmitate oxidation were corrected for label fixation by using previously a determined acetate correction factor of 1.6 for both the G and I groups (25). Oxygen and glucose uptake as well as lactate release were calculated by multiplying perfusate flow by the respective arteriovenous difference in concentration and are expressed per gram of perfused muscle, which was previously determined to be 5.6% of body weight for unilateral hindquarter perfusion (25). Malonyl-CoA concentrations were calculated from the radioactive incorporation of acetyl-CoA into palmitic acid, corrected for unspecific labeling, and are expressed in picomoles per milligram of tissue (25). Muscle triglyceride synthesis rate was calculated as the product of muscle triglyceride fractional synthesis rate and postperfusion muscle triglyceride concentration (24).

Statistical evaluation of muscle triglyceride, glycogen, and malonyl-CoA data was performed using two-way ANOVA (StatSoft Statistica version 5.0, Tulsa, OK). A two-way ANOVA with repeated measures was used when determining changes over time, and a Newman-Keuls test for post hoc multiple comparisons was performed when appropriate. All other data were analyzed by one-way ANOVA. In all instances, an alpha  value of 0.05 was used to determine significance.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Substrate exchange across the hindquarter. Arterial perfusate glucose concentration did not vary significantly over time in either the G or I group (P > 0.05) but was threefold higher in the G than the I group (P < 0.05; Fig. 1). The difference in arterial glucose concentration was necessary to obtain similar glucose uptake rates in the G and I groups in the absence and presence, respectively, of insulin. Thus, as dictated by the protocol, glucose uptake was similar between the G and I groups (P > 0.05; Fig. 1). Arterial perfusate lactate concentration was not significantly different between groups at any time point (P > 0.05; Table 1) but increased by 35% over time in both the G and I groups (P < 0.05). Lactate release did not vary significantly over time in either group and was not significantly different between groups (P > 0.05; Table 1). Resting oxygen uptake did not vary over time and was not significantly different between the G (25.2 ± 1.0 µmol · g-1 · h-1) and I (29.1 ± 7.9 µmol · g-1 · h-1) groups (P > 0.05).


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Fig. 1.   Arterial glucose concentration and glucose uptake in rat hindquarters perfused at rest. Values are means ± SE for the glucose (G; n = 10) and insulin (I; n = 10) groups. Because there were no significant changes in values measured after 20, 30, and 40 min of perfusion, average values were used for each animal. * Significantly different compared with G, P < 0.05.


                              
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Table 1.   Effects of insulin on substrate concentration and exchange in rat hindquarters perfused at a predetermined rate of glucose uptake

Palmitate metabolism. As dictated by the protocol, perfusion palmitate concentration and delivery to the hindquarters were not significantly different between groups (P > 0.05; Table 1). Fractional (G: 0.083 ± 0.009 vs. I: 0.084 ± 0.008) and total uptake of palmitate (G: 10.1 ± 1.0 vs. I: 10.2 ± 0.6 nmol · min-1 · g-1) were not significantly different between the G and I groups (P > 0.05; Fig. 2) and did not vary significantly over time in either group (P > 0.05). Similarly, the percentage of palmitate oxidized (G: 9.9 ± 1.5 vs. I: 13.9 ± 2.6%) and total rate of palmitate oxidation (G: 0.8 ± 0.1 vs. I: 1.1 ± 0.2 nmol · min-1 · g-1) were not significantly different between the G and I groups (P > 0.05; Fig. 2) and did not vary significantly over time in either group (P > 0.05).


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Fig. 2.   Effects of insulin on total palmitate uptake and oxidation in rat hindquarters perfused at a predetermined rate of glucose uptake. Values are means ± SE for the glucose (G; n = 10) and insulin (I; n = 10) groups. Because there were no significant changes in values measured after 20, 30, and 40 min of perfusion, average values were used for each animal. Total palmitate oxidation was corrected for label fixation, as described in MATERIALS AND METHODS.

Muscle metabolite levels. Preperfusion muscle triglyceride concentrations were not significantly different between the G and I groups and did not vary significantly during the perfusion period (from 4.6 ± 0.8 to 4.1 ± 1.2 and from 4.5 ± 0.8 to 3.0 ± 0.4 µmol/g wet wt in the G and I groups, respectively, P > 0.05). Triglyceride synthesis was not significantly different between G and I groups (0.3 ± 0.03 vs. 0.3 ± 0.07 nmol · g-1 · min-1, respectively; P > 0.05). Preperfusion malonyl-CoA levels were not significantly different between the G and I groups and did not change significantly during the perfusion period (from 0.42 ± 0.1 to 0.43 ± 0.1 and from 0.42 ± 0.04 to 0.28 ± 0.05 pmol/mg wet wt in the G and I groups, respectively; P > 0.05).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Our results demonstrate that, at a predetermined rate of glucose uptake and palmitate delivery, the presence of insulin failed to alter muscle LCFA metabolism in resting perfused muscle. Thus, under these conditions, similar rates of LCFA uptake and oxidation as well as triglyceride synthesis were measured in perfused muscle at rest. In addition to the similarities observed in LCFA metabolism and in accordance with the proposed role of malonyl-CoA in the regulation of LCFA oxidation, there was no significant difference in muscle malonyl-CoA levels between groups. The similar malonyl-CoA levels in the presence or absence of insulin suggest that a mechanism associated with glucose flux may prevail over the independent effect of insulin in the regulation of malonyl-CoA production under these conditions.

The lack of change in LCFA uptake in muscle perfused at a predetermined rate of glucose uptake agrees with our previous results showing a positive correlation between glucose uptake and palmitate uptake (25). This correlation is also in agreement with previous studies in which glucose flux was manipulated by changes in insulin or glucose levels or both (8, 23, 27). Indeed, in muscle perfused under conditions of extreme carbohydrate and glucose depletion, extremely low glucose uptake was associated with a significant drop in LCFA uptake in contracting muscle (23). Likewise, in studies that have demonstrated an insulin-induced increase in LCFA uptake, glucose uptake, although not always measured, was probably increased by the use of supraphysiological insulin concentrations (8, 27). The insulin-induced increase in glucose uptake may have been critical for the increase in LCFA uptake measured in those studies. Thus, in these studies, one consistent observation is that LCFA uptake changed in parallel with glucose uptake under conditions of both low and high glucose uptake, and these results would be in line with the proposed relationship between glucose uptake and LCFA uptake. Although an acute effect of supraphysiological levels of insulin on LCFA uptake capacity cannot be disregarded (8), our results suggest that an intracellular regulatory mechanism, independent of insulin but intricately tied to glucose flux, may regulate LCFA uptake in muscle. This cellular mechanism may respond to changes in metabolic demand. Thus the metabolic demand imposed by a set rate of glucose uptake could elicit a specific demand for LCFA and regulate LCFA uptake via an increase in the production of glycerol 3-phosphate, the backbone of glycerolipid synthesis (9). Alternatively, regulation of LCFA uptake by glucose flux could also occur via activation of selective intracellular protein kinase-signaling cascades, such as those that activate protein kinase C-zeta /lambda (3).

Previous studies have demonstrated a decline in LCFA oxidation under hyperinsulinemic conditions in the presence or absence of maintained LCFA availability, and this has led to the well accepted conclusion that insulin has potent antioxidative effects in muscle (8, 13, 15, 21, 27). However, in these previous studies, the impact of elevated glucose flux per se on LCFA oxidation was generally overlooked. As suggested above, hyperinsulinemia is associated with an increase in glucose uptake, and the effects attributed to insulin could be due, in part, to the concomitant increases in glucose uptake, which can sometimes be dramatic. In a recent study using incubated soleus muscle, an increase in glucose supply in the absence of insulin was shown to decrease LCFA oxidation, demonstrating a possible role of glucose availability per se in the regulation of skeletal muscle LCFA oxidation (14). Our results of no change in LCFA oxidation under conditions of similar glucose uptake agree with this hypothesis and suggest that glucose flux may be critically linked to a regulatory mechanism for LCFA oxidation. It has been suggested that malonyl-CoA, a potent inhibitor of carnitine palmitoyltransferase I (CPT-I), could be a potential regulator of LCFA oxidation in muscle under some, but not all, experimental conditions (4, 5, 16, 17, 26). Under our conditions of similar glucose uptake, malonyl-CoA levels did not demonstrate any difference between groups. This lack of difference in malonyl-CoA concentrations is possibly due to comparable activation of acetyl-CoA carboxylase induced by the presence of similar intracellular citrate levels (1, 18). The lack of difference in malonyl-CoA levels between groups would also suggest that the role of insulin on acetyl-CoA carboxylase activation in muscle is limited under conditions of matched glucose uptake (11, 27).

The lack of change in intramuscular triglyceride content over time and the equal rates of triglyceride synthesis among groups suggest that the previously reported lipogenic effects of insulin may be secondary to the effects of glucose availability in perfused muscle (8, 14). Past studies have demonstrated insulin-induced increases in the rate of triglyceride synthesis in incubated soleus muscle and in the activity of glycerol-3-phosphate acyltransferase in BC3H-1 myocytes (6, 8, 14). Here again, this insulin-induced increase in triglyceride synthesis could have been related to a concomitant unreported increase in glucose uptake. In fact, in flexor digitorum brevis muscle incubated with insulin but no glucose, there was no independent effect of insulin on fatty acid incorporation into triglycerides (10). Alternatively, the lack of change in triglyceride synthesis does not exclude the possibility that other pathways of glycerolipid synthesis were directly affected by the presence of insulin.

In summary, our results demonstrate that the presence of insulin under conditions of similar glucose uptake does not alter muscle LCFA uptake or disposal toward oxidative and nonoxidative metabolism. Our results further suggest that cellular mechanisms induced by carbohydrate availability, but independent of insulin, may be important in the regulation of muscle LCFA metabolism. The effects of glucose flux on LCFA metabolism could be induced by glucose-mediated metabolic demand or by activation of putative glucose-sensitive signaling cascades.


    ACKNOWLEDGEMENTS

The present study was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR-45168 and the American College of Sports Medicine Foundation Grant FRG26.


    FOOTNOTES

Address for reprint requests and other correspondence: Address for reprint requests and other correspondence: L. P. Turcotte, Dept. of Kinesiology, University of Southern California, 3560 Watt Way, PED 107, Los Angeles, CA 90089-0652.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published March 12, 2002;10.1152/ajpendo.00553.2001

Received 18 December 2001; accepted in final form 5 March 2002.


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ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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